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PMC4086132 | aside from the symmetry of homomeric complexes , many protein domains are made of repeating units that are arranged in a symmetric manner .
proteins with such internal symmetry are interesting objects to study . since gene duplication is a relatively frequent event , it is possible that proteins with repeating units were the first complex structures to arise in the evolution of protein structures .
internally symmetric proteins have a diverse array of function ( 1 ) and are probably good starting structures for a de novo design of protein structures with a desired function .
the interface between the repeating units serves as a prototype of general protein protein interaction interface .
generally , detecting internal symmetry enhances our understanding of the protein structure , folding and function .
the recent study on the proton - gated urea channel from helicobater pylori ( 2 ) is an example in which symd was used to detect and characterize the internal symmetry of the molecule .
many algorithms have been described for detecting the internal symmetry of a protein structure ( 37 ) .
rcsb pdb server ( http://source.rcsb.org/jfatcatserver/symmetry.jsp ) allows users to examine internal pseudo - symmetry from structures in pdb ( 8) or scop ( 9,10 ) , but not the users own structural files .
the gangsta+ server ( http://agknapp.chemie.fu-berlin.de/gplus/index.php?page=symmetry ) takes structures from pdb , scop or the local drive , but does not output the symmetry axis nor does it give the order ( fold ) of the symmetry .
the symd webserver is a web application we built on the galaxy platform , which runs the symd algorithm .
a brief description of the symd algorithm is given below and a full description can be found in our previous publication ( 1 ) .
symd algorithm works by trying many different transformations ( rotation and translation ) that will result in a large number of residues structurally aligned between the original and the transformed structures .
the structure is judged to be symmetric if one or more of these trial transformations result in a large number of residues superimposed .
more specifically , the algorithm first duplicates the structure of interest , circularly permutes the sequence numbering by k residues and generates the k - th initial sequence alignment by aligning residue i of the original structure with residue i of the permuted structure for all i or all i greater than k. this initial alignment is then fed into the rse program ( 11 ) to obtain the optimal structure - based sequence alignment .
symd repeats this procedure for each initial shift k , from 1 to n-3 , ( alignment scan ) where n is the number of residues of the structure .
the goodness of the alignment is measured by t - score , which is a weighted number of aligned residue pairs ( 12 ) :
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\begin{equation * }
t = \sum\limits_{ij,|i - j| { > } s\ ; } { \frac{1}{{1 + \left ( { \frac{{d_{ij } } } { { d_0 } } } \right)^2 } } }
\end{equation * }
\end{document}where dij is the c distance between the aligned residue pair i and j , which are more than s residues apart where s = 3 to avoid self - alignments .
the distance cut - off d0 can be varied by the user ( the default value is 2 ) and the summation is over all aligned residue pairs .
symd reports the transformation matrix and the sequence alignment that produced the best t - score for each initial shift k. it also calculates the position and orientation of the ( potential ) symmetry axis , as well as the rotation angle and the translation along the symmetry axis from the calculated transformation matrix .
symd webserver is accessed in a standard web browser and is therefore independent of any operating system and requires no installation .
it allows users to upload their own structure files in pdb format as well as to retrieve structures from pdb ( http://www.rcsb.org/pdb/ ) or domain databases such as cath ( 13 ) or scop as inputs .
symd webserver uses the galaxy ( 14 ) platform , which has been used extensively in genomics research .
here we take advantage of the tool for running a workflow and of the extensibility the galaxy platform provides and apply it on protein symmetry studies .
the symd webserver is hosted in a vmware vsphere virtualized ubuntu 12 server with an apache webserver front - end proxy .
jmol ( http://www.jmol.org ) is used for structure visualization and jalview ( 15 ) for sequence alignment visualization and analysis .
similar to other galaxy systems , the symd webserver always has three panels : tools on the left for selecting input , initiating a computation , and viewing instructions ; details in the middle for managing tool settings and displaying data and history on the right for a list of input and output files as well as visualization tools .
users can upload their own structures of interest in pdb format or retrieve a chain or a domain from the scop , cath or pdb database , by selecting one of the options under provide data for computation in the left tools panel .
structures that are successfully fetched will be listed in the history panel on the right .
the content of the file can be displayed in the middle detail panel by clicking on the eye icon in the history panel .
the structure can be viewed in jmol by clicking the jmol link in the history panel .
, the user can choose the option perform symd calculation in the left tools panel .
the names of the stored structures are then shown in the drop - down list in the middle , detail , panel , from which the user can choose one structure for symd computation .
users may input different d0 cut - off ( range from 0.0 to 6.0 ) for t - score calculation .
when the initial shift value , which is the number of residues initially shifted for circular permutation and initial alignment , is explicitly specified , optimal alignment using only the specified initial shift will be calculated .
otherwise , symd will perform the alignment scan and report the best shift alignment which has the highest z - score .
the color changes from grey to green if computation is done successfully , or red if a problem is encountered . symd program generates three output files , which are listed in the history panel on the right when the calculation is finished .
they are the alignment scan information file ( x-info.txt ) , two superposed structures , one original and the other best ( highest scoring ) transformed structure , with the corresponding symmetry axis ( x-trfm.pdb ) , and the sequence alignment file for the best transformation ( x-best.fasta ) , where x is the name of the structure .
each of these files can be viewed directly ( by clicking the eye icon ) or downloaded ( by clicking the disc icon ) .
users can always retrieve the data later by opening the symd webpage and clicking the file name in the history panel .
the alignment scan information file ( figure 1a ) gives information on the optimal alignment obtained from each initial shift .
the items included in the default output are the t - score , z - score , the number of residues aligned , and the rotation angle and the translation along the rotation axis obtained from the optimal transformation matrix .
the rotation axis of the transformation that produced the best t - score overall is considered the symmetry axis of the molecule .
transformations from all other initial shifts in the alignment scan are considered as signal or noise depending on whether the rotation axis of the transformation aligns with that of the symmetry axis within a certain tolerance ( angle between the rotation axes is less than 18 = arcos(0.95 ) ) .
( b ) clicking the scatterplot hyperlink displays the z - score versus initial shift and the z - score versus angle interactive scatter plots .
the six signal peaks in the example plot indicate that the domain has a 7-fold symmetry . hovering the cursor over a point in the scatter plot
in addition to the information in the table format , symd webserver also displays two interactive scatter plots : z - score versus initial shift and z - score versus rotation angle ( figure 1b ) .
the number of signal peaks shown in the z - score versus rotation angle plot is equal to the number of repeating units of the symmetric protein minus one .
upon rotating every 90 degrees , the structure superposes to the original structure and produces a high z - score .
the x and y values of each point in the scatter plot can be displayed by hovering the cursor over the point . for proteins with open , helical symmetry , such as superhelices , e.g. 1bk5a
, the signal peaks show more distinctly in the z - score versus initial shift plot .
the transformed structure and the symmetry axis ( figure 2a ) can be downloaded in pdb format by clicking the disc icon in the x-trfm.pdb section in the history panel . to visualize the symmetry of the structure directly ,
the symmetry axis along with the original and the superposed transformed structures can be displayed in jmol ( figure 2b ) .
( a ) clicking the eye icon displays the pdb - format file in the detail panel .
( b ) clicking the jmol hyperlink displays the original and transformed structures and the symmetry axis in jmol .
the coordinates of the structures and the axis can also be downloaded by clicking the disc icon .
the sequence alignment generated by rse between the original and transformed structures is displayed in jalview for examination ( figure 3b ) .
the fasta format alignment file can also be viewed by clicking the eye icon under x-best.fasta section and downloaded by clicking the disc icon ( figure 3a ) .
( a ) clicking the eye icon displays the fasta format file in the detail panel .
in addition to the text explanation of different parameters and different results , the example run video in help section also provides interactive instruction for symd users .
the webserver also has a link where users can download the symd executable to run off - line .
the symd webserver is hosted in a vmware vsphere virtualized ubuntu 12 server with an apache webserver front - end proxy .
jmol ( http://www.jmol.org ) is used for structure visualization and jalview ( 15 ) for sequence alignment visualization and analysis .
similar to other galaxy systems , the symd webserver always has three panels : tools on the left for selecting input , initiating a computation , and viewing instructions ; details in the middle for managing tool settings and displaying data and history on the right for a list of input and output files as well as visualization tools .
users can upload their own structures of interest in pdb format or retrieve a chain or a domain from the scop , cath or pdb database , by selecting one of the options under provide data for computation in the left tools panel .
structures that are successfully fetched will be listed in the history panel on the right .
the content of the file can be displayed in the middle detail panel by clicking on the eye icon in the history panel .
the structure can be viewed in jmol by clicking the jmol link in the history panel .
, the user can choose the option perform symd calculation in the left tools panel .
the names of the stored structures are then shown in the drop - down list in the middle , detail , panel , from which the user can choose one structure for symd computation .
users may input different d0 cut - off ( range from 0.0 to 6.0 ) for t - score calculation .
when the initial shift value , which is the number of residues initially shifted for circular permutation and initial alignment , is explicitly specified , optimal alignment using only the specified initial shift will be calculated .
otherwise , symd will perform the alignment scan and report the best shift alignment which has the highest z - score .
the color changes from grey to green if computation is done successfully , or red if a problem is encountered .
symd program generates three output files , which are listed in the history panel on the right when the calculation is finished .
they are the alignment scan information file ( x-info.txt ) , two superposed structures , one original and the other best ( highest scoring ) transformed structure , with the corresponding symmetry axis ( x-trfm.pdb ) , and the sequence alignment file for the best transformation ( x-best.fasta ) , where x is the name of the structure .
each of these files can be viewed directly ( by clicking the eye icon ) or downloaded ( by clicking the disc icon ) .
users can always retrieve the data later by opening the symd webpage and clicking the file name in the history panel .
the alignment scan information file ( figure 1a ) gives information on the optimal alignment obtained from each initial shift .
the items included in the default output are the t - score , z - score , the number of residues aligned , and the rotation angle and the translation along the rotation axis obtained from the optimal transformation matrix .
the rotation axis of the transformation that produced the best t - score overall is considered the symmetry axis of the molecule .
transformations from all other initial shifts in the alignment scan are considered as signal or noise depending on whether the rotation axis of the transformation aligns with that of the symmetry axis within a certain tolerance ( angle between the rotation axes is less than 18 = arcos(0.95 ) ) .
( b ) clicking the scatterplot hyperlink displays the z - score versus initial shift and the z - score versus angle interactive scatter plots .
the six signal peaks in the example plot indicate that the domain has a 7-fold symmetry . hovering the cursor over a point in the scatter plot displays the x- and y - coordinates of the point .
in addition to the information in the table format , symd webserver also displays two interactive scatter plots : z - score versus initial shift and z - score versus rotation angle ( figure 1b ) .
the number of signal peaks shown in the z - score versus rotation angle plot is equal to the number of repeating units of the symmetric protein minus one .
for example , 1gena is a 4-bladed -propeller with a 4-fold internal symmetry . upon rotating every 90 degrees , the structure superposes to the original structure and produces a high z - score .
the x and y values of each point in the scatter plot can be displayed by hovering the cursor over the point . for proteins with open , helical symmetry , such as superhelices ,
e.g. 1bk5a , the signal peaks show more distinctly in the z - score versus initial shift plot .
the transformed structure and the symmetry axis ( figure 2a ) can be downloaded in pdb format by clicking the disc icon in the x-trfm.pdb section in the history panel . to visualize the symmetry of the structure
directly , the symmetry axis along with the original and the superposed transformed structures can be displayed in jmol ( figure 2b ) .
( a ) clicking the eye icon displays the pdb - format file in the detail panel .
( b ) clicking the jmol hyperlink displays the original and transformed structures and the symmetry axis in jmol .
the coordinates of the structures and the axis can also be downloaded by clicking the disc icon .
the sequence alignment generated by rse between the original and transformed structures is displayed in jalview for examination ( figure 3b ) .
the fasta format alignment file can also be viewed by clicking the eye icon under x-best.fasta section and downloaded by clicking the disc icon ( figure 3a ) .
( a ) clicking the eye icon displays the fasta format file in the detail panel .
in addition to the text explanation of different parameters and different results , the example run video in help section also provides interactive instruction for symd users .
the webserver also has a link where users can download the symd executable to run off - line .
symd webserver is the first web - based application allowing users to upload proteins of interest or retrieve them from various databases , and analyze the symmetry property of the protein .
it provides not only the numerical data in different formats but also a visual analysis of the symmetry axis in jmol , and interactive text and video tutorials .
the webserver is useful for synthetic biological design and for studying protein structure , function and evolution .
intramural research program of the nih , national cancer institute , center for cancer research . | internal symmetry of a protein structure is the pseudo - symmetry that a single protein chain sometimes exhibits .
this is in contrast to the symmetry with which monomers are arranged in many multimeric protein complexes .
symd is a program that detects proteins with internal symmetry .
it proved to be useful for analyzing protein structure , function and modeling .
this web - based interactive tool was developed by implementing the symd algorithm . to the best of our knowledge ,
symd webserver is the first tool of its kind with which users can easily study the symmetry of the protein they are interested in by uploading the structure or retrieving it from databases .
it uses the galaxy platform to take advantage of its extensibility and displays the symmetry properties , the symmetry axis and the sequence alignment of the structures before and after the symmetry transformation via an interactive graphical visualization environment in any modern web browser .
an example run video displays the workflow to help users navigate .
symd webserver is publicly available at http://symd.nci.nih.gov . | INTRODUCTION
SYSTEM DESCRIPTION
Hardware and software
User interface
Input
Output
CONCLUSIONS
FUNDING |
PMC3788586 | the binding of igg
fc to cell surface and soluble serum ligands
triggers a variety of immunological processes including phagocytosis ,
cytotoxicity , inflammation , and immunosuppression .
the
structure of the fc domain is stabilized by the n - linked glycan , attached
to asn297 of each igg heavy chain .
this glycan is critical for fc function :
genetic or enzymatic removal leads to an almost complete loss of antibody
effector functions .
moreover , composition of the glycan
is a key parameter in determining the balance between pro - inflammatory
or anti - inflammatory effects .
for example , removal of the core 1,6-linked
fucose residue of the igg1 fc glycan enhances binding for fcriiia , while elevation of the levels of terminal 2,6-sialylation
leads to decreased natural killer cell activation and other potent cell - mediated immunosuppressive effects .
in addition to natural
variations in fc glycosylation , a growing number of enriched and engineered
fc glycoforms are finding application in therapeutic monoclonal antibodies
when a particular balance of effector functions is desirable .
the three - dimensional structure of the fc glycoforms has been investigated as have the independent effects of glycan and protein engineering
on receptor binding . by comparison
, however , relatively little is known about the interdependence
of glycan composition and protein structure on receptor binding .
the n - linked glycans in igg1 fc are complex , mostly core - fucosylated ,
biantennary - type structures with varying amounts of bisecting glcnac ,
terminal galactose , and sialic acid residues .
levels of sialylation are low with < 10% of total fc glycans from
serum igg being sialylated .
tri- or tetra - antennary
glycans are generally not found in serum igg fc .
the absence of larger ,
branched , and/or sialylated structures is notable , especially when
compared to the glycosylation of other serum or cell - surface glycoproteins .
x - ray crystallographic and nmr studies of the igg fc domain have defined the conformation of the n - linked
glycans at asn297 . in the complex - type igg fc glycoforms ,
the conformation
of the oligosaccharide is well conserved and contacts over 500 of the surface of each c2 domains .
the
six arms of the glycan chain makes several stable interactions with
hydrophobic amino acid residues of the c2 domain .
the terminal
gal6 ( see legend to figure 1 for terminology ) on the six arm has been shown to restrict glycan
flexibility through interaction with the fc protein backbone .
key amino acid residues that interact with the
6-arm glycans through hydrogen bonds and hydrophobic interactions
include : phe241 , phe243 , val262 , val264 , asp265 , lys246 , and arg301 .
aromatic rings of phe241 and phe243 form ch interactions
with the glcnac2 and glcnac5 residues
of the fc glycan and contribute to the stability of the fc domain .
the three arm on the other hand makes fewer contacts with the protein
backbone with hydrophobic interactions between man4 and
lys334 being the only observable protein glycan interaction .
while the presence of extensive protein glycan interactions
suggests a relatively immobile carbohydrate conformation and reduced
enzymatic processing , recent nmr spectroscopic studies indicate a
more dynamic and mobile role for the fc glycans .
nonetheless , the relatively limited processing of the fc glycan
indicates a reduced accessibility to glycan reactive enzymes in the
golgi apparatus .
mass spectrometric analysis of n - glycans released from
igg fc - f241a .
( b ) the data from panel a were processed
with the maximum entropy 3 function of masslynx to convert multiply
charged ions to singly charged ions . the position of the fucose residue
in the triantennary glycans was not determined .
the ion at m / z 3169 gave a composition corresponding
to the tetrasialylated triantennary glycan , but this was not confirmed
by fragmentation .
( c ) an example of negative ion collision - induced
dissociation spectrum of the monosialylated , fucosylated biantennary
glycan .
( d ) spectra showing
trigalactosylated structures with three ( triply charged ) and four
( quadruply charged ) sialic acids attached , respectively .
residue labeling follows that of vliegenhart
et al . with the additional modifications
of 7 for sialic acid , 1 for 16-linked
core fucose .
the symbolic representation
of glycans follows that of harvey et al . with residues in both the schematic diagrams and molecular graphics
following the color scheme of the consortium for functional glycomics .
the influence of hydrophobic residues
in the protein glycan
interface of the fc on glycan processing was first observed in mutational
studies on mouse human chimeric igg3 antibodies , where replacement
of phe241 , phe243 or val264 with ala resulted in elevated levels of
mono- and disialylated glycans and decreased binding to c1q and fcr
activity .
in addition , ribosomal display
has been used to discover site - specific fc mutants with enhanced fcriiia
binding .
this approach identified the
f243l mutant which , in addition to enhanced fcriiia binding ,
also exhibited altered glycan processing including decreased fucosylation .
two possible explanations have been proposed
to explain these observations : either
the alteration of the protein carbohydrate interaction directly
affected the fc protein conformation or the increased glycan processing
affected fcr binding . to discriminate between these possibilities ,
these glycoform - controlled mutants still
exhibited similar reduced
fcr binding indicating that the conformation of the c2
domain is modulated by glycan
furthermore we show that the effects of these glycan - destabilizing
mutations on fcr binding are independent of mutations which
directly affect the fc fcr interface .
thus , combinatorial
mutagenesis can yield antibodies with novel effector properties . for
example , we combine fc fcr interface mutations known
to selectively enhance fcriib binding , with a glycan
protein
interface mutation that decreases binding to all fcrs , to provide
an igg which more broadly eliminates activatory ( but not inhibitory )
receptor binding .
this study therefore clarifies the role of the fc
glycan in maintaining antibody structure and provides routes to the
development of antibody therapeutics with bespoke effector functions .
glycan
interface , we determined the crystal structure of recombinant igg1
fc f241a ( table 1 ) .
this mutant has been previously
reported by lund et al . to reduce fcr binding and increase
glycan processing .
prior to crystallization ,
we performed electrospray ionization ( esi ) mass spectrometric analysis
of the n - linked glycans which confirmed extensive branching and terminal
sialylation ( figure 1 ) .
the igg1 fc f241a
crystallized in the primitive orthorhombic spacegroup , p212121 , with one molecule of the
fc homodimer in the asymmetric unit .
data were collected to a resolution
of 1.9 ( table 1 ) . as observed in many
crystal structures of the fc ,
the protein and glycan moieties of the c2
domain from one chain ( chain b ) exhibited a high degree of disorder
( figure 2d ) .
i(hkl)|/hkl ii(hkl;i ) , where i(hkl;i ) is the intensity of an individual
measurement and i(hkl
is the average intensity from multiple observations .
k|fcalc||/hkl |fobs| rfree is calculated as for rwork but using
only 5% of the data , which were sequestered prior to refinement .
interpretable electron density was observable for
seven monosaccharide
residues on chain a ( figure 2e , f ) . in the structure
of the wild - type fc , phe241 packs with the glcnac2 and
man3 residues ( figure 2b , c ) .
in contrast , electron density in our structure of the
f241a mutant , revealed the f241a site - directed substitution and a
destabilization of the protein carbohydrate interface ( figure 2e , f ) .
the n - linked glycan rests in the established
position along the c2 domain with defined electron density
corresponding to the 6-arm , while only diffuse density corresponding
to the 3-arm is observed in the 2fo fc map .
packing of n - link glycans in native ( a c )
and f241a mutant
( d f ) igg fc .
glycans are displayed as blue ( glcnac ) , red ( fuc ) ,
and green ( man ) sticks .
protein is displayed as a gray cartoon with
four hydrophobic residues at the protein
. overall structure of ( a ) native ( pdb i d 3ave ) and ( b ) f241a mutant
igg1 fc .
the c2 domains from chain a ( b and e ) are shown with
close - ups of the protein - glycan interfaces ( c and f ) .
electron density corresponding to carbohydrate is
depicted as a blue mesh ( 2fo fc map contoured at 1 ) around the carbohydrate
moiety of the mutant fc reported herein .
this weak density indicates that
the 3-arm is either conformationally
disordered or glycoforms differing in 3-arm composition are adopting
different conformations where the x - ray scattering does not sum to
yield consensus electron density .
previous reports had suggested that
the intrinsic flexibility of the 3-arm causes the lack of electron
density of the galactose in native galactosylated fc .
consistent with a loss of electron density of the 3-arm , detailed
negative - ion esi mass spectrometry with fragmentation analysis revealed
extensive branching on the 3-arm but not the 6-arm ( figure 1 ) .
the localized induction of disorder at
the 3-arm does not fully
account for the apparent increase in accessibility of the glycans
to golgi - resident glycosyltransferases ( e.g. , increase in 6-arm galactosylation )
or the reduction in fcr binding affinity ( figure 2 ) .
protein interface not sufficiently
captured by low - temperature x - ray crystallographic methods . for example , the hydrophobic interface mutations
may affect the position of equilibrium between the protein
bound
and free conformations proposed by nmr studies to
yield more accessible glycans and potentially more widely spaced c2
domains .
interestingly , nmr studies have
indicated that 2,6-sialylation has minimal impact on glycan
dynamics in the fc domain .
this finding
is entirely consistent with our observation of an elevation in 6-arm
sialylation by electrospray mass spectrometry and the conserved conformation
of the 6-arm observed by x - ray crystallography ( figure 2c , f ) .
protein
interface can induce at least localized glycan disorder , we next sought
to determine to what extent the reduction in fcriiia binding
can be attributed to disruption of the glycan protein interface
or to changes in glycan processing . to this end
a series of human
igg1 fc mutants was generated containing mutations that modulated
the protein carbohydrate interactions within the fc : f241a ,
f243a , v262e , and v264e .
the panel of igg1 b12 mutants was expressed
in either human embryonic kidney ( hek ) 293 t or glcnac transferase
( gnt ) i - deficient hek 293s cells .
the
glycans were released from the purified antibody via protein n - glycosidase
f ( pngase f ) .
the free sugars were fluorescently labeled and resolved
via normal - phase high - performance liquid chromatography ( hplc ) using
a tsk - amide column .
the hplc spectra from igg1 fc mutants expressed
in hek 293 t ( black spectra ) or gnt i - deficient hek 293s cells ( blue
spectra ) are shown in figure 3 .
the glycans
from igg b12 expressed in hek 293 t cells are composed of a series
of fucosylated , biantennary , complex - type carbohydrates , typical of
the protein - directed glycosylation observed for igg ( figure 3a ; black spectrum ) .
the most abundant species observed
were agalactosylated structures with smaller amounts of mono and digalactosylated
structures .
a small population of sialylated material was also present ,
showing the typical glycan profile for recombinant igg1 fc as reported
previously .
consistent with previous analyses
of igg3 , mutations interrupting the hydrophobic
protein glycan interface led to dramatic increases in terminal
1,4 linked galactose levels , with digalactosylated species
representing the most abundant glycan populations for all mutants
generated from hek 293 t cells ( figure 3b e ;
black spectra ) .
tri- and tetra - antennary species , not normally observed
on fc , were detected , most notably on v262e and v264e . additionally ,
increased bisecting glcnac and terminal sialylation were also evident
for these mutants .
an unusual digalactosylated , trisialylated species
was also detected in the hplc spectra of all of the mutants .
this structural assignment was
confirmed by electrospray mass spectrometry of recombinant igg1 fc
( figure 1 ) and b12 mutants ( figures s1 and s2 ) .
further matrix - assisted laser desorption / ionization
time - of - flight mass spectrometry ( maldi - tof - ms ) ( figure s3 ) and hplc analysis of desialylated mutants ( figures s4 and s5 ) were carried out to validate
the hplc assignments .
in contrast to the heterogeneous complex - type
glycan spectra observed
when the igg panel was expressed in hek 293 t cells , expression in
gnt i - deficient hek 293s cells led to a purely oligomannose - type glycan
profile composed of man5glcnac2 together with
a small population of fucosylated man5glcnac2 ( figure 3 ; blue spectra ) . the latter structure
has previously been shown to arise via the inefficient gnt i - independent
fucosylation pathway .
protein interface affected neither
oligomannose processing to man5glcnac2 nor the
proportion of gnt i - independent fucosylation .
in addition to glycan
analysis , the n - glycosylation site occupancy at the asn297 was examined ,
as previous report showed decreased glycan site occupancy for the
hydrophobic mutants .
a combination of
pngase f treatment and trypsin digestion , followed by maldi - tof - ms ,
showed that the n - glycosylation site on fc asn297 is fully occupied
for all the mutants ( figures s6 and s7 ) .
the discrepancy might be due to the different antibody isotypes used
or methods used to examine site occupancy .
hplc analysis of 2aa - labeled
n - linked glycans from monoclonal igg1
b12 mutants expressed in hek 293 t and hek 293s cells .
normal - phase
hplc analysis of 2-aa - labeled n - linked glycans released from target
antibody glycoforms by in - gel protein pngase f digestion .
glycan profile
of igg1 b12 expressed in hek 293 t ( black ) and hek 293s ( blue ) for
the following variants : ( a ) wild type ; ( b ) f241a ; ( c ) f243a ; ( d ) v262e ;
and ( e ) v264e .
the y - axis displays relative fluorescence
( rf ) . to investigate the impact of sialylation
on fcr binding , in vitro glycosyltransferase
reactions were used to generate
hyper-2,6-sialylated igg ( figure 4 ) .
as there is a potential for 2,3-linked sialic acid to be present
in glycoproteins derived from hek 293 t cells , we first performed a
sialidase digestion .
sialylation was not detected by hplc analysis
( figure 4a , b ) . the sialidase - treated material
was then subjected to sequential 1,4-galactosylation and 2,6-sialylation
( figure 4c , d , respectively ) .
hplc analysis
revealed that a trace population ( 5% ) of monogalctosylated
glycans remained after the galactosyltransferase reaction ( figure 4c ) .
generation of differentially glycosylated igg1 fc . normal - phase
hplc analysis of 2-aa - labeled n - linked glycans , released from target
antibody glycoforms by in - gel pngase f digestion . ( a ) glycan profile
of monoclonal igg1 b12 .
( b ) glycan profile of igg1 incubated with
50 u / ml clostridium perfringens neuraminidase
for 48 h at 37 c .
( c ) glycan profile of igg1 incubated with
25 g / ml 1,4-galactosyltransferase ( b4galti ) and 80 m
uridine 5-diphosphogalactose in 50 mm hepes , 10 mm mncl2 , ph 7.5 for 48 h at 37 c .
( d ) glycan profile of igg1
sequentially treated with b4galti and 2,6-sialyltransferase
i ( st6gali ) as described above .
fcrs by determining the binding of
our panel of igg mutants to the extracellular regions of fcria ,
fcriia , fcriib , fcriiia , and fcriiib ( figure 5 ) .
elisa of monoclonal igg variants binding human fcria ,
fcriia ,
fcriib , fcriiia , and fcriiib .
the fcrs
were plated at 5 g / ml overnight at 4 c , igg variants
f241a , f243a , v262e , and v264e were incubated for 1.5 h , and binding
was detected by hrp - conjugated goat antihuman fab antibody
. symbolic
representation of igg mutation and glycovariants : solid black square
= wild - type native ; solid blue square = wild - type man5glcnac2 ; open black triangle = mutant native ; open blue triangle
= mutant man5glcnac2 ; solid red square = wild - type
hypergalactosylated and hypersialylated , elisa binding curves of the
four igg hydrophobic mutants for ( a ) fcria , igg variant starting
concentration at 10 g / ml . ( b ) fcriia , igg variant starting
concentration at 100 g / ml . ( c ) fcriib , igg variant starting
concentration at 300 g / ml . ( d ) fcriiia , igg variant
starting concentration at 100 g / ml . ( e ) fcriiib , igg
variant starting concentration at 300 g / ml . ( f ) fcriiia ,
igg variant starting concentration at 100 g / ml . all data points
represent the calculated mean of two independent measurements from
a total of at least two experiments .
consistent with studies with fcri , our data show that
abolition
of the fc glycan protein hydrophobic interaction resulted in
a decreased fc affinity for the fcria ( figure 5d ) . a similar pattern , albeit
to different extents
, was observed in the binding analysis with fcriiia ,
fcriia , fcriib , and fcriiib ( figure 5 ) . the high affinity , activatory fcria and
low - affinity , inhibitory fcriib were least prone to such modulation .
interestingly , the v264e mutant contains the highest level of sialylated
structures among mutants tested ( figure 3e ) .
. the v264e mutation would be
predicted to perturb the overall trajectory of the glycan away from
the surface of the c2 domain and may account for the extensive
glycan terminal processing due to increased steric accessibility . to investigate the effect of these hydrophobic mutations on fcr
binding independent of the differential fc glycoforms , we compared
fcr binding to the panel of uniformly glycosylated igg1 fc
mutants expressed in the gnt i - deficient hek 293s cells ( figure 3 ; blue spectra ) .
consistent with previous
reports , wild - type igg1 fc with man5glcnac2 glycosylation
exhibits increased affinity
for fcriiia ( figure 5d ) and decreased
affinity for the fcriib ( figure 5e )
compared with fc with native biantennary complex glycans .
we also demonstrate that the man5glcnac2 glycoform
exhibits decreased fc affinity for fcriia , which is highly
homologous to fcriib ( figure 5b , c ) .
mutations that disrupt the hydrophobic interface significantly
decrease the fc affinity for fcriia , fcriiia , and fcriiib ,
largely independently of the glycoform .
this effect is evident by
comparison of the wild - type and mutants expressed in gnti - deficient
hek 293s cells ( figure 3 ; blue spectra ) .
mutations
affecting the hydrophobic interface resulted in significantly decreased
fc affinity for fcriia , fcriiia , and fcriiib
( figure 5b , d , e ) , while interestingly , no significant
changes were observed for fcriib binding ( figure 5c ) . for the high affinity fcria ,
together ,
our results show that the hydrophobic mutations disrupt the fc binding
to the activatory fcrs independently of the fc glycan , indicating
that the productive engagement of fc fcr requires the
protein glycan interaction at the fc c2 domain . unlike f241a , v262e , and v264e ,
the f243a mutant expressed as the
man5glcnac2 glycoform has a minimal effect on
fcrs binding ( figure 5 ) .
this minimal
effect can be explained by the different protein glycan interfaces
of oligomannose- , hybrid- , and complex - type antibody glycoforms as
revealed by x - ray crystallography . in
contrast to the other residues , f243 exhibits minimal van der waals
contacts with the 6-arm mannose residues in the predicted structure
of the man5glcnac2 glycoform .
therefore , in contrast to the significant effect of f243a
mutation on the mobility of complex - type glycans , f243a would be predicted
to have a minimal impact on the mobility of man5glcnac2 structures .
the relative affinity of the single mutants for
fcrs is summarized in table s2 .
remarkably , the interaction between fcriib and igg1 fc is
relatively unperturbed by the disruption of fc protein
we hypothesized that
further enhancements to the fcriib selectivity could be achieved
by combining our glycan
protein interface mutations with previously
reported hinge - proximal c2 mutations ( s267e / l328f ) that exhibit
selective fcriib - binding .
we generated
two novel igg1 fc mutants , v262e / s267e / l328f and v264e / s267e / l328f ,
which exhibited enhanced affinity for the fcriib and , by comparison
to the double mutant alone , significantly decreased
affinity for fcria , fcriia , and fcriiib ( figure 6 ; table s3 ) .
the double
mutant s267e / l328f shows significantly increased affinity for the
inhibitory fcriib and decreased affinity for the activatory
fcriiia , while the binding for the other fcrs remains
similar to the wild type ( figure 6 ) , consistent
with published data . the increased levels
of glycan terminal processing and bisection of these triple mutants
are comparable to those of the single v262e and v264e mutants ( figure s8 ) .
elisa of monoclonal igg variants binding
human fcria , fcriia ,
fcriib , fcriiia and fcriiib .
the fcrs
were plated at 5 g / ml overnight at 4 c , igg variants
s267e / l328f , v262e / s267e / l328f , and v264e / s267e / l328f were incubated
for 1.5 h and binding was detected by hrp - conjugated goat antihuman
fab antibody .
symbolic representation of igg mutation and glycovariants :
solid black square = wild - type native ; solid blue square = wild - type
man5glcnac2 ; open black triangle = mutant native ;
open blue triangle = mutant man5glcnac2 , elisa
binding curves of the four igg hydrophobic mutants for ( a ) fcria ,
igg variant starting concentration at 10 g / ml . ( b ) fcriia ,
igg variant starting concentration at 100 g / ml . ( c ) fcriib ,
igg variant starting concentration at 300 g / ml . ( d ) fcriiia ,
igg variant starting concentration at 100 g / ml . ( e ) fcriiib ,
igg variant starting concentration at 300 g / ml . all data points
represent the calculated mean of two independent measurements from
a total of at least two experiments .
protein
interactions modify the fc structure relevant for fcr binding
( figure 5 ) .
however , it has previously been
shown that hypersialylation of igg1 fc , regardless of the linkage
type , decreases fc binding to the fcria , fcriib , and
fcriiia , which also reduces antibody - mediated cytotoxicity
both in vivo and in vitro . therefore , we generated hypergalactosylated
and hypersialylated human igg1 fc ( figure 4 ) and examined their affinity for fcriiia , a critical determinant
of natural killer cell - mediated adcc .
usually , the fc fcr
affinity strongly correlates with effector functions measured by cellular
assays .
however , our data indicate that the hypersialylated
fc binds to the fcriiia with very similar affinity to the wild
type , supported by both elisa ( figure 5f ) and
surface plasmon resonance ( spr ) data ( figure 7 ) .
one possible explanation for the reduced cytotoxicity is that
terminal sialic acid might interact with inhibitory sialic acid binding
ig - like lectins ( siglec ) present on the surface of immune cells including
macrophages and natural killer cells .
this minimal effect of fc sialylation on fcriiia binding
is also supported by the structural analysis presented here , which
reveals that the protein
glycan interface , rather than glycan
terminal processing , modulates c2 domain conformation ( figure 5 ) .
the igg variants were injected at 5 different concentrations
at a flow rate of 30 l / min : igg and igg hypersialylated ( 0.67 ,
0.33 , 0.17 , 0.083 , and 0.042 m ) ; igg man5glcnac2 ( 0.33 , 0.17 , 0.083 , 0.042 , and 0.021 m ) .
sensorgrams were fitted with a global
1:1 interaction , and the ka , kd , and kd were calculated ,
all using bia evaluation software 2.0.3 .
kd values are reported as mean sd , and sensorgrams are representative
a total of three independent experiments .
modulation of the multiple hydrophobic
interactions between the
protein surface and the glycan within the igg fc strongly influences
fcr binding , presumably by altering the dynamics of the fc
and affecting the adoption of receptor binding conformations .
while
these mutations also modulate glycan processing , expression of fc
domains with homogenized glycoforms revealed that the interaction
between the glycan and the protein independently influences fcr
binding . by combining interface mutations with glycan engineering ,
new portfolios of effector activities can be generated .
the pfuse vector
with the human igg1 fc insert was obtained from invivogen , u.k .
the
vectors encoding igg1 b12 light and heavy chains were kindly provided
by prof .
protein mutagenesis was performed using the
quikchange kit ( agilent technology , u.k . ) to generate the igg1 fc
mutant f241a and the full - length igg1 b12 mutants f241a , f243a , v262e ,
and v264e .
the mutated fc ( residues 225447 , swiss - prot accession
number p01857.1 ) encompassing hinge , c2 and c3 domains was cloned
into the mammalian expression vector , phlsec .
the vectors containing
full - length fcria , fcriia ( his131 variant ) , fcriib ,
fcriiia ( val158 variant ) , fcriiib , and mouse -1,4-galactosyltransferase
i ( b4galti ) were all purchased from open biosystems , u.k .
the vector
containing full - length rat 2,6-sialyltransferase i ( st6gali )
was a gift from prof .
the soluble extracellular regions of each fcr , b4galti and
st6gali were cloned into the phlsec vector as described for the fc :
fcria ( residues 16288 ; swiss - prot accession number bc152383 ) ; fcriia
( residues 34217 ; swiss - prot accession number bc020823 ) ; fcriib
( residues 42225 ; swiss - prot accession number nm_001190828 ) fcriiia ( residues 16288 ; swiss - prot accession number bc033678 ) ; fcriiib
( residues 17200 ; swiss - prot accession number bc128562 ) ; st6gali
( residues 89403 ; swiss - prot accession number np_001106815 ) ; and b4galti ( residues 127399 ; swiss - prot accession number bc053006 ) . for
b4galti , the residue cys339 was mutated to thr to minimize the potential
for aggregation .
the fc , fcrs , b4galti , and
st6gali were expressed in hek 293 t cells as previously described .
briefly , hek 293 t cells ( atcc number crl-1573 )
were grown to 90% confluence and transiently transfected with polyethyleneimine
( pei ) , using a transfection mix with
dna and pei in ratio of 1:1.5 . following transfection ,
cells were
grown in dmem/1% fetal bovine serum at 37 c and 5% co2 for 5 days .
protein was purified from cell supernatant by immobilized
metal affinity chromatography using chelating sepharose fast flow
ni - agarose beads ( ge healthcare , u.k . )
followed by size
exclusion chromatography using a superdex s-200 column equilibrated
in phosphate buffered saline ( pbs ) ( for fcrs ) or 10 mm hepes
ph 7.4 , 150 mm nacl ( for fc ) .
full - length igg1 b12 was transiently
expressed in hek 293 t or gnt i - deficient hek 293s cells .
prior to transfection , light and heavy chain plasmids were mixed
in a mass ratio of 4:1 , and the total dna was mixed with pei in a
mass ratio of 1:1.5 . after incubation for 4 days at 37 c , cell
culture supernatant was harvested and igg1 b12 was purified using
protein a sepharose ( ge healthcare , u.k . ) according to the manufacturer s
directions .
oligosaccharides
were released from coomassie blue - stained reducing sds - page gel bands
containing 40 g of igg fc as previously reported .
gel bands were rehydrated with 30 l of
30 mm nahco3 ph 7.0 containing 100 units / ml pngase f ( new
england biolabs , u.k . ) and incubated for 12 h at 37 c .
desialylation was carried out using linkage
nonspecific neuraminidase from clostridium perfringens ( new england biolabs , u.k . ) for 48 h at 37 c .
glycans were
labeled with anthranilic acid ( 2-aa ) as previously described and separated using tsk amide column ( sigma - aldrich ,
u.k . ) .
the released n - glycans , dissolved in water and 2-aa labeling
buffer ( 3/8 , v / v ) were mixed with 2-aa and sodium cyanoborohydride
and incubated for 1 h at 80 c ; excess 2-aa dye was removed using
a spe - ed amide-2 column ( systematic systems , u.k . ) .
solvent
a was acetonitrile , solvent b was milli - q water , and solvent c was
800 mm ammonium hydroxide adjusted to ph 3.85 using acetic acid .
the gradient
was a constant 71.6% a for 6 min at a flow rate of 0.8 ml / min , followed
by a linear gradient of 71.635% a over 80 min at 0.8 ml / min .
afterward , the gradient was a linear 3571.6% a for 1 min at
a flow rate of 0.8 ml / min ; then at the same gradient , the flow rate
increased from 0.8 ml / min to 1.2 ml / min over 1 min and followed by
the same gradient and flow rate for 13 min .
the run finished by returning
the flow rate to 0.8 ml / min over 1 min .
fluorescence was detected
at 425 nm , and the excitation wavelength was 360 nm .
chromatography
data were processed by the empower software ( all instruments and software
from interlink scientific services limited , u.k . ) .
assignments were
consistent with previously reported serum igg n - linked glycan profiles and were confirmed by maldi - tof ms and exoglycosidase
analysis .
hyper-2,6-sialylated igg was generated by incubating with
b4galti in the presence of 80 m uridine 5-diphosphogalactose
( sigma - aldrich , u.k . ) in 50 mm hepes , 10 mm mncl2 , ph 7.5
for 48 h at 37 c .
the hyper-1,4-galactosylated igg was
treated with st6gali in the presence of 70 m cytidine-5-monophospho - n - acetylneuraminic acid ( sigma - aldrich , u.k . ) in 50 mm hepes ,
10 mm mncl2 , ph 6.5 for 48 h at 37 c .
the composition
of the glycoform was verified by hplc analysis after each enzymatic
treatment .
recombinantly
expressed mutant igg1 fc ( f241a ) was concentrated to 7.0 mg / ml and
crystallized after 10 days using the sitting drop vapor diffusion
method using 100 nl protein plus 100 nl precipitant equilibrated against
95 l reservoirs .
crystals of f241a igg1 fc grew at room temperature
in a precipitant containing 28% polyethylene glycol monomethyl ether
2000 in 0.1 m bis - tris buffer at ph 6.5 .
crystals were flash frozen
by immersion in a cryoprotectant containing the mother liquor diluted
in 30% polyethylene glycol and then rapidly transferred to a gaseous
nitrogen stream .
crystallographic data were collected to 1.9
resolution at beamline i04 at the diamond light source ( oxfordshire ,
u.k . ) .
the structure was solved using molecular replacement
with the program phaser using native
fc ( pdb accession no .
model building was performed with
coot and iteratively refined using restrained
refinement in the ccp4 supported program , refmac5 , with the incorporation
of translation - libration - screw ( tls ) parametrization and automatically
generated local noncrystallographic symmetry restraints.model quality was validated with molprobity .
recombinant fcrs at
5 g / ml in pbs were coated on high - binding microtiter plates
( 3690 , corning , ny , u.s.a . ) overnight at 4 c .
coated plates
were washed with pbs containing 0.05% tween 20 ( sigma - aldrich , u.s.a . )
and blocked for 1 h at room temperature with 5% bovine serum albumin
( bsa ) in pbs .
recombinant igg1 b12 expressed from hek 293 t cells or
from gnt i - deficient hek 293s cells were then added and allowed to
bind for 1.5 h at room temperature .
plates were washed five times
with pbs containing 0.05% tween and binding was detected using a horse
radish peroxidase ( hrp)-conjugated fab fragment specific for human
igg fab ( abcam , u.k . ) .
the 3,3,5,5-tetramethylbenzidine
substrate ( tmb ; thermo scientific , u.s.a . ) was used for development
according to the manufacturer s directions and was stopped
by the addition of 2 m h2so4 .
absorbance was
measured at 450 nm on a spectramax m5 ( molecular devices , california ,
u.s.a . )
apparent affinity was calculated as
the concentration of igg1 b12 corresponding to half - maximal binding
on the elisa binding curve .
spr experiments
were carried
out using the biacore t100 instrument ( ge healthcare , u.k . ) .
briefly ,
the fcriiia ( val158 variant ) was immobilized onto the surface
of cm5 sensor chip ( ge healthcare , u.k . ) to about 1000 ru during each
independent experiment .
all experiments were carried out in the hbs - ep
running buffer ( 10 mm hepes , 150 mm nacl , 3 mm edta and 0.005% surfactant
p20 ) , at a flow rate of 30 l / min .
the monoclonal igg1 b12 protein
and the hypersialylated and high - mannose glycol variants were injected
at 5 different concentrations , allowing 2 min for association and
3 min for dissociation .
after each run , the sensor chip was regenerated
using 10 mm glycine - hcl , ph1.7 .
the sensorgrams were fitted to a global
1:1 interaction , and the ka , kd , and kd were calculated ,
all using biaevaluation software 2.0.3 ( ge healthcare , u.k . ) . | biologically active
conformations of the igg1 fc homodimer are
maintained by multiple hydrophobic interactions between the protein
surface and the n - glycan .
the fc glycan modulates biological effector
functions , including antibody - dependent cellular cytotoxicity ( adcc )
which is mediated in part through the activatory fc receptor , fcriiia .
consistent with previous reports , we found that site - directed mutations
disrupting the protein carbohydrate interface ( f241a , f243a ,
v262e , and v264e ) increased galactosylation and sialylation of the
fc and , concomitantly , reduced the affinity for fcriiia .
we
rationalized this effect by crystallographic analysis of the igg1
fc f241a mutant , determined here to a resolution of 1.9 , which
revealed localized destabilization of this glycan protein interface .
given that sialylation of fc glycans decreases adcc , one explanation
for the effect of these mutants on fcriiia binding is their
increased sialylation .
however , a glycan - engineered igg1 with hypergalactosylated
and hypersialylated glycans exhibited unchanged binding affinity to
fcriiia .
moreover , when we expressed these mutants as a chemically
uniform ( man5glcnac2 ) glycoform , the individual
effect of each mutation on fcriiia affinity was preserved .
this effect was broadly recapitulated for other fc receptors ( fcri ,
fcriia , fcriib , and fcriiib ) .
these data indicate
that destabilization of the glycan protein interactions , rather
than increased galactosylation and sialylation , modifies the fc conformation(s )
relevant for fcr binding .
engineering of the protein carbohydrate
interface thus provides an independent parameter in the engineering
of fc effector functions and a route to the synthesis of new classes
of fc domain with novel combinations of affinities for activatory
and inhibitory fc receptors . | Introduction
Results
and Discussion
Conclusions
Experimental Procedures |
PMC5426155 | fused tooth can be defined as a single enlarged or joined tooth in which the tooth count reveals a missing tooth when the anomalous tooth is counted as one .
bilateral fusion has an incidence ranging from 0.01% to 0.04% in the primary dentition , and 0.05% in the permanent dentition , out of which , incisors and canines are mostly affected .
fusion may be classified as complete or incomplete depending on the developmental stage of the teeth .
if it occurs before the beginning of calcification , the union will be complete with the formation of a single large tooth .
complete fusion may be characterized by a single pulp chamber and root canal , or a single pulp chamber and two separate root canals , or separate pulp chambers as well as root canals .
fusion of teeth is known to result from physical force or pressure causing contact of the developing tooth germs , hypervitaminosis a , viral infection during pregnancy and the use of thalidomide , heredity , aberration of the ectoderm and mesoderm during morphodifferentiation stage of tooth development , and syndromes such as achondrodysplasia , chondroectodermal dysplasia , focal dermal hypoplasia , and osteopetrosis .
a 10-year - old boy visited the department of pedodontics and preventive dentistry with the chief complaint of an unaesthetic large bifid anterior right tooth .
there was no family history of dental anomalies , and neither was there any history of trauma to the teeth or jaws .
intraoral examination revealed unusually large maxillary central incisors , out of which , the maxillary right incisor ( mri ) was bifurcated with a groove traversing the labial and palatal aspects of the crown [ figures 1 and 2 ] . there was the absence of any caries and both incisors responded normally to thermal and electric pulp testing ( c pulse pulp tester , foshan coxo medical instrument co. ltd ) .
an orthopantomogram revealed the absence of maxillary lateral incisors ( mli ) [ figure 3 ] . a cone - beam computed tomography ( cbct ) scan revealed two distinct roots that were connected through the groove , containing two distinct connected root canals , in relation to the mri
however , the mli had a single large root and root canal [ figures 46 ] .
a diagnosis of incomplete fusion of the mri ( mesiodistal diameter of 16.5 mm ; crown length of 12 mm ) and complete fusion of the mli ( mesiodistal diameter of 12.5 mm ; crown length of 12 mm ) was made , based on the appearance of the incisors and the fact that the mlis were missing .
cone - beam computed tomography with three - dimensional reconstruction cone - beam computed tomography ( longitudinal ) cone - beam computed tomography ( cross - section ) after informed consent was obtained for carrying out treatment , upper and lower alginate impressions were made and poured with dental stone .
a diagnostic mock - up was done using mockup wax on the mri that matched with the size and shape of the adjacent mli [ figure 7 ] .
a putty index was made of the mock up wax , following which , bis - acryl composite ( kettenbach , germany ) was placed into the index and transferred to the mri , intraorally .
pretreatment esthetic evaluation was carried out to check for adequate phonetics , lip support and smile line , and for approval from the patient and his parents .
an incisal overlap veneer preparation ( facial reduction of 0.5 mm ) with a shoulder finish line was carried out for the mri [ figure 8 ] .
a digital mock - up was done using adobe photoshop 7.0 software so that adequate information regarding the size , shape , and extent of the veneer could be sent to the laboratory . in consisted of the following steps : tooth preparation for veneer
the free selection tool was used to select and trace the outline of the adjacent central incisor.the outline was converted into a smart object.the inversion tool was used to derive a mirror image of the same.the image was saved as a jpeg file.the jpeg file was imported into the coral draw software and placed over the image of the fused tooth.the cloning tool was used to mimic the gingiva to create an illusion of distinction between the central and the lateral incisors that constituted the mri .
the free selection tool was used to select and trace the outline of the adjacent central incisor .
the jpeg file was imported into the coral draw software and placed over the image of the fused tooth .
the cloning tool was used to mimic the gingiva to create an illusion of distinction between the central and the lateral incisors that constituted the mri .
the digital mock - up helped in providing adequate information to the laboratory with regard to the addition of gingival porcelain on the veneer .
the fabricated ips e - max press veneer ( ivoclar vivadent ag , schaan , liechtenstein ) , with an incisal lapping preparation , was cemented using a translucent shade of dual cure composite resin luting cement ( rely - x arc , 3m - espe , germany ) [ figures 9 and 10 ] .
there has only been one case of bilateral fusion of permanent maxillary incisors without the involvement of supernumerary teeth , reported in literature . moreover ,
ours is the first case where there has been incomplete and complete fusion , bilaterally .
the mri was classified as incomplete fusion since there was a bifurcation of the crown along a groove running labially and lingually till the cervical margin , without the confluence of dentin between the two sections .
however , the mli was classified as complete fusion ( between the central and lateral incisors ) because it consisted of only a single large crown and root structure .
intraoral periapical radiographs are insufficient for understanding the morphology of roots and its canal systems in fused teeth .
moreover , it was difficult to interpret the same with the help of the orthopantomograph .
cbct that can produce three - dimensional images of oral structures are helpful particularly in such cases . since the volume of the scan is smaller , the resolution of the image is higher with little effective radiation dose to the patient .
the cbct scans obtained provided us with accurate information with regard to the number of roots , canals and the region of the divide , both occlusally and labially , in the mri . since the cbct scan showed a connection between the canals in the mri , it was decided to place a veneer to avoid endodontic treatment , hemisectioning , and full crown placement on both the separated teeth .
endodontic treatment was not preferred for the mri because the tooth was asymptomatic and responded favorably to pulp tests .
orthodontic movement was not planned either because there was no shift in the midline , proclination , or spacing .
although the mli had a primary canine adjacent to it , the child or his parents had no complaint with regard to its esthetics .
however , when the primary canine would get replaced with the succedaneus canine , its reshaping with composite resin or a veneer may be necessary to make it resemble a lateral incisor . in a case reported by sammartino et al .
, involving fusion of both upper central incisors , surgical sectioning was carried out , and both teeth were restored with all - ceramic crowns after orthodontic alignment and endodontic treatment . in another case of geminated central incisors , esthetic rehabilitation was carried out using all ceramic crowns for both the incisors . however , in this case report , a conservative approach using ips e - max press veneer ( ivoclar vivadent ag , schaan , liechtenstein ) was carried out . in a case reported by samimi et al .
, a new lateral incisor tooth was fixed between the fused teeth ( maxillary central and lateral incisors ) and the canine using fiber - reinforced composite resin , after creating space by stripping the fused teeth .
the contours of the teeth were designated by lines made with a thin tapered bur and restored with composite veneering .
the pink composite was used to mimic gingival papillae between teeth . however , in this case , the mri was made to appear like two teeth by veneering with ips e - max tooth colored and gingival ceramic , instead of pink composite .
the contours of the divided teeth that constituted the mri were designated using the adobe photoshop software .
the groove that persists lingual to the mri may be susceptible to caries and periodontal disease .
however , since the groove was found to be shallow , periodic topical fluoride application and maintenance of oral hygiene should be sufficient to prevent caries or periodontal disease .
the patient is on a follow - up schedule every 6 months and has been asymptomatic for over 2 years . | this case report highlights the management of a case of bilateral complete and incomplete fusion of maxillary incisors in a 10-year - old child .
a mock - up was done on the diagnostic cast .
pretreatment esthetic evaluation was done using bis - acryl composite temporaries which were transferred intraorally from the diagnostic cast using a putty index .
an incisal overlap veneer preparation was done , following which , an ips e - max veneer was cemented .
a digital mock - up was carried out using the adobe photoshop and corel draw softwares to aid in laboratorial fabrication of the veneer . | Introduction
Case Report
Discussion
Financial support and sponsorship
Conflicts of interest |
PMC4565810 | experimental animals : six intact purpose - bred cats ( 3 males and 3 females ) , 1 to 3 years of age [ 1.8 1.0 ( mean standard deviation ) years ] and weighing from 3.0 to 5.4 kg [ 4.3 0.8 ( mean standard deviation ) kg ] , were used in the present study .
all cats were judged to be in good to excellent health based upon a physical examination .
food was withheld for at least 12 hr before drug administration , but the cats were allowed free access to water prior to each treatment .
guide for the care and use of laboratory animals prepared by rakuno gakuen university .
the animal care and use committee of rakuno gakuen university approved this study ( approved no .
study design : the cats received 5 treatments with a minimum 14-day washout period ( 14 to 685 days ) between treatments . in each treatment , the cats were administered one of the following 5 doses of alfaxalone - hpcd ( alfaxan , jurox pty .
rutherford , nsw , australia ) : an i m dose of 1 mg / kg ( im1 ) , 2.5 mg / kg ( im2.5 ) , 5 mg / kg ( im5 ) , 10 mg / kg ( im10 ) or an iv dose of 5 mg / kg ( iv5 ) .
the total volumes of injection of alfaxalone - hpcd were 0.45 0.08 , 1.0 0.2 , 2.1 0.4 , 4.5 0.8 and 1.9 0.4 ml for the im1 , im2.5 , im5 , im10 and iv5 treatments , respectively .
the i m doses were injected into the dorsal lumbar muscle of the cats by using a 23-gauge , 1-inch needle ( top injection needle , top co. , ltd . ,
the iv dose was administered for over approximately 60 sec through a 22-gauge catheter ( supercath , medikit co. , ltd . ,
sedative effects and cardio - respiratory function were evaluated in the cats before ( baseline ) and at 2 , 5 , 10 , 15 , 20 , 30 , 45 , 60 , 90 , 120 , 150 and 180 min after starting the drug administration .
evaluation of sedative effects : the sedative effects of alfaxalone - hpcd were subjectively evaluated using a composite measure scoring system modified from a sedative scale previously used in dogs .
the observer ( j. t. ) was not blinded , but responsible for the evaluation using this scoring system throughout the present study .
the scoring system consisted of 5 categories : spontaneous posture , placement on side , response to noise , jaw relaxation and general attitude .
these categories were rated in scores 0 to 2 for jaw relaxation , 0 to 3 for placement of side and general attitude , or 0 to 4 for spontaneous posture and response to noise based on responsiveness expressed by the cats ( table 1table 1.composite measure scoring system for evaluating sedative effects in catsscorespontaneous posturestanding0tired and standing1lying but can rise2lying with difficulty rising3unable to rise4placement on sideresists strongly0modest resistance1slight resistance2no resistance3response to noisejump0hears and moves1hears and twitches ear2barely perceives3no response4jaw relaxationpoor0slight1good2general attitudeexcitable0awake and normal1tranquil2stuporous3sedation score*016this scoring system consisted of 5 categories ( spontaneous posture , placement on side , response to noise , jaw relaxation and general attitude ) .
these categories were rated in scores 0 to 2 , 0 to 3 or 0 to 4 based on responsiveness expressed by the cats . *
the sedation score was calculated as a sum of scores for the 5 categories : spontaneous posture , placement on side , response to noise , jaw relaxation and general attitude . ) .
the sedation score was calculated as a sum of scores in the 5 categories ( a maximum of 16 ) .
in addition , times to onset of lateral recumbency , the first appearance of spontaneous movement , head lift and unaided standing after starting the drug administration , and the durations of lack of spontaneous movement and maintenance of lateral recumbency were recorded .
this scoring system consisted of 5 categories ( spontaneous posture , placement on side , response to noise , jaw relaxation and general attitude ) .
these categories were rated in scores 0 to 2 , 0 to 3 or 0 to 4 based on responsiveness expressed by the cats . *
the sedation score was calculated as a sum of scores for the 5 categories : spontaneous posture , placement on side , response to noise , jaw relaxation and general attitude .
measurements of cardio - respiratory valuables : lead ii electrocardiography ( ecg ) , heart rate ( hr ; beats / min ) , rectal temperature ( rt;c ) , mean arterial blood pressure ( mabp ; mmhg ) and percutaneous oxygen saturation of hemoglobin ( spo2;% ) were recorded before and after the drug administration until reliable data were collected .
ecg , hr and spo2 were recorded by a patient monitoring system ( ds-7210 , fukuda denshi co. , ltd . ,
. respiratory rate ( rr ; breaths / min ) was counted by observing thoracic movements .
rt was measured with a digital thermometer ( thermo flex for animal , astec co. , ltd . ,
mabp was indirectly measured by an oscillometric method ( pet map , ramsey medical , inc . ,
hudson , oh , u.s.a . ) using a blood pressure cuff approximately 40% circumference of the measuring site in width placed around the clipped tail base of each cat .
the arterial blood pressure was
measured three times at each assessment , and the average of these measurements was defined as arterial blood pressure . statistical analysis :
the sedation score was reported as median quartile deviation and analyzed by the kruskal - wallis test to assess the changes with time for each treatment .
differences in the sedation score among the treatments were compared by the kruskal - wallis test with the steel - dwass test for post hoc comparisons .
times related to the sedative effects and cardio - respiratory variables were reported as mean standard deviation .
times related to the sedative effects were compared by one - way ( treatment ) factorial anova with the bonferroni test for post hoc comparisons among treatments .
the cardio - respiratory variables were analyzed using one - way ( time ) factorial anova with the dunnet test for post hoc comparisons for each treatment .
observations and/or perceived adverse effects related to drug administration were compared between treatments by use of chi square test .
the times required for the i m injection were 5 5 , 11 9 , 8 5 and 35 18 sec in the im1 , im2.5 , im5 and im10 treatments , respectively . in the im10 treatment , the time required for the i m injection was longer compared to the im1 , im2.5 and im5 treatments ( p<0.001 , p=0.006 and p=0.002 , respectively ) .
vocalization during the i m administration was observed in 3 cats ( 50% ) , 2 cats ( 33% ) , 4 cats ( 67% ) and 4 cats ( 67% ) receiving the im1 , im2.5 , im5 and im10 treatments , respectively . on the other hand , no cat receiving the iv5 treatment showed discomfort ( vocalization , attempting to bite at injection site or aggressively excited behavior ) during the administration .
there was no significant difference in the incidence of vocalization during the i m administration between the treatments ( p=0.605 ) .
no cats showed discomfort ( excessive grooming at injection site or avoiding touching the injection site ) , and
swelling , redness and/or other inflammatory changes around the i m injection site after the experiment on each occasion .
the sedative effects : times associated with the sedative effects of each treatment are shown in table 2table 2.times related to sedative effects after starting intramuscular ( i m ) or intravenous ( iv ) administration of alfaxalone - hpcd in catsalfaxalone administration1 mg / kg im2.5 mg / kg im5 mg / kg im10 mg / kg im5 mg / kg ivtime to onset of lateral recumbency ( sec)451 129 * 213 143152 85128
2227 14time to the first appearance of spontaneous movement ( min)15 10 * 27 742 1274 1229 12time to head lift ( min)22 10 * 40 1575 18100 1848 6time to unaided standing ( min)30 10 * 60 1095 24121 2259 6duration of lack of spontaneous movement ( min)10 11 * 21 835 1672 1229 12duration of maintenance of lateral recumbency ( min)16 12 * 40 1576 21115 2250 5data are expressed as mean standard deviation .
* statistical analysis was performed among three treatments ( im2.5 , im5 and im10 ) , because of small samples in the im1 treatment .. the cats ceased to move after being recumbent , except for one cat each after the im1 and im2.5 treatments .
all cats after the im2.5 , im5 and im10 treatments showed lateral recumbency within 7 min after starting the injection . on the other hand ,
the im1 treatment produced lateral recumbency in only 3 cats ( 50% ) , and consequently , it was impossible to attempt statistical comparison using this treatment group . therefore , the times related to the sedative effects were only statistically compared amongst the im2.5 , im5 , im10 and iv5 treatments . the times to head lift and unaided standing and duration of maintenance of lateral recumbency were significantly longer in the im5 ( p=0.004 , p=0.014 and p=0.009 , respectively ) and im10 treatments ( p<0.001 , p<0.001 and p<0.001 , respectively ) ,
compared to the im2.5 treatment .
the times to first appearance of spontaneous movement and durations of lack of spontaneous movement were markedly longer in the im10 treatment , compared to the im2.5 ( p<0.001 and p<0.001 ) and im5 treatments ( p<0.001 and p=0.005 ) .
the times to head lift and unaided standing were significantly longer in the im5 than those in the iv5 treatment ( p=0.033 and p=0.011 ) .
all observations were completed at 45 min after the im1 treatment , at 90 min after the im2.5 and iv5 treatments and at 120 min after the im5 treatment , because the cats fully recovered and could walk normally .
* statistical analysis was performed among three treatments ( im2.5 , im5 and im10 ) , because of small samples in the im1 treatment .
1.median ( quartile deviation ) sedation score in 6 cats before and after starting intramuscular ( i m ) or intravenous ( iv ) administration of alfaxalone - hpcd . based on responsiveness expressed by the cats ,
the categories of a composite measure scoring system were rated in scores 0 to 2 for jaw relaxation , 0 to 3 for placement of side and general attitude , or 0 to 4 for spontaneous posture and response to noise ( see table 1 ) .
the sedation score was calculated as a sum of scores in these 5 categories . a : significant difference from 1
e : significant difference from 5 mg / kg iv ( p<0.05 ) .. each treatment produced a higher sedation score compared to each baseline value ( p<0.001 ) .
a maximum sedation score of 16 was observed in 2 cats ( 33% ) after the im2.5 treatment and all cats ( 100% ) each after the im5 , im10 and iv5 treatments .
the median sedation scores peaked at 10 min after the im1 treatment , 15 min after the im2.5 treatment , 10 min after the im5 treatment , 10 to 20 min after the im10 treatment and 2 to 5 min after the iv5 treatment .
the sedation scores in cats administered the im5 and im10 treatments were significantly higher compared to the im1 ( p<0.001 and p<0.001 ) and im2.5 treatments ( p=0.029 and p<0.001 ) .
there was no significant difference in the sedation score between the im5 and iv5 treatments ( p=0.984 ) .
median ( quartile deviation ) sedation score in 6 cats before and after starting intramuscular ( i m ) or intravenous ( iv ) administration of alfaxalone - hpcd . based on responsiveness expressed by the cats ,
the categories of a composite measure scoring system were rated in scores 0 to 2 for jaw relaxation , 0 to 3 for placement of side and general attitude , or 0 to 4 for spontaneous posture and response to noise ( see table 1 ) .
the sedation score was calculated as a sum of scores in these 5 categories . a : significant difference from 1
e : significant difference from 5 mg / kg iv ( p<0.05 ) . during the recovery period
, ataxia was observed in all cats ( 100% ) administered the im2.5 , im5 , im10 and iv5 treatments .
transient muscular tremors were observed in 2 cats ( 33% ) , 4 cats ( 67% ) , 6 cats ( 100% ) , 6 cats ( 100% ) and 5 cats ( 83% ) administered the im1 , im2.5 , im5 , im10 and iv5 treatments , respectively .
opisthotonus - like posture was also observed in one cat ( 17% ) , 4 cats ( 67% ) , 3 cats ( 50% ) and 3 cats ( 50% ) administered the im2.5 , im5 , im10 and iv5 treatments , respectively ( fig .
2.opisthotonus-like posture observed in cats during the recovery period . during the recovery period , opisthotonus - like posture was observed in one cat ( 17% ) received 2.5 mg / kg of intramuscular ( i m ) alfaxalone , 4 cats ( 67% ) received 5 mg / kg of i m alfaxalone , 3 cats ( 50% ) received 10 mg / kg of i m alfaxalone and 3 cats ( 50% ) received 5 mg / kg of intravenous ( iv ) alfaxalone .
a cat showed opisthotonus - like posture from 60 to 90 min after the im5 treatment ( a ) .
another cat showed opisthotonus - like posture from 90 to 120 min after the im10 treatment ( b ) . ) .
in addition , paddling of the forelimbs was observed in 4 cats ( 67% ) and one cat ( 17% ) administered the im10 and iv5 treatments , respectively , and running was observed in one cat ( 17% ) each administered the im2.5 , im5 and iv5 treatments .
in addition , one cat ( 17% ) vomited during the recovery period after the im10 treatment .
opisthotonus - like posture observed in cats during the recovery period . during the recovery period , opisthotonus - like posture was observed in one cat ( 17% ) received 2.5 mg / kg of intramuscular ( i m ) alfaxalone , 4 cats ( 67% ) received 5 mg / kg of i m alfaxalone , 3 cats ( 50% ) received 10 mg / kg of i m alfaxalone and 3 cats ( 50% ) received 5 mg / kg of intravenous ( iv ) alfaxalone .
a cat showed opisthotonus - like posture from 60 to 90 min after the im5 treatment ( a ) .
another cat showed opisthotonus - like posture from 90 to 120 min after the im10 treatment ( b ) .
changes in cardio - respiratory valuables : the cardio - respiratory variables are summarized in table 3table 3.changes in cardio - respiratory variables before and after starting intramuscular ( i m ) or intravenous ( iv ) administration of alfaxalone - hpcdminutes after starting administration of alfaxalonebaseline2510152030456090120150180rt ( c)im138.1 0.438.4 0.438.1 0.238.2 0.338.0 0.338.0 0.337.8 0.437.7 0.6n.d.n.d.n.d.n.d.n.d.im2.538.4 0.338.5 0.338.5 0.238.3 0.238.0 0.237.9 0.2 * 37.5 0.1 * 37.4 0.2 * 37.7 0.4 * 37.8 0.3*n.d.n.d.n.d.im538.3 0.238.8 0.4 * 38.7 0.438.5 0.338.2 0.338.1 0.337.7 0.2 * 37.2 0.2 * 37.0 0.2 * 37.3 0.5 * 38.2 0.4n.d.n.d.im1038.3 0.438.5 0.438.4 0.538.3 0.538.1 0.5 * 37.9 0.537.6 0.437.1 0.4 * 36.6 0.4 * 35.7 0.7 * 36.3 1.0 * 36.9 1.0 * 36.9iv538.5 0.538.3 0.538.1 0.437.8 0.437.7 0.4 * 37.4 0.4 * 37.0 0.4 * 36.8 0.4 * 37.2 0.8 * 37.7 0.4*n.d.n.d.n.d.hr ( beats / min)im1171 29180 23188 46178 31177 30176 29179 47177 38n.d.n.d.n.d.n.d.n.d.im2.5172 21178 29179 42161 31157 30155 35167 43207 44211 36218 34n.d.n.d.n.d.im5169 11191 25188 24185 15173 19172 14154 29171 29210 34 * 203 38179 25n.d.n.d.im10161 9200 17 * 194 9 * 175 5168 6161 7152 5142 8142 16192 36203 49 * 188 32192iv5160 13180 12167 11148 13137 11143 24152 43189 32216 33 * 185 12n.d.n.d.n.d.rr ( breaths / min)im148 2180 2444 1433 1340 2738 2447 1978 59n.d.n.d.n.d.n.d.n.d.im2.564 3165 3033 7 * 28 7 * 28 11 * 27 15 * 22 4 * 27 8 * 31 9 * 46 16n.d.n.d.n.d.im557 1446 735 11 * 26 8 * 24 5 * 21 5 * 19 4 * 19 5 * 25 7 * 33 8 * 38 13*n.d.n.d.im1055 1456 2727 14 * 21 9 * 18 6 * 17 5 * 16 4 * 14 5 * 19 14 * 30 25 * 29 9 * 30 8 * 32iv546 1117 2 * 21 2 * 19 3 * 19 3 * 17 3 * 21 9 * 22 8 * 33 12 * 40 9n.d.n.d.n.d.data are expessed as mean standard deviation . rt : rectal temperature , hr : heart rate , rr : respiratory rate .
im1 : i m administration of 1 mg / kg alfaxalone , im2.5 : i m administration of 2.5 mg / kg alfaxalone , im5 : i m administration of 5 mg / kg alfaxalone , im10 : i m administration of 10 mg / kg alfaxalone , iv5 : iv administration of 5 mg / kg alfaxalone .
the observation was completed at 45 min after the im1 treatment , at 90 min after the im2.5 and iv5 treatments , and at 120 min after the im5 treatments , because the cats recovered from the sedation and could walk up and down normally . *
significant difference from baseline value ( p<0.05 ) .. the rt significantly decreased from baseline at 20 min , at 30 min , at 45 min and at 15 min after the m2.5 , im5 , im10 and iv5 treatment , respectively .
a mild hypothermia was detected in 4 cats ( 34.9 to 35.9c ) from 90 to 150 min after the im10 treatment . a transient increase in hr from 2 to 5 min after the im10 treatment was observed . a transient increase in hr during the recovery period was also detected in the im5 , im10 and iv5 treatments .
in particular , clinically relevant hypotension ( mabp < 60 mmhg ) was observed in each one cat from 10 to 30 min after the im10 treatment ( 50 to 57 mmhg ) and from 2 to 20 min after the iv5 treatment ( 55 to 58 mmhg ) .
spontaneous breathing was maintained in all cats , but the rr significantly decreased from baseline after the im2.5 , im5 , im10 and iv5 treatments .
the iv5 treatment produced a transient hypoxemia in 2 cats ( spo2 87% and 88% ) early after
injection .
im1 : i m administration of 1 mg / kg alfaxalone , im2.5 : i m administration of 2.5 mg / kg alfaxalone , im5 : i m administration of 5 mg / kg alfaxalone , im10 : i m administration of 10 mg / kg alfaxalone , iv5 : iv administration of 5 mg / kg alfaxalone . n.d . : not done .
the observation was completed at 45 min after the im1 treatment , at 90 min after the im2.5 and iv5 treatments , and at 120 min after the im5 treatments , because the cats recovered from the sedation and could walk up and down normally . * significant difference from baseline value ( p<0.05 ) .
in the present study , the i m administration of alfaxalone - hpcd produced a dose - dependent sedation and immobilization with mild cardio - respiratory depression in healthy cats .
the i m alfaxalone - hpcd at 10 mg / kg produced a similar degree , but longer lasting sedation and immobilization , compared to the i m alfaxalone - hpcd at 5 mg / kg .
the i m alfaxalone - hpcd at 5 mg / kg produced a similar degree of sedation and immobilization to the iv alfaxalone - hpcd at 5 mg / kg . the duration of sedation , however , was longer lasting after the i m alfaxalone - hpcd at 5 mg / kg , compared to that after the iv alfaxalone - hpcd at 5 mg / kg . during recovery , tremors , ataxia ,
opisthotonus - like posture and transient paddling were observed in cats administered i m or iv alfaxalone - hpcd .
one cat vomited during recovery from the i m alfaxalone - hpcd at 10 mg / kg .
based on the dosing recommendations from the product information sheet , an i m administration of alfaxalone - hpcd ( 10 mg / kg ) is expected to induce deep sedation or light anesthesia .
thus , we chose four increasing i m doses of alfaxalone - hpcd up to 10 mg / kg in the present study .
some cats vocalized during the i m administration of alfaxalone - hpcd , but there was no significant difference in the incidence of vocalization between the i m treatments . in the iv5 treatment ,
alfaxalone - hpcd has a neutral ph and does not cause pain and tissue irritation after iv administration or perivascular injection .
in addition , grubb et al . mentioned that the i m alfaxalone - hpcd at 10 mg / kg seemed to cause moderate to profound discomfort in cats because of its excessive injection volume ( 1
ml / kg ) .
therefore , we propose that the discomfort observed in the study was a volume associated effect and not a property of the alfaxalone - hpcd formulation itself . actually , there was no swelling , redness or changes in the skin observed around the site of injection during and after the experiment .
the european federation of pharmaceutical industries associations and the european centre for the validation of alternative methods provide a guideline for the administration i m volumes .
the reported i m volume considered as good practice is 0.25 ml / kg and the maximal dose volume is 0.5 ml / kg in the guideline , although there is no description for dose volume in cats . according to this guideline , the i m alfaxalone - hpcd dose considered as good practice is 2.5 mg / kg , and the maximal dose is 5 mg / kg based on the
concentration of alfaxalone in the approved product ( 10 mg / ml ) .
we considered that the im10 treatment was not a practical volume because the im10 treatment ( 1 ml / kg of the approved alfaxalone - hpcd product ) produced discomfort during injection and required longer duration of i m administration .
the development of a more concentrated alfaxalone - hpcd product is desirable in order to reduce the discomfort associated with large i m injection volumes and promote practical convenience .
the objective of the present study was to investigate the sedative effects of i m alfaxalone - hpcd in cats without any other medicants .
therefore , we adopted the experimental design that had the least amount of nociceptive stimulation ( i.e. manipulations ) during the assessment of sedation effect and the cardio - respiratory variables . for similar reasons , we did not perform tracheal intubation during the experiment .
we adopted the composite measure scoring system for the evaluation of sedative or anesthetic effect .
the scoring system was modified from the existing scoring system used for evaluating sedative and analgesic effects of medetomidine in dogs that consisted of 6 categories ( spontaneous posture , placement on side , response to noise , jaw relaxation , general attitude and nociceptive response to interdigital pad pinch ) .
these categories seemed to be suitable for the evaluation of the extent of sedation in cats , however , it was also
anticipated that nociceptive stimulation would have some influence on the evaluation of sedative effect produced by alfaxalone - hpcd because of its poor analgesic property .
therefore , we modified the existing scoring system by removing the category of nociceptive response to interdigital pad pinch . in the present study , the i m administration of alfaxalone - hpcd at 2.5 , 5 and 10 mg / kg produced a clinically relevant sedative effect
the i m alfaxalone - hpcd at 1 mg / kg did not provide reliable sedation for handling in healthy cats .
the sedative scores after the im5 and im10 treatments were higher than that after the im2.5 treatment .
in addition , the times to first appearance of spontaneous movement and durations of lack of spontaneous movement were markedly longer in the im10 treatment , compared to the im2.5 and im5 treatments .
muir et al . reported that iv alfaxalone - hpcd produced a dose - dependent unresponsiveness in cats .
the observations in the present study show that i m alfaxalone - hpcd produced a dose - dependent sedative effect in cats .
the i m alfaxalone - hpcd at higher dose ( 5 and 10 mg / kg ) produced a marked and prolonged sedation and immobilization in cats .
an i m dose of alfaxalone - hpcd at 2.5 mg / kg may provide
enough sedation for securing vascular access in healthy cats .
the iv administration of alfaxalone - hpcd at 5 mg / kg is a recommended dose for anesthetic induction in cats [ 23 , 30 ] .
whittem et al . reported that all the 8 cats receiving 5 mg / kg of iv alfaxalone - hpcd became anesthetized shortly after the start of injection and the times to first head lift , and the times to unaided standing were 45 and 69 min , respectively .
these findings are consistent with our results in the cats administered the iv5 treatment . in the present study ,
the im5 and im10 treatments produced slower onset , but achieved a maximum sedation score ( score 16 ) in all cats equivalent to the iv5 treatment .
it is possible that i m alfaxalone - hpcd at 5 or 10 mg / kg may achieve anesthetic induction as well as reliable sedation in cats .
further investigation including an examination of the degree of difficulty in tracheal intubation will be
necessary to determine the proper i m anesthetic induction doses of alfaxalone - hpcd in cats .
it has been reported that an iv administration of alfaxalone - hpcd resulted in uneventful recovery in dogs [ 2 , 22 ] and cats [ 23 , 29 ] . on the other hand
, it has also been reported that the iv alfaxalone - hpcd produced a poor quality of recovery in dogs and cats [ 20 , 33 ] .
reported that twitching , paddling , face rubbing , opisthotonus and tremors during recovery were observed in cats receiving iv alfaxalone - hpcd or propofol .
it has also been reported that an iv alfaxalone - hpcd induction was associated with more episodes of paddling and trembling during recovery , compared to the propofol induction .
these results may be related to the differences in drug clearance and the proportion of occupied gabaa receptor between alfaxalone and propofol .
abnormal behavior including ataxia , increased motor activity , hyperreflexia , sensitivity to touch and violent recovery may also occur during the recovery from ketamine anesthesia , and these reactions are probably attributable to depression of the inferior colliculus and medial geniculate nucleus leading to misperception of auditory and visual stimuli . in the present study , almost the cats receiving the i m treatment at 2.5 to 10 mg / kg or the iv treatment at 5 mg / kg of alfaxalone - hpcd on its own exhibited tremors , ataxia and opisthotonus - like posture during the earlier period of recovery , but
no cat was excited and behaved aggressively . on the other hand , less frequency of ataxia and tremors during recovery was observed after
the i m treatment of alfaxalone - hpcd at 1 mg / kg .
in addition , the i m alfaxalone - hpcd at the highest dose ( 10 mg / kg ) was associated with paddling and vomiting .
further studies are needed to clarify whether the perceived adverse effects during recovery are regarded as a direct side effect of alfaxalone - hpcd or not .
the incidences of these episodes during recovery in the present study were higher than those in previous studies [ 20 , 33 ] .
reported that twitching , paddling , face rubbing , opisthotonus and tremors were observed during recovery from anesthesia in 14.3 , 21.3 , 4.3 , 12.8 and 21.3% of 47 client owned cats receiving iv alfaxalone - hpcd for induction of anesthesia , respectively . in that study
, all the cats were premedicated with acepromazine ( 0.05 mg / kg i m ) and buprenorphine ( 0.01 mg / kg i m ) before the iv alfaxalone - hpcd induction .
zaki et al . showed that premedication ( acepromazine 0.03 mg / kg and butorphanol 0.3 mg / kg administered subcutaneously ) improved the quality of recovery after the
iv alfaxalone - hpcd induction .
similarly , it was reported that the quality of recovery from anesthesia with alfaxalone - hpcd was superior in cats premedicated with acepromazine or medetomidine compared to those in cats without premedication .
these findings indicate that premedication with sedative and analgesic drugs would improve the quality of recovery from alfaxalone - hpcd in cats .
in contrast , grubb et al . reported that premedication with dexmedetomidine ( 0.01 mg / kg i m ) or dexmedetomidine plus hydromorphone ( 0.1 mg / kg i m ) did not improve the quality of recovery from the i m alfaxalone - hpcd ( 5 mg / kg ) and mentioned that the sedative effect of dexmedetomidine might have dissipated before the recovery period .
further studies will be necessary to determine the influence of premedication on the quality of recovery from the i m alfaxalone - hpcd in cats .
muir et al . reported that iv administration of alfaxalone - hpcd produced dose - dependent cardio - respiratory depression and an iv dose of 5 mg / kg produced hypoxia associated with hypoventilation mainly caused by decreases in respiratory rate and a transient apnea in cats .
these findings are consistent with our results in the cats receiving the iv5 treatment . in the present study ,
spontaneous breathing was maintained , but the rr decreased in all the cats receiving the im2.5 , im5 , im10 and iv5 treatments .
the cardio - respiratory changes were within normal ranges in all the cats receiving the im1 , im2.5 and im5 treatments .
the i m alfaxalone - hpcd at the highest dose ( 10 mg / kg ) produced clinically relevant hypotension in one cat .
these findings suggest that the i m administration of alfaxalone - hpcd produces a mild dose - dependent cardio - respiratory depression which is not clinically relevant up to doses of 5 mg / kg .
however ,
there was a limitation in the present study , because we adopted indirect methods to measure cardio - respiratory valuables in order to achieve less nociceptive stimulation . in cats ,
the oscillometric method may result in an underestimation of the arterial blood pressure compared with the direct arterial blood pressure measurement [ 12 , 25 ] .
tremors during the recovery phase may affect the accuracy of the arterial blood pressure measured by the oscillometric method .
in addition , inadequate light transmission and animal movement are the greatest limitations of spo2 measurement .
we applied the spo2 sensor to the tongue during the lack of spontaneous movement and changed periodically .
further sophisticated cardiorespiratory measurement including arterial blood gas analysis and measurement of cardiac output or tidal volume will be required to
confirm the cardiopulmonary depression produced by the i m alfaxalone - hpcd in cats . in conclusion ,
i m alfaxalone - hpcd at doses of 2.5 , 5 and 10 mg / kg produced dose - dependent sedation and immobilization in cats .
the 10 mg / kg dose rate produced a similar degree of sedation , but longer lasting sedation compared to the i m alfaxalone - hpcd at 5 mg / kg .
i m alfaxalone - hpcd caused a mild dose - dependent cardio - respiratory depression at doses up to 5 mg / kg . at 10 mg / kg ,
i m alfaxalone - hpcd caused clinical hypotension in one cat and was associated with vomiting in another cat .
some cats vocalized during the i m administration of alfaxalone - hpcd , however , it was thought that the vocalization was related to the act of administering an i m injection and not a pain response . during the early recovery period ,
undesirable effects including tremors , ataxia and opisthotonus - like posture were associated with i m alfaxalone - hpcd at 2.5 to 10 mg / kg .
nevertheless , we do not recommend the i m alfaxalone - hpcd at 10 mg / kg , because of the excessive i m volume ( 1 ml / kg ) .
premedication with sedatives and/or analgesics should be a focus of future investigations in order to reduce the injection volume of i m alfaxalone - hpcd and improve the quality of recovery . | the sedative effects of intramuscular ( i m ) alfaxalone in 2-hydroxypropyl - beta - cyclodextrin ( alfaxalone - hpcd ) were evaluated in cats .
the cats were treated with alfaxalone - hpcd in five occasions with a minimum 14-day interval between treatments : an i m injection of 1.0 mg / kg ( im1 ) , 2.5 mg / kg ( im2.5 ) , 5 mg / kg ( im5 ) or 10 mg / kg ( im10 ) , or an intravenous injection of 5 mg / kg ( iv5 ) .
the sedative effects were evaluated subjectively using a composite measurement scoring system ( a maximum score of 16 ) .
cardio - respiratory variables were measured non - invasively .
the median sedation scores peaked at 10 min ( score 9 ) , 15 min ( score 14 ) , 10 min ( score 16 ) , 10 to 20 min ( score 16 ) and 2 to 5 min ( score 16 ) after the im1 , im2.5 , im5 , im10 and iv5 treatments , respectively .
the im5 treatment produced longer lasting sedation , compared to the iv5 treatment .
durations of maintenance of lateral recumbency after the im10 treatment ( 115 22 min ) were longer than those after the im2.5 ( 40 15
min ) , im5 ( 76 21 min ) and iv5 treatments ( 50 5 min ) .
cardio - respiratory variables remained within clinically acceptable ranges , except for each one cat that showed hypotension ( < 60 mmhg ) after the im10 and iv5 treatments .
tremors , ataxia and opisthotonus - like posture were observed during the early recovery period after the im2.5 , im5 , im10 and iv5 treatments . in conclusion ,
i m alfaxalone - hpcd produced dose - dependent and clinically relevant sedative effect at 2.5 to 10 mg / kg in healthy cats .
hypotension may occur at higher i m doses of alfaxalone - hpcd . | MATERIALS AND METHODS
RESULTS
DISCUSSION |
PMC5217772 | respiratory chain defects ( rcd ) are usually phenotypically related to heterogeneous clinical features , ranging from fatal infantile multisystem syndromes to encephalomyopathies or isolated myopathies sometimes associated with cardiomyopathies .
hypotonia , lactic acidosis , cardiorespiratory failure and severe psychomotor delay are the most frequently reported features in paediatric patients , while myopathy , associated to central nervous system involvement ( hearing loss , pigmentary retinopathy , seizures , ataxia , polyneuropathy , rarely movement disorders ) is a main characteristic of the adult - onset pathologies .
respiratory chain defects are either related to mitochondrial dna mutations , or to abnormalities in nuclear genes linked to mitochondrial function .
complex i ( ci , nadh dehydrogenase ubiquinone ubiquinol reductase ) is the largest complex of the respiratory chain .
it catalyses the transfer of electrons from nadh to coenzyme q10 , and consists of 45 subunits , seven of which ( nd1nd6 , nd4l ) encoded by the mitochondrial genome . in mitochondrial disorders ,
isolated ci deficiency is relatively frequent , usually associated with severe , early - onset , multisystem phenotype . due to the enzyme complexity , in almost half of the cases of ci defect ,
only few mutations in the mitochondrial dna ( mtdna)-encoded nd2 subunit ( ec:1.6.5.3 ) have been reported , usually associated with leigh syndrome , and leber 's hereditary optic neuropathy ; a single patient has been reported carrying a 2-bp deletion in mtnd2 gene and suffering from severe exercise intolerance . here
we report a new mutation in the mtnd2 gene in a patient with a severe and isolated ci defect showing a relatively mild phenotype characterized by exercise intolerance and lactic acidosis .
the patient is a 21-year - old man , the first born after uncomplicated pregnancy and delivery from healthy unrelated parents .
first symptoms became evident at the age of 7 years , when he began to complain of overall fatigability presenting exclusively during physical activities , and worsened by exposure to cold temperatures . in the late five years , the exercise intolerance became so severe he could not keep up with his schoolmates when playing and running , requiring 30 to 60 min to regain the overall strength .
the progressive fatigability eventually prevented him from riding a bike , then he became unable to carry ordinary burdens ( i.e. backpack , books ) while the tolerable walking distance gradually shortened to 150 m. neither cognitive nor behavioural changes were described ; he did not report any hearing , vision , speech impairment , he never lost consciousness , and he never experienced any selective muscle group weaknesses nor myoglobinuria . starting from the age of 17 years , he began to exhibit frequent vomiting while exercising , symptoms that urged the patient 's family to search for medical help .
the first clinical evaluation performed at 20 years of age showed asthenic habitus , no dysmorphic facial features but he showed high - arched palate and malocclusion of teeth , normal head circumference , low body mass index ( bmi , 18
clinical tests for myasthenia , prostigmine test , anti - achr and anti - musk antibodies were negative .
however , he referred general weakness during arm uplifting > 60 s. and five squats .
the psychological testing and psychiatric status were normal ( full - scale iq = 97 , wais - iv ) .
electrophysiological assessment ( electroencephalogram , multimodal evoked potentials , eng , emg with repetitive stimulation ) and brain mri with spectroscopy were normal .
thyroid status and routine blood tests for renal and liver function were within normal ranges , so as ck values ( 190 u / l ) .
lactate at rest were increased in the blood ( 9.9 mmol / l , n.v .
< 2 mmol / l ) increasing at 14,4 mmol / l after exercise , while normal in cerebrospinal fluid .
electrocardiogram and echocardiogram were normal , while 24 h - holter ecg detected a supraventricular tachycardia ( 190/min ) during minor physical activity ( e.g. slow walking ) , with normal heart rate at rest ( 80/min ) and ergometry test .
respiratory assessment ( clinical examination , spirometry , arterial blood gas analysis ) was normal .
since a mitochondrial disorder was suspected , the patient underwent a muscle biopsy and was afterwards put on oral antioxidant therapy ( coenzyme q10 180 mg per day and vitamin b complex ) .
after the starting of antioxidant therapy , he showed an improvement of his condition , with a better tolerance of physical activity better and a decrease of his overall fatigability with shortening of the post - exercise recovery time ; no effects were reported on exercise tolerance .
histological and ultrastructural assessment of the muscle biopsy were performed using standard histological , histochemical and electron microscopy techniques .
mitochondrial respiratory chain ( mrc ) enzymes activity was assayed by standard spectrophotometric techniques in muscle homogenate and in digitonin - treated cultured fibroblasts obtained from skin biopsy .
each mrc enzyme specific activity was normalized to that of citrate synthase ( cs ) , a standard marker of cellular mitochondrial content .
normal activity range is expressed as mean value standard deviation ; residual activity was expressed in percentage .
the entire mtdna was pcr - amplified and sequenced as described in bugiani et al . .
restriction fragment length polymorphism ( rflp ) analysis was used to confirm and quantify the mutation using the nlaiv enzyme ( neb ) .
quadriceps muscle biopsy disclosed the presence of numerous ragged red - cox - positive fibres , with accumulations enlarged mitochondria and mitochondrial proliferations ( fig .
the electron microscopy confirmed the presence of numerous and enlarged mitochondria , often with abnormal proliferating cristae and rounded osmiophilic inclusions ( fig .
biochemical assays of mrc complexes performed on muscle homogenate showed a severe isolated ci deficiency equal to 10% of control mean with very high cs activity equal to 345% of control mean ; all mrc activities was normal on patient 's fibroblasts ( table 1 ) .
we performed the sequence analysis of entire mtdna , classified into haplogroup j1c3 by haplogrep2 inherited from the mother , as confirmed by the same analysis performed on her dna , and identified a new mutation in mtnd2 gene ( m.4831 g > a ) that causes the substitution of the glycine in position 121 with an aspartic acid ( p.gly121asp ) in the protein , in a site between two intermembrane domains .
the mutation has never been reported , the p.gly121asp change scored very highly for likelihood to be deleterious according to ad - hoc softwares for pathogenicity prediction ( damaging for polyphen2 : p = 1.000 ; panther : 0.96 ; mutpred : 0.969 ; sift and mutationtaster ) and the amino - acid involved is highly conserved in the phylogenies ( fig .
pcr - rflp analysis performed to quantify the mutation in different tissues using the nlaiv enzyme ( fig .
2b ) showed the presence of the m.4831 g > a change in the 95% of mitochondrial genomes from patient 's muscle , in the 40% of genomes from urinary tract cells and only in < 5% of genomes from patient 's peripheral blood lymphocytes .
the mutation was absent in fibroblasts obtained from skin biopsy as in the tissues ( blood and urine ) from the healthy mother and younger brother ( fig .
isolated ci deficiency is a frequent cause of mitochondrial dysfunction , usually related to severe and early - onset multisystem phenotype and associated with mutations in nuclear genes coding for ci structural proteins .
we report a novel mutation in mitochondrial mtnd2 gene coding for a ci subunit , characterized by a mild phenotype with exclusive muscular involvement presented as exercise intolerance and high blood lactate at rest .
vomiting while exercising might be due to frequent physiological causes or rare somatic causes , and it is often a hallmark of exercise intolerance . in our patient coenzyme q10 therapy could ameliorate the capability to endure physical activity , but was unable to treat the overall fatigability .
the severe ci deficiency observed in patient 's muscle homogenate is related to the almost homoplasmic mutation in this tissue , while in fibroblasts , where the mutation was absent , the activity was normal .
this evidence confirms the pathogenicity of the mutation which , by changing an amino - acid highly preserved between species that links two intermembrane domains , probably affects their stability and function .
furthermore , the high mutation load on muscle tissue can explain the onset in childhood and the relatively benign course of the disease involving only skeletal muscle , but raises the question of how and when the specific tissue segregation ensued in this patient .
the absence of the mutation in the healthy mother and brother suggests the exclusive presence of a lower mutation load in the ovarian cells or a de novo genesis in our patient .
extreme exercise intolerance and isolated mitochondrial myopathy has been reported in mitochondrial dna mutations in at least 5 genes : mtcytb mtnd2 , mtnd4 , mtnd5 , and trnas .
our case resembles that of several mutations in mtcytb , another mtdna gene that encodes for the cytochrome b , a subunit of complex iii , which are often sporadic , in which a pure myopathy with exercise intolerance has been described , , . since now only few patients showing an exclusively mild muscular phenotype have been described carrying mutations in mtnd4 and mtnd5 genes coding for ci , and only one patient has been reported with a phenotype similar to our patient , characterized by severe exercise intolerance and lactic acidosis , due to a deletion of 2 bp in mtnd2 gene . in all this patients
the muscle biopsy showed ragged - red and cox - positive fibres ; the mutations were present at high level only in muscle tissue , as seen in our patient .
the accumulation of mitochondria is present in a large number of muscle fibres in particular in type 2 fibres , which show a peculiar large accumulation of mitochondria positive for cox and sdh staining that correlate with the biochemical data of a significative increment of citrate synthetase activity and a normal cox activity .
nevertheless the presence of mitochondrial proliferation with enlarged organelles containing osmiophilic inclusions and abnormal cristae confirm the severe involvement of mitochondria in muscle in conclusion , in the presence of isolated exercise intolerance with high blood lactate , histopathological signs of mitochondrial myopathy and defects of ci activity , the sequence analysis of mitochondrial dna should be performed . | to date , only few mutations in the mitochondrial dna ( mtdna)-encoded nd2 subunit of complex i have been reported , usually presenting a severe phenotype characterized by early onset encephalomyopathy and early death . in this report
, we describe a new mutation in the mtnd2 gene in a 21-year - old man with a mild myopathic phenotype characterized by exercise intolerance and increased plasma lactate at rest .
electromyography and brain nmr were normal , and no cardiac involvement was present .
muscle biopsy showed a massive presence of ragged red cox - positive fibres , with enlarged mitochondria containing osmiophilic inclusions .
biochemical assays revealed a severe isolated complex i deficiency .
we identified a novel , heteroplasmic mutation m.4831 g > a in the mtnd2 gene , causing the p.gly121asp substitution in the nd2 protein .
the mutation was present in the 95% of mitochondrial genomes from patient 's muscle tissue , at a lower level in cells from the urinary tract and at a lowest level in lymphocytes from patient 's blood ; the base substitution was absent in fibroblasts and in the tissues from proband 's healthy mother and brother .
the specific skeletal muscle tissue involvement can explain the childhood - onset and the relatively benign , exclusively myopathic course of the disease . | Introduction
Case report
Materials and methods
Results
Discussion |
PMC4392974 | spain is currently the country with the highest last month prevalence of cannabis use among those aged 1524 in europe , with consistently high figures since 2005 ( 17.218.6% , ) and with the highest prevalence of regular ( 339 times within the past 12 months ) and heavy ( 40 times or more within the past 12 months ) use according to 2009/2010 hbsc ( health behaviour in school - aged children , ) survey of 15 - 16-year olds .
a range of negative effects of regular cannabis use on adolescent health and psychosocial status have been identified , including adverse effects on psychosocial development and mental health ( including developing psychotic symptoms ) , decrease in academic performance ( which can lead to academic failure ) , and other negative outcomes later in life [ 59 ] . since 2009 , cannabis as a primary drug has overtaken heroin among those requesting drug treatment for the first time in their lives in the european union .
its mention as a primary drug further increased in 2010 ( accounting for more than 100 000 treatment demands ) . in this year , 76% of reported treatment entrants aged 1519 years cited cannabis as their primary drug as did 86% of those younger than 15 years .
chiefly from the perspective of planning public health interventions , like secondary / targeted drug prevention or drug treatment , there is considerable value in implementing screening instruments that are capable of detecting ( probable ) cannabis dependence or problematic use at the population level .
population or youth surveys in several european countries have recently started to incorporate screening instruments to measure cannabis dependence or problematic cannabis use [ 1117 ] . in order to better understand these instruments ,
the european monitoring centre for drugs and drug addiction ( emcdda ) has recommended methodological studies . in the context of general population or school surveys ,
there is often shortage of space for inclusion of new items , as this may considerably increase the cost of the survey .
moreover , studies confirm that well - constructed short screening scales can be strong predictors of the same results found in more lengthy instruments or interviews [ 19 , 20 ] .
there are few epidemiological studies conducted in europe to date applying psychometric scales to assess problematic forms of cannabis use in the general population of adolescents [ 13 , 1517 ] .
the aims of the present study were ( 1 ) to explore the feasibility of administration of short cannabis instruments within a wide school survey of spanish adolescents , ( 2 ) to obtain insight into the psychometric performance of cast , sds , and dsm - iv cannabis abuse criteria , ( 3 ) to find out whether the instruments identify the same groups of cannabis users by exploring their overlaps and associations of positivity on them with key variables .
data collection was carried out on a sample of spanish students within a biennial national school survey conducted by the spanish national programme on drugs ( dgpnsd ) in 2006 .
the reference population for this survey was students from 7 spanish regions , selected by convenience , between the ages of 14 and 18 years who attended secondary schools .
two - stage cluster sampling was used , by randomly selecting 322 schools as first stage units and 644 classrooms as second stage units . in order to select the schools
, the sampling frame was firstly stratified by autonomous region ( seven strata ) and school type ( two strata , public and private schools ) .
9.6% of the registered students did not attend class on the date and time of the survey .
0.2% of students refused to participate in the study . the final sample consisted of 14,589 school attendees born between 1987 and 1992 .
the numbers of cannabis users in the studied sample were : lifetime use n = 5002 , last 12 months use n = 4089 , and last 30 days use n = 2735 . the participation of students in the survey was based on a passive parental consent .
parents ' associations of schools , school administrations , and regional educational authorities were informed about the nature , objectives , and characteristics of the study .
all selected students were informed that participation in the survey was voluntary . to ensure confidentiality ,
a standardized questionnaire was self - completed with paper and pencil by all of the students in the selected classrooms during a normal class ( 4560 minutes ) , in the presence of the teacher who remained at the lectern throughout .
the questionnaire included questions related to sociodemographic characteristics , drug use , perception of risk of different drug use behaviours , leisure time , level of perceived availability of different psychoactive drugs , social and health - related problems , sources of information on drugs , drug use by friends and classmates , and the attitudes of parents toward drug use .
frequency of cannabis use in the last 12 months and in the last 30 days was measured in categories of 0 , 1 , 2 , 3 , 4 - 5 , 69 , 1019 , 2039 , and 40 or more days ( in the case of last 12 months use ) .
alcohol bingeing in the past 30 days was defined as drinking 5 or more glasses of alcohol in one single occasion .
short cannabis disorders screening instruments were sought , which have been tested in similar settings .
dsm - iv cannabis abuse criteria : a 5-item instrument ( corresponding to four abuse criteria ) , which has been incorporated in the us national survey on drugs since the year 2000 and administered in those who used the respective substance on 6 or more days in the past year . by fulfilling at least one of the four abuse criteria
, a person meets the criteria for abuse , which is diagnosed in the absence of dependence ( dsm - iv , ) . for the purpose of the present epidemiological survey ,
abuse criteria are used without excluding dependence cases . the severity of dependence scale ( sds ) : a 5-item scale that measures psychological components of dependence .
it has been successfully used to assess cannabis dependence in germany , australia , and brazil [ 11 , 25 , 26 ] , including adolescent populations of cannabis users , where a cut - off point of 4 for dependence was established .the cannabis abuse screening test ( cast ) is a 6-item scale screening for problematic forms of cannabis use , validated using dsm - iv cannabis dependence and cannabis use disorders criteria as a gold standard .
it has been used successfully with teenagers , and a cutoff of 2 has recently been proposed to screen for either cannabis dependence or cannabis use disorders in this population .
binary scoring procedure was used , as the resulting total score has a more intuitive interpretation , while the full scoring offers only modest measurement advantages .
dsm - iv cannabis abuse criteria : a 5-item instrument ( corresponding to four abuse criteria ) , which has been incorporated in the us national survey on drugs since the year 2000 and administered in those who used the respective substance on 6 or more days in the past year . by fulfilling at least one of the four abuse criteria
, a person meets the criteria for abuse , which is diagnosed in the absence of dependence ( dsm - iv , ) . for the purpose of the present epidemiological survey ,
abuse criteria are used without excluding dependence cases . the severity of dependence scale ( sds ) : a 5-item scale that measures psychological components of dependence .
it has been successfully used to assess cannabis dependence in germany , australia , and brazil [ 11 , 25 , 26 ] , including adolescent populations of cannabis users , where a cut - off point of 4 for dependence was established .
the cannabis abuse screening test ( cast ) is a 6-item scale screening for problematic forms of cannabis use , validated using dsm - iv cannabis dependence and cannabis use disorders criteria as a gold standard .
it has been used successfully with teenagers , and a cutoff of 2 has recently been proposed to screen for either cannabis dependence or cannabis use disorders in this population .
binary scoring procedure was used , as the resulting total score has a more intuitive interpretation , while the full scoring offers only modest measurement advantages .
see table 1 for all instruments ' items , answer options , and the respective scoring .
the instruments were adapted into spanish and catalan in a process including translation , back - translation , cognitive debriefing with young cannabis users , and discussion at an expert meeting .
they were placed at the end of the questionnaire and addressed only to those adolescents who had indicated that they had used cannabis in the past 12 months .
the assessment frame of all three instruments was last 12 months . in the case of one missing item on a particular scale , in all analyses that included positivity on the instrument , its value was replaced by mean score of the rest of the scale items .
analyses of missing items and their relationship with gender , age , and frequency of use in the past year and past month were performed using the chi - square test .
an analysis of psychometric properties of the three cannabis screening instruments was carried out including item analysis .
internal consistency was assessed by age , to examine possible age - related lack in response consistency .
instruments ' total scores were tested for their relationship with frequency of cannabis use in the past 12 months . in this correlation analysis ,
averages of the above - mentioned categories were used to compute the pearson coefficient and order of these categories to compute spearman 's rho .
a series of bivariate analyses ( chi - square test and one - way anova ) and a multinomial logistic regression analysis was run to explore the associations of being positive / negative on the different combinations of instruments ( in mutually exclusive categories ) with key demographic , substance use , and drug - related problems variables .
of the 4089 cannabis users in the past 12 months , 3569 fully completed the cast scale , 3546 the sds scale , and 3573 the dsm - iv abuse criteria .
between 10.5% and 12.3% of answers per item were missing ( in most of these cases the entire scale was left blank ) . leaving blank a substantial number of items
was associated with reporting lower frequency of cannabis use in the past 12 months and in the past 30 days ( chi - square test , p < 0.001 in both cases ) .
there were , however , 136 students who reported that they had used cannabis on at least 20 days during the past year , and their total scores of at least one scale could not be computed due to missing items .
no association between the number of missing items and gender or age was found .
table 1 shows results of item analysis as well as some results for the composite scores of the instruments .
some items had a higher share in the total score than others : the first two items in the case of dsm - iv abuse , the third item of cast , and item 4 of sds .
the importance of these items was generally confirmed by analysis of proportion positive if the item was deleted .
principal components analysis revealed unidimensionality in each of the instruments with relatively high factor loadings .
less than 50% of variance was explained by the first component in all cases ( 46 - 47% ) .
the only item which would provide ( small ) improvement of alpha when deleted was item 4 of the sds scale .
analysis of internal consistency by age group did not reveal a clear age - related pattern with the exception of cast , where those aged 14 showed a somewhat lower internal consistency ( results not shown ) .
at least one dsm - iv criterion for abuse was fulfilled by 28.6% ( 1023 ) of the instrument completers .
34.3% ( 1273 ) of students who completed at least five items of cast had 2 or more points and would thus be assigned possible cannabis dependence / cannabis use disorders .
sds positive cases ( possible dependence on cannabis ) at a cutoff of 4 points or more were 16.3% ( 592 ) .
if only those , who used cannabis on six or more days in the past twelve months were considered for positivity , the figures would be 21.6% , 28.7% , and 11.7% , respectively .
students positive on the respective instruments constituted 7.0% , 8.7% , and 4.1% of the entire sample , or 5.3 , 7.2 , and 2.9% , if six or more days users only were considered .
sensitivity analysis was performed taking into account the 136 daily or near - daily users whose total score could not be computed due to missing items . assuming that all of these frequent users would cross the cut - off point on the respective instruments , the original range of prevalence estimates based on crossing the thresholds for positivity of the different instruments ( see above ) would change slightly to 2.97.9% .
out of the 1708 positive on at least one instrument , most cases , 70.9% , were positive on cast criteria with 26.1% only on cast ; the second largest group , 59% , was positive on the dsm - iv abuse with 18.6% only on dsm - iv abuse .
a smaller proportion , 33.4% , was positive on the sds scale with 8.2% only on sds ( see figure 1 ) .
pearson correlations between the instruments ' total scores were moderate : 0.363 between dsm - iv abuse and sds , 0.456 between cast and sds , and 0.500 between cast and dsm - iv abuse ( r between 0.13 and 0.25 ) .
cast total score had the highest correlation of all the instruments with frequency of cannabis use in the past twelve months ( 0.567 and 0.588 , spearman and pearson correlation coefficient , resp . ) .
number of fulfilled dsm - iv abuse criteria correlated with frequency of use in the past year only moderately ( 0.326 and 0.339 , correlations in the same order ) , and sds 's total score correlations were the lowest ( 0.283 and 0.251 ) . in bivariate analyses ,
there were significant associations between the mutually exclusive categories of all combinations of positivity / lack of positivity on the tested instruments and all of the following variables : years of cannabis use , gender , frequency of cannabis use in the past 30 days and in the past year , friends ' consumption of cannabis , having an accident requiring medical help in the past 12 months within 6 hours after cannabis use , being under police arrest in the past 12 months within 6 hours after cannabis use , thinking that habitual cannabis smoking causes quite some problems or
a lot of problems , smoking tobacco on some days a week or more during the past month , age at survey , not going to school for two or more days in the past month , because of not feeling like to , and two or more days of alcohol bingeing in the past month and going out one or more nights per week ( see table 2 ) . in a multinomial logistic regression analysis
( see table 3 ) , those being positive only on sds were not significantly different from the referent category ( negative on all instruments ) , with the exception of a higher proportion of those thinking that habitual cannabis use poses quite some problems or a lot of problems and a significantly higher proportion of those belonging to the youngest age group ( 14 - 15 years old ) . out of the remaining categories of instrument positivity , those categories , which contained positivity on cast ( positive only on cast or in combination with sds , dsm - iv abuse or both ) had the highest average number of years of cannabis use .
they also had a higher prevalence of truancy , a higher prevalence of frequent cannabis use in the past month ( 10 or more days of use ) , higher , but mostly not statistically significant , prevalence of alcohol bingeing in the past month , a higher proportion of the majority ( or all ) friends consuming cannabis , and the highest prevalence of regular tobacco smoking in the past month ( at least some days a week ) . students who were positive on cast also had a higher prevalence of suffering an accident requiring medical help and/or detention by police within six hours after cannabis use in the past year , but this was further strengthened if positivity on cast was combined with positivity on dsm - iv abuse .
the present study confirmed that it was feasible to use short psychometric instruments for problematic forms of cannabis use in a large national probabilistic survey on substance use in spanish adolescents . even though the instruments were placed at the end of the questionnaire , 8890% of items were filled in .
sensitivity analysis was performed which did not find a significant influence of missing scale results in those reporting frequent cannabis use on the overall prevalence estimate .
we have studied overlap between the three instruments and found that it was relatively small ( 16.1% ) with more than half of the respondents positive on only one instrument .
this is partially due to the different concepts of problem cannabis use , which are behind the three instruments .
the expected relationships in this respect would be a high overlap between dsm - iv abuse and cast ( the latter designed to screen for cannabis use disorders , that is , cannabis abuse and/or cannabis dependence ) and a subgroup of abuse and/or cast positive persons being identified as cannabis dependent by sds . however , this was clearly not the case .
moreover , focusing only on cannabis dependence gives a result which is similar to comparing all three instruments .
if we were to explore the overlap between cast and sds , both validated against gold standard representing cannabis dependence , in the present study , we would still find only 28.1% of cases identified by both instruments , while almost 60% would be positive only according to cast and 12.9% only according to sds .
swift et al . studied three measures of cannabis dependence , among them the sds and found 56% of cases identified by all three measures .
one possible explanation of low overlap between screening instruments in the present study might be the existence of different types of problematic cannabis users , with different characteristics and different kinds of problems experienced by them .
another possibility is the presence of measurement bias , with varying extent in the three studied instruments .
the multinomial logistic regression analysis discussed sheds some light on this possibility . according to the results of this analysis , students positive only on sds scale were similar to those who were negative on all instruments with the exception of being a younger age and more likely to think that habitual cannabis use causes quite some problems or a lot of problems . on the other hand , those groups which included positivity on cast had the longest history of cannabis use , higher prevalence of different problems , some of which were directly related to cannabis use . in this group
, there was a lower proportion of girls , higher tobacco smoking , markedly higher prevalence of high frequency cannabis use in the past month , more of their friends smoked cannabis , while they thought less that habitual cannabis use caused substantial problems .
dsm - iv abuse positivity has further strengthened cast 's association with such problems as being detained by the police or having an accident shortly after cannabis use .
this is consistent with two abuse criteria legal problems and using the substance in physically hazardous situations and confirms concurrent validity of the instrument . fulfilling dsm - iv abuse criteria
was associated less strongly than cast with history and frequency of cannabis use , and other problems potentially related to cannabis use , but more strongly than sds positivity .
this suggests that the usefulness of sds in screening for problematic cannabis use in the general population of adolescents is limited , as it may identify a considerable proportion of
this finding from the multinomial logistic regression analysis is further corroborated by the weak association of the scale with frequency of use in the past year
an important indicator within the diagnostic approach [ 30 , 31 ] and by the fact that item 4 of the scale ( wish to stop ) contributed to the total score considerably while its removal would improve the internal consistency of the instrument .
this weaker performance might be associated with the concept behind the scale , namely psychological components of dependence .
for example , other authors have found a response bias in adolescents in the direction of overreporting dependence , mainly its symptoms being unable to cut down and tolerance .
another study suggested that substance use disorders diagnostic criteria might have different meaning for adolescents , who may experience rapid development of tolerance after initiation of use and may be confused about a question on using more or longer than intended as they typically do not foresee a limit of consumption but intend to become intoxicated .
the scales administration was based on self - assessment , moreover of relatively complex concepts .
the sampling frame of the present study was adolescents aged 1418 who were schooled ; 18% of this age group were estimated to be unschooled and thus outside of the sampling frame . moreover , almost 10% of the students of the selected classrooms were not attending school on the day of survey .
there are associations of dropping out of school and truancy with substance use and related problems shown by numerous studies ( e.g. , and [ 16 , page 172 ] ) . however , the analysis of psychometric properties of the explored instruments may be less biased by these factors than , for example , prevalence estimation .
the placing of the screening instruments ( almost at the end of the questionnaire ) , and , possibly , the order of their presentation may have affected the number of missing responses and/or the reliability of responses due to such factors as running out of time , or tiredness of the respondent and possibly the measures of psychometric properties of the instruments .
a study comparing the effects of different orders of presentation would be necessary for clarification of this question .
furthermore , as establishing clinical diagnoses was not part of this study , its findings have to be interpreted cautiously in relation to these concepts .
the reported psychometric properties of the applied instruments might not be transferable to clinical or targeted samples of heavy cannabis users , owing to spectrum bias in those samples .
nevertheless , it is important to know how instruments to detect problematic cannabis use perform in the general adolescent population . in conclusion , sds ,
cast , and dsm - iv abuse criteria have performed moderately well in the present sample , but identified largely different groups of users with only modest overlaps , even between scales validated against the same gold standard . given the fact that a researcher planning an epidemiological survey may decide to choose any of these instruments to represent a measure of problematic cannabis use , this finding may be highly relevant .
while one possible explanation for these differences might be the existence of different subtypes of problematic cannabis use , measurement bias of varying extent may also play a role .
the present analyses put in question the validity of the sds scale in the general adolescent population . the concept of psychological components of dependence represented by the scale may be difficult to understand for adolescent cannabis users .
this may be the reason for high scoring on sds items operationalizing this concept in the youngest lower frequency users who have few cannabis - related problems and who think that habitual cannabis use causes quite some problems or a lot of problems .
on the other hand , cast scale had generally strong relationship with frequency of cannabis use in the last year and other variables suggesting its concurrent validity .
dsm - iv abuse was less strongly associated with intensive and long - term use , but its association with legal problems and an item indicating possible use in physically hazardous situations corroborated the validity of the instrument in relation to the concept behind it .
the findings of the present study are relevant also from the broader perspective of using short , self - report - based screeners of complex , more abstract concepts in the population of adolescents in psychiatry , general practice , and clinical psychology .
further research , especially qualitative , is needed to shed light on adolescents ' understanding of psychometric instruments ' items related to cannabis dependence symptoms in order to improve the ascertainment of problematic cannabis use in this population . |
the aim of this study was to examine the feasibility of problem cannabis use screening instruments administration within wide school surveys , their psychometric properties , overlaps , and relationships with other variables .
students from 7 spanish regions , aged 1418 , who attended secondary schools were sampled by two - stage cluster sampling ( net sample 14,589 ) .
standardized , anonymous questionnaire including dsm - iv cannabis abuse criteria , cannabis abuse screening test ( cast ) , and severity of dependence scale ( sds ) was self - completed with paper and pencil in the selected classrooms .
data was analysed using classical psychometric theory , bivariate tests , and multinomial logistic regression analysis .
not responding to instruments ' items ( 10.512.3% ) was associated with reporting less frequent cannabis use .
the instruments overlapped partially , with 16.1% of positives being positive on all three .
sds was more likely to identify younger users with lower frequency of use who thought habitual cannabis use posed a considerable problem .
cast positivity was associated with frequent cannabis use and related problems .
it is feasible to use short psychometric scales in wide school surveys , but one must carefully choose the screening instrument , as different instruments identify different groups of users .
these may correspond to different types of problematic cannabis use ; however , measurement bias seems to play a role too . | 1. Introduction
2. Methods
3. Results
4. Discussion |
PMC4181276 | over the past decade , minimally invasive approaches to mitral valve surgery have been commonly used at many centers around the world with excellent short- and long - term outcomes .
there has been great enthusiasm about their performance because they have proven to be at least as good and safe as the standard sternotomy approach , even in elderly patients .
a variety of approaches have been reported , aimed at reducing surgical trauma and postoperative morbidity while remaining safe and effective .
some approaches have been aided by the use of thoracoscopy , specially designed retractors and surgical clamps as well as special instruments for long - distance knot - tying .
the right thoracotomy approach has been the most appealing , for reasons of cosmesis and reduced trauma .
however minimally invasive mitral valve surgery ( mimvs ) is performed , the most important consideration is that the approach must yield results equal to or better than those of the approaches it modifies or replaces .
there has been increasing interest in simplifying the operation so that it can be widely applied to benefit patients .
this report illustrates a simplified , reproducible minimally invasive approach with optimal access to and exposure of the mitral valve ( mv ) under direct vision .
we applied this approach to all patients with moderate and severe mv insufficiency and/or stenosis of various etiologies , with no concomitant coronary artery disease or aortic valve regurgitation .
even complex repair procedures for severe bileaflet prolapse in patients with barlow s disease can be successfully performed through this approach .
mv surgery using any commercially available prostheses can be performed with the same reliability in patients in whom the mitral valve is not amenable to repair .
it is also a useful alternative for patients requiring mv procedures after a previous cardiac operation , particularly in those with patent coronary artery bypasses or previous aortic valve replacement .
this may also be applied in patients who had had surgeries via right thoracotomy approach .
assessment of mitral valve lesions the degree of mv insufficiency or stenosis is estimated by means of standard echocardiographic measurements .
assessment of mv function includes measurement of the mitral annulus , evaluation of leaflet mobility and coaptation , determination of mitral valve orifice area , mitral flow assessment using continuous wave doppler , and valve anatomy evaluation as to valve thickness and pliability and morphology of the subvalvular apparatus .
standard guideline in mimvs using the simplified approach because the mitral valve is a posterior structure , excellent exposure can be established through the right anterolateral thoracotomy .
a simplified right anterolateral thoracotomy approach includes a 10 - 12 cm incision , either direct aortic or peripheral arterial cannulation and direct bicaval canulation , with standard retractors .
under general anesthesia using a double lumen endotracheal tube , the patient is placed in left lateral position with the chest elevated to about 45 - 60 ( figure 1a ) .
the right arm is placed over the head at approximately 120 with the elbow joint in the right - angle position .
the operating table is rotated leftwards and the patient is bent at the 12 thoracic vertebra .
transcutaneous defibrillation pads ( philips multifunction electrode pads , philips , amsterdam , the netherlands ) are placed at the left lateral chest wall and right shoulder .
a 10 - 12 cm skin incision over the fifth anterolateral intercostal space is made beginning in the skin fold below the right breast ( figure 1b ) .
the right lung is deflated and the chest cavity is entered without division or resection of any bone .
standard chest retractors are used and , under direct vision , the pericardium is incised parallel and approximately 3 - 4 cm anterior to the phrenic nerve .
this provides optimal and excellent visualization and access to the ascending aorta and superior vena cava .
heparin is administered and arterial cannulation is performed either directly in the ascending aorta or in the common femoral artery ( only in patients with a small and narrow chest ) with direct bicaval cannulation with caval snares ( figure 1c ) .
adequate myocardial protection is achieved with intermittent blood cardioplegia through a cardioplegia needle in the aortic root .
the ease and handling of myocardial protection provides no difficulty and is comparable when the approach is through a standard median sternotomy . after clamping the ascending aorta with a straight clamp having a flexible handle ,
the left atrium is opened by incising the interatrial groove with a vent cardiotomy sucker placed directly towards the left pulmonary veins .
a retractor is placed to elevate the interatrial septum , exposing the mitral valve , and the valve is meticulously inspected to determine the precise nature of the lesion .
leaflet coaptation is assessed with transvalvular saline injection under pressure . using a nerve hook , leaflet coaptation and the presence of sufficient tissues along the coaptation plane
depending on the results of this evaluation , mitral valve repair or replacement is performed under direct vision , using precisely the same approach as in a conventional median sternotomy .
modified gerbode plication plastic is applied for posterior leaflet prolapse , ruptured chordae and in ischemic mitral insufficiency ( mi ) .
prolapse can occur anywhere along the posterior leaflet but is most commonly found in the region of p2 , which may lead to chordal rupture.in this technique , the flail segment is plicated towards the left ventricle in a v - shaped fashion with interrupted mattress sutures using double - ended 3 - 0 polypropylene with untreated autologous pericardial pledgets .
when competence and size are satisfactory a strip of untreated autologous pericardium is sutured continuously onto the posterior annulus without further annular narrowing . modified paneth - hetzer posterior annulus shortening technique
this is performed by running a pericardial - pledgeted 3 - 0 polypropylene suture through the fibrous body of the trigone and tying it
. then it is run along the annulus from one trigone towards the middle of the posterior annulus .
these sutures are then tied over an appropriately sized ziemer - hetzer valve sizer to prevent over - narrowing of the valve orifice .
then , with a continuous suture , the pericardial strip is attached to the posterior annulus from the midsegment towards the trigone .
the leaflet coaptation is tested by a forceful injection of saline through the valve , to look for residual regurgitation .
we also use this technique in anterior leaflet prolapse ; the then wider coaptation plane will eliminate prolapse .
evaluation of the adequacy of repair after mv repair , it is obligatory to assess the valve function before closure of the atrium and separation from cardiopulmonary bypass ( cpb ) .
any remaining areas contributing to significant incompetence must be attended to before closure of the atrium .
once de - airing has been completed and extracorporeal circulation is discontinued , the repair result must be further evaluated with intraoperative transesophageal echocardiography ( tee ) in order to test for inadequate mitral opening area , residual incompetence , myocardial ischemia due to coronary kinking and presence of the systolic anterior motion ( sam ) phenomenon .
immediate and prompt correction must be made if the repair is shown to be unsatisfactory .
regardless of the underlying pathology and techniques used , no patient should be discharged from the operating room with more than minimal mi .
mitral valve replacement if it is established that the mitral valve lesion is not amenable to repair , the valve is replaced , with either a mechanical or biological prosthesis , in accordance with the patient s wishes . in both procedures ,
no specially designed instruments are required , and the knots may be tied by hand or with a knot pusher .
several strategies of knot tying have been learned with experience , such as having the assistant hold up the annular sutures during knot tying . after completion of the procedure ,
the left ventricle is vented with a catheter positioned across the valve and the atriotomy is closed .
caval snares are snugged tight when tricuspid valve reconstruction ( double - orifice - valve technique ) or closure of patent foramen ovale are performed , and these procedures are done through right atriotomy . throughout the procedure , a vacuum - assisted venous drain in the heart - lung machine is used and carbon dioxide is infused into the operative field to decrease the chance of air embolism .
complete evacuation of intracardiac air is performed through the aortic root and left atrium and confirmed by tee .
defibrillation , when necessary , is administered through the external defibrillator pads . once the hemodynamic status is stable , cardiopulmonary bypass is discontinued and decannulation is performed .
transesophageal echocardiography is mandatory at the moment to document the repair results or prosthetic function .
the right pleural space and mediastinum are drained through the 7 intercostal space with two chest tubes and the intercostal spaces are closed with five or six pericostal sutures .
this simplified minimally invasive approach to mitral valve surgery with optimal access to all cardiac structures and optimal exposure of the mitral valve under direct vision offers distinct advantages , including direct aortic root and caval cannulation performed with ease and without obscuring the operative field , optimal exposure and overview of the operative field , controlled myocardial protection , and adequate de - airing .
although the skin incision is 5 - 8 cm longer than in the conventional minimally invasive technique , besides providing good cosmesis and an acceptable postoperative scar , it outweighs the placement of additional skin incisions for the other cannulae or the aortic clamp , and obviously avoids the potential complications related to femoral vessel cannulation .
additionally , standard aortic cross - clamping and antegrade cardioplegia delivery obviate the need for specialized endovascular occlusive balloons , hence avoiding the potential complications associated with balloon malposition or migration .
this simplified approach combines good cosmesis with optimal exposure of the mitral valve and all the cardiac structures , is readily applicable and is not associated with a steep learning curve because one employs conventional cannulation and clamping techniques . | with increasing enthusiasm in minimally invasive surgery , several approaches and access are being performed with great precision . in this report , we illustrate and describe a minimal invasive approach to mitral valve surgery with optimal access under direct vision , the indications and patient selection , the surgical techniques , its advantages over the other approaches , and its simplicity and reproducibility . | Introduction
Conclusion |
PMC4352481 | the reason why exposure to common environmental antigens induces allergic responses in some people and not others remains undetermined .
atopy and asthma have a complex genetic background , and multiple genes can contribute to their development through main effect , gene - gene , and gene - environmental interactions . allergen - specific cd4 + helper t - cell ( th ) generation is the initial event leading to the development of allergic disease .
th2 subtypes are pivotal to the inflammatory cascade through the production of il-4 , il-5 , il-13 , and il-9 .
th1 cells ( secreting mainly il-12 and ifn- ) may contribute to the chronicity and effector phase of these diseases .
several genetic polymorphisms of these genes have been studied in relation to allergic diseases and asthma [ 13 ] . however , other important molecules have been associated with phenotypes of asthma , including tumor necrosis factor- ( tnf- ) . this proinflammatory cytokine has been found in increased concentrations in asthmatic airways and the inhalation of tnf- has been shown to cause airway hyperresponsiveness and increased sputum neutrophil counts in healthy volunteers .
tnf- is a member of the tnf gene superfamily located within the human major histocompatibility complex on chromosome 6p , linked to atopic asthma in several studies [ 5 , 6 ] .
several polymorphisms have been reported in the promoter region of the tnfa gene , with tnf-308g > a being the polymorphism most widely studied in relation to this disease .
in addition to the inflammatory events , regulatory t - cell dysfunction is associated with development of complex genetic conditions such as atopy and asthma .
peripheral t - cell tolerance is characterized by functional inactivation of the cells in contact with the antigen , which in turn eliminates both proliferate response and cytokine secretion [ 8 , 9 ] . in humans ,
several t - cell subtypes with an immune - suppressive function , generically named regulatory t cells ( treg ) , have been extensively studied [ 10 , 11 ] .
the main role of all these cell subsets is to maintain the integrity of the body by avoiding excessive immune responses that may result in harmful immune pathology , as well as preserve a state of tolerance to innocuous substances . il-10 and
tgf- secreted by these tregs play an important role in the immune regulatory response [ 1315 ] .
several common polymorphisms have been identified in the promoter region of both cytokines , including 1082g > a and 592c > a for il10 and 509c > t for tgfb genes , some of these polymorphisms being associated with allergic diseases [ 1619 ] .
olive tree pollen is one of the most important causes of respiratory allergy in the mediterranean area .
olea europaea pollen induces mainly nasal and conjunctive symptoms , although it may also induce asthma exacerbations in areas with high levels of o. europaea pollen in the atmosphere . in jan , a region in southern spain
, there is a very high level of pollen ( 500 to 1000 grains / m during pollen season , with peaks of more than 5000 grains / m ) and a high prevalence of asthma . to date , at least 20 proteins with allergenic activity have been described in olive pollen . among them , ole e 1 is the most frequent sensitizing allergen .
besides ole e 1 , twelve additional allergens have also been isolated and purified from olea europaea pollen extract , some of which are major allergens in areas with high levels of pollen exposure , such as ole e 2 and ole e 10 [ 21 , 22 ] .
previously our group described specific genetic and environmental factors associated with olive pollen allergy [ 2327 ] , as also a strong association between ole e 10 and ole e 2 specific sensitizations and bronchial asthma clinical phenotype , being these sensitizations restricted by different hla class ii antigens .
more recently we have described how , during the pollen season , olive pollen allergic patients showed a statistically significant decrease of tgf- ( regulatory cytokine ) .
this result was consistent with a significant decrease in relative foxp3 mrna expression ( marker of regulatory t - cell cytokines ) and with the lower number of regulatory t cells , indicating a lack of regulatory mechanisms in olive pollen allergic subjects during the pollen season .
the subjects were selected from a region in southern spain with particularly high pollen counts during the pollen season and a high prevalence of asthma .
taking into account all of these previous results , we analyzed the role of 6 polymorphisms of genes previously associated with allergy and asthma , in a population of olive pollen allergic patients , with an extremely high incidence of asthma : tnfa ( g-308a , c-857 t , and c-1031 t ) as proinflammatory gene and il10 ( c-571-a and a-117 g ) and tgfb ( c-509 t ) as genes related to regulatory response .
one hundred forty - six unrelated patients were recruited at the allergy service of the complejo hospitalario de jan .
all patients fulfilled the following criteria : seasonal rhinitis and/or asthma from april to june , a positive skin prick test for o. europaea pollen extract ( alk abell , madrid , spain ) , and no previous o. europaea immunotherapy .
fifty healthy subjects from the same geographic area were recruited as a control group . clinical assessment performed in these patients
patients recorded symptom scores , drug requirements , and peak expiratory flow rates ( pefr ) every day from april to june ( pollen season ) .
individual nasal and eye symptoms ( sneezing , blockage , running , redness , and itching ) and chest symptoms ( breathlessness , wheezing , and tightness ) were recorded on a scale of 03 ( 0 = no symptoms ; 1 = mild symptoms ; 2 = moderate symptoms ; 3 = severe symptoms ) .
an asthma day fulfilled at least one of the following criteria : asthma symptoms score 2 , morning pef 20% lower than the mean morning pef of the last 7 days before pollination , and daytime use of salbutamol twice or more compared with the mean use of the last 7 days before pollination .
olive tree pollen and olive pollen allergens were isolated as previously was described [ 29 , 30 ] .
total serum ige levels and specific o. europaea extract ige antibodies were determined by pharmacia systems ( ige enzyme immunoassay and uni - cap , uppsala , sweden ) .
allergen - specific ige antibody measurements ( ole e 1 , ole e 2 , and ole e 10 ) of allergic sera were performed by enzyme - linked immunoassay ( elisa ) .
all 146 patients and 50 controls were tested for olive pollen allergy by a skin prick test with o. europaea whole extract and olive pollen purified allergens , following the recommendations of the european academy of allergy and clinical immunology ( eaaci ) .
the patients were also tested with a panel of common aeroallergens , including dermatophagoides pteronyssinus , blattella germanica , dog and cat dander , alternaria alternata , aspergillus fumigatus , cladosporium herbarum , lolium perenne , cynodon dactylon , cupressus sempervirens , platanus acerifolia , artemisia vulgaris , chenopodium album , salsola kali , and parietaria judaica ( abell , madrid , spain ) .
tnf- and il-10 sera levels were analyzed by flow cytometry , using the bd cytometric bead array ( cba ) , human th1/th2 cytokine kit ( becton dickinson , bd , san diego , ca ) .
flow cytometric analysis was performed using a facscalibur flow cytometer ( bd immunocytometry systems , san jose , ca ) .
tgf- sera levels were analyzed by a commercial elisa ( bd , san diego , ca , usa ) .
genomic dna from all individuals was genotyped for 6 polymorphisms : tnfa ( g-308a , c-857 t , and c-1031 t ) , il10 ( c-571-a and a-1117 g ) , and tgfb ( c-509 t ) .
polymorphisms were analyzed according to the applied biosystems allelic discrimination assay - by - design protocol ( ab-7500 real time pcr system ) .
single nucleotide polymorphisms ( snps ) in research and their identifier names , locations , alternative names , and dbsnp identification are shown in table 1 .
comparisons of phenotypic ( olive pollen sensitized versus controls , asthmatic versus rhinitis ) or genotypic frequencies between groups were measured by fisher 's exact test or by chi - square test .
cochran and mantel - haenszel and 2-factor anova tests were used for the statistical comparison of clinical parameters ( qualitative and quantitative , resp . ) between sensitized and nonsensitized groups .
multiple regression analysis was used to examine quantitative traits ( total ige , specific ige to o. europaea whole extract , specific olive pollen allergens ige , and soluble cytokines serum levels ) .
the control group included 33 females ( 66% ) and 17 males ( 34% ) , with a mean age of 37.6 years ( range 2358 years ) .
these individuals were selected from the same geographical area and were free from any allergic symptoms .
all the allergic subjects ( n = 146 ) showed significant ige antibody levels against pollen crude extract , but different frequencies of sensitization were observed for the purified o. europaea allergens ( table 2 ) .
ole e 1 , ole e 2 , and ole e 10 are major allergens in our population ( > 50% of recognition frequency ) .
genotype and allelic frequency distribution in the patient and control groups and the genotype and allelic distribution according to asthma or rhinitis in our patient population are shown in table 3 .
the only statistically significant polymorphism associated with o. europaea allergy was related to the tgfb - c509-t polymorphism .
we found a statistically significant increase in the phenotypic percentage of the ct heterozygous genotype in patients compared with controls ( 56.8% versus 40% , p = 0.03 , odds ratio , or , 1.97 ; 95% confidence interval , ci , 1.023.8 ) ( table 3 ) .
the sera of ct heterozygous patients showed the lowest levels of soluble tgf- ( 10849.71 12613
pg / ml ) homozygous patients , but the differences were not statistically significant ( data not shown ) . when we analyzed the genotype distribution of the 6 polymorphisms according to the clinical phenotype of asthma or rhinitis , no relevant associations were found ( table 3 ) . however , the analysis according to specific ole e 2 and ole e 10 sensitizations showed several relevant results which are summarized in table 4 .
firstly , the study of 2 polymorphisms of il10 ( c-571-a and a-1117 g ) in patients with ole e 2 sensitization showed a statistically significant decrease of cc homozygous il10 - 571c > a genotype compared with ole e 2 nonsensitized patients ( 50% versus 70.5% , p = 0.029 , or , 0.41 ; 95% confidence interval , ci , 0.190.82 ) . this protection was higher when patients with asthma and ole e 2 sensitization were compared with ole e 2 nonsensitized patients with asthma ( 49.4% versus 78.6% , p = 0.008 , or , 0.26 ; 95% ci , 0.090.72 ) .
this genotype showed intermediate levels of il-10 cytokine ( 7.77 5.4 pg / ml ) compared with the other 2 genotypes ( aa homozygous : 5.9 6.1 pg / ml , ca heterozygous : 8.76 5.3 pg / ml ) .
ca heterozygous il10 - 571c > a genotype was increased in patients with asthma and ole e 2 sensitization compared with ole e 2 nonsensitized patients with asthma ( 45.7% versus 21.4% , p = 0.02 , or , 3.08 ; 95% ci , 1.138.4 ) .
secondly ( table 4 ) , we have found a statistically significant increase of ga heterozygous tnfa-308g > a genotype in ole e 10 sensitized patients compared with olive pollen allergic patients without sensitization to ole e 10 allergen ( 25.3% versus 11.9% , p = 0.04 , or , 2.5 ; 95% ci , 1.026.1 ) .
this polymorphism showed intermediate levels of tnf- cytokine ( 7.77 5.4 pg / ml ) compared with the other 2 genotypes ( gg homozygous : 7.01 4.8 pg / ml , aa homozygous : 9.3 6.1 pg / ml ) .
sensitization to specific allergens is a complex response controlled by both genetic and environmental factors .
preliminary studies reported that the allergograms of olive - allergic patients living in areas with high level of pollen production ( such as some regions as andalusia in spain ) are notably different from those obtained in areas of low - level exposure ( e.g. , madrid in the central region of the country ) . besides , the level of each allergenic protein in the pollen grain could be a second parameter to be taken into consideration .
in fact , a great variability in the allergen composition of o. europaea pollen has been described .
the reasons behind this variability are unknown , but they could be due to the existence of several genetic strains , climatic conditions , and specific culture techniques .
previous works showed how , in areas with high levels of antigenic load , olive pollen allergic patients were sensitized to allergens that are minor allergens ( frequency of recognition lower than 50% ) in areas with low antigenic load [ 21 , 22 ] .
this sensitization ( mainly related to ole e 2 and ole e 10 ) was associated with a worse clinical prognosis , with a very high presence of asthma and more days of symptomatology .
this population was analyzed for inflammatory cytokine polymorphisms ( il-13 , il4ra , il5 , and2adr genes ) finding some relevant associations , mainly related to risk factor for olive pollen allergic sensitization .
very recently we demonstrated how several immuneregulatory elements are decreased ( tgf- and foxp3 ) in olive pollen allergic patients exposed to extremely high olive pollen antigenic load . .
with these previous results , the aim of this work was to determine whether some of the genes previously associated with asthma and immune regulation are important in these specific sensitizations .
to do so , we decided to analyze the relationship between 6 genetic polymorphisms of tnfa ( g-308a , c-857 t , and c-1031 t ) as a proinflammatory gene and il-10 ( c-571-a and a-117 g ) and tgfb ( c-509 t ) as being related to regulatory response and specific sensitization to o. europaea pollen in a well - characterized spanish population of patients with olive pollen allergy and a high prevalence of asthma ( 74.7% ) . in spite of the small size of our control population
firstly , we have shown that the ct genotype of tgfb c-509 t was the only polymorphism ( of the 6 analyzed ) associated with the genetic regulation of o. europaea allergy ( table 3 ) .
tgf- is a multifactorial cytokine that plays key roles in normal cellular processes and diseases , such as t - cell activation and proliferation , and may be essential in modulation of allergic airway inflammation and airway remodeling .
the tgf- gene is located on chromosome 19q and there are a number of polymorphisms within this gene that are believed to have a role in tgf- expression .
tgfb ( c-509 t ) is presented within a proximal negative regulatory region , and the t allele has been associated with higher tgf-1 plasma levels . due to the inconclusive results on the association of tgf- polymorphisms and asthma , 3 years ago , a meta - analysis was reported studying the role of tgf- and asthma , based on 16-case control sets .
the authors concluded that the c-509 t polymorphism ( carriers of the t allele ) could be a risk factor for asthma , mainly in the asian population and adults .
our results are in agreement with the association of tt homozygous and the highest levels of soluble tgf- , but this genotype was infrequent in our population . however , the heterozygous ct genotype , which showed the lowest levels of tgf- , was a risk factor for olive pollen allergy in our population .
these data support our previous results in which we reported a tgf- decrease in the sera of olive pollen allergic patients , in an independent cohort of subjects , selected from the same area .
all of these results reinforce our previous idea that olive pollen allergic patients have a decreased tgf- dependent regulatory response , thereby pointing to the importance of evaluating , when feasible , the combination of levels and cytokine polymorphisms . according to ole e 2 and ole e 10 specific sensitizations
firstly , the cc homozygous il10 - 571c > a genotype could be a protective factor for ole e 2 sensitization and mainly for patients with asthma and ole e 2 sensitization compared with asthmatic - patients nonsensitized to ole e 2 .
this polymorphism showed intermediate levels of il-10 cytokine ( 7.77 5.4 pg / ml ) compared with the other 2 polymorphisms ( aa homozygous : 5.9 6.1 pg / ml , ca heterozygous : 8.76 5.3 pg / ml ) .
in contrast , the ca heterozygous il10 - 571c > a genotype was increased in patients with asthma and ole e 2 sensitization compared with ole e 2 nonsensitized patients with asthma ( 45.7% versus 21.4% , p = 0.02 , or , 3.08 ; 95% ci , 1.138.4 ) .
a recent meta - analysis suggested that il-10 promoter polymorphisms were associated with asthma risk .
this work revealed significant associations between 1082a / g and 592a / c polymorphisms and asthma .
however , there was no significant association between 819t / c polymorphism and asthma risk .
other studies showed that a polymorphism at the 592 position of il-10 is associated with its regulation of expression and recently the association of this polymorphism and immune - related diseases has been studied including type 2 diabetes with and without nephropathy , multiple sclerosis , and asthma . secondly
, we have found a statistically significant increase of the ga heterozygous tnfa-308g > a genotype in ole e 10 sensitized patients compared with olive pollen allergic patients without sensitization to the ole e 10 allergen ( 25.3% versus 11.9% , p = 0.04 , or , 2.5 ; 95% , ci , 1.026.1 ) ( table 4 ) .
this genotype showed intermediate levels of the tnf- cytokine ( 7.77 5.4 pg / ml ) compared with the other 2 genotypes ( gg homozygous : 7.01 4.8 pg / ml , aa homozygous : 9.3 6.1 pg / ml ) .
the 308g / a polymorphism in the tnf- gene has been extensively investigated for its association with asthma ; however , the results of different studies have been inconsistent .
two years ago a meta - analysis was carried out to analyze the association between the 308g / a polymorphism of the tnf- gene and asthma risk .
the results suggested that the tnf- 308g / a polymorphism contributes to susceptibility to asthma and , specifically , significantly elevated risks of asthma were associated with a allele carriers in the atopic population but not in the nonatopic population .
more recently , it has been described that the genetic polymorphisms of tgf-1 and tnf- are associated with asthma .
this same study also described tgf-1 as being a modulator of atopy , and both tgf-1 and tnf- were found to be elements that modulate clinical severity and airway obstruction in an additive manner .
overall , our results suggest a role of tgf-1 in olive pollen sensitization and tnf- and il-10 genotypes in the asthma induced by specific olive pollen allergens .
combined analysis of multiple genetic polymorphisms with different allergic phenotypes could be a useful tool for identifying profiles of risk or prevention of these disorders . | sensitization to specific olive pollen - allergens ( ole e 2 and 10 ) has been correlated with a clinical pattern of asthma .
this study analyzes the association between several polymorphims of tnfa ( g-308a , c-857 t , and c-1031 t ) , il10 ( c-571a and a-1117 g ) , and tgfb ( c-509-t ) and these sensitizations .
these polymorphisms were genotyped by allelic discrimination , in olive pollen - allergic patients ( phenotyped for specific ole e 2 and 10 sensitizations ) and healthy controls .
levels of serum - soluble cytokines were correlated with specific genotypes and clinical phenotypes .
the results showed that heterozygous tgfb c-509 t genotype , besides having the lowest sera tgf- levels , was significantly increased in olive pollen - allergic patients compared with controls . according specific sensitizations , cc genotype of il10 c-571a could be a protective factor for ole e 2 sensitization and mainly for asthmatic ole e 2 sensitized patients compared with asthmatic non - ole e 2 sensitized patients ( or : 0.26 , p = 0.008 ) .
in contrast , heterozygous ca genotype was increased in ole e 2 asthmatic subjects compared to asthmatic non - ole e 2 sensitized patients .
lastly , heterozygous tnfa g-308a genotype was associated with ole e 10 sensitization ( or : 2.5 , p = 0.04 ) . in conclusion ,
these results suggest a role of tgf-1 in olive - pollen sensitization and tnf- and il-10 genotypes in the asthma induced by specific olive - pollen allergens . | 1. Introduction
2. Material and Methods
3. Results
4. Discussion |
PMC4358666 | rapidly developing countries such as the kingdom of saudi arabia ( ksa ) have experienced a dramatic decrease in physical activity mostly because of economic prosperity and industrialization , and especially in urban areas such as riyadh , ksa .
data from the world health organisation ( who ) report on the ksa in 2005 show overall low percentages of high ( 16% ) and moderate ( 16% ) physical activity , and high percentages of low ( 68% ) activity among men and women 1565 years old.1 others have also described high prevalence of physical inactivity among saudis.2,3 data from a comparative study on 20 countries showed that the ksa was among the countries that reported the highest amounts of time spent sitting.4 studies have shown that women in the ksa are more inactive than men in all age groups,1,3,5 and this is also the case for other countries in the gulf cooperation council.6 a considerable number of studies have been performed on saudi adolescents711 and young adults , including university students,12,13 and they show similar results in relation to sex .
in addition , in cross - cultural studies , saudi female youth appear to be more sedentary and to have lower physical activity levels compared to female british youth.14,15 the reasons for the sedentary behaviors and low physical activity levels among saudi women compared with both saudi men and women from other countries are poorly explored .
saudi arabian society , especially in urban areas , shows a combination of modernity mixed with traditional roles , values , and conservative social norms , especially for women . this is a unique socioeconomic , environmental , and cultural context that needs to be further understood
there are certain sociocultural factors that appear to be barriers to women s physical activity , such as low education levels,16 wearing an abaya outside , depending on a driver , limited freedom of movement outside of the house , and employing domestic helpers . however , high socioeconomic status might , as in western countries , contribute to access to health clubs ( especially in the large cities where they are available ) , having physical activity equipment at home , and being more liberal both the women themselves and their families .
in addition , hot weather is an important barrier for exercising outdoors both for men and women
. men also experience significant barriers such as low education , employing domestic helpers , and frequent use of automobiles , but they have more freedom of movement outside of the home .
very few studies have addressed these issues,13,17,18 and those that have , did so from a limited perspective . to our knowledge
, no study has extensively explored the sociocultural factors that contribute to low physical activity among women in the ksa .
this would aid the understanding of which factors are the most important and , therefore , allow the implementation of effective health promotion strategies in the future , particularly for young saudi adults .
university students are an interesting population to study and to target for change , for multiple reasons : 1 ) they are relatively easy to access ; 2 ) the university is an open milieu that is susceptible to change ; and 3 ) universities can support health promotion initiatives through appropriate infrastructures .
university students are an important part of the young adult population , which is the largest segment of young societies such as the ksa s .
based on these criteria , we selected university students with the aim of exploring different physical activity parameters , including self - efficacy , as well as perceived barriers and benefits for physical activity in saudi women .
the study participants were first - year university students at princess nora bint abdul rahman university ( pnu ) , faculty of rehabilitation and health sciences , riyadh , ksa , recruited for the study in 2014 .
pnu is a female - only university in the ksa that aims to educate saudi women .
the study population was taken from seven bachelor s programs in health and well - being : health education and promotion , epidemiology , physiotherapy , occupational therapy , audio and speech , clinical nutrition , and radiology .
the current study is cross - sectional and involved 94 female students who were 1822 years old .
the questionnaire was self - administered and had five parts : 1 ) questions related to anthropometric and sociodemographic characteristics ; 2 ) the arab teens lifestyle ( atls ) questionnaire;19 3 ) the barriers self - efficacy scale ( barse ) ; 4 ) questions related to sex , social factors , and self - image ; and 5 ) the exercise benefits / barriers scale ( ebbs ) .
the first part involved standard questions on sex , weight , height , parents educational levels , and professions , number of housekeepers , number of family members , and illnesses among close family members .
the second part was the atls questionnaire , a questionnaire that was developed for arabic youth and young adults.19 it was chosen as the most suitable scale for obtaining detailed information on the physical activity types , intensities , frequencies , and durations for this sample of young saudi females .
the atls has been used in studies on physical activity in the ksa812 and in other countries in the arab world2022 among adolescents and young adults .
in addition , the atls includes ten questions on dietary habits and four questions on behaviors related to physical activity ( where , with whom , when , and why the physical activities were performed ) .
the barse scale is used to assess self - efficacy , health beliefs , and motivation , which have all been shown to be related to sustained health behavior , including physical activity.23,24 the scale is built on the likelihood of a person s perceived ability to engage in physical activity three times / week in the next 3 months in the face of barriers . the barse is based on 13 questions with a ranking of 0100 ( at intervals of 10 ) ranging from not at all confident to highly confident ; the higher the mean scale score ( closer to 100 ) , the higher the person s self - efficacy .
the fourth part was developed based on literature and information from focus group discussions organized as part of the present study ( conducted prior to the present study ) , and refers to social and cultural factors .
more precisely , the fourth part considered five main social milieus ( university , family , the islamic community , girlfriends , and social networks ) with regard to their hindering or encouraging physical activity ; these questions have a ranking of 3 to + 3 , ranging from hindering to encouraging .
in addition , a number of questions related to media , self - image , and sex were added , using 5-point likert scales for each item .
finally , the ebbs is a scale that has been previously used to identify perceived barriers to and facilitators of physical activity.25 the instrument is built on 43 questions divided into two dimensions , barriers and benefits .
the range for the overall score is 43172 ( the higher the number , the more positively physical activity is perceived ) ; for the benefits , the score is 29116 , and for the barriers , it is 1456 ( the higher the score , the more negatively physical activity is perceived ) .
some minor changes were needed for the atls for it to fit the needs of this specific population .
the qualitative data were generated in a subgroup of students from four focus group discussions of five to eight students each .
the focus group discussion sessions showed that students are very inclined to dance , and thus dancing was added to the questionnaire in the category of moderate - intensity physical activity ; these changes were approved by the original author of the atls .
cronbach s alpha was used to calculate the reliability of the barse and ebbs scales .
the internal consistency ( alpha ) for the barse scale was 0.79 , and it was 0.83 for the ebbs scale , indicating good consistency for group comparisons .
the study participants were first - year university students at princess nora bint abdul rahman university ( pnu ) , faculty of rehabilitation and health sciences , riyadh , ksa , recruited for the study in 2014 .
pnu is a female - only university in the ksa that aims to educate saudi women .
the study population was taken from seven bachelor s programs in health and well - being : health education and promotion , epidemiology , physiotherapy , occupational therapy , audio and speech , clinical nutrition , and radiology .
the current study is cross - sectional and involved 94 female students who were 1822 years old .
the questionnaire was self - administered and had five parts : 1 ) questions related to anthropometric and sociodemographic characteristics ; 2 ) the arab teens lifestyle ( atls ) questionnaire;19 3 ) the barriers self - efficacy scale ( barse ) ; 4 ) questions related to sex , social factors , and self - image ; and 5 ) the exercise benefits / barriers scale ( ebbs ) .
the first part involved standard questions on sex , weight , height , parents educational levels , and professions , number of housekeepers , number of family members , and illnesses among close family members .
the second part was the atls questionnaire , a questionnaire that was developed for arabic youth and young adults.19 it was chosen as the most suitable scale for obtaining detailed information on the physical activity types , intensities , frequencies , and durations for this sample of young saudi females .
the atls has been used in studies on physical activity in the ksa812 and in other countries in the arab world2022 among adolescents and young adults .
in addition , the atls includes ten questions on dietary habits and four questions on behaviors related to physical activity ( where , with whom , when , and why the physical activities were performed ) .
the barse scale is used to assess self - efficacy , health beliefs , and motivation , which have all been shown to be related to sustained health behavior , including physical activity.23,24 the scale is built on the likelihood of a person s perceived ability to engage in physical activity three times / week in the next 3 months in the face of barriers .
the barse is based on 13 questions with a ranking of 0100 ( at intervals of 10 ) ranging from not at all confident to highly confident ; the higher the mean scale score ( closer to 100 ) , the higher the person s self - efficacy .
the fourth part was developed based on literature and information from focus group discussions organized as part of the present study ( conducted prior to the present study ) , and refers to social and cultural factors .
more precisely , the fourth part considered five main social milieus ( university , family , the islamic community , girlfriends , and social networks ) with regard to their hindering or encouraging physical activity ; these questions have a ranking of 3 to + 3 , ranging from hindering to encouraging .
in addition , a number of questions related to media , self - image , and sex were added , using 5-point likert scales for each item .
finally , the ebbs is a scale that has been previously used to identify perceived barriers to and facilitators of physical activity.25 the instrument is built on 43 questions divided into two dimensions , barriers and benefits .
the range for the overall score is 43172 ( the higher the number , the more positively physical activity is perceived ) ; for the benefits , the score is 29116 , and for the barriers , it is 1456 ( the higher the score , the more negatively physical activity is perceived ) . a pilot test was performed with 30 students .
some minor changes were needed for the atls for it to fit the needs of this specific population .
the qualitative data were generated in a subgroup of students from four focus group discussions of five to eight students each .
the focus group discussion sessions showed that students are very inclined to dance , and thus dancing was added to the questionnaire in the category of moderate - intensity physical activity ; these changes were approved by the original author of the atls .
cronbach s alpha was used to calculate the reliability of the barse and ebbs scales .
the internal consistency ( alpha ) for the barse scale was 0.79 , and it was 0.83 for the ebbs scale , indicating good consistency for group comparisons .
statistical analyses were performed using the sas software package version 9.3 ( sas institute , cary , nc , usa ) .
descriptive statistics ( means and frequencies ) were calculated to describe the sociodemographic characteristics as well as individual atls items , sociocultural items , and the barse and ebbs scales . for the atls questionnaire ,
variables were created for the total minutes for individual activities and for moderate , high , and total physical activity .
the atls allows for calculating all the above , because it gives information on times / week and minutes / day for all individual activities .
met calculations are based on total physical activity.26 activities are also grouped as moderate or
high based on the mets for each of the activities . based on who recommendations,27 a minimum of 150 minutes / week of moderate - intensity physical activity is required for adults ( equivalent to 600 met minutes / week ) .
physical activity was used as a continuous variable and was also based on the cut - off of 600 met minutes / week of moderate physical activity . for the latter ,
the threshold for each individual was based solely on moderate physical activity levels ( met minutes from high physical activity were not included ) .
sedentary behavior was defined based on two questions ( time spent watching tv and/or dvds / videos per day and time spent on the computer and/or the internet ) .
the combined time for being sedentary was truncated at 16 hours / day to ensure a reasonable and realistic level of physical activity ( as advised by the atls guidelines ) .
body mass index ( bmi ) was calculated as follows : weight ( kg / height m ) . for dietary habits , a cut - off of > 3 times / week was set for unhealthy food and of < 7 times / week for healthy food .
individuals who had one or more missing values for a specific activity were completely excluded in the calculations of total minutes for moderate , high , and total physical activity , and for met minutes of moderate , high , and total physical activity . for the barse and the ebbs ,
if more than 5% of the items were unanswered , the individuals were considered missing and were excluded ( more than one item for the barse and more than two items for the ebbs ) .
otherwise , the missing values for specific items were replaced by the mean values calculated from the remaining items .
pearson s correlation coefficients were calculated to evaluate the association between physical activity and bmi with various variables .
the student s t - test was used to test the differences between the active and inactive students ( based on the cut - off value of 600 points for met ) for the barse and ebbs scales .
the barse scale , ebbs overall scale , ebbs benefits subscale , and ebbs barriers subscale scores were normally distributed ( graphic representation , skewness , and kurtosis features ) , and therefore were fit for the student s t - test .
the average bmi was 22.44.4 kg / m , and the average age was 20.00.7 years .
both fathers and mothers of the students had relatively high educational levels ( 53.2% and 40.3% , respectively ) , but only a small percentage of the mothers ( 23.4% ) worked outside of the home compared with the fathers ( 79.2% ) .
one - third of the participants had at least one ill family member within the previous year because of chronic diseases such as diabetes , cancer , heart disease , etc .
regarding daily dietary habits , 36.4% of the participants had breakfast , 22.1% had vegetables , 5.2% had fruit , and 33.8% had dairy .
with regard to unhealthy dietary habits , fast food ( including international and local fast food chains ) and soft drink consumption of more than three times / week were both at 41.6% , and french fry consumption more than three times / week was 46.8% . a high consumption of cakes ( including biscuits , donuts , etc ) and sweets was also observed ( 45.5% and 64.9% , respectively ) .
table 2 shows the levels of physical activity for individual activities and for activities categorized as moderate or high in total minutes and met minutes .
walking , household work , and moderate intensity sports ( dancing was often chosen ) were the most preferred activities , with 101114 , 90150 , and 68175 minutes / week , respectively .
overall , students reported more time spent in moderate than in high physical activity during the week ( 267269 vs 4753 minutes / week ) . among high - intensity physical activities , running /
jogging was the only activity on which a relatively high percentage of the students spent some time during the week ( approximately 53% of students spent > 0 minutes / week ) .
based on the cut - off of 600 met minutes / week of moderate physical activity . regarding sedentary levels , an average of 6.23.5 hours / day sitting still was observed . when physical activity was studied as a continuous variable ( total minutes of physical activity / week ) ,
it was not associated with bmi or with being sedentary ( r=0.08 , p=0.52 ; and r=0.15 , p=0.23 , respectively ) .
being sedentary was positively significantly associated with bmi ( r=0.35 , p=0.0014 ) , and moderate physical activity was positively significantly associated with high physical activity ( r=0.34 , p=0.0041 ) .
table 3 describes behaviors related to physical activity as part of the atls questionnaire , as percentages . for each question ,
the data show that students exercise at home ( 92.3% ) and alone ( 79.5% ) , and that their main reasons were for health ( 71.8% ) or for losing weight ( 50.0% ) . the students exercised mostly during the morning ( 41.0% ) or the evening ( 32.0% ) .
table 4 displays the means for the 13 individual items on the barse scale as well as for the overall scale .
the highest point scores appeared for vacation ( 6129 ) and exercise alone ( 5931 ) ; being self - conscious about one s appearance had a high score as well ( 4631 ) .
the overall point scores showed low self - efficacy , with the average total score being < 50 ( 4214 ) .
all other factors had an average score of < 50 , showing low self - efficacy .
when the students were categorized as active or inactive ( based on the 600 met minutes / week cut - off ) , the student s t - test showed significant differences for the overall barse scores ( 4613 vs 3715 , p=0.02 ) .
information relating to social factors showed that among different social settings ( university , family , the islamic community , girlfriends , online ) , the university was the only setting that hindered physical activity ; the results showed that 32% of the students selected the university , compared with 19% for family , 15% for the islamic community , 7% for girlfriends , and 13% for online networks and social media .
table 5 shows the students responses as frequencies for the categories agree , neutral , and disagree on questions related to self - image , attitudes toward physical activity , and physical activity and sex .
forty - four percent of the students disagreed that they would change their diets rather than engage in physical activity if they wanted to lose weight .
sixty percent of the students agreed that a healthy body shape has curves , but 98% of them agreed that it is important for a woman to have a fit and healthy body .
with regard to sex , 66% agreed that it is easier for men to be physically active than for women , and 60% agreed that men are more physically active than women . regarding the ebbs , the overall scale mean score was 13110 ; for the benefits and barriers subscales , the point scores were 9510 and 345 , respectively , showing that physical activity is positively perceived by the students . with the individual items for benefits , the students agreed or strongly agreed on most of them , thus showing awareness of the effects of physical activity for health and well - being .
the students had high percentages of disagreement for the following : 1 ) exercising lets me have contact with friends and persons i enjoy ( 41% ) ; 2 ) exercising is a good way for me to meet new people ( 33% ) ; and 3 ) exercising increases my acceptance by others ( 33% ) . regarding barriers ,
the students considered one important barrier to be that locations for exercise were too far away ( 52% agreed ) and that there were too few places to exercise ( 75% agreed ) .
they mostly disagreed that family members ( 66% disagreed ) or feeling embarrassed to exercise were barriers to physical activity ( 71% disagreed ) .
no association was found when students were categorized as active vs inactive for the overall scale ( 13211 vs 13010 , p=0.44 ) , for the benefits scale ( 959 vs 9510 ,
to our knowledge , this is the first study to consider in depth a number of sociocultural factors related to physical activity among saudi females . in the current study , among first - year university students
, we found that female saudi students had low levels of physical activity and high levels of sedentariness .
there was an observed overall low self - efficacy that was associated with physical activity .
in addition , the young women perceived physical activity as beneficial , but their physical activity levels were low .
the students showed awareness of the health - related and well - being benefits of physical activity , and they considered the lack of physical activity facilities a more important barrier than their families or society .
the main focus of the present study was not physical activity levels but physical activity - related sociocultural factors . however , our results are in agreement with those of previous studies with regard to the low levels of physical activity ( and also with moderate physical activity levels being more common than high levels),8,10,14 the high sedentariness levels,4,8,10,14 and dietary habits ( high percentages of unhealthy food consumption).8 for specific activities , our study showed a preference for walking , household activities , and moderate - intensity sports , including dance .
another study also showed a preference for household activities and walking but not moderate - intensity physical activity such as dancing , very likely because dance is a modification that we added to the atls in our study.10 in a previous study , jogging and swimming were also reported as preferred activities.14 finally , in our study , moderate - intensity physical activity was positively associated with high - intensity physical activity , suggesting that students who are more active tend to be active at both moderate and high levels .
students reported that they exercised mostly alone , and that factor did not appear to decrease their self - efficacy . in the same line of evidence , meeting new people or getting together with friends was not a benefit related to physical activity .
in addition , the students exercised at home , and in another study , saudi adolescents also reported that they exercised alone and at home.11 if this is a genuine preference , then assumed barriers for physical activity for saudi women related to isolation and restrictions on going outside do not appear to be a major concern .
however , these results should be interpreted with caution , because they might also reflect the lack of choice and/or habits rather than preferences .
interestingly , our results demonstrated that the students chose physical activities for their health and for losing weight .
the results from the ebbs scale also showed that the students are aware of the benefits of physical activity , for their health and their well - being .
in addition , the students appear to consider physical activity to be as important as diet for losing weight and think that having a healthy body is crucial .
highly scored perceived benefits of physical activity were observed in another study on saudi students ( the only other study that used a benefits / barriers scale , to our knowledge).17 it must be noted that in our sample , the parents had relatively high educational backgrounds , which could be significant in encouraging overall health knowledge .
young saudis from different socioeconomic backgrounds might have lower levels of awareness of physical activity.7 in addition , our students are part of health science programs and therefore might be more sensitive to and knowledgeable about health ( even though they are first - year students ) . with regard to sociocultural barriers , information from our questions about different social factors showed encouragement toward physical activity from families , the islamic community , friends , and social media , but less from the university . among the barriers to physical activity , family discouragement was one of the least considered .
another study , however , showed that norms and traditions can be significant barriers to physical activity in the ksa.18 it must be emphasized that in this study , older saudi females and those from different socioeconomic backgrounds were also studied , which implies that at least some of them were conservative .
regarding the role of universities , it is important to mention that pnu is a newly established university that does not yet have available organized physical facilities . the schedule for students does not allow for any involvement in physical activity (
students are picked up after study hours and do not spend additional time at pnu ) .
based on this , the students would benefit enormously from pnu physical activity facilities if these were to be developed and offered in a systematic way .
the students being self - conscious about their appearance did not show low self - efficacy , and feeling embarrassed about exercising was also not a barrier to physical activity .
sixty percent of students agreed that a healthy body should have curves , and arabic women appear to be comfortable with having curvy bodies , as has also been reported by others.28 interestingly , the greatest barrier for students was the lack of facilities and their distance , which also agrees with other studies.17,18,29 however , in two of these studies,17,18 both men and women were included , and limited facilities was a common barrier .
that is , this appears to be a significant barrier for men as well ; in our study , the students did not clearly disagree regarding whether it was easier for men to be active or that men were more active than women , suggesting that sex differences are not particularly significant .
overall , students perceived physical activity positively , just as in other studies.17 however , the overall scores were not associated with physical activity when the students were categorized as active or inactive .
this can likely be explained by the fact that positive opinions about physical activity and health in general are not necessarily accompanied by appropriate actions . in other words , a high level of awareness
self - efficacy regarding performing physical activities in our study was overall relatively low , requiring further study .
self - efficacy was , however , significantly positively associated with physical activity , suggesting that students who were more motivated were more active as well.30 some of the strengths and limitations of the current study need to be considered .
a major strength is that this study is the first to provide a considerable amount of information on sociocultural factors related to physical activity among young saudi women .
an additional strength is the use of the atls , a validated questionnaire for arab youth and therefore an excellent tool for assessing physical activity and factors related to physical activity in our population .
the main limitation of this study is that it involved university students from the capital , who do not represent all students in the ksa or all women of different ages and socioeconomic backgrounds .
in addition , the students had health - related study backgrounds , which likely led to their acknowledging the benefits of physical activity .
in conclusion , the lack of facilities and lack of encouragement from pnu appear to be the major barriers to greater physical activity in female students .
however , there does appear to be sufficient knowledge about physical activity itself and also encouragement from families and social surroundings . in that context , developing more culturally sensitive exercise facilities for women , as well as creating more facilities and implementing university health promotion programs for physical activity would be of great value , at least for educated saudi females . | purposethe kingdom of saudi arabia is experiencing a dramatic increase in physical inactivity , with women having higher levels of inactivity than men among all age groups .
it is assumed that factors such as dress codes , restrictions on going outdoors , and conservative norms are the main reasons for women s low physical activity .
our aim was to explore the different parameters related to physical activity , including self - efficacy , as well as the perceived barriers to and benefits of physical activity in young saudi females.patients and methodsninety - four first - year female saudi university students in riyadh , kingdom of saudi arabia , participated in the present study in 2014 .
the students were from eight bachelor s programs in health and well - being , and each completed a questionnaire with questions divided into five parts as follows : 1 ) socioeconomic status , 2 ) physical activity , 3 ) self - efficacy 4 ) social factors , and 5 ) barriers and facilitators related to physical activity.resultsthe students exercised at home and alone , and there was low self - efficacy for physical activity ( mean score = 4214 ) . among social factors , attending university was the only factor that hindered physical activity ( 32% ) .
physical activity was positively perceived overall ( mean score = 13110 ) .
students showed awareness of the benefits of physical activity for health and well - being .
the most important barrier was the lack of designated areas available for physical activity .
students disagreed that family or the islamic community were barriers to physical activity.conclusionthe lack of facilities and lack of encouragement from the university , but not a lack of knowledge ( a high level of knowledge is to be expected given their health and well - being studies backgrounds ) and/or restrictions from families and society , seem to hinder female students physical activity , at least young saudi students . | Introduction
Materials and methods
Population
Questionnaire
Statistical analyses
Results
Discussion
Conclusion |
PMC2915807 | the construction of specific surface architectures via controlled self - assembly is a key goal in the design of nanoscale molecular electronic devices . in this respect ,
particularly , attractive candidates are guanine ( g ) and its derivatives , which are unique among the dna constituents for their propensity to form quadruplex structures , known to be stabilized through hoogsteen and watson - crick base pairing [ 1 , 2 ] .
moreover , guanine has a low ionization potential due to which it plays a key role in electrical conductivity of dna - based materials .
various research groups have recently reported on the evolution of the art of making surface deposited 1-dimensional ( 1d ) structures known as guanosine wires ( g - wires ) [ 312 ] . however , despite the promising experimental results , basic processes responsible for wire formation are still far from being fully understood .
two main steps are fundamental to drive the topic from a laboratory curiosity towards the technologically relevant g - wire engineering : ( a ) achieving control of self - folding and self - stimulated end - to - end fusion of the macromolecular quadruplexes in solution as a function of their molecular composition ( i.e. , base sequence ) and solution conditions , and ( b ) understanding the effect of surface interactions on material deposition from solution phase onto the solid substrate . in this work
, we report on how particular modifications of the base sequence of a g - rich oligonucleotide , which are supposed to affect the folding geometry of the quadruplex , affect its ability to form g - wires in solution phase .
the idea of wire formation is based on the previously established finding that gc sticky ends may be used to link quadruplexes into longer 1d aggregates .
a series of oligonucleotide sequences with one or two gc ends and central sequences of different length were designed for this purpose ( see table 1 ) .
the folding topology [ 14 , 15 ] has been previously investigated , and quadruplex formation has been recently studied by mergny et al . .
in these studies , length and nature of propeller loops were studied , but end - to - end fusion of the quadruplexes was not considered . here , we focused on end - to - end fusion of these quadruplex folds .
polyacrilamide gel electrophoresis ( page ) is a conventional tool to differentiate between macromolecular objects of different length . during migration through
an alternative noninvasive method for determining the size of macromolecular objects is dynamic light scattering ( dls ) .
this method was extensively applied to study dna molecules over a wide range of sizes [ 1721 ] .
specifically , it has been used to characterize g - quartet stacking in solutions of single guanosine molecules [ 2226 ] , and for studying formation of g - quadruplexes [ 2730 ] .
protozanova and macgregor compared the use of dls and page on sequences with long terminal tracks .
these tracks were based on sequences such as d(a15g15 ) and d(a13g15tc ) , which form frayed wires with a g - quadruplex stem and nonguanine portions reaching out as single - stranded arms .
we investigated the formation and dimensions of the supramolecular objects formed in aqueous solution of specially designed g - rich dna sequences ( table 1 ) by combining dls and page measurements .
we studied the effects of temperature , oligonucleotide concentration , method of preparation , and base sequence to explore the basic principles of g - wire formation .
oligonucleotides ( see table 1 ) were ordered from eurogentec ( belgium ) as 40 nm desalted syntheses and reconstituted in water .
dialysis was performed at a concentration of 100 m dna in the presence of 100 mm nacl buffered with 10 mm napi at ph 6.8 , then diluted in same concentration of buffer or h2o to specified concentrations immediately prior to measurements . where heat treatment is specified , oligonucleotides at 100 m dna in presence of 100 mm nacl , 10 mm napi ( ph 6.8 ) were heated to 93c for ten minutes and left to cool in heating block to room temperature prior to experiment . for dls measurements ,
the oligonucleotides with a 0.4 mm concentration were used . for the concentration dependence studies
gel electrophoresis experiments were performed on 15% native bis / acrylamide gels , utilising 1x tbe running buffer supplemented with 5 mm nabo2 to retain folded architectures .
oligonucleotide samples were prepared to a final concentration of 2 m dna in presence of 100 mm nacl buffered with 10 mm napi at ph 6.8 with 5% sucrose to facilitate sample loading .
gels were run at 120 v and 4c for a maximum of 2 hours and stained with 1x sybr gold ( invitrogen , paisley , uk ) in 1x tbe buffer . in order to compare migration rates between different oligonucleotide samples , a generuler ultra low range ( 10200 bps ) dna ladder ( fermentas , york , uk ) was used .
dynamic light scattering experiments ( dls ) were performed using a digital correlator ( alv - laser vertriebgesellschaft ) , a goniometer , and a photomultiplier detector .
the light source was a frequency doubled nd : yag laser with a wavelength of 532 nm .
the incident laser beam was linearly polarized in the direction perpendicular to the scattering plane .
the scattered light of the same polarization was detected at scattering angles 50 130. the capillary containing the sample had an inner diameter of 5 mm and was immersed in an index matching bath with a diameter of 10 cm to minimize stray light from the outer capillary wall .
we measured the autocorrelation function g2(t ) = i(0)i(t)/i of the average scattered light intensity i .
most of the measurements were performed in a mixed regime , in which the intensity autocorrelation function is related to the field correlation function g1(t ) by the relation
( 1)g2(t)=1 + 2(1jd)jdg1(t)+jd2g12(t ) ,
with jd being the ratio between the intensity of the light that is scattered inelastically and the total scattered intensity .
the field correlation function g1(t ) for systems with a polydisperse size distribution can be expressed by stretch exponential functions
( 2)g1(t)=iaiexp ( ti)i ,
where ai is the amplitude of the ith relaxation mode and the stretch exponent lies in the range 0 i 1 .
the average relaxation time of the ith relaxation mode is given by
( 3)i=ii(1i ) ,
where (1/i ) is the gamma function .
the parameter i is a measure of the width of the distribution of relaxation times .
very narrow distributions correspond to i ~ 1 , while smaller values of i indicate broader distributions .
the characteristic relaxation times of the observed dynamic modes were obtained by fitting the measured autocorrelation curves g2(t ) to ( 1 ) and ( 2 ) . according to the polarized scattering ( so called vv scattering ) detected in our experiments ,
the translational diffusion coefficients were calculated as
( 4)di=1iq2 ,
where q is the scattering wave vector given as q = ( 4n/)sin ( /2 ) with n = 1.33 being the solution refractive index , the laser wavelength and the scattering angle . in most cases ,
several correlation curves were measured for every solution and averaged values of the fitting parameters were taken for further consideration . from the translational diffusion coefficients ,
the corresponding hydrodynamic radius is calculated as
( 5)rh = kbt6d ,
with kb being the boltzmann constant , t the absolute temperature , and the solvent viscosity . for dilute solutions of rod - like particles the hydrodynamic theory of tirado and garcia de la torre [ 3436 ] can be applied as long as the ratio of length to diameter p = l / d is in the range 2 p 30 . the translational diffusion coefficient in this theory is given by
( 6)d = kbt3l(ln p+ ) ,
where is the end - effect correction term given as = 0.312 + 0.565/p 0.100/p .
knowing the diameter of the studied rod - like assembly , thus the length of the objects can be estimated .
the sequences were first investigated by page to check for folding of oligonucleotides into higher order architectures .
samples prepared by heat treatment show a lesser degree of macromolecular assembly as compared to dialysed samples for all investigated oligonucleotides ( figure 1 ) .
the band with the highest mobility in each lane corresponds to a bistranded monomeric unit .
the shortest dialysed sequences ( sequences 2 and 3 ) show only few albeit sharp bands pointing to the formation of smaller discrete stable species and a very low degree of macromolecular self - assembly .
the longer sequences 1 , 5 and 6 , on the other hand , indicate possible stepwise assembly into large aggregates . in all investigated samples , we detected a slow diffusive mode with correlation times in the range of 110 ms ( at = 90 ) ( figures 2 and 3 ) .
such a mode corresponds to translational motion of large globular aggregates with hydrodynamic radii in the range of micrometers .
they are usually attributed to the presence of loose multichain associates formed due to electrostatic interactions , but their nature is still not quite resolved [ 20 , 37 , 38 ] .
as these aggregates are to our opinion not connected with g - wire formation , they will not be further considered . in all heat
treated samples , only the above mentioned slow mode exists , so they will not be further discussed .
solely the slow mode is observed also in the dialysed sequence 2 ( figure 3(a ) ) , which is the only sequence containing adenine base . in dialysed sequence 3
, an additional fast diffusive mode can be faintly resolved at large scattering angles ( > 90 ) .
the corresponding diffusion coefficient calculated from ( 4 ) has a value of df = 3.0 0.3 10 m / s ( table 2 ) , which indicates fast translational motion of very small scattering objects , most probably single oligonucleotides . in dialysed sequence 4 ,
the fast mode is more profound and can be clearly resolved ( figure 3(b ) ) .
the dls autocorrelation functions of dialysed sequences containing gggg repeats ( sequences 1 , 5 , and 6 ) all show two relaxation modes .
the angular dependence of the inverse relaxation time of the fast mode ( figure 4 ) reveals quadratic dispersion as given by ( 4 ) .
the resultant diffusion coefficients df obtained for solution concentration of 0.4 mm are listed in table 2 .
the obtained values suggest that the fast mode is most probably associated with translational motion of oligonucleotides assembled into g - quadruplex structures .
the dialysed sequence 4 at 0.4 mm dna was also used to investigate the temperature dependence of the fast diffusive mode ( figure 5 ) .
a relative amplitude of the fast mode decreased with increasing temperature and for t > 338 k the fast mode could not be resolved anymore .
to reveal possible temperature - induced structural changes of the scattering objects , the values of diffusion coefficient df obtained at different temperatures were divided by temperature t and multiplied by the corresponding solvent viscosity (t ) .
the resulting values of df(t)/t as a function of temperature are shown in figure 5 .
one can notice that they remain constant throughout the investigated temperature range , which indicates that no structural modification of the scattering objects ( quadruplexes ) takes place .
the effect of oligonucleotide concentration on the value of df was investigated for dialyzed sequence 1 ( figure 6 ) .
although it appears that the values decrease with increasing concentration , no precise conclusion can be drawn due to the large uncertainty of the measured data points . at low solution concentrations and short relaxation times
however , for dna concentrations below 1 mm , which was the case in all our measurements , the effect of electrostatic interactions on solution dynamics is usually negligible and so the solution can be considered as infinitely dilute .
both , page and dls analysis , reveal that the method of sample preparation is essential for the level of self - assembly in solution .
we observe formation of slower migrating species for all oligonucleotides in page for the dialysis treated samples .
sequences 1 , 2 , 5 , and 6 appear to form larger assemblies , whilst sequences 3 and 4 do not .
the most striking results are for sequences 2 and 6 , where one or two species of lower molecular weight were formed in the annealing treatment versus numerous larger species through dialysis .
page comparison between sequences 2 and 3 shows that there are sequence effects in the kinetics of formation for the same topology .
when comparing dialysis , a band appearing at nominal 15 bp in sequence 3 appears only very faintly in sequence 2 , while a faster , and a slower , migrating bands appear quite distinctively in both .
the effect of sticky ends is also shown when comparing sequences 1 and 6 . while between nominal 10 and 35 bp they have the same number of bands appearing in approximately the same positions , their intensities vary substantially . for these particular sequences ,
the difference in the annealing treatment is greater , with sequence 6 showing a very strong fast migrating band at nominal 16 bp . in contrast ,
the fastest migrating band for seq1 is at nominal 12 bp . in general , and as expected , it is apparent that the greater the oligonucleotide length the longer the species formed .
indeed , it is apparent for the longest oligonucleotide sequence ( sequence 5 ) that the macromolecular dna objects break into smaller units in a less discrete manner than for the other sequences .
the smear for low migrating dna in sequence 5 may thus be due to steric hindrance by the pore size of the gel . for the shortest of the dialysed sequences , sequences 2 and 3 ,
the absence of any measurable fast dls mode for the former and the observation of a faint extremely fast mode for the latter , can be explained only by the presence of very small scattering objects , most probably single oligonucleotide molecules .
this finding is not consistent with page results ( figure 1 ) , which reveal additional slower diffusing species resulting in sharp bands , probably signifying multimers .
similar discrete bands are seen also in the picture of page for dialysed sequence 4 . for this sequence ,
the diffusion coefficient of the fast dls mode gives quadruplex length of 8.8 0.8 nm ( table 2 ) , which is considerably larger than the length of a single oligonucleotide strand ( about 4.8 nm ) .
the two dialysed intermediate - length sequences ( sequences 1 and 6 ) both exhibit the same page band attributed to fast migrating monomers and a strong band at about 35 bps ( accordingly to the reference duplex dna ladder ) .
the main difference between the two oligonucleotides is in the formation of two intermediate bands for sequence 6 , while sequence 1 forms no intermediate species .
interestingly , the two strong bands ( at 35 bps ) very well agree with the quadruplex length l estimated from the dls measurements , which is 5.0 0.4 nm for sequence 6 and 6.1 0.5 nm for sequence 1 , respectively .
the smaller apparent length of sequence 6 can be explained by the presence of the intermediate species contributing to the scattering . the diffusion coefficient measured for sequence 1 ( df = 1.06 0.037 ) is in very good agreement with the values obtained for similar folded structures by hydrodynamic modelling .
in page , the majority of the material is so slowly migrating through the gel , that just a broad smearing is observed .
dls measurements , on the other hand , reveal the presence of supramolecular assemblies with the length of 11.0 0.2 nm , which is only slightly larger than the length of a single strand .
in contrast to page , dls also reveals a relatively narrow distribution of aggregate lengths .
this is seen from the dls stretch exponent factor for the fast mode , which is in the range 0.9 < f < 1 , thus excluding a broad distribution of the dimensions of the scattering objects . also for other sequences ,
the origin of the differences between the page and the dls results is not clear . on the basis of the page results , sequences 1 , 5 , and 6
are all supposed to form some long wire - like assemblies , but dls measurements do not support this expectations .
accordingly to the dls results , the highest level of self - assembly is expected for sequence 4 , which exhibits the largest length of the scattering objects with respect to the length of the single strand .
one possible reason for the discrepancies might be quite different solution concentrations used for the two experiments .
page is usually performed with concentrations c ~ 10 m , while dls gives reasonable signals . yet for c > 100 m , this is not consistent with what we intuitively know , that is , higher concentrations tend to favour multimer formation .
another aspect arises from the slow dls mode , which signifies that in addition to wire - like assemblies there are also some other aggregate types present in solution .
these aggregates , most probably loose multipolyion associates , might correspond to the slow diffusing objects observed in page .
dls experiments bring also information on temperature and concentration dependence of the self - assembly .
the temperature dependence ( figure 5 ) shows that up to t = 65c the length of the self - assembled objects remains constant .
nevertheless , the amplitude of the fast dls mode strongly decreases by increasing temperature , while the overall scattering intensity is only slightly reduced .
this signifies that the slow mode , which mainly contributes to the scattering intensity , is not much affected by heating .
but relatively to it , the scattering intensity related to the fast mode becomes more and more weak .
this can be explained by temperature - driven dissociation of the self - assembled structures of well - defined size into much smaller single oligonucleotides , which are practically invisible by dls .
dependence of the fast dls mode on the concentration of the solution ( figure 6 ) also supports the idea of formation of the self - assembled structures with well - defined length , which are not affected by modifications of the solution concentration . on the contrary to wire - like assemblies formed from single guanosine molecules ( gmp )
, it seems that formation of supramolecular assemblies of g - rich oligonucleotides , does not exhibit any critical solution concentration , at which the assembling would become profound .
a series of g - rich oligonucleotides was studied by page and dls to investigate the formation and dimensions of g - quadruplexes .
both methods show that thermal annealing induces much less macromolecular self - assembly than the dialysis method .
this demonstrates that not only the base sequence , but also the folding kinetics play an important role in the self - assembly process . on one hand , this makes the phenomenon very complex , but on the other hand it provides a possibility for fine tuning of the assembling features via external stimuli .
further studies are needed to find the source of the differences and how they can be modulated .
page and dls show the best agreement on quadruplex dimensions for sequences of intermediate length ( sequences 1 , 4 and 6 ) .
the last band of sequence 4 coincides with the dls signal arising from aggregates of a 9 nm length .
the two similar sequences 1 and 6 give also aggregates of similar length 56 nm and these agree with a strong page band observed for both sequences .
for the shortest sequences 2 and 3 , page suggest the formation of multimers not detected by dls .
the only dls signal comes from very fast diffusing objects with effective dimensions below 1 nm pointing to single oligonucleotides . for the largest sequence , on the other hand , page suggests very long aggregates giving a broad smeared band at the beginning of the lane . in dls instead , a well defined fast mode is attributed to a species with the length of about 11 nm .
because we are able to assess intermediate lengths , we are currently investigating the mechanism of self - assembly of these wires by combining both methods .
thus , the combination provides valuable information on the g - quadruplex formation towards control of its length . | the formation and dimensions of g - wires by different short g - rich dna sequences in solution were investigated by dynamic light scattering ( dls ) and polyacrilamide gel electrophoresis ( page ) . to explore the basic principles of wire formation
, we studied the effects of base sequence , method of preparation , temperature , and oligonucleotide concentration .
both dls and page show that thermal annealing induces much less macromolecular self - assembly than dialysis .
the degree of assembly and consequently length of g - wires ( 5 - 6 nm ) are well resolved by both methods for dna sequences with intermediate length , while some discrepancies appear for the shortest and longest sequences .
as expected , the longest dna sequence gives the longest macromolecular aggregates with a length of about 11 nm as estimated by dls .
the quadruplex topologies show no concentration dependence in the investigated dna concentration range ( 0.1 mm0.4 mm ) and no structural change upon heating . | 1. Introduction
2. Experimental
3. Results
4. Discussion
5. Conclusions |
PMC4370847 | many of the difficulties of higher - order calculations in qft can be traced back to the treatment , in the framework of dimensional regularization ( dr ) , of the infinities arising in the intermediate steps of the computation .
ultraviolet ( uv ) , infrared ( ir ) , and collinear ( cl ) divergences are first dimensionally regulated , and then renormalized away in the uv case or canceled by combining virtual and real contributions , or reabsorbed in the collinear behavior of the initial state parton densities . in order to attack the problem numerically , it is often necessary to subtract and add back approximations of the ir / cl singular structures . at one loop ,
several well - tested subtraction procedures have been introduced in the last two decades [ 28 ] . at two loops and beyond , the situation is more involved , but progress is under way [ 914 ]
. the first obvious ingredient , which may lead to a significant simplification in the above picture , is a computational procedure in which all parts of the calculation can be directly treated in four dimensions . as for the virtual contribution ,
the fdr approach has been recently introduced in reference , which allows a subtraction of the uv divergencesat the level of the integrand , leaving a four - dimensional integration over the loop momenta . in the same work , the use of fdr as an ir regulator in qed with massive fermions is also suggested .
in this paper ,
i present the first application of the fdr ideas in the context of fully massless qcd , where the issues related to gauge invariance are much more subtle than in the qed case .
i concentrate , in particular , on the calculation of the \documentclass[12pt]{minimal }
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\begin{document}$$m_\mathrm{top } \rightarrow \infty $ $ \end{document}mtop limit , and re - derive the well - known fully inclusive result [ 16 , 17]1\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \vargamma ( h \rightarrow gg ) = \vargamma ^{(0)}(\alpha _ s(m_h^2 ) ) \left [ 1+\frac{95}{4}\,\frac{\alpha _ s}{\pi } \right ] , \end{aligned}$$\end{document}(hgg)=(0)(s(mh2))1 + 954s,where2\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \vargamma ^{(0)}(\alpha _ s(m_h^2))= \frac{g_f \alpha _
s^2(m_h^2)}{36 \sqrt{2 } \pi ^3 } m^3_h \end{aligned}$$\end{document}(0)(s(mh2))=gfs2(mh2)3623mh3is the lowest order contribution , with \documentclass[12pt]{minimal }
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\begin{document}$$\alpha _ s(m_h^2)$$\end{document}s(mh2 ) , since only gluons are considered . despite its simplicity , all key ingredients of massless qcd are present in this process , such as the simultaneous occurrence of ir / cl divergences and uv renormalization .
the fact that the correct expression is reproduced shows that fdr is a valid and consistent approach in massless qfts , and it gives confidence in its potential to simplify multi - leg / loop computations .
section 2 provides the set - up of the calculation . in sect .
3 , i review the fdr treatment of the uv divergences and discuss its interplay with the ir and cl infinities .
section 4 presents the fdr computation of the virtual part , while sect .
5 deals with the real contribution and its merging with the one - loop piece .
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\begin{document}$$h$$\end{document}h with two , three and four gluons mediated by an infinitely heavy top loop is described by the lagrangian [ 18 , 19]3\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \mathcal{l}_\mathrm{eff } = -\frac{1}{4 } a h g^{a}_{\mu \nu } g^{a,\mu \nu } , \end{aligned}$$\end{document}leff=-14ahgaga,,where4\documentclass[12pt]{minimal }
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\begin{document}$$\vargamma ( h \rightarrow gg)$$\end{document}(hgg ) are drawn in fig .
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\begin{document}$$r_1(p_i , p_j , p_k)$$\end{document}r1(pi , pj , pk ) corresponds to three diagrams with permuted gluons there are five graphs contributing to the virtual part \documentclass[12pt]{minimal }
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\begin{document}$$\vargamma _ { r}$$\end{document}r . in the following ,
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\begin{document}$$\begin{aligned } \vargamma _ { v}(h \rightarrow gg ) + \vargamma _ { r}(h \rightarrow ggg ) .
consider the one - loop quadratically divergent rank - two tensor6\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \int d^4q \frac{q_\alpha q_\beta } { d_0 d_1 } , \end{aligned}$$\end{document}d4qqqd0d1,with7\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } d_i ~=~ q^2-d_i,~~~d_i ~=~ m^2_i - p^2_i- 2 ( q\cdot p_i),~~~p_0=0.\nonumber \\ \end{aligned}$$\end{document}di = q2-di , di = mi2-pi2 - 2(qpi),p0=0.its uv convergence can be improved by first deforming the propagators by a vanishing amount \documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \frac{1}{\bar{d}_i } = \frac{1}{\bar{q}^2 } \bigg ( 1+\frac{d_i}{\bar{d}_i } \bigg ) , \end{aligned}$$\end{document}1di=1q2(1+didi),where10\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \frac{q_\alpha q_\beta } { \bar{d}_0 \bar{d}_1}&= q_\alpha q_\beta \left ( \left [ \frac{1}{\bar{q}^4 } \right ] + \left [ \frac{d_0+d_1}{\bar{q}^6 } \right ] + \left [ \frac{d_1 ^ 2}{\bar{q}^8 } \right ] + \frac{d_1 ^ 3}{\bar{q}^8 \bar{d}_1 } \right .
+ \frac{d_0 d_1}{\bar{q}^6 \bar{d}_1 } + \frac{d_0 ^ 2}{\bar{q}^4 \bar{d}_0\bar{d}_1 } \right ) , \end{aligned}$$\end{document}qqd0d1=qq1q4+d0+d1q6+d12q8+d13q8d1+d0d1q6d1+d02q4d0d1,where the terms in square brackets are uv divergent but depend only on \documentclass[12pt]{minimal }
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\begin{document}$$\mu ^2$$\end{document}2 . the fdr definition of the integral in eq .
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\begin{document}$$\begin{aligned}&b_{\alpha \beta } ( p_1 ^ 2,m_0 ^ 2,m_1 ^ 2 ) = \int [ d^4q ] \frac{q_\alpha q_\beta } { \bar{d}_0 \bar{d}_1 } \nonumber \\&\quad \equiv \lim _ { \mu \rightarrow 0 } \int d^4q \ , q_\alpha q_\beta \left ( \frac{d_1 ^ 3}{\bar{q}^8 \bar{d}_1 } + \frac{d_0 d_1}{\bar{q}^6 \bar{d}_1 } + \frac{d_0 ^ 2}{\bar{q}^4 \bar{d}_0\bar{d}_1 } \right ) \!.\nonumber \\ \end{aligned}$$\end{document}b(p12,m02,m12)=[d4q]qqd0d1lim0d4qqqd13q8d1+d0d1q6d1+d02q4d0d1.the r.h.s . of eq .
( 12 ) corresponds to a well - defined four - dimensional integral , in which all uv divergences are explicitly subtracted .
the gauge - invariance properties of this definition are discussed in detail in [ 15 , 21 ] . in the rest of this section ,
i mostly concentrate on cl and ir infinities , and , in particular , on the matching between virtual and real contributions . a convenient starting point to study the cl singularities
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\end{aligned}$$\end{document}b(0,0,0)=[d4q]qd0d1=0,b(0,0,0)=[d4q]1d0d1=0.those results coincide with dr in which scale - less integrals are zero and are due to a cancelation between two \documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } b(p^2,0,0)&= -i \pi ^2 \lim _ { \mu \rightarrow 0}\int _ 0 ^ 1 \mathrm{d}x\ , \nonumber \\&\times \,\,\left [ \ln ( \mu ^2 -p^2 x ( 1-x ) ) -\ln ( \mu ^2 ) \right ] \ ! , \end{aligned}$$\end{document}b(p2,0,0)=-i2lim001dxln(2-p2x(1-x))-ln(2),where the first logarithm develops a cl singularity in the limit \documentclass[12pt]{minimal }
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the one - gluon cut in a contributes to the virtual part , the two - gluon cut in b to the real radiation .
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\begin{document}$$\begin{aligned } c(s)= \int [ d^4q ]
\frac{1}{\bar{d}_0 \bar{d}_1 \bar{d}_2 } = \lim _ { \mu \rightarrow 0 } \int d^4q \frac{1}{\bar{d}_0 \bar{d}_1 \bar{d}_2 } , \end{aligned}$$\end{document}c(s)=[d4q]1d0d1d2=lim0d4q1d0d1d2,with18\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } m_0 ^ 2 = m_1 ^ 2 = m_2 ^ 2 = p_1 ^ 2= p_2 ^ 2= 0,~~~s= -2(p_1 \cdot p_2).\nonumber \\
\end{aligned}$$\end{document}m02=m12=m22=p12=p22=0,s=-2(p1p2).by denoting19\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \mu _ 0=\frac{\mu ^2}{s } , \end{aligned}$$\end{document}0=2s , one computes20\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } c(s)&= \lim _ { \mu \rightarrow 0 } \frac{i \pi ^2}{2 s } \ln ^2\bigg ( \frac{\sqrt{1 - 4\mu _ 0}+1}{\sqrt{1 - 4\mu _ 0}-1}\bigg ) \nonumber \\&= \frac{i \pi ^2}{s } \left [ \frac{\ln ^2(\mu _ 0)-\pi ^2}{2 } + i\,\pi \ln ( \mu _ 0 ) \right ] , \end{aligned}$$\end{document}c(s)=lim0i22sln2(1 - 40 + 11 - 40 - 1)=i2sln2(0)-22+iln(0),which is indeed fully matched by the inclusive real contribution in fig .
i use the described approach to uv / cl / ir infinities to compute \documentclass[12pt]{minimal }
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\begin{document}$$\vargamma _ { v}(h \rightarrow gg)$$\end{document}v(hgg ) and \documentclass[12pt]{minimal }
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\begin{document}$$\vargamma _ { r}(h \rightarrow ggg)$$\end{document}r(hggg ) .
the calculation is greatly simplified by eqs . ( 13 ) and ( 14 ) .
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\begin{document}$$v_1$$\end{document}v1 and \documentclass[12pt]{minimal }
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\begin{document}$$v_2$$\end{document}v2 in fig . 1
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\begin{document}$$\begin{aligned } \vargamma _ { v}(h \rightarrow gg)=-3 \frac{\alpha _ s}{\pi } \,\vargamma ^{(0)}(\alpha _ s)\,m^2_h\ , \mathcal{r}e \left [ \frac{c(m^2_h)}{i \pi ^2}\right ] .\nonumber \\
veltman decomposition , the only subtlety being the fdr treatment of \documentclass[12pt]{minimal }
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\begin{document}$$\mu ^2$$\end{document}2 [ 15 , 21 ] : for consistency with eq .
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\begin{document}$$q^2$$\end{document}q2 appearing in the numerator of a diagram should also be deformed,22\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } q^2 \rightarrow \bar{q}^2 , \end{aligned}$$\end{document}q2q2,and integrals involving \documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } { \tilde{b}}(p_1 ^ 2,m_0 ^ 2,m_1 ^ 2 ) = \int [ d^4q ]
\frac{\mu ^2}{\bar{d}_0 \bar{d}_1 } , \end{aligned}$$\end{document}b~(p12,m02,m12)=[d4q]2d0d1,require the same integrand expansion as if\documentclass[12pt]{minimal }
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\begin{document}$$\mu ^2= q^2$$\end{document}2=q2 .
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\begin{document}$$\begin{aligned}&{\tilde{b}}(p_1 ^ 2,m_0 ^ 2,m_1 ^ 2)\nonumber \\&\quad = \lim _ { \mu \rightarrow 0 } \int d^4q \ , \mu ^2 \left ( \frac{d_1 ^ 3}{\bar{q}^8 \bar{d}_1 } + \frac{d_0 d_1}{\bar{q}^6 \bar{d}_1 } + \frac{d_0 ^ 2}{\bar{q}^4 \bar{d}_0\bar{d}_1 } \right ) \nonumber \\&\quad = \frac{i \pi ^2}{2}\left ( m_0 ^ 2+m_1 ^ 2-\frac{p_1 ^ 2}{3}\right ) .
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\begin{document}$$\begin{aligned } \vargamma _ { v}(h \rightarrow gg)=\frac{3}{2 } \frac{\alpha _ s}{\pi } \,\vargamma ^{(0)}(\alpha _ s)\ , \left ( \pi ^2-\ln ^2\frac{m^2_h}{\mu ^2 } \right ) .
the unpolarized matrix element squared , derived from the real emission diagrams in fig . 1 , reads26\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned}&|m|^2 = 192\,\pi \alpha _
sa^2 \bigg [ \frac{s^3_{23}}{s_{12}s_{13 } } + \frac{s^3_{13}}{s_{12}s_{23 } } + \frac{s^3_{12}}{s_{13}s_{23 } } \nonumber \\&\quad + \,\ , \frac{2(s^2_{13}+s^2_{23})+3 s_{13}s_{23}}{s_{12 } } + \frac{2(s^2_{12}+s^2_{23})+3 s_{12}s_{23}}{s_{13 } } \nonumber \\&\quad + \,\ , \frac{2(s^2_{12}+s^2_{13})+3 s_{12}s_{13}}{s_{23}}+ 6 ( s_{12 } + s_{13 } + s_{23})\bigg ] , \end{aligned}$$\end{document}|m|2=192sa2[s233s12s13+s133s12s23+s123s13s23 + 2(s132+s232)+3s13s23s12 + 2(s122+s232)+3s12s23s13 + 2(s122+s132)+3s12s13s23 + 6(s12+s13+s23)],where \documentclass[12pt]{minimal }
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\begin{document}$$s_{ij}= ( p_i+ p_j)^2$$\end{document}sij=(pi+pj)2 .
this expression is obtained from the massless result with the replacement \documentclass[12pt]{minimal }
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\begin{document}$$2(p_i \cdot p_j ) \rightarrow s_{ij}$$\end{document}2(pipj)sij , in accordance with eq .
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\begin{document}$$|m|^2$$\end{document}|m|2 should be integrated over a massive three - gluon phase space with \documentclass[12pt]{minimal }
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\begin{document}$$p_i^2= \mu ^2$$\end{document}pi2=2 , which can be parametrized as27\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \int d \phi _ 3 = \frac{\pi ^2}{4s } \int ds_{12 } ds_{13 } ds_{23}\ , \delta ( s - s_{12}-s_{13}-s_{23}+3\mu ^2 ) , \end{aligned}$$\end{document}d3=24sds12ds13ds23(s - s12-s13-s23 + 32),where \documentclass[12pt]{minimal }
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\begin{document}$$\sqrt{s}$$\end{document}s is the higgs mass .
it is convenient to introduce the dimensionless variables28\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } x= \frac{s_{12}}{s}-\mu _ 0,~~y= \frac{s_{13}}{s}-\mu _ 0,~~z= \frac{s_{23}}{s}-\mu _ 0 , \end{aligned}$$\end{document}x = s12s-0,y = s13s-0,z = s23s-0,with \documentclass[12pt]{minimal }
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\begin{document}$$\mu _ 0$$\end{document}0 given in eq .
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\begin{document}$$\begin{aligned } x+y+z = 1 , \end{aligned}$$\end{document}x+y+z=1,all ir / cl divergent bremsstrahlung integrals can be reduced to the following ones:30\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned}&i(s ) = \int _ { r } \mathrm{d}x \mathrm{d}y\ , \frac{1}{(x+\mu _ 0 ) ( y+\mu _ 0 ) } , \nonumber \\&j_{p}(s ) = \int _ { r } \mathrm{d}x \mathrm{d}y\ , \frac{x^p}{(y+\mu _ 0)}\,\,~~(p \ge 0 ) , \end{aligned}$$\end{document}i(s)=rdxdy1(x+0)(y+0),jp(s)=rdxdyxp(y+0)(p0),where the integration region reads , in the fully inclusive case,31\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \int _ { r } \mathrm{d}x \mathrm{d}y \equiv \int _ { 3 \mu _ 0}^{1 - 2\sqrt{\mu _ 0 } } \mathrm{d}x \int
_ { y_-}^{y_+ } \mathrm{d}y , \end{aligned}$$\end{document}rdxdy301 - 20dxy - y+dy , with32\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } y_{\pm } & = \frac{1}{4(x + \mu _ 0 ) } \left [ ( 1-\mu _ 0)^2-(r_0 \mp r_1)^2\right ] -\mu
\mu _ 0}.\nonumber \\ \end{aligned}$$\end{document}y=14(x+0)(1-0)2-(r0r1)2-0,r0=(x-0)2 - 402,r1=(1-x)2 - 40.thus33\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned}&\vargamma _ { r}(h \rightarrow ggg)= 3 \frac{\alpha
_ s}{\pi } \,\vargamma ^{(0)}(\alpha _ s)\ , \nonumber \\&\quad \times \left ( \frac{1}{4}+i(m^2_h)-\frac{3}{2}j_0(m^2_h)-j_2(m^2_h ) \right ) .
\end{aligned}$$\end{document}r(hggg)=3s(0)(s)14+i(mh2)-32j0(mh2)-j2(mh2).finally , one computes , up to terms which vanish in the limit \documentclass[12pt]{minimal }
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\begin{document}$$\mu _ 0 \rightarrow 0$$\end{document}00,34\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } i(s ) = \frac{\ln ^2(\mu _ 0)-\pi ^2}{2 } \end{aligned}$$\end{document}i(s)=ln2(0)-22and35\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned}&j_{p}(s ) = -\frac{1}{p+1 } \ln ( \mu _ 0 ) + \int _ 0 ^ 1 \mathrm{d}x \,x^p\left [ \ln ( x)+ 2 \ln ( 1-x ) \right ] \nonumber \\&\quad = -\frac{1}{p+1 } \ln ( \mu _ 0 ) \!-\!\frac{1}{p+1}\left [ \frac{1}{p+1}\!+\!2 \sum _ { n=1}^{p+1}\frac{1}{n } \right ] ( p \ge 0),\nonumber \\ \end{aligned}$$\end{document}jp(s)=-1p+1ln(0)+01dxxpln(x)+2ln(1-x)=-1p+1ln(0)-1p+11p+1 + 2n=1p+11n(p0),so that36\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned}&\vargamma _ { r}(h \rightarrow ggg)= \frac{3}{2 } \frac{\alpha _ s}{\pi } \,\vargamma ^{(0)}(\alpha _ s)\ , \nonumber \\&\quad \times \left ( \ln ^2\frac{m^2_h}{\mu ^2}-\pi ^2+\frac{73}{6 } -\frac{11}{3}\ln \frac{m^2_h}{\mu ^2 } \right ) .
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\begin{document}$$\begin{aligned } \vargamma ( h \rightarrow gg ) = \vargamma ^{(0)}(\alpha _ s ) \left [ 1+\frac{\alpha _ s}{\pi } \left ( \frac{95}{4}-\frac{11}{2 } \ln \frac{m^2_h}{\mu ^2 } \right ) \right ] .\nonumber \\ \end{aligned}$$\end{document}(hgg)=(0)(s)1+s954 - 112lnmh22.all cl / ir \documentclass[12pt]{minimal }
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\begin{document}$$\ln ( \mu ^2)$$\end{document}ln(2 ) and \documentclass[12pt]{minimal }
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\begin{document}$$\ln ^2(\mu ^2)$$\end{document}ln2(2 ) cancel in eq . ( 37 ) , so that the remaining \documentclass[12pt]{minimal }
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\begin{document}$$\mu $ $ \end{document} is directly interpreted as the renormalization scale .
this is a typical procedure in fdr : since the uv infinities are subtracted from the very beginning , the unphysical left - over \documentclass[12pt]{minimal }
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\begin{document}$$\mu $ $ \end{document} dependence is eliminated , on the perturbative level one is working at , by a finite renormalization , which fixes the bare parameters in terms of the observables .
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\begin{document}$$\vargamma ^{(0)}(\alpha _ s ) \rightarrow \vargamma ^{(0)}(\alpha _
then the logarithm is reabsorbed in the gluonic running of the strong coupling constant,38\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha _
s(\mu ^2)}{1+\frac{\alpha _ s}{2 \pi } \frac{11}{2 } \ln \frac{m_h^2}{\mu ^2 } } , \end{aligned}$$\end{document}s(mh2)=s(2)1+s2112lnmh22,and eq .
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\begin{document}$$\alpha _ s$$\end{document}s and parton densities , conventionally derived in dr .
i consider uv , cl , and ir divergences in turn , showing the equivalence of fdr with the dimensional reduction version of dr , widely used in supersymmetric theories .
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\begin{document}$$1/\epsilon $ $ \end{document}1/ dr regulator and the \documentclass[12pt]{minimal }
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\begin{document}$$\ln ( \mu ^2)$$\end{document}ln(2 ) appearing in fdr . as for the uv infinities ,
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\begin{document}$$\begin{aligned } \int d^nq \frac{1}{q^2(q+p)^2}=
i \pi ^2 \int _ { 0}^{1 } \mathrm{d}x\ , [ \delta -\ln ( -p^2 x ( 1-x))],\nonumber \\ \end{aligned}$$\end{document}dnq1q2(q+p)2=i201dx[-ln(-p2x(1-x))],where40\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } n = 4 + \epsilon \,~~~\mathrm{and}~~~\delta = -\frac{2}{\epsilon } - \gamma _ e -\ln \pi .
\end{aligned}$$\end{document}n=4+and=-2-e - ln.thus , dr and fdr uv regulators are linked through the simple \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{ms}}$$\end{document}ms replacement41\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \delta \rightarrow \ln ( \mu ^2 ) .
virtual singularities follow the same pattern , as can be inferred from the exact uv / cl cancelation in eqs .
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\begin{document}$$j_{p}$$\end{document}jp reads42\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned}&j^\mathrm{dr}_{p}(s ) \!=\ ! \frac{(\pi s)^{\frac{\epsilon } { 2}}}{\vargamma \left ( 1+\frac{\epsilon } { 2}\right ) } \int \mathrm{d}x\,\mathrm{d}y\,\mathrm{d}z\,\frac{x^p}{y}\delta ( 1\!-\!x\!-\!y\!-\!z ) ( xyz)^{\frac{\epsilon } { 2 } } \nonumber \\&\quad = -\frac{1}{p+1 } ( \delta -\ln ( s ) ) -\frac{1}{p+1}\left [ \frac{1}{p+1}+2 \sum _ { n=1}^{p+1}\frac{1}{n } \right ] , \nonumber \\ \end{aligned}$$\end{document}jpdr(s)=(s)21+2dxdydzxpy(1-x - y - z)(xyz)2=-1p+1(-ln(s))-1p+11p+1 + 2n=1p+11n , which indeed coincides with eq . ( 35 ) if \documentclass[12pt]{minimal }
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\begin{document}$$\delta = \ln ( \mu ^2)$$\end{document}=ln(2 ) . finally , the \documentclass[12pt]{minimal }
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\begin{document}$$\ln ^2(\mu ^2)$$\end{document}ln2(2 ) terms generated by overlapping ir / cl singularities drop , together with the full constant part , when adding virtual and fully inclusive real contributions , which can be traced back to the following relation:43\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \mathcal{r}e \left [ \frac{c(s)}{i \pi ^2}\right ] = \frac{1}{s } i(s)\ , \end{aligned}$$\end{document}rec(s)i2=1si(s)between eqs .
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\begin{document}$$\begin{aligned}&\mathcal{r}e \left [ \frac{1}{i \pi ^2 } \int d^nq \frac{1}{q^2(q+p_1)^2(q+p_2)^2 } \right ] \nonumber \\&\quad = \frac{1}{s } ( \pi s)^{\frac{\epsilon } { 2}}\ , \vargamma \left ( 1-\frac{\epsilon } { 2}\right ) \left [ \frac{4}{\epsilon ^2}-\frac{2}{3 } \pi ^2 \right ] , \end{aligned}$$\end{document}re1i2dnq1q2(q+p1)2(q+p2)2=1s(s)21-242 - 232,where \documentclass[12pt]{minimal }
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\begin{document}$$p_1 ^ 2= p_2 ^ 2= 0$$\end{document}p12=p22=0 and \documentclass[12pt]{minimal }
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\begin{document}$$s= -2(p_1\cdot p_2)$$\end{document}s=-2(p1p2 ) , and45\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } i^\mathrm{dr}(s)&= \frac{(\pi s)^{\frac{\epsilon } { 2}}}{\vargamma \left ( 1+\frac{\epsilon } { 2}\right ) } \int \mathrm{d}x\,\mathrm{d}y\,\mathrm{d}z\,\frac{1}{xy}\delta ( 1\!-\!x\!-\!y\!-\!z ) ( xyz)^{\frac{\epsilon } { 2 } } \nonumber \\&= ( \pi s)^{\frac{\epsilon } { 2}}\,\vargamma \left ( 1-\frac{\epsilon } { 2}\right ) \left [ \frac{4}{\epsilon ^2}-\frac{2}{3 } \pi ^2 \right ] .
however , an important difference between dr and fdr follows from self - contractions of metric tensors coming from the feynman rules . in dr \documentclass[12pt]{minimal }
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\begin{document}$$g_{\alpha \beta } g^{\alpha \beta } = n$$\end{document}gg=n , while \documentclass[12pt]{minimal }
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\begin{document}$$g_{\alpha \beta } g^{\alpha \beta } = 4$$\end{document}gg=4 in fdr .
( 41 ) , and the fdr treatment of \documentclass[12pt]{minimal }
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\begin{document}$$\mu ^2$$\end{document}2 discussed in sect .
4 , makes explicit the equivalence between fdr and dimensional reduction in the \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{\mathrm ms}}$$\end{document}ms scheme . having established this , all the well - known transition rules between dimensional reduction and dr [ 26 , 27 ] can be directly applied to fdr . in the case of eq .
( 37 ) , it turns out that the expression is the same in both dimensional reduction ( or fdr ) and dr .
therefore , the correct strong coupling constant to be used is the customary \documentclass[12pt]{minimal }
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\begin{document}$$\overline{\mathrm{ms}}$$\end{document}ms scheme , proving that the fdr result coincides with eq .
i have presented an fdr calculation of the gluonic qcd corrections to \documentclass[12pt]{minimal }
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\begin{document}$$h \rightarrow gg$$\end{document}hgg in the large top effective theory , demonstrating that ultraviolet , collinear , and infrared divergences can be simultaneously and successfully regulated in four dimensions .
i have proved the equivalence , at the one - loop level , of dimensional reduction and fdr , making the latter approach attractive also in supersymmetric calculations , where the fermionic and bosonic sectors must share the same number of degrees of freedom .
the advantage of directly working in the four - dimensional minkowsky space is expected to lead to considerable simplifications in higher - order qft computations , especially in connection with numerical techniques .
this issue , together with the extension of fdr to more loops , is currently under study . | i apply fdr a recently introduced four - dimensional approach to quantum field theories ( qfts)to the computation of the nlo qcd corrections to \documentclass[12pt]{minimal }
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\begin{document}$$h \rightarrow gg$$\end{document}hgg in the large top mass limit .
the calculation involves all key ingredients of qcd namely ultraviolet , infrared , and collinear divergences , besides \documentclass[12pt]{minimal }
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\begin{document}$$\alpha _ s$$\end{document}s renormalization and paves the way for successful use of fdr in massless one - loop qft computations .
i show in detail how the correct result emerges in fdr , and discuss the translation rules to dimensional regularization . | Introduction
The model for
FDR versus infinities
The virtual part
The real radiation
FDR versus DR
Conclusions |
PMC4800001 | graphene was mechanically exfoliated from highly - oriented pyrolytic graphite onto si / sio2 substrates .
surface hybrids were obtained by immersion in a solution of [ fe4(l)2(dpm)6 ] in 1,2-dichloroethane : dichloromethane ( 9/1 , v / v ) at room temperature , followed by washing with isopropanol .
bulk samples of the hybrids were prepared by first sonicating 8.4 mg of graphite powder in 20 ml of the solvent mixture for 48 hrs , followed by adding 11.5 mg of [ fe4(l)2(dpm)6 ] in 20 ml of the same mixture , followed by stirring for 45 min and final filtering through ptfe membranes ( 0.2 m pore size ) .
magnetic measurements were corrected for the diamagnetism of the sample and sample holder , as independently determined .
a home - built micro - squid susceptometer inside a dilution refrigerator was used for zero - field measurements , with extremely small ac field amplitudes ( 50 moe ) and the data corrected by the bare graphene signal .
afm images were acquired with a bruker icon system in tapping mode , using olympus ac200ts cantilevers .
maldi - tof spectra were recorded using a laser pulsed at ~4 hz , while the xps spectra were obtained with a monochromatized al - k source ( 1486.6 ev ) , see si for details .
electronic devices were fabricated using a pmma resist , e - beam lithography and thermally evaporated au / ti . | controlling the dynamics of spins on surfaces is pivotal to the design of spintronic1 and quantum computing2 devices .
proposed schemes involve the interaction of spins with graphene to enable surface - state spintronics3,4 , and electrical spin - manipulation4 - 11 .
however , the influence of the graphene environment on the spin systems has yet to be unraveled12 . here
we explore the spin - graphene interaction by studying the classical and quantum dynamics of molecular magnets13 on graphene .
while the static spin response remains unaltered , the quantum spin dynamics and associated selection rules are profoundly modulated .
the couplings to graphene phonons , to other spins , and to dirac fermions are quantified using a newly - developed model .
coupling to dirac electrons introduces a dominant quantum - relaxation channel that , by driving the spins over villain s threshold , gives rise to fully - coherent , resonant spin tunneling .
our findings provide fundamental insight into the interaction between spins and graphene , establishing the basis for electrical spin - manipulation in graphene nanodevices . | Methods
Supplementary Material |
PMC3894773 | the advent of computed tomography ( ct ) has started a revolution of information in health
studies and has contributed to planning , diagnosis , treatment , and prognosis analysis of
several diseases .
cone - beam computed
tomography ( cbct ) is a recently developed technology with potential for
applications in different areas of research and clinical dentistry . in endodontics ,
conventional radiographic images provide a two - dimensional ( 2d ) rendition of
a three - dimensional ( 3d ) structure , which may result in interpretation errors .
periapical lesions of endodontic origin may be present but not visible on conventional
2d radiographs .
these lesions
are visible more often on cbct images , and new methods using cbct scans to investigate
apical periodontitis and root resorption have been developed , as new imaging
tools are now used in several endodontic research areas
artifacts can cause low image
quality and poor image contrast leading to limited interpretation of the 3d volumes .
there is a concern with artifacts and the search for beam hardening corrections have
been the focus of several studies . according to ketcham and carlson
( 2001 ) beam hardening
beam
hardening causes the edges of an object to appear as cupping , streaks , dark bands , or
flare artifacts , and is caused by preferential absorption of low - energy photons by
absorbing materials with higher atomic numbers ( e.g. metals ) .
( 2006 ) related that the
cbct image defects often appeared in images of solid non anatomical objects placed on
the dental arch , such as diagnostic stents ( guide splint ) for the accurate positioning
of dental implants and rectangular radiopaque reference markers for the assessment of
periodontal disease .
this was more frequent when radiopaque materials , such as
hydroxyapatite containing resin and aluminum , were used .
it is essential to understand , however , that image artifacts are likely to occur because
of the density of several materials used in root canal treatment . alterations from true
dimension of rcf may offer false interpretation on real dentin remnants .
thus ,
evaluating the dimension of endodontic materials on cbct images may warn the clinicians
about the potential risks of misdiagnosis .
this study evaluated the discrepancy of rcf
measurements obtained from original root specimens and cbct images .
seventy - two human maxillary anterior teeth , extracted for different reasons , were
obtained from the dental urgency service of the federal university of gois - school
of dentistry , goinia , go , brazil .
this study was approved by the ethics committee of
the federal university of gois , brazil .
preoperative radiographs of each tooth were
taken to confirm the absence of calcified root canal , internal or external
resorption , and the presence of a fully formed apex .
the teeth were removed from storage in 0.2% thymol solution and immersed in 5% sodium
hypochlorite ( fitofarma , goinia , go , brazil ) for 30 min to remove external organic
tissues .
the crowns were removed to set the remaining tooth length to a standardized
13 mm from the root apex . after taking the initial radiographs , standard access
cavities were prepared , and the cervical third of each root canal was enlarged using
iso # 50 up to # 90 gates - glidden drills ( dentsply / maillefer , ballaigues , switzerland ) .
teeth were prepared up to an iso#50k - file ( dentsply / maillefer ) 1 mm short of the
apical foramen . during instrumentation ,
the root canals were irrigated with 3 ml of
1% naocl ( fitofarma ) at each change of files .
root canals were dried and filled with
17% edta ( ph 7.2 ) ( biodinmica , ibipor , pr , brazil ) for 3 min to remove the smear
layer .
after that , the root canals were irrigated again with 3 ml of 1% naocl , and
dried with paper points ( dentsply / maillefer ) .
the teeth were randomly divided into 8 experimental groups with 9 specimens each ,
according to different sealers : group 1 - sealapex ( sybron endo ,
glendora , ca , usa ) ; group 2 - sealapex + gutta - percha points
( dentsply / maillefer ) ; group 3 - sealer 26 ( dentsply ind .
,
petrpolis , rj , brazil ) ; group 4 - sealer 26+gutta - percha points ; group 5
- ah plus ( dentsply / maillefer ) ; group 6 - ah
plus+gutta - percha points ; group 7 - grossman sealer ( endofill ,
dentsply ) ; group 8 - grossman sealer+gutta - percha points .
the chemical composition of sealers used are : sealapex ( calcium oxide , bismuth
trioxide , zinc oxide , sub - micron silica , titanium dioxide , zinc stearate , tricalcium
phosphate , and blend - ethyl toluene sulfonamide , polymethylenemethylsalicylate
resin , isobutyl salicylate and a pigment ) ; sealer 26 ( bismuth trioxide , calcium
hydroxide , tetraminehexamethilene , titanium dioxide and bisphenol epoxy resin ) ; ah
plus ( paste a - epoxy resins , calcium tungstate , zirconium oxide , silica , iron oxide
pigments , and paste b - amines , calcium tungstate , zirconium oxide , silica , silicone
oil ) ; grossman sealer ( zinc oxide , hydrogenated resin , bismuth subcarbonate , barium
sulfate , sodium borate , eugenol and almond oil ) . after root canal preparation , groups 1 ,
3 , 5 , and 7 were filled with the
corresponding sealers prepared according to the manufacturer 's directions and taken
using a lentulo spiral . in groups 2 , 4 , 6 , and 8
, teeth were filled with the
corresponding sealer and gutta - percha points using the conventional lateral
condensation technique .
the teeth were wrapped in wet gauze and placed in an
incubator at 37c for 72 h to allow complete set of the root canal filling materials .
the coronal and the apical portion of the teeth were rendered waterproof with a layer
of cyanoacrylate adhesive ( super bonder , itapevi , sp , brazil ) to protect against the
influence of the water on materials .
teeth were positioned in the center of a bucket filled with water to simulate soft
tissue and supported by a plastic platform , based on the model used in previous
studies .
cbct
images were acquired with the first generation i - cat cone beam 3d imaging system
( imaging sciences international , hatfield , pa , usa ) .
exposure time was 40 s. after obtaining the cbct scans , the roots of all teeth were carefully sectioned in
axial , sagittal or coronal planes using a high - speed endo z bur ( dentsply / maillefer )
under water spray cooling .
the axial cuts were obtained at 6.5 mm from the root apex ;
and for sagittal and coronal planes , the roots were sectioned longitudinally , in the
center of the root canal ( figure 1 )
. schematic representation of sectioning root method and post length , showing the
sagittal , axial , and coronal views all measurements of the sectioned roots were made by two endodontists using a digital
caliper accurate to 0.01 mm ( fowler / sylvac ultra - cal mark iv electronic caliper ,
crissier , switzerland ) .
the calibrated examiners measured all the specimens and cbct
images , and assessed rcf dimensions in the directions described below .
all measurements made on the cbct images were acquired by two dental radiology
specialists with the scanner 's proprietary software ( xoran version 3.1.62 ; xoran
technologies , ann arbor , mi , usa ) in a pc workstation running microsoft windows xp
professional sp-2 ( microsoft corp , redmond , wa , usa ) , with processor
intelcore 2 duo-6300 1.86 ghz ( intel corporation , usa ) ,
nvidia geforce 6200 turbo cache videocard ( nvidia corporation , usa ) and monitor eizo
- flexscan s2000 , resolution 1600x1200 pixels ( eizo nanao corporation hakusan ,
japan ) .
all cbct scans were reformatted using 0.2 mm , 0.6 mm , 1.0 mm , 3.0 mm and 5.0
mm slice thickness . to determine the discrepancy between the rcf measurements made on the original root
specimens and those made on the cbct images , the same sites were evaluated on the
cbct scans using the same orientation ( axial , sagittal and coronal ) . in all planes ,
on axial images , the rcf
measurement was made in the buccal / palatal direction ; on sagittal images , in the
mesial / distal direction ; and on coronal images , in the buccal / palatal direction .
one - way analysis of variance ( anova ) and tukey tests were used for statistical
analyses .
seventy - two human maxillary anterior teeth , extracted for different reasons , were
obtained from the dental urgency service of the federal university of gois - school
of dentistry , goinia , go , brazil .
this study was approved by the ethics committee of
the federal university of gois , brazil .
preoperative radiographs of each tooth were
taken to confirm the absence of calcified root canal , internal or external
resorption , and the presence of a fully formed apex .
the teeth were removed from storage in 0.2% thymol solution and immersed in 5% sodium
hypochlorite ( fitofarma , goinia , go , brazil ) for 30 min to remove external organic
tissues .
the crowns were removed to set the remaining tooth length to a standardized
13 mm from the root apex . after taking the initial radiographs , standard access
cavities were prepared , and the cervical third of each root canal was enlarged using
iso # 50 up to # 90 gates - glidden drills ( dentsply / maillefer , ballaigues , switzerland ) .
teeth were prepared up to an iso#50k - file ( dentsply / maillefer ) 1 mm short of the
apical foramen . during instrumentation ,
the root canals were irrigated with 3 ml of
1% naocl ( fitofarma ) at each change of files .
root canals were dried and filled with
17% edta ( ph 7.2 ) ( biodinmica , ibipor , pr , brazil ) for 3 min to remove the smear
layer .
after that , the root canals were irrigated again with 3 ml of 1% naocl , and
dried with paper points ( dentsply / maillefer ) .
the teeth were randomly divided into 8 experimental groups with 9 specimens each ,
according to different sealers : group 1 - sealapex ( sybron endo ,
glendora , ca , usa ) ; group 2 - sealapex + gutta - percha points
( dentsply / maillefer ) ; group 3 - sealer 26 ( dentsply ind .
,
petrpolis , rj , brazil ) ; group 4 - sealer 26+gutta - percha points ; group 5
- ah plus ( dentsply / maillefer ) ; group 6 - ah
plus+gutta - percha points ; group 7 - grossman sealer ( endofill ,
dentsply ) ; group 8 - grossman sealer+gutta - percha points .
the chemical composition of sealers used are : sealapex ( calcium oxide , bismuth
trioxide , zinc oxide , sub - micron silica , titanium dioxide , zinc stearate , tricalcium
phosphate , and blend - ethyl toluene sulfonamide , polymethylenemethylsalicylate
resin , isobutyl salicylate and a pigment ) ; sealer 26 ( bismuth trioxide , calcium
hydroxide , tetraminehexamethilene , titanium dioxide and bisphenol epoxy resin ) ; ah
plus ( paste a - epoxy resins , calcium tungstate , zirconium oxide , silica , iron oxide
pigments , and paste b - amines , calcium tungstate , zirconium oxide , silica , silicone
oil ) ; grossman sealer ( zinc oxide , hydrogenated resin , bismuth subcarbonate , barium
sulfate , sodium borate , eugenol and almond oil ) . after root canal preparation , groups 1 ,
3 , 5 , and 7 were filled with the
corresponding sealers prepared according to the manufacturer 's directions and taken
using a lentulo spiral . in groups 2 , 4 , 6 , and 8
, teeth were filled with the
corresponding sealer and gutta - percha points using the conventional lateral
condensation technique .
the teeth were wrapped in wet gauze and placed in an
incubator at 37c for 72 h to allow complete set of the root canal filling materials .
the coronal and the apical portion of the teeth were rendered waterproof with a layer
of cyanoacrylate adhesive ( super bonder , itapevi , sp , brazil ) to protect against the
influence of the water on materials .
teeth were positioned in the center of a bucket filled with water to simulate soft
tissue and supported by a plastic platform , based on the model used in previous
studies .
cbct
images were acquired with the first generation i - cat cone beam 3d imaging system
( imaging sciences international , hatfield , pa , usa ) .
after obtaining the cbct scans , the roots of all teeth were carefully sectioned in
axial , sagittal or coronal planes using a high - speed endo z bur ( dentsply / maillefer )
under water spray cooling .
the axial cuts were obtained at 6.5 mm from the root apex ;
and for sagittal and coronal planes , the roots were sectioned longitudinally , in the
center of the root canal ( figure 1 )
. schematic representation of sectioning root method and post length , showing the
sagittal , axial , and coronal views
all measurements of the sectioned roots were made by two endodontists using a digital
caliper accurate to 0.01 mm ( fowler / sylvac ultra - cal mark iv electronic caliper ,
crissier , switzerland ) .
the calibrated examiners measured all the specimens and cbct
images , and assessed rcf dimensions in the directions described below .
all measurements made on the cbct images were acquired by two dental radiology
specialists with the scanner 's proprietary software ( xoran version 3.1.62 ; xoran
technologies , ann arbor , mi , usa ) in a pc workstation running microsoft windows xp
professional sp-2 ( microsoft corp , redmond , wa , usa ) , with processor
intelcore 2 duo-6300 1.86 ghz ( intel corporation , usa ) ,
nvidia geforce 6200 turbo cache videocard ( nvidia corporation , usa ) and monitor eizo
- flexscan s2000 , resolution 1600x1200 pixels ( eizo nanao corporation hakusan ,
japan ) .
all cbct scans were reformatted using 0.2 mm , 0.6 mm , 1.0 mm , 3.0 mm and 5.0
mm slice thickness . to determine the discrepancy between the rcf measurements made on the original root
specimens and those made on the cbct images , the same sites were evaluated on the
cbct scans using the same orientation ( axial , sagittal and coronal ) . in all planes ,
on axial images , the rcf
measurement was made in the buccal / palatal direction ; on sagittal images , in the
mesial / distal direction ; and on coronal images , in the buccal / palatal direction .
one - way analysis of variance ( anova ) and tukey tests were used for statistical
analyses .
the variation of rcf dimensions on cbct images
ranged from 9% to 100% greater than those measured on the original root specimens .
the
lowest rcf dimensions ( percentage values ) corresponded to sealer 26 and sealer 26 plus
gutta - percha points .
groups that were filled with sealers alone showed the greatest
dimensional values in cbct images when compared with groups filled with sealer and
gutta - percha points , with statistically significant difference among the groups . when
slice thicknesses varying from 0.2 mm to 5.0 mm were measured , an increase of 46.16% to
50.53% in rcf dimensions
the
different visualization planes analysis revealed an increase in rcf dimensions ranging
from 35.48% ( sagittal slice ) to 59.28% ( axial slice ) ( with statistically significant
difference ) .
figures 2 - 3 illustrate the sagittal , axial and coronal views of the rcf using
cbct . percentage ( % ) of root canal filling dimension increase from original specimens to
cone - beam computed tomography images according to slice thickness and planes for
each endodontic material ( =5% ) interaction between type of cut and slice thickness significantly by tukey
test .
gpp - gutta - percha points percentage ( % ) of root canal filling dimension increase from original specimens to
cone - beam computed tomography images for each group according to sealers , slice
thickness and planes , and statistical analysis ( =5% ) different letters in horizontal demonstrate statistically significant
difference with p<0.05 .
* p=0.0001 by anova test and p=0.0001 by tukey
test ; p=0.647 by anova test and p=0.272 by tukey test ; p=0.0001 by anova test and p=0.0001 by tukey test . gpp - gutta - percha
points .
cone - beam computed tomography images of root canal filling with sealapex ( a ) ,
sealapex+gutta - percha ( b ) , sealer 26 ( c ) , and sealer 26+gutta - percha ( d ) in
different slice thickness and planes ( sagittal , axial and coronal ) cone - beam computed tomography images of root canal filling with grossman sealer
( a ) , grossman sealer 26+gutta - percha ( b ) , ah plus ( c ) , and ah plus+gutta - percha
( d ) in different slice thickness and planes ( sagittal , axial and coronal )
a new standard of contemporary endodontics has been created with the advent of
cbct . for several
years , rcf quality was evaluated in clinical practice according to a 2d image of 3d
structures .
the radiographic appearance of the filled root canal
space is used to evaluate its sealing quality and to indicate the presence of apical
periodontitis .
however , the limitations of a radiographic assessment as a reference and
study method have been demonstrated in several studies . there is a possibility of interference from artifact caused by different densities from
endodontic materials , which can cause errors of interpretation .
our main purpose was to
determine the discrepancy of rcf measurements between original root specimens and cbct
images , and our findings showed that measurements obtained from cbct images of rcf with
sealers and sealers plus gutta - percha were greater than those obtained on the original
root specimens ( tables 1 and 2 ) .
these results bring important implications for
the clinical evaluation of rcf and anatomic dental structures .
special attention should
be paid depending on the density of the endodontic material and slice
thickness / orientation which can lead to misdiagnosis .
( 2008 ) compared the accuracy of cbct and
multislice ct for linear jaw bone measurements , and found that both methods were
accurate when used to evaluate an ex vivo specimen .
( 2008 ) determined the
geometric accuracy of cbct scans in comparison with a multidetector computed tomography
( mdct ) scanner .
their results showed that the cbct devices provide satisfactory
information about linear distances and volumes .
mdct scans proved slightly more accurate
in both measurement categories , but this difference may be irrelevant for most clinical
applications .
recent studies using cbct images detected voids in root filling
and incomplete removal of filling material during endodontic retreatment .
( 2007 ) showed that image
quality of storage phosphor images was subjectively as good as conventional film images
and superior to limited - volume cbct images for the evaluation of both homogeneity and
length of root fillings in single - rooted teeth .
( 2009 ) analyzed voids in root fillings
using intraoral analogue , intraoral digital and cbct images .
voids larger than 30 m
were detected by all imaging techniques . for small void detection , all digital intraoral
techniques performed better than intraoral analogue and cbct images .
the difference in density of rcf materials may have affected the results of several
studies , and was also found in our specimens .
the endodontic
sealers and gutta - percha used in our study have different physical and chemical
properties because of different radiopaque substances ( bismuth oxide , barium sulfate ,
zinc oxide ) .
( 2011 )
evaluated the effect caused by intracanal posts ( glass - fiber post , carbon fiber root
canal , pre - fabricated post - metal screws , silver alloy post and gold alloy post ) on the
dimensions of cbct images of endodontically treated teeth .
gold alloy and silver alloy
post dimensions were greater on cbct scans than on the original root specimens .
cbct reconstructions may show higher rcf dimensional values , as well as lack of image
homogeneity and definition .
( 2007 ) conducted an in vitro study about the
effect of projection data discontinuity - related artifacts in limited - volume cbct imaging
of jaws .
the effects of artifacts were scored as the difference in relative density
between the lingual and buccal soft tissue .
the intensity of artifacts increased when
more objects were outside the area being imaged .
fewer artifacts were noted in images
produced by the particular flat cbct panel detector used in this investigation .
( 2009 ) analyzed
the relationship between density values and cbct volume size using an alphard cbct
system capable of providing different - size imaging volumes .
the authors found that the
data discontinuity - related effect was different in limited - volume cbct scanning .
considering that the higher density of rcf materials may produce image artifacts ,
special attention should be paid to the rcf evaluation of endodontically treated teeth .
any change from true dimension of rct may favor false interpretation on real dentin
remnants , and constitute potential risks of misdiagnosis .
the implication of density
artifacts on diagnostic procedures seems to be obvious , and different correction methods have been
investigated .
( 2008 ) studied the effect of incident radiation energy on the
amount and extent of image artifacts over adjacent anatomic structures .
the use of a
harder energy beam during scanning appears to result in less extensive artifact
formation .
a strong correlation between gray scale values on cbct images and bone densities has
been found by haristoy , et al .
this has implications for potential quantitative radiological approaches to
determine bone density from cbct images .
however , given the variation of gray scale
values despite normalization , it may be necessary to use calibration phantoms scanned
simultaneously with the patient to ensure an accurate determination of bone
density . hunter and
mcdavid ( 2009 )
showed that
additional copper filtration suppresses beam hardening artifacts . in micro - computed
tomography scanning based on bone mineral density measurements , the effects of beam
hardening - induced cupping artifacts may be also minimized by beam filtration .
cbct provides a three - dimensional image , in which a new plane has been added : depth .
its
clinical application results in greater accuracy and may be used in nearly all areas of
dentistry : surgery , implants , dentistry , orthodontics , endodontics , periodontics ,
temporomandibular dysfunction , image diagnosis , and so on .
few studies investigated the dimensional alterations of endodontic materials observed on
cbct images , and further studies should include other variables , such as the effect of
artifacts on intracanal post alloys .
the evolution of the new softwares certainly can
reduce metallic artifact and dimensional alterations in future reconstructions of cbct
images .
periapical radiographs should be used as a reference standard , together with
cbct image interpretation , when making endodontic diagnoses .
the results of this study showed that rcf dimensions were greater on cbct images than on
the original root specimens , especially when only sealer was used . | objective to evaluate the discrepancy of root canal filling ( rcf ) measurements obtained
from original root specimens and cone - beam computed tomography ( cbct ) images .
material and methods seventy - two human maxillary anterior teeth were prepared up to an iso # 50 k - file
1 mm short of the apical foramen .
thus , the teeth were randomly divided into 8
groups , according to the root canal filling material : sealapex ,
sealapex+gutta - percha points , sealer 26 , sealer
26+gutta - percha points , ah plustm , ah plustm+gutta - percha points ,
grossman sealer , and grossman sealer+gutta - percha points .
after root canal
preparation and rcf , cbct scans were acquired and the specimens were sectioned in
axial , sagittal and coronal planes .
the rcf measurements were obtained in
different planes and thicknesses to determine the discrepancy between the original
root specimens ( using a digital caliper ) and the cbct images ( using the scanner 's
proprietary software ) .
one - way analysis of variance and tukey tests were used for
statistical analyses .
the significance level was set at =5% .
results measurements of the different endodontic filling materials were 9% to 100%
greater on the cbct images than on the original root specimens .
greater rcf
dimensions were found when only sealers were used , with statistically significant
difference among the groups .
conclusions rcf dimensions were greater on cbct images than on the original root specimens ,
especially when only sealer was used . | INTRODUCTION
MATERIAL AND METHODS
Tooth preparation
CBCT image acquisition
Sectioning of roots
Measurement of specimens and CBCT slices
RESULTS
DISCUSSION
CONCLUSIONS |
PMC3857379 | nguyen et al . ( 1 ) reported an increasing trend in the prevalence of knee pain in the united states after they evaluated data from the national health and nutrition examination surveys between 1971 and 2004 and from the framingham osteoarthritis study between 1983 and 2005 .
kim et al . ( 2 ) investigated 504 community residents of chuncheon , korea , aged 50 yr , and reported that the prevalence of knee pain was 46.2% ( 32.2% in men , 58.0% in women ) and increased with age in women .
the prevalence of knee osteoarthritis ( oa ) in elderly koreans has been reported by several researchers .
cho et al . ( 3 ) reported that the prevalence of radiographic oa was 38.1% in residents aged 65 yr in seongnam , korea .
kim et al . ( 4 ) reported that prevalences of radiographic oa and symptomatic oa were 37.3% and 24.2% , respectively , in residents aged 50 yr in chuncheon .
although oa is the most common cause of knee pain in elderly people ( 5 ) , disorders other than oa may also cause knee pain in this population .
it has also been reported that radiographic changes correlate poorly with pain and that knee pain is a better predictor of disability than radiographic changes ( 6 ) .
previous studies that investigated the epidemiology of knee pain or oa in elderly koreans were limited to some local areas in korea .
thus , arguments could be raised against the representativeness of these results . additionally , there is no reported study presenting severity profiles of knee pain in koreans .
we investigated epidemiological indices of knee pain and its severity - prevalence , risk factors , and impact on quality of life - in a representative sample of elderly koreans .
the korea center for disease control and prevention has conducted the korea national health and nutrition examination survey ( knhanes ) on a random sample of the general korean population to assess health and nutritional status through interviews and health examinations .
the fifth knhanes was conducted from 2010 to 2012 , assessing the whole nation each year without overlapping of survey areas or participants .
a multistage stratified probability sampling was used according to geographical area , gender , and age groups , based on the statistics korea registries .
1 is a flowchart that shows the inclusion of subjects in this study . of the 8,958 participants in the fifth knhanes conducted in 2010 ,
we excluded 203 persons who did not respond to the question about the presence of knee pain and 16 persons who did not report the severity of knee pain or reported a pain score of 0 even though they reported that they had knee pain .
a questionnaire was distributed to the participants to evaluate demographic , socioeconomic , lifestyle , and health status information , including presence of knee pain and its severity .
subjects were divided into three age groups : 50 - 59 , 60 - 69 , and 70 yr .
for smoking status , subjects were classified into former or current smokers vs non - smokers .
alcohol consumption was classified according to frequency by modifying the definitions used by anttila et al .
those who drank alcohol at least once per month in the past year were defined as frequent drinkers . those who drank alcohol less than once per month or who did not drink in the past year were defined as infrequent or non - drinkers .
regular exercise was defined as practicing high - intensity physical activities ( strenuous or gasping activities such as running , high - speed cycling , and swimming ) for at least 20 min at one session and at least 3 days per week , or practicing moderately intense physical activities ( slightly strenuous or gasping activities such as slow swimming , badminton , and table tennis ) for at least 30 min at one session and at least 5 days per week , or walking for at least 30 min at one session and at least 5 days per week .
the presence of hypertension was defined as a systolic blood pressure 140 mmhg , a diastolic pressure 90 mmhg , or taking anti - hypertensive medication(s ) .
the presence of diabetes mellitus was defined as a fasting glucose level 126 mg / dl , a history of taking oral hypoglycemic agent(s ) or insulin injection , or physician - diagnosed diabetes mellitus .
the presence of obesity was defined as a body mass index ( bmi ) 25 kg / m , calculated by dividing weight ( kg ) by the square of height ( in meters ) .
participants aged 50 yr underwent knee radiography ; the radiographs were evaluated using the kellgren - lawrence grading scale : grade 1 , doubtful narrowing of joint space and possible osteophytic lipping ; grade 2 , definite osteophytes and possible narrowing of joint space ; grade 3 , moderate multiple osteophytes , definite narrowing of joint space , some sclerosis , and possible deformity of bone contour ; and grade 4 , large osteophytes , marked narrowing of joint space , severe sclerosis , and definite deformity of bone contours ( 8) . to maintain quality control of the radiographic examinations , the radiology technicians were taught how to use the digital x - ray machine and the standard radiography procedures before they participated in the survey .
two independent radiologists examined the radiographs ; if the two reported different kellgren - lawrence grading scales for a radiograph and the difference was greater than 2 , then the radiograph was transferred to a third radiologist for final determination of the scale value ( 9 ) .
radiographic oa was defined by a kellgren - lawrence grade 2 or higher radiographic change .
knee pain in the fifth knhanes was defined as the presence of pain in the knee joint lasting 30 or more days during the most recent 3 months .
participants with knee pain were asked to rate their pain severity on a 10-point numerical rating scale ( nrs ) , where 0 was the absence of pain and 10 was the most severe pain .
more than half of the subjects reported a score of 0 ( i.e. , no knee pain ) .
the extreme skew creates a potential problem with treating this variable as an interval level variable and modeling it with linear regression .
thus , we transformed the severity score into an ordered variable with four levels : none , mild , moderate , and severe
. the cutoff points between mild , moderate , and severe pain levels were based on the study by jones et al .
they assigned a nrs to a four - level metric after extensively reviewing literature , consultations with several pain experts , and consideration of pain guidelines and recommendations .
the assignments were as follows : 0=no pain , 1 - 3=mild , 4 - 6=moderate , and 7 - 10=severe . in the fifth knhanes ,
it consists of five questions on mobility , self - care , pain , usual activities , and psychological status , with three possible answers for each item ( 1=no problem , 2=moderate problem , 3=severe problem ) ( 11 ) . a summary index with a maximum score of 1 ( eq-5d index )
the maximum score of 1 indicates the best health state , in contrast to the scores of individual questions , where higher scores indicate more severe or frequent problems ( 12 ) . the prevalence of knee pain and profiles of its severity in elderly koreans were calculated and represented diagrammatically .
logistic regression analysis was used to estimate crude and adjusted odds ratio ( or ) for knee pain with regard to risk factors . the proc logistic procedure in the sas statistical package ( ver . 9.3 ; sas institute , inc . , cary , nc , usa ) was used to conduct logistic regression analysis for knee pain .
the proportional odds model is an extension of logistic modeling and is used for assessing proportionality where the response variable takes on values in a set of ordered categories .
it was used to estimate crude and adjusted proportional odds ratio ( por ) for severity of knee pain with regard to risk factors .
if por for a specific risk factor is 2.0 , the subjects are 2.0 times more likely to report a greater severity of knee pain .
the proc genmod procedure was used to calculate pors for severity of knee pain , with a multinomial distribution and cumulative logit link function ( 13 ) .
the eq-5d index was analyzed and compared between subjects with knee pain and without knee pain , and between subjects with individual severity profiles and without knee pain using student 's t - test .
crude and adjusted ors for belonging to the worst group of eq-5d indices with regard to knee pain or severity profiles were calculated .
the proc logistic procedure was used to conduct logistic regression analysis for belonging to the worst group of eq-5d indices .
the study protocol was reviewed and approved by the institutional review board of jeju national university hospital ( jejunuh 2013 - 05 - 036 ) . as this study used open - source data not containing personal information ,
the korea center for disease control and prevention has conducted the korea national health and nutrition examination survey ( knhanes ) on a random sample of the general korean population to assess health and nutritional status through interviews and health examinations .
the fifth knhanes was conducted from 2010 to 2012 , assessing the whole nation each year without overlapping of survey areas or participants .
a multistage stratified probability sampling was used according to geographical area , gender , and age groups , based on the statistics korea registries .
1 is a flowchart that shows the inclusion of subjects in this study . of the 8,958 participants in the fifth knhanes conducted in 2010 ,
we excluded 203 persons who did not respond to the question about the presence of knee pain and 16 persons who did not report the severity of knee pain or reported a pain score of 0 even though they reported that they had knee pain .
a questionnaire was distributed to the participants to evaluate demographic , socioeconomic , lifestyle , and health status information , including presence of knee pain and its severity .
subjects were divided into three age groups : 50 - 59 , 60 - 69 , and 70 yr .
for smoking status , subjects were classified into former or current smokers vs non - smokers .
alcohol consumption was classified according to frequency by modifying the definitions used by anttila et al .
those who drank alcohol at least once per month in the past year were defined as frequent drinkers . those who drank alcohol less than once per month or who did not drink in the past year were defined as infrequent or non - drinkers .
regular exercise was defined as practicing high - intensity physical activities ( strenuous or gasping activities such as running , high - speed cycling , and swimming ) for at least 20 min at one session and at least 3 days per week , or practicing moderately intense physical activities ( slightly strenuous or gasping activities such as slow swimming , badminton , and table tennis ) for at least 30 min at one session and at least 5 days per week , or walking for at least 30 min at one session and at least 5 days per week .
the presence of hypertension was defined as a systolic blood pressure 140 mmhg , a diastolic pressure 90 mmhg , or taking anti - hypertensive medication(s ) .
the presence of diabetes mellitus was defined as a fasting glucose level 126 mg / dl , a history of taking oral hypoglycemic agent(s ) or insulin injection , or physician - diagnosed diabetes mellitus .
the presence of obesity was defined as a body mass index ( bmi ) 25 kg / m , calculated by dividing weight ( kg ) by the square of height ( in meters ) .
participants aged 50 yr underwent knee radiography ; the radiographs were evaluated using the kellgren - lawrence grading scale : grade 1 , doubtful narrowing of joint space and possible osteophytic lipping ; grade 2 , definite osteophytes and possible narrowing of joint space ; grade 3 , moderate multiple osteophytes , definite narrowing of joint space , some sclerosis , and possible deformity of bone contour ; and grade 4 , large osteophytes , marked narrowing of joint space , severe sclerosis , and definite deformity of bone contours ( 8) . to maintain quality control of the radiographic examinations , the radiology technicians were taught how to use the digital x - ray machine and the standard radiography procedures before they participated in the survey .
two independent radiologists examined the radiographs ; if the two reported different kellgren - lawrence grading scales for a radiograph and the difference was greater than 2 , then the radiograph was transferred to a third radiologist for final determination of the scale value ( 9 ) .
radiographic oa was defined by a kellgren - lawrence grade 2 or higher radiographic change .
knee pain in the fifth knhanes was defined as the presence of pain in the knee joint lasting 30 or more days during the most recent 3 months .
participants with knee pain were asked to rate their pain severity on a 10-point numerical rating scale ( nrs ) , where 0 was the absence of pain and 10 was the most severe pain . although the pain severity score was a numeric , the distribution was extremely skewed .
more than half of the subjects reported a score of 0 ( i.e. , no knee pain ) .
the extreme skew creates a potential problem with treating this variable as an interval level variable and modeling it with linear regression .
thus , we transformed the severity score into an ordered variable with four levels : none , mild , moderate , and severe
. the cutoff points between mild , moderate , and severe pain levels were based on the study by jones et al .
they assigned a nrs to a four - level metric after extensively reviewing literature , consultations with several pain experts , and consideration of pain guidelines and recommendations .
the assignments were as follows : 0=no pain , 1 - 3=mild , 4 - 6=moderate , and 7 - 10=severe . in the fifth knhanes ,
it consists of five questions on mobility , self - care , pain , usual activities , and psychological status , with three possible answers for each item ( 1=no problem , 2=moderate problem , 3=severe problem ) ( 11 ) . a summary index with a maximum score of 1 ( eq-5d index )
the maximum score of 1 indicates the best health state , in contrast to the scores of individual questions , where higher scores indicate more severe or frequent problems ( 12 ) .
the prevalence of knee pain and profiles of its severity in elderly koreans were calculated and represented diagrammatically .
logistic regression analysis was used to estimate crude and adjusted odds ratio ( or ) for knee pain with regard to risk factors . the proc logistic procedure in the sas statistical package ( ver . 9.3 ; sas institute , inc . , cary , nc , usa ) was used to conduct logistic regression analysis for knee pain .
the proportional odds model is an extension of logistic modeling and is used for assessing proportionality where the response variable takes on values in a set of ordered categories .
it was used to estimate crude and adjusted proportional odds ratio ( por ) for severity of knee pain with regard to risk factors .
if por for a specific risk factor is 2.0 , the subjects are 2.0 times more likely to report a greater severity of knee pain .
the proc genmod procedure was used to calculate pors for severity of knee pain , with a multinomial distribution and cumulative logit link function ( 13 ) .
the eq-5d index was analyzed and compared between subjects with knee pain and without knee pain , and between subjects with individual severity profiles and without knee pain using student 's t - test .
crude and adjusted ors for belonging to the worst group of eq-5d indices with regard to knee pain or severity profiles were calculated .
the proc logistic procedure was used to conduct logistic regression analysis for belonging to the worst group of eq-5d indices .
the study protocol was reviewed and approved by the institutional review board of jeju national university hospital ( jejunuh 2013 - 05 - 036 ) . as this study used open - source data not containing personal information ,
the average age of the subjects was 63.69.1 yr . of the subjects , 56.3% were women ; 10.2% were college graduates ; 34.2% had manual occupations ; 15.9% were current smokers ; 43.5% consumed alcohol ; 50.7% performed regular exercise ; 48.8% had hypertension ; 16.9% had diabetes mellitus ; 34.6% had obesity ( bmi25 kg / m ) ; 31.1% had hyperlipidemia ; 39.0% had radiographic oa , defined as the presence of kellgren - lawrence grade 2 or more radiographic change ( table 1 ) .
the prevalence of knee pain in all subjects was 23.1% ( 11.7% in men , 31.9% in women ) .
as age increased , the prevalence of knee pain increased . in every age group ,
the prevalences of mild , moderate , and severe knee pain in all subjects were 4.3% , 9.1% , and 9.7% , respectively .
those in men were 2.8% , 5.4% , and 3.5% , respectively ; those in women were 5.4% , 12.0% , and 14.4% , respectively ( fig .
crude ors showed significantly higher risk of knee pain in those 60 yr old , women , those with a low level of education , hypertension , obesity , or radiographic oa .
current and former smokers or those who frequently consumed alcohol had significantly lower risks for knee pain .
the adjusted ors revealed that those 60 yr old , women , and those who had a low level of education ( less than college ) , a manual occupation , obesity , or radiographic oa had significantly higher risks for knee pain , compared with those without ( table 2 ) .
based on the crude pors , those 60 yr old , women , those with a low level of education , hypertension , diabetes mellitus , obesity , or radiographic oa reported greater severity of knee pain .
former and current smokers or those who frequently consumed alcohol were more likely to report lower severity of knee pain .
the adjusted pors revealed that those 60 yr old , women , and those who had a low level of education ( less than college ) , a manual occupation , obesity , or radiographic oa had a risk for reporting greater severity of knee pain , compared with those without ( table 3 ) .
all subjects , all men , or all women with knee pain had significantly lower eq-5d index scores than all subjects , all men , or all women without knee pain . all subjects or all women with mild knee pain had significantly lower eq-5d index scores than all subjects or all women without knee pain .
men with mild knee pain had lower eq-5d index scores than men without knee pain , but the difference was not significant .
all subjects , all men , or all women with moderate knee pain or severe knee pain had significantly lower eq-5d index scores than all subjects , all men , or all women without knee pain ( table 4 ) .
all subjects , all men , or all women with knee pain had significantly higher risks for belonging to the worst eq-5d index quartile compared with all subjects , all men , or all women without knee pain , unadjusted or after adjusting for other risk factors .
all subjects or all women with mild knee pain had significantly higher risks for belonging to the worst eq-5d index quartile compared with all subjects or all women without knee pain .
men with mild knee pain had a higher risk for belonging to the worst eq-5d index quartile than men without knee pain , but the difference was not significant .
all subjects , all men , or all women with moderate or severe knee pain had significantly higher risks for belonging to the worst eq-5d index quartile compared with all subjects , all men , or all women without knee pain ( table 5 ) .
the knhanes is a nationwide survey , and data are obtained from subjects who are randomly sampled from the general korean population .
thus , we consider that our study is representative of the prevalence of knee pain and its severity profile in the general korean elderly population .
the prevalence of knee pain in the present study ( 23.1% in all subjects , 11.7% in men , and 31.9% in women ) was lower than that reported by kim et al .
( 2 ) ( 46.2% in all subjects , 32.2% in men , and 58.0% in women ) , who investigated the prevalence of knee pain in community residents of chuncheon , aged 50 yr . the difference in prevalence between the two studies may be attributable to the definition of knee pain and the residence distribution ( community residents vs nationwide sample ) .
the mean age of subjects in the present study was significantly lower than that reported by kim et al .
old age , female gender , a low level of education , manual occupation , obesity , and radiographic oa were significant risk factors for knee pain . a prospective study of knee pain and its risk factors revealed that age , previous knee injuries , being overweight , and knee - straining work were risk factors for knee pain ( 14 ) .
no association with age , education , manual occupation , or obesity was reported by kim et al .
differences in study design , subjects , and adjustment of variables may explain the discrepancies between studies .
further studies to identify risk factors for knee pain are required . according to the crude ors , smoking and alcohol consumption significantly reduced the risk of knee pain . however , the adjusted ors indicated that their influence on the study variables was insignificant .
the prevalence of musculoskeletal disorders is lower in men than in women ( 15 ) .
thus , the apparently lower risk for knee pain associated with smoking and alcohol consumption , based on the crude ors , probably resulted from the confounding effects of gender .
the association of smoking and alcohol consumption with joint pain in previous reports is inconsistent .
( 16 ) found a protective association between smoking and oa when studying knee oa in the first u.s .
they suggested that smoking , or some unidentified factor associated with smoking , modestly protects against development of knee oa .
however , amin et al . ( 17 ) reported that men with knee oa who smoke have greater cartilage loss and more severe knee pain than men who do not smoke .
( 18 ) reported that alcohol consumption is inversely associated with the risk and severity of rheumatoid arthritis .
however , adamson et al . ( 19 ) reported an inconsistent association between alcohol consumption and joint pain among men and women in the west of scotland twenty-07 study . to our knowledge , this is the first reported study to present a severity profile of knee pain in elderly koreans , risk factors that worsen the severity of knee pain , and the influence of the severity of knee pain on quality of life .
this study revealed that a considerable number of elderly koreans suffer from mild , moderate , or severe knee pain .
this study showed that the risk factors for knee pain were also risk factors for worse pain severity .
creamer et al . ( 20 ) reported that helplessness , education , and bmi were important factors in determining pain severity in knee oa .
people with a lower level of education generally work in manual occupations . compared with office work , manual
work usually requires high physical loads , resulting in various musculoskeletal disorders , including knee pain , and worsening severity of musculoskeletal disorders .
obesity is an emerging epidemic , and weight loss has been shown to improve pain and function in knee joints with osteoarthritis ( 21 ) . among the risk factors for knee pain and its severity , manual occupation and obesity are modifiable factors .
thus , workplace environment modifications , and weight loss programs for patients with knee pain , focusing especially on those with manual workers and those with high bmis , may reduce the prevalence of knee pain or at least the severity of knee pain . excluding men with mild knee pain , participants with knee pain had significantly lower quality of life than participants without knee pain .
thus , the management of knee pain , even mild knee pain , is important for improving quality of life in the elderly population . considering the trend toward an ageing population in korea ,
early interventional approaches are needed to reduce the medical , social , and economic burdens of knee pain . | this study investigated the epidemiology ( prevalence , risk factors , and impact on quality of life ) of knee pain and its severity in elderly koreans . the subjects ( n=3,054 ) were participants aged 50 yr from the fifth korea national health and nutrition examination survey , conducted in 2010 .
knee pain was defined as pain in the knee lasting 30 days during the most recent 3 months ; severity was categorized as mild , moderate , or severe .
eq-5d was used to measure quality of life .
the prevalence of knee pain was 23.1% ( 11.7% in men , 31.9% in women ) .
the prevalences of mild , moderate , and severe knee pain were 4.3% , 9.1% , and 9.7% , respectively ( 2.8% , 5.4% , and 3.5% in men and 5.4% , 12.0% , and 14.4% in women ) .
old age , female gender , a low level of education , a manual occupation , obesity , and radiographic osteoarthritis were risk factors for knee pain , and were associated with increased severity of knee pain . excluding men with mild knee pain , people with knee pain had significantly lower quality of life than those without knee pain .
early interventional approaches are needed to reduce the medical , social , and economic burden of knee pain in elderly koreans . | INTRODUCTION
MATERIALS AND METHODS
Subjects
Data collection
Data analysis
Ethics statement
RESULTS
DISCUSSION |
PMC3660129 | hypernatremia ( serum sodium concentration > 146 millimoles per liter [ mmol / l ] ) is a common electrolyte disorder that can occur in very young or elderly patients.1,2 severe hypernatremia is defined as a serum sodium concentration > 160 mmol / l.3 hypernatremia may be caused by simple excess of sodium , often resulting from dehydration , due to excessive water loss or the ingestion of hypertonic fluids .
large losses of low - salt body fluids can occur during episodes of diarrhea or after the administration of diuretics or may result from renal tubular damage in patients with nephrogenic diabetes insipidus.1 hypernatremia may occur with the use of certain drugs , such as lithium and valproate , which may lead to renal impairment.4,5 hypernatremia can also occur in patients treated with excess sodium bicarbonate ( nahco3 ) to correct metabolic acidosis , which may increase the concentration of sodium in the serum.6 particular attention must be paid to hypernatremic episodes occurring in elderly patients , which are usually caused by high - salt loads due to poor oral fluid intake .
multiple myeloma is one of the common hematological diseases in elderly patients . in the past
, reports are available on igg - kappa - type multiple myeloma and proximal renal tubular acidosis ( fanconi syndrome),615 but the occurrence of hypernatremia in such cases has rarely been described.16 here , we report the case of an elderly patient presenting with multiple myeloma and underlying renal tubular damage , who developed extreme hypernatremia ( peak serum sodium concentration , 183 mmol / l ) and hyperchloremia ( peak serum chloride concentration , 153 mmol / l ) after intravenous treatment with bortezomib / dexamethasone / elcatonin and nahco3 in normal saline .
a 77-year - old man ( height , 170 cm ; body weight , 55 kg ; blood pressure , 172/66 mmhg ; and heart rate , 84 beats per minute ) was transferred to our care after being injured in a road traffic accident . on admission
a computed tomography scan revealed a fracture of the left pelvic bone ( figure 1 ) .
the laboratory findings were : white blood count , 5600/l ; hemoglobin , 9.9 g / dl ; platelet count , 142,000/l ; aspartate transaminase , 42 ( normal range ; 1337 ) units / l ; alanine aminotransferase , 21 ( 845 ) units / l ; lactate dehydrogenase , 402 ( 122228 ) units / l ; ammonium , 61 ( < 100 ) g / dl ; total protein , 9.1 ( 6.78.3 ) g / dl ; albumin , 3.1 ( 4.15.2 ) g / dl ; blood urea nitrogen , 18.1 ( 7.818.9 ) mg / dl ; creatinine , 1.73 ( 0.641.11 ) mg / dl ; uric acid , 9.3 ( 3.07.0 ) mg / dl ; estimated glomerular filtration rate , 41.5 ( > 90 ) ml / min / l ; ca , 11.9 ( 8.710.3 ) mg / dl ; inorganic phosphate ( ip ) , 2.6 ( 2.94.9 ) mg / dl ; na , 140 ( 138146 ) mmol / l ; k , 2.8 ( 3.65.1 ) mmol / l ; cl , 102 ( 99108 ) mmol / l ; and serum beta 2-microglobulin , 3.1 ( 0.91.9 ) mg / l .
urinalysis revealed a ph of 5.0 with occult blood ( 2 + ) and protein ( 2 + ) but glucose ( ) .
because the patient showed high levels of total protein in the serum , we considered a differential diagnosis of multiple myeloma .
further tests revealed serum igg levels of 4389 ( 8701700 ) mg / dl , with igg - kappa - type m protein .
, the patient was diagnosed with multiple myeloma ( durie salmon stage 2a ) .
clinically , the patient showed the complete range of multiple myeloma symptoms ( hypercalcemia , renal dysfunction , anemia , and bone disease , such as osteolytic pelvic bones and ribs ) , which had been unrecognized until hospital admission . during the first week after admission ,
the patient was able to communicate but was somehow in a state of a mild disturbance of consciousness ( japan coma scale ; jcs - i-2 ; disoriented ) and had difficulty imbibing oral fluids ; thus , he was hydrated with 10001500 ml of peripheral parenteral nutrition solution ( soldem 3 : sodium [ na ] concentration , 35 mmol / l ; chloride [ cl ] concentration , 35 mmol / l ) .
after the diagnosis of multiple myeloma with hypercalcemia , specific treatment was instigated , according to the following protocol ( figure 2 ) : intravenous normal saline ( na , 154 mmol / l ; cl , 154 mmol / l ; 500 ml 2/day ; daily ) , dexamethasone ( 8 mg per day 2 consecutive days ) , nahco3 ( 80 mmol / l per day ; daily ) , bortezomib ( 1.3 mg / m per dose , weekly ) , elcatonin ( 40 units per day for 5 consecutive days ) , and zoledronic acid hydrate ( 4 mg per dose , monthly ) . all drugs were intravenously administered in normal saline ( n / s ) ; thus , the patient received 200 mmol / l of sodium per 24 hours over a period of 6 days .
as shown in figure 2 , the patient experienced a gradual increase in serum sodium and chloride levels , which peaked at 183 mmol / l and 153 mmol / l , respectively . at this point ,
the patient was then switched to an intravenous infusion ( 1500 ml ) of soldem 1 ( na , 90 mmol / l ; cl , 70 mmol / l ) , soldem 3 , and potassium chloride ( kcl ) in 5% glucose , which helped gradually resolve the hypernatremia / hyperchloremia over the next 7 days , but the patient continued to have difficulty in swallowing and a low oral fluid intake . the patient s disturbed consciousness persisted for almost a month , then the situation improved significantly thereafter . during the entire period of hypernatremia / hyperchloremia ,
the former was associated with the excretion of high levels of potassium ( 38 mmol / l ; normal levels , < 20 mmol / l ) , although ip excretion was normal ( 1.14 grams per day ; normal range , 0.5 grams2.0 grams per day ) .
there was no evidence of glucosuria , hypouricemia , or generalized aminoaciduria . while examining other factors that might contribute to the electrolyte disorders in this patient , we observed that , although the levels of adrenocorticotropic hormone ( 19.9 pg / ml ) , plasma renin ( 2.4 ng / ml per hour ) , and aldosterone ( 56 pg / ml ) were all within the normal range , his serum cortisol levels ( 22.4 g / dl ) were slightly elevated .
arterial blood gas analysis , which was performed not in the critical period , but in the convalescent phase of the disease , revealed the following : ph , 7.481 ; pao2 , 84.0 mmhg ; paco2 , 28.2 mmhg ; hco3 , 20.8 ( normal ; 2331 ) mmol / l base excess , 1.0 mmol / l ; and anion gap , 8.9 ( normal ;
we later confirmed that the patient s low - serum potassium and plasma hco3 levels eventually normalized after a month .
taken together , these data suggested that the patient was probably suffering from distal renal tubular damage17 during the period of severe hypernatremia / hyperchloremia .
thereafter , the patient has fully recovered with no further disturbances of consciousness and with normal oral fluid intake , and continues to receive treatment for multiple myeloma .
severe hypernatremia ( defined as a serum sodium concentration > 160 mmol / l)3 has been described in various case reports , as high as a serum na concentration of 196 mmol / l , 201 mmol / l , 202 mmol / l , or 211 mmol / l.3,5,18,19 in addition , in a reported case of fatal ingestion of sodium hypochlorite bleach , the patient showed hypernatremia ( serum na , 169 mmol / l ) and hyperchloremia ( cl , 143 mmol / l ) in association with metabolic acidosis,20 and a patient with acute salt poisoning , due to an overdose of sodium chloride , showed a na load of 400 mmol / l over 12 hours.21 here , we report a case of severe hypernatremia ( na , 183 mmol / l ) in a patient with multiple myeloma . to date , however , few cases of hypernatremia in association with multiple myeloma have been described.16 in the present case , hypernatremia occurred after the patient was hydrated intravenously mainly with n / s as well as all necessary drugs ( bortezomib , dexamethasone , elcatonin , and nahco3 ) were given in n / s , as he was unable to take fluids orally .
besides the high - saline burden , nahco316 and dexamethasone administration might have accelerated the hypernatremia . in calculation , he received a salt load of 200 mmol / l over 24 hours , which was nearly comparable to one - fourth of the dose in a fatal case of household bleach ingestion reported by ross et al.20 the peak serum concentrations of sodium and chloride in the current patient reached 183 mmol / l and 153 mmol / l , respectively . although the patient suffered some mild disturbances of consciousness , he made a full recovery within approximately 1 month , with no residual adverse effects . in our case , the cause of the hypernatremia / hyperchloremia was likely to be linked to the renal tubular damage due to multiple myeloma , as the laboratory data showed a normal blood anion gap and a high urine anion gap , both of which are indicative of hyperchloremic acidosis ( base excess
< 2 mmol / l ; anion gap < 17 mmol / l).22 in general , decreases in plasma hco3 levels are associated with hyperchloremic acidosis and lactic acidosis .
both type 1 ( distal ) and type 2 ( proximal ) renal tubular damage have been reported in patients with multiple myeloma and other related diseases.615 in particular , distal renal tubular acidosis is characterized by hyperchloremic metabolic acidosis23 and hypokalemia.24 on the other hand , sakaue et al and uchida et al described igg - kappa - induced proximal renal tubular acidosis , as observed in fanconi syndrome , in patients with igg - kappa type myeloma.14,15 although the patient in the present study showed persistently low levels of hco3 , hypokalemia , and hypophosphatasia , he did not show evidence of glucosuria or generalized aminoaciduria , indicating that he did not have fanconi syndrome .
therefore , we concluded that the extreme hypernatremia / hyperchloremia was due to a combination of excess saline and nahco3 , dexamethasone administration in association with poor oral fluid intake , on top of the distal renal tubular damage caused by multiple myeloma .
considering the rarity of hypernatremia regardless of renal tubular damage in patients with multiple myeloma , it remains unclear how much myeloma - associated renal damage played a role in causing severe hypernatremia in this patient .
minemura et al8 reported that renal tubular acidosis was not improved by treating the myeloma with chemotherapy agents ; indeed , in their case , the patient died from an exacerbation of the disease . on the other hand , uchida et al15 reported a case in which renal tubular acidosis was corrected after chemotherapy for multiple myeloma , and sakaue et al14 reported that serum igg levels and urine sugar levels decreased , and serum potassium levels returned to normal , in a patient receiving chemotherapy for multiple myeloma . in the current case
, the patient recovered from the electrolyte disturbances and acidosis within a month , responding to the myeloma therapy .
the improvement in the electrolyte imbalance was associated with a return to normal levels of consciousness .
in summary , there appears to be an increased risk of hypernatremia in elderly patients with renal tubular damage associated with multiple myeloma ; therefore , clinicians must exercise caution and not prescribe excess salt , particularly in elderly patients who have difficulty taking oral fluids .
written , informed consent to publish this case report was obtained from the caregiver of the patient involved .
si , nk , and kk contributed equally to this work ; si , nk , and kk managed the patient ; kk performed bone marrow studies ; all authors prepared the manuscript ; all authors read and approved the final manuscript . | a 77-year - old male was admitted to hospital after suffering a pelvic bone fracture in a road traffic accident and was incidentally found to have igg - kappa - type multiple myeloma with hypercalcemia .
the patient was also noted to be hypokalemic and had low hco3 , with possible damage to the distal tubules in the kidneys . when the treatment was begun with
bortezomib / dexamethasone / elcatonin and sodium bicarbonate ( nahco3 ) in normal saline ( equivalent to a daily sodium dose of 200 millimoles per liter [ mmol / l ] ) , the patient was in a state of poor oral fluid intake .
the patient developed hypernatremia and hyperchloremia , with a peak serum sodium and chloride levels of 183 mmol / l and 153 mmol / l , respectively , at the sixth day after the start of treatment .
following the switch of the intravenous infusions from normal saline to soldem 1 and soldem 3 solutions , these high - electrolyte levels gradually returned to normal over the next 7 days .
although the patient showed disturbed consciousness ( japan coma scale = jcs - i-3 ) during the period of electrolyte abnormality , he eventually fully recovered without sequelae . in this patient , we successfully managed the severe hypernatremia / hyperchloremia , caused by the combined effects of intravenous saline burden in a state of poor oral fluid intake , during the treatment for igg - kappa type multiple myeloma . | Introduction
Case report
Discussion
Conclusion
Consent
Author contributions |
PMC3236484 | cardiorespiratory fitness ( crf ) maintenance is important for functional independence and physical capacity throughout aging [ 1 , 2 ] .
substantial declines in the ability to tolerate physical exertion generally predict mobility problems and cardiovascular morbidity and mortality , particularly in the sedentary elderly [ 3 , 4 ] . despite the importance of crf assessment , very low functional capacity and frailty
may hinder the use of exercise tests in this population [ 5 , 6 ] . in this context ,
nonexercise prediction models become practical alternatives to estimate crf and may have important applications both in clinical and epidemiological settings .
these models are developed by means of regression - based equations that usually include variables of simple and fast assessment , such as anthropometric measures , demographic characteristics , and daily habits .
cross - validated an equation developed primarily in middle - aged adults by jurca et al . and suggested that nonexercise models could be used to estimate the crf of older adults .
however , the prevalence of chronic diseases such as cardiovascular disease and diabetes increases dramatically with age , and is associated to lower physical capacity , inactivity , and limitations in the ability to exercise
. it would be therefore important to take into account that elderly populations are not always healthy and free of cardiovascular and metabolic diseases , which on the contrary , are common in the later life . notwithstanding to date nonexercise models to assess the crf in elderly subjects with chronic diseases
hence the present study aimed to develop a crf nonexercise prediction linear model of cardiorespiratory fitness and test its validity in elderly men presenting chronic cardiovascular and metabolic diseases .
the sample consisted initially of 108 subjects admitted to the elderly care center of the open university of the third age ( unati ) of the rio de janeiro state university ( uerj ) .
these subjects went through clinical exams in order to detail their medical history and completed a brief questionnaire providing demographic information , which was used to determine whether they could perform maximal aerobic exercise testing .
the inclusion criteria were asymptomatic patients , stable disease , and no abnormalities in rest electrocardiogram for at least six months .
tests interrupted due to clinical reasons were not considered as maximal and therefore have been excluded from the study .
twelve participants did not complete the exercise testing ( five were not allowed by the physicians to perform the test , four asked to stop before achieving maximal effort , and three exhibited high blood pressure ) . from the initial sample ,
a total of 96 subjects remained in the study ( 6091 years ) , being randomly assigned into two groups : validation ( 70% ) and cross - validation ( 30% ) .
the command sample 70 from stata statistical package version 10.1 ( stata , college station , tx ) was used with this purpose .
the validation group was used to develop the statistical model while the cross - validation group was used to confirm the generalization potential of the obtained model .
the experimental protocol was approved by institutional ethical committee and participants provided a written informed consent for the use of their data for research prior to the commencement of the study , as stated in the declaration of helsinki .
individual data on anthropometry , self - reported physical fitness , and physiological measurements were assessed .
anthropometric measurements included weight and height from which the body mass index ( bmi ) was calculated .
the physical activity history was assessed by the self - reported physical activity index ( srpa ) .
the subjects were asked to choose one of five activity categories that best described their usual pattern of daily physical activities .
self - rated fitness was evaluated by means of the rating of perceived capacity scale ( rpc ) , a 1 - 20-scale previously adapted and translated to the brazilian portuguese language , in which the subject chooses the most strenuous activity that can be sustained for at least 30 min . the rpc score is expressed in mets and the listed activities include walking , jogging , running , and cycling at different paces . instead of asking of the physical activity history ,
the blood pressure at rest and during exercise was measured by auscultation with a sphygmomanometer welchalln ( tycos , arden , mn , usa ) .
a 12-lead electrocardiogram was used to assess the resting heart rate ( rhr ) and maximal heart rate ( micromed , brasilia , df , brazil ) .
handgrip strength ( hg ) was measured with subjects keeping their shoulder adducted and neutrally rotated , with the arm fully extended and being encouraged to exert maximal grip force on a lafayette dynamometer 78010 ( lafayette , in , usa ) .
the highest value in kilograms ( kg ) was determined after four trials in the dominant and nondominant hands and the relative handgrip strength ( i.e. , handgrip strength normalized to body weight and represented as the ratio handgrip / weight ) was then used for further analyses .
all subjects performed a clinically supervised maximal exercise test in an electromagnetically braked cycloergometer ( cateye ec-1600 , osaka , japan ) using an individualized ramp protocol .
subjects were submitted to a familiarization trial to get used to the cycloergometer and mouthpiece on the day prior to the exercise test .
prediction equation was used to estimate the test incremental workload in watts in order to achieve maximal exertion in approximately 10 minutes .
paul , mn , usa ) was used for gas exchange measures , using a medium flow pneumotachometer ( 10120 lmin ) .
peak oxygen uptake ( vo2 peak ) was determined as the maximal oxygen uptake at the point of test termination due to volitional exhaustion .
the borg cr-10 perceived exertion scale was used to estimate the degree of exertion and standard clinical criteria for terminating exercise testing have been applied . before each test
, the equipment was calibrated as recommended by the manufacturer , using standard reference gases .
the test was considered maximal when at least two of the following criteria were observed : ( a ) respiratory exchange ratio ( rer ) > 1.1 , ( b ) vo2 plateau despite an increase in workload ( increase < 2.0 mlkgmin between the last two loads ) , and ( c ) maximum volitional exhaustion .
differences between the validation and cross - validation groups were analyzed by t - test student 's and chi - square tests .
age , relative strength index ( handgrip strength / body weight ) , an anthropometric measure ( bmi ) , self - related fitness ( rpc scale ) , self - related physical activity ( srpa index ) , and resting heart rate ( rhr ) were entered , respectively , into the first , second , third , fourth , fifth , and sixth blocks of the hierarchical linear regression procedure to develop a model to predict metpeak ( i.e. , the number of mets achieved at vo2 peak ) . the coefficient of determination ( r ) and the standard error of estimate ( see ) were calculated and the prediction equation generated
was then cross - validated using the press method ( predicted residual sum of squares ) , a statistical jack - knife procedure that consists of refitting the proposed model many times , leaving each observation out of the model fit in turn , so as to predict and then calculate the residual for that observation .
the second series of cross - validation analyses based on the independent sample was performed using the pearson correlation between the metpeak predicted by the model and the metpeak measured , as well as the difference between the crf estimated by the prediction model and measured crf ( defined in the maximal exercise testing ) was analyzed by the t - test .
a bland - altman analysis of measurement differences plotted versus mean values was used to assess the degree of agreement . in order to evaluate the accuracy of the model in screening individuals that present very low cardiorespiratory fitness , and
therefore are at higher risk for mortality and morbidity , a disability index was used to dichotomize crf .
basically , the crf level below which successful independent living is significantly compromised ( i.e. , 5 mets ) , was used as a cutoff to calculate the model sensitivity , specificity , and the area under the receiver operation curve ( auc ) . a 95% confidence interval ( 95% )
the auc is a measure of diagnostic power of the model and describes the probability that the model will correctly identify subjects with low crf .
the predicted and measured crf categories from the validation and cross - validation groups were cross - tabulated to detect classification accuracy .
table 1 presents the descriptive statistics , as well as values for cardiorespiratory variables at rest and in response to maximal exercise for subjects in both validation and cross - validation groups .
these groups did not significantly differ in any of the variables studied ( p > 0.05 ) .
all subjects attended at least two of the criteria adopted to consider the cardiopulmonary exercise testing as maximal : respiratory exchange ratio ( rer ) > 1.1 ( 87% of the sample ) , vo2 plateau despite an increase in workload ( 75% ) , and ( c ) maximum volitional exhaustion ( 100% ) .
three levels of physical activity were detected by the srpa index : inactivity or little activity other than usual daily activities ( 58% of the whole sample ) , participation in physical activities requiring low levels of exertion that result in slight increases in breathing and heart rate for at least 10 minutes at a time ( 38% ) , and participation in aerobic exercises such as brisk walking , jogging or running , cycling , swimming , or vigorous sports at a comfortable pace or other activities requiring similar levels of exertion for 20 to 60 minutes per week ( 4% ) .
only three of four variables were significantly associated ( p < 0.01 ) with crf after the linear regression procedure ( table 2 ) .
age , relative handgrip strength , and rpc explained 44% , 22% , and 13% of crf variance , respectively .
the bmi ( r = 0.003 , p = 0.44 ) , srpa index ( r = 0.003 , p = 0.37 ) , and rhr ( r = 0.001 , p = 0.53 ) were not significant predictors .
based on the r value , the obtained model explained 79% of the variability in vo2 peak .
the low shrinkage of adjusted r and the r - press conferred a good generalization to the model .
the following prediction equation was generated from the coefficients presented in table 2 : metpeak = 6.095 0.096
( age ) + 8.84 ( handgrip strength / weight ) + 0.67 ( rpc ) .
once the statistical model was established it was then further tested in an independent sample for cross - validation .
there were no significant differences between predicted crf mean and actual crf mean in both validation and cross - validation groups ( figure 1 ) .
the pearson correlation coefficient between predicted and actual crf in the cross - validation group was 0.85 ( p < 0.01 ) , ratifying that the model was appropriate to estimate crf in elderly subjects with cardiovascular and metabolic diseases .
the bland altman analysis ( figure 2 ) showed that few values fell outside the ranges established by the 2 sds , which suggests a good agreement between the values estimated by the statistical model and the actual met values .
table 3 exhibits the model accuracy from the validation and cross - validation groups after diagnostic tests .
the model was almost equally efficient in identifying subjects with crf 5 mets and with crf > 5 mets .
the auc further confirmed the high accuracy of the model in screening subjects presenting crf either below or above 5 mets .
the negative effect of aging on crf as well as the use of self - reported fitness are both well documented in the literature and are frequently included in prediction models [ 8 , 20 ] . although not previously used in other models , the relative handgrip strength was a very significant predictor of crf in the present study .
one could argue that the inclusion of handgrip strength may limit the widespread applicability of this equation , given that it requires equipment that may not be routinely used in epidemiological studies .
in fact , the handgrip dynamometry has been a very important tool to assess the functional status of elderly samples , and the inclusion of this measurement in epidemiological settings should be reconsidered .
the handgrip strength test is very simple and inexpensive , and has been previously related to mortality , mobility , functional capacity , and correlated with walking speed and overall strength , which are variables strongly related to the functional independence and health in older persons [ 1 , 21 , 22 ] . despite of the fact that 79% ( r ) of variance in the prediction of crf was explained by the obtained statistical model , the see ( table 2 )
suggests that more precise methods as maximal tests are recommended if the exactly crf value is needed .
on the other hand , the auc values ( table 3 ) suggested that the model has good accuracy to stratify elderly men with very low ( i.e. , crf 5 mets ) and higher cardiorespiratory capacity ( i.e. , crf > 5 mets ) .
in other words , the model was capable to identify elder subjects whose functional and exercise capacity are compromised .
based on this finding , the proposed model can be useful for population - based investigations or epidemiological studies , especially those searching for associations between crf and other physical and mental health outcomes , such as cognitive function and wellbeing . in clinical studies our model
mailey et al . tested the validity of the nonexercise model proposed by jurca et al .
in old adults , and reported a multiple r of 0.72 ( p < 0.001 ) for the regression in a sample composed mainly of old women , which were somewhat more physically active than our subjects .
our model was developed in a sample of elderly men with cardiovascular and metabolic diseases and produced a multiple r of 0.89 ( p < 0.001 ) .
it is also worthy to note that metabolic and cardiovascular diseases in the elderly are highly prevalent , reinforcing the relevance of the present model for this population .
although comparisons between the two models are difficult because they were developed in different populations , we have included variables from mailey 's model in our regression model , and they were not significant .
we have also compared the crf estimation accuracy of the models , and the results were favorable to the present equation ( mailey 's model in the validation group : r = 0.47 ; epe = 1.7 mets , and in the cross - validation group : r = 0.33 ; epe = 1.6 mets ) . the adoption of a cycloergometer maximal exercise testing protocol to assess the metpeak must be justified .
although treadmill tests are known to engage larger muscle mass and therefore may elicit higher peak vo2 [ 16 , 23 ] , some authors have proposed that cycloergometer tests would be more appropriate to assess the cardiorespiratory fitness in older subjects , mainly for safety reasons .
for instance , it has been suggested that high - intensity treadmill exercise should be avoided in older subjects with balance restrictions or joint problems . moreover , the poor mechanical efficiency while running seems to reduce the performance of older compared to younger subjects during treadmill exercise , which would very likely limit the peak vo2 in maximal cardiopulmonary tests .
such limitation has been considered by previous research that adopted cycloergometer protocols to assess the cardiorespiratory fitness in older populations [ 4 , 26 ] .
the main finding of this study was that crf of elderly men with cardiovascular and metabolic diseases and low physical capacity can be classified without aerobic tests using a combination of information on the subject 's daily activity levels , relative handgrip strength , and a self - report of physical fitness level .
maximal aerobic tests have a higher cost , demand familiarization with ergometers , and are frequently difficult to perform in old adults due to poor balance and coordination , gait problems , and fear of exercising .
it is also worthy to mention that the accuracy of some submaximal exercise testing models to estimate crf [ 28 , 29 ] could be comparable to the accuracy of the present prediction model .
it also represents an alternative to some walking tests because it does not require encouragement , which can be a source of disparity across trials , and does not require fatigue and dyspnea measurements .
this study has some limitations , namely , the relatively small sample size , the prediction model restricted to unhealthy men .
it is worthy to mention that since running tests may potentially produce higher metpeak values , the reproducibility of our findings in treadmill exercise testing should also be addressed , despite the fact that cycloergometer tests are frequently indicated due to safety and mechanical efficiency reasons .
probably , changes in age , handgrip strength , and self - related fitness would influence the functional capacity and the mets levels , but only longitudinal studies could confirm this possibility .
additionally , the main objective of the present study was similar to other studies that developed nonexercise models , which were not conceived to detect slight longitudinal variations . in brief , the model aims to classify and compare individuals within a given population , and does not intend to replace cardiopulmonary exercise testing to precisely assess the crf .
therefore , it is possible that small changes in crf due to training can not be detected by our equation .
however , the crf assessment is usually limited due the mobility issues and the risk of cardiovascular events especially in older people with chronic conditions .
the present study presented an accurate fitness prediction model for elderly men with cardiovascular and metabolic diseases .
the model provides a very fast and safe assessment of fitness , without any chance for cardiovascular events during assessment , which could be very feasible in many healthcare settings to estimate crf and stratify elderly subjects accordingly , and very attractive for epidemiological studies . | low cardiorespiratory
( crf ) is associated with health problems in elderly people , especially cardiovascular and metabolic disease .
however , physical limitations in this population frequently preclude the application of aerobic tests .
we developed a model to estimate crf without aerobic testing in older men with chronic cardiovascular and metabolic diseases .
subjects aged from 60 to 91 years were randomly assigned into validation ( n = 67 ) and cross - validation ( n = 29 ) groups .
a hierarchical linear regression model included age , self - reported fitness , and handgrip strength normalized to body weight ( r2 = 0.79 ; see = 1.1 mets ) .
the press ( predicted residual sum of squares ) statistics revealed minimal shrinkage in relation to the original model and that predicted by the model and actual crf correlated well in the cross - validation group ( r = 0.85 ) .
the area under curve ( auc ) values suggested a good accuracy of the model to detect disability in the validation ( 0.876 , 95% ci : 0.7930.959 ) and cross - validation groups ( 0.826 , 95% ci : 0.6770.975 ) .
our findings suggest that crf can be reliably estimated without exercise test in unhealthy elderly men . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion |
PMC1874174 | in times of increasing environmental and industrial standards for technologies and innovation toward safer substances closely connected to subsequent legal surveillance in most european union ( eu ) countries , it may strike us as strange to communicate that we are confronted with a group of new problematical substances in the environment .
long forgotten are the impressions of great environmental disasters , fish dying in running waters , the disappearance of predatory mammals and birds scenarios such as these belong to bygone days . for quite some time now , in many eu countries water condition maps indicate a mainstream trend : the status of water quality in europe seems to be improving ( nixon et al .
2003)although one should bear in mind that these statements do not refer to the biological status of water quality as required by the newly introduced eu water framework directive ( european parliament and the council of the european union 2000 ) . today
s problematical pollutants are not characterized by high environmental concentrations but occur in the ultratrace range . in many cases even modern analytical techniques
are not well enough developed to detect those substances in the subnanogram per liter range .
daughton and ternes ( 1999 ) coined the term lingering environmental toxicants , identifying personal care products , pharmaceuticals , and endocrine disruptors as integral parts of this substance group . regarding their common distribution in the environment , optimized biological activity , and effectiveness at comparatively low concentrations , they obviously show some similarities .
therefore , the class of substances possessing hormonal activities incorporates diverse groups such as man - made and natural chemicals ( e.g. , pesticides , plastisizers , alkylphenol ethoxylates as industrial surfactants , organotin compounds , polychlorinated biphenyls , phytoestrogens , natural hormones ) and pharmaceuticals ( e.g. , ethinylestradiol , methyltestosterone , trenbolone ) .
there is a wide range of biophysical properties , from persistent to rapidly degraded , lipophilic to hydrophilic , and nontoxic to very toxic , that can be attributed to endocrine modulators .
even from a mechanistic point of view , one can not define a unique mode of action .
although some of the substances take effect by binding but not activating the receptor ( antagonistic effect ) , others may mimic biological activity by attaching to the receptor to produce hormonelike action to an inflationary extent ( agonistic effect ) .
furthermore , endocrine toxicants may act indirectly in a non - receptor mediated manner when binding to transport proteins ( e.g. , corticosteroid - binding globulin , steroid hormone binding globulin , thyroid hormone binding globulin , growth hormone binding protein ) or by blocking , increasing , and decreasing enzyme activities or other metabolic pathways to affect the synthesis of endogenously produced hormones .
there are a number of less well - known endocrine tissues and hormones other than those producing sexual steroid hormones ( e.g. , those affecting the hypothalamus and pituitary gland , thyroid and parathyroid glands , adrenal glands , and endocrine pancreas ) . in any case , a particularly large number of publications on the impairment of reproduction , sexual differentiation , and development caused by hormonally active substances ( colborn et al .
2004 ) drove research toward a strong focus on sex steroids produced by the gonads .
endocrine disruption is a global phenomenon . across the world , endocrine - related effects have been reported in wildlife causing reproductive failure and in some cases population declines ( candia carnevali et al .
these effects range from feminization of males and virilization of females to suppressed thyroid and immune functions and altered development .
nevertheless , the greatest attention to endocrine disruption has been paid to estrogenic effects , although a clear cause effect relationship has been established for the androgenic activities of organotin compounds in mollusks ( bettin et al .
1996 ; horiguchi et al . 1997 ; oehlmann and schulte - oehlmann 2003 ) . in prosobranch snails , tributyltin and
triphenyltin compounds interfere with key enzymes of the sexual steroid metabolism . moreover , these compounds are known also to affect comparable or the same molecular targets in other taxa , including humans ( doering et al . 2002 ; heidrich et al .
therefore , it was self - evident that research within the framework of the eu project comprendo ( comparative research on endocrine disrupters
phylogenetic approach and common principles focussing on androgenic / antiandrogenic compounds ) would offer the unique opportunity to study a real wildlife
human connection when zooming in on effects mediated by androgen- and antiandrogen - mimicking compounds .
comprendo is one of the four projects that form the core of the credo ( coordinating european environmental and human health research into endocrine disruption ) cluster and is funded by the european commission s fifth framework programme for research , technological development and demonstration activities in the european community .
the overall goal of comprendo is to improve the understanding of the effects of endocrine - disrupting chemicals ( edcs ) on aquatic wildlife and humans to improve environmental quality standards and public health in europe . to this end
the special issue of edcs and their potential to cause serious health problems in wildlife species make it questionable whether drinking water and food are still without impact on normal development and sexual differentiation and also whether they allow a normal reproduction and the aging of individuals without avoidable health effects .
a major concern for human health is the suspected intake of edcs via the diet . to give consideration to the latter in comprendo ,
human exposure to androgenic and antiandrogenic compounds ( aacs ) is assessed in all participating european partner countries via food basket analyses ( identification of background concentrations in human food ) . in parallel ,
an exemplary aac residue determination in human samples ( blood , urine , placenta ) in european areas with higher and lower environmental and industrial standards is accomplished .
environmental samples ( sediment and biota ) are additionally taken and chemically analyzed in these regions .
this is to assess the exposure of wildlife populations to aacs as well , and to characterize potential effects in highly contaminated and reference areas .
presently , the organisms that are used to assess effects of chemicals and in turn infer potential health and ecosystem - level effects are very limited . as new problems arise , we need to be mindful of what chemical tests are the most appropriate and fit for purposes of environment protection rather than necessarily being constrained by existing test systems .
this is why comprendo aims to identify new test species and toxicological end points to help safeguard the protection of organisms living in aquatic ecosystems . to do this a wide range of organisms from invertebrates ( echinoderms , crustaceans , mollusks ) to vertebrates ( fish , amphibians ) and human models
( human cell lines , tissues , rodents ) are under investigation to develop an understanding of the commonalities and differences in responses to aacs across the animal kingdom .
initially , the application of positive controls for aacs known to cause direct receptor - mediated or indirect functional effects [ e.g. , methyltestosterone and mibolerone as androgen receptor agonists ; letrozole as steroidal aromatase inhibitor ; cyproterone acetate , flutamide , and prochloraz as androgen receptor antagonists ; 4,7-dimethyl-4-azacholestan-3-one ( mk 386 ) and finasteride as reductase type 1 and 2 inhibitors ] provides a basis for the assessment of the potential vulnerability of representative aquatic wildlife groups and humans .
second , test species are exposed to several compounds [ organotins , e.g. , mono- , di- , tri - butyltin and triphenyltin compounds , and pesticides such as fenarimol , vinclozolin , linuron , diuron , p , p-dde ( dichlorodiphenyl - dichloroethylene ) ] at environmentally relevant concentrations to identify sensitive and easily measurable end points ( genetic , biochemical , physiological , histological , morphological ) and critical stages of exposure ( via early - life stage and life - cycle tests ) .
these monosubstance tests are linked to sediment exposure studies ( sediments originating from environmental sampling ) representing complex environmental mixtures . by means of comparison ,
the results are used to determine whether pure substances or mixtures adversely affect communities in ecosystems .
most of the compounds suspected as endocrine disruptors end up in aquatic ecosystems by sewage effluents .
aquatic organisms are almost continually exposed to these chemicals and , as a matter of plausibility , therefore provide sentinel organisms for detecting adverse biological effects .
determining whether a chemical , especially at sublethal concentrations , can affect endocrine - mediated functions in wildlife populations is difficult and may require a prolonged and profound understanding of the animal s life history , morphology , and the influence of the local environmental conditions ( tagatz 1968 ) .
although it is unquestionable that invertebrates are in principle sensitive to edcs , their use as standard biotest organisms has been impeded by the lack of laboratory cultures and detailed insights into invertebrate endocrinology .
therefore , the establishment of long - term cultures of selected species of mollusks ( marisa cornuarietis , potamopyrgus antipodarum ) and crustaceans ( hyalella azteca , arcatia tonsa ) will be used for studies on endocrine disruption to potentially provide new standard test organisms on the international scale , such as tests mandated by the organisation for economic cooperation and development ( 2006 )
inter alia robustness , synchronicity of development , number of generations per year , ease of handling , diet preferences and the possibilities to achieve a standardization of the diet , cost of culture , and sensitivity of species in the exposures have been important evaluation criteria for checking their suitability for the establishment of laboratory cultures .
the invertebrate reproductive cycle can be highly complex ( sastry 1968 , 1970 ) and is controlled by many environmental stimuli , including light intensity , temperature , desiccation , and diet ( ansell and trevallion 1967 ; copeland and bechtel 1974 ; largen 1967 ; tessier et al .
this is why laboratory cultures will finally be optimized in terms of favorable culture conditions ( temperate and light / dark regimen , water chemistry , standardized food , animal densities in tanks ) and a characterization of the species - specific biology under the chosen culture conditions [ reproductive phase(s ) , time and length of spawning , duration of different development phases , time until puberty , length of sexually active life phase ] .
on the level of sexual development , reproduction , and differentiation , endocrine modulators may produce a variety of deviations compared with normogenesis and unaffected progeny .
therefore , test species will be checked for pathomorphoses and histopathological aberrations of target organs ( e.g. , sexual glands , gonads , midgut gland ) during exposure .
tracing of cell cycle kinetics , apoptosis , protein content , and mitochondrial respiration provides additional information on disturbances of cytologic processes .
sex steroid concentrations ( androstenedione , testosterone , dihydrotestosterone , 11-ketotestosterone , estrone , and estradiol ) are checked in all species under investigation during an unspoiled annual reproductive cycle to be linked with comparative data generated during the exposure s run . the same approach is applied for the determination of phase i and ii metabolism . in this regard ,
the activities of cytochrome p450 aromatase and 3- , 11- , and 17-hydroxysteroid dehydrogenase are analyzed for phase i. because interferences with hormone phase ii metabolism will alter hormone excretion rates and may lead to hormonal imbalance in exposed organisms , the activities of several enzymes , such as sulfotransferase , acyl - coenzyme a acyltransferase , and sulfotransferases , are analyzed in human cell lines and tissue ( placenta , prostate , liver ) , digestive gland , liver , and gonad of vertebrates and invertebrates in parallel . in the development of new tests for aacs , innovative approaches are being employed , including the development of a multispecies gene array . here ,
a suite of key genes that control sexual function is being isolated and applied to a matrix , and the array subsequently used to screen for the effects of aacs on target gene expression .
it is envisioned that this approach will offer new perspectives in the application of modern molecular techniques in our understanding of endocrine disruption .
the comprendo work program reflects a holistic attempt that will have relevance far beyond the chosen group of compounds .
environmental and human health aspects are simultaneously addressed by using an interdisciplinary and comparative phylogenetic approach .
studies of this nature require a comprehensive understanding of the endocrinology of sentinel organisms and where there are fundamental gaps of knowledge in baseline endocrinology ( notably in invertebrates ) .
a common and distinguishing mark of the endocrine system of all animal phyla consists of a widely distributed system of glands , tissues , and diffuse groups of widespread cells that synthesize and release chemical messengers into the circulatory system .
doubtless the regulation of many parts of biological processes has been widely conserved , although individual components of the endocrine system have passed through profound evolutionary processes .
consequently , remarkable divergences resulting in distinct differences in the endocrine systems of the diverse taxa can be expected ( de fur et al .
1999 ) , but on the other hand , we have reason to suppose that in the animal kingdom many similarities and analogues of metabolic pathways also exist .
it is likely that genes , proteins , or receptors differ in structural configuration across taxa , and even if the differences are very small , they may constitute the basis for different effects and reactions in test species when exposed to the same substances and concentrations .
for example , conventional radioreceptor assays have provided evidence for androgen- and estrogen - specific binding sides in mollusks , crustaceans , and echinoderms ( oehlmann et al .
nevertheless , their binding affinity to direct - acting aacs varies enormously compared with those of vertebrates .
although parts of vertebrate - type sex steroid metabolism can be identified in the different invertebrate test species , the conversion rates from androgens to estrogens might be identified to be much lower compared with those of vertebrates .
different activity patterns of the enzymes involved ( occurrence , function , biosynthesis ) or other interspecies differences with respect to the metabolic pathways ( e.g. , conjugating activities ) may turn out to be responsible for this .
however , these and other findings make it worthwhile to clone and sequence the androgen and estrogen receptors in the different species under investigation and to focus on deciphering their endocrine systems
. finally , this could pave the way toward an extrapolation of data derived from animal experiments to human health based on factual knowledge and improve our knowledge on the extent and nature of endocrine disruption by aacs with a critical evaluation of their role as disruptors of physiological functions in wildlife and human populations ( risk assessment ) . | tens of thousands of man - made chemicals are in regular use and discharged into the environment .
many of them are known to interfere with the hormonal systems in humans and wildlife . given the complexity of endocrine systems , there are many ways in which endocrine - disrupting chemicals ( edcs ) can affect the body s signaling system , and this makes unraveling the mechanisms of action of these chemicals difficult .
a major concern is that some of these edcs appear to be biologically active at extremely low concentrations .
there is growing evidence to indicate that the guiding principle of traditional toxicology that the dose makes the poison may not always be the case because some edcs do not induce the classical dose response relationships .
the european union project comprendo ( comparative research on endocrine disrupters
phylogenetic approach and common principles focussing on androgenic / antiandrogenic compounds ) therefore aims to develop an understanding of potential health problems posed by androgenic and antiandrogenic compounds ( aacs ) to wildlife and humans by focusing on the commonalities and differences in responses to aacs across the animal kingdom ( from invertebrates to vertebrates ) . | Endocrine Disruptors: The Issue
Characterization of Human and Environmental Exposure to Androgenic and Antiandrogenic Compounds
Impacts of AACs at Environmentally Relevant Concentrations on Human-Relevant Models and Aquatic Species
Establishment of Laboratory Cultures for Suitable Aquatic Invertebrates and Their Baseline Endocrinology
Species-Specific Critical End Points, Including a Molecular Screen for Genomic Effects of AACs
Identification of Common Principles of an AAC Action across Taxa |
PMC4071669 | inhaled foreign bodies ( fbs ) are a common paediatric emergency and perhaps among the greatest cause of accidental death at home among nigerian children , although the exact incidence is not known because many are not recognised especially in rural communities.1 it is equally a major source of morbidity and mortality in even the most sophisticated of centres .
this is particularly so in the paediatric age group especially the under - fives.2 this is said to be due to their inquisitiveness , adventurous nature , lack of molars for proper mastication , playing and running with food in the mouth with consequent incoordination in swallowing and glottic closure.3 with the advent of the ventilating bronchoscope , skilled personnel and better anaesthetic techniques , the mortality rate is said to have reduced from 24 to 3% or even less in developed countries.4 this , however , is not the case in developing countries especially sub - saharan africa , where the mortality rate is variable but reported cases range from 2.7 to 8.3%567 with majority probably under reported as most die before getting to a definitive point of care .
the classical symptoms during fb inhalation are a sudden onset of paroxysms of cough , stridor and dyspnoea and occasionally this history is unavailable .
the commoner signs are respiratory difficulty , reduced air entry in an affected lung , wheezing and/or crepitations .
different types of foreign bodies ( fbs ) have been reported from the ordinary to the most bizarre .
these range from inorganic materials such as needles , nails , whistles , toy parts and beads / rosary to organic substances such as groundnuts , maize seeds , water melon seeds , beans , variety of grains , etc .
nonetheless , one thing is sure they are all a major source of anxiety and fear to the patient , caregiver and perhaps the attending physician as well .
this broad classification becomes important when considering obstruction to airflow , amount of mucosal reaction , inflammation / infection and abscess formation akin to organic foreign bodies in the tracheobronchial tree . on the other hand ,
inorganic fbs may cause total or partial obstruction which may be tolerated for some time with mild signs and symptoms leading to formation of granulation tissue after a period of time.8 the clinical course and outcome of inhaled foreign bodies largely depends on the nature / type of foreign body , the site of arrest or impaction along the tracheobronchial tree and perhaps availability of skilled manpower especially in developing countries .
foreign bodies can get impacted at any point from the laryngeal inlet to the terminal bronchioles , but more often these fbs get lodged in the right main bronchus .
this is due to the right main stem bronchus being more in line with the trachea , thereby , creating a relatively straight path from the larynx to bronchus .
despite this anatomical arrangement some controversies exist to suggest that more fbs are lodged in the left main bronchus compared to the right bronchus . in the same vain , some studies also posit that the site of election for impaction has an almost equal incidence in children , while others claim that this arrangement becomes relevant only after 15 years of age.1910 however , the site of impaction of fbs may ultimately depend on the position of the patient at the time of inhalation .
the right main bronchus is commoner in the erect position and right lateral position while fbs which are small enough preferentially lodge in the left main bronchus in the left lateral position .
this may also account for the difference in distribution of site of lodgment between adults / adolescent and children.1
this is a 6-year retrospective review of case notes of patients who have had bronchoscopies performed for foreign body inhalation at the otorhinolaryngology department of aminu kano teaching hospital , kano from january 2005 to december 2010 .
all the cases were referrals from the accidents and emergency unit , emergency paediatric unit and direct referrals from peripheral hospitals as well .
the hospital is the main tertiary centre serving a population of over 10 million nigerians with three other states as catchment areas ( katsina , jigawa and bauchi states ) .
a total of 39 patient case notes were retrieved for the study and only 35 were included for analysis , one case note had incomplete information / missing pages , two case notes revealed a failed / negative bronchoscopy , with one recorded mortality due to cardiac arrest intra - operatively .
basic information such as date of birth , age groups , gender , indication for bronchoscopy , type of fb , sites of fb impaction , and surgical outcome were recorded and analysed . in our centre
ibm spss ( for windows , version 21 ) software was used to analyse this data and for ease of interpretation presented in the form of tables and charts .
statistically significant association between variables was explored using a two - tailed chi - square test and a confidence interval of 95.0% .
ibm spss ( for windows , version 21 ) software was used to analyse this data and for ease of interpretation presented in the form of tables and charts .
statistically significant association between variables was explored using a two - tailed chi - square test and a confidence interval of 95.0% .
there were 18 ( 51.4% ) males and 17 ( 48.6% ) females with a male : female ratio of 1.1:1 .
the most affected age group is 3 - 5 years ( 31.4% ) , followed by 0 - 2 years ( 25.7% ) old age group [ table 1 and figure 1 ] .
age group by sex distribution of foreign bodies among our patient groups chart depicting percentage of foreign body inhalation by age groups with highest among the under - five 's groundnuts and whistles were the most frequently inhaled fbs in our series with 9 ( 25.7% ) each [ figure 2 and table 2 ] .
subdivision of types of fbs into two broad groups revealed organic 18 ( 51.4% ) foreign bodies and 17(48.6% ) inorganic foreign bodies [ figures 3 and 4 , respectively ] .
chart to show the frequency of foreign body types as seen in our patient population variety of foreign bodies removed at bronchoscopy during the period under review distribution of organic foreign bodies by ( groundnut with highest ) percentage distribution of inorganic foreign bodies by ( whistles with highest ) percentage with regard to the bronchus , overall fbs were more preferentially lodged in the left main bronchus in 8 ( 22.9% ) patient against the right main bronchus in 6 ( 17.1% ) patients .
the 9 - 11-year and 3 - 5-year - old age groups were especially noted to have fb impaction involving the left main bronchus 3 ( 42.9 ) and right main bronchus 2 ( 18.2% ) ( p = 0.592 ) , respectively . however , the majority of extracted fbs from the airways had no label for the specific site of impaction 15 ( 42.9% ) .
[ figures 5 and 6 ] distribution of impaction sites by percentages for foreign bodies in the tracheobronchial tree in our patient population frequency of impaction site by age group cluster bar chart analysis to determine relationship between age groups of these patients and types of inhaled foreign bodies revealed a strong association between these variables [ exact test p = 0.001 ; phi and cramer 's v = 1.255 and 0.725 , respectively , and contingency coefficient 0.782 ] further attempts to find relationships within variables such as sex of patients and nature of inhaled fbs , age groups and site of impaction , organic and inorganic fbs did not reveal any significant associations ( p > 0.05 ) including the effect of age and gender in this study .
our study clearly revealed that children below 5 years of age were most susceptible with a slight male preponderance .
this was also the finding by gulshan et al . , and is probably due to the over active nature of male children compared to their female counterparts.11 a higher male preponderance ratio of atleast 2:1 or more , has been reported by several authors this may be due to variations in sample size when compared to ours.6111213 in contrast , from available literature we did not find a higher female preponderance .
moreover , with regard to variety of fb 's , groundnuts and whistles were the most common fb 's accidently inhaled with equal frequencies in our series [ table 2 ] . to our knowledge , no recent study has reported an equal frequency of these fbs , much less reporting whistles as high as 25.7% .
a study in pakistan showed that whistles were the commonest inhaled foreign body with a frequency of 57.7%.14 however , two recent studies show a frequency of 5.26% and 17.3% , respectively,1011 which is still less than findings from our series .
children under 5 years are said to be generally more susceptible to fb inhalation due to their oral tendency , playing or running around with food pieces in the oral cavity and also parental nonchalance with regard to close supervision .
many studies allude to this same finding.11516 children between the ages of 3 and 5years were the highest in our series [ figure 1 ] , this was also the finding of onotai et al ; however , other studies report a higher incidence in children less than or equal to 3 years.2121718 these periods coincide with the periods of inquisitiveness , adventure and exploration in a child 's developmental milestone , and by implication require closer attention and supervision from parents / caregivers . by and large organic fbs are commoner than inorganic fbs ; our study revealed 51.4% and 48.6% respectively and this was the finding by other authors.1920 the commonest organic fb inhaled in children is the groundnut and this concurs with our finding ( 25.7% ) and is corroborated by other studies as well.1011162122 in contrast , a recent study in kenya revealed that the commonest organic fb in their series was beans while the second were peanuts / groundnuts.6 this was closely followed by seeds , although the particular type of seed was not documented in some instances due to its fragmented nature , but in addition to maize seeds , these still made up a large portion of inhaled organic fbs [ figure 3].1618 kolanut and vegetable matter were the least ( < 10% ) but what we found , however , was the feeding , in some cases , of kolanut to children either by older siblings or accidently inhaled by the child while the parents were looking away or were not around .
kolanut is a commonly chewed nut by adults in nigeria especially in the north , and requires molars to adequately grind them enough to be swallowed , but how they end up in the mouths of children since they lack molars says a lot about their care , or perhaps due to careless storage by the parents .
other studies have revealed fishbone as the commonest type of fb seen especially in south - south nigeria.12 generally , inorganic fbs portend grave danger with acute asphyxiation from total obstruction or respiratory difficulty from partial obstruction . with regard to whistles , in our part of the country during festivities or ceremonies sweet candies are frequently giving to children . however , a variety of sweet candy is now available which is made combined with a whistle embedded within the sweet candy and after sucking out the candy bit , the children then go ahead to indulge in the reward by blowing the whistle as part of the fun . and as such , older children with whistles as fbs ( 25.7% ) accounted for a greater portion of this group and this coincided with the islamic festive periods of eid - el - fitr or -kabir and in the wet season [ figure 4 ] .
, reported the same observation during the wet season in ibadan south - eastern nigeria,1 a similar turkish study also reported similar findings highlighting an association between fbs with seasonal , geographic locality and sociocultural environment of affected patients.23 the seasonal significance of this finding at this time is unclear and may require further observations .
the finding of whistles as part of other toys or snacks is , however , not uncommon , as this was also reported in abbottabad , where they mostly accounted for ( 17.3% ) second highest after groundnuts.10 in one study in saudi arabia , the whistle could not be removed transglottically and tracheostomy with tracheal slit had to be performed , in our series all the whistle were successfully extracted transglottically via a rigid bronchoscope .
this further emphasises the danger posed by these toys of sweetness. other varieties of fbs are more straightforward in terms of extraction with the appropriate forceps at hand .
ball bearings in particular , had to be removed with a ventilating bronchoscope via the tracheostome .
this obviously requires cooperation between the anaesthetist and endoscopist in addition to relevant skill and experience . by and large ,
most studies show organic fbs to be most prevalent in comparison to inorganic fbs116 and this was our finding as well [ figures 3 and 4 ] . with regard to site of impaction or lodgment of foreign bodies ,
most studies have reported the preferential lodgment of fbs in children and adults to the right main bronchus as compared to the left main bronchus.15162425 however , in this review we did not record fb in both bronchi at the same seating as reported by a previous study.16 location of impacted fb is somewhat contentious ; most authors have argued that there is a preferential lodgment of fb in the right main bronchus than to the left main bronchus .
this is said to be dependent on the patient 's age and relative physical position at the onset of inhalation .
up until 15 years of age the angle made by the bronchi with the trachea is about the same , by implication an equal frequency is thereby recorded .
however , after this age with continued growth and development , the left main bronchus assumes a more acute angle than the right and as such , the right bronchus is therefore more in line with the trachea .
this may account for the relative higher frequency of fbs in the right main bronchus than in left as alluded to by many authors.41021 conversely , a recent study using tracheobronchial widths and angles from a large database of paediatric radiographs tested this hypothesis using standardised techniques with good reproducibility , and they concluded that an inhaled foreign body is more likely to enter the right bronchial tree than the left in children of
all ages . however , in another recent study the authors claim that variability in the position of the carina with respect to the mid - trachea may explain why this right - sided preference is less marked in children compared to adults .
this is because , in most children , the carina was positioned to the left of the mid - trachea , but in 34% of cases ( 40% of infants ) , it was to the right of the mid - trachea.24 in another related study the authors posit that aspirated foreign bodies are equally distributed between the left and right main bronchus
that in children aged 3 and older foreign bodies were more commonly found in the right main bronchus.1826 in our review of these paediatric cases , we found a preferential lodgement of fbs in the left bronchus than the right with 22.9% and 17.1% , respectively [ figures 5 and 6 ] .
, as well.27 although , as previously highlighted , the lack of specific labelling for site of impaction might have affected this result inadvertently . however , it is not uncommon.27 it suffices to say then that the bronchi , irrespective of laterality are the commonest areas of impaction along the tracheobronchial tree .
our study showed a combined frequency of 40% ( right and left ) of bronchi involvement [ figure 5 ] and this was the finding of some authors as well.172328 with regard to the rest of the tracheobronchial tree , the larynx and trachea have the lowest prevalence except in children less than 1 year,17 [ figure 5 ] this was our finding as well .
finally , our finding of left bronchial lodgment of fbs in specific age groups of 9 - 11 and 3 - 5 years was not statistical significant and as such no reliable comment can be made at this time .
it suffices to say that proper and/or specific identification of extracted seeds and accurate delineation of impaction sites may have affected significantly the overall outcome measures in this study .
possible biases may have existed in the recorded information , and some patient information was missing or not reported by the attending surgeon .
we also recognise that perhaps the sample size may be too small to make population - based conclusions .
it suffices to say that proper and/or specific identification of extracted seeds and accurate delineation of impaction sites may have affected significantly the overall outcome measures in this study .
possible biases may have existed in the recorded information , and some patient information was missing or not reported by the attending surgeon .
we also recognise that perhaps the sample size may be too small to make population - based conclusions .
children particularly infants will always be curious leading to accidents and as such parents / caregivers have a fundamental role to play by providing constant supervision .
the government must organise regular campaigns and proper education of the public both in the news / print media on the risk of foreign body aspiration especially among the under - five 's .
furthermore , the left main bronchus is a common area for impaction of fbs as well , although this may ultimately depend on the position of the patient at the time of inhalation .
we suggest that a strong legislation be put in place to regulate sweet and/or toy - producing companies on whistles ( toys ) manufactured with candies / sweets , and if possible be totally withdrawn from the market . | background : inhaled foreign bodies are common paediatric emergencies and a major cause of accidental deaths at home among nigerian children especially among children below the age of 5 years .
different types of foreign bodies have been reported from the ordinary to the most bizarre .
we evaluated the types of foreign bodies and sites of possible impaction of these foreign bodies along the tracheobronchial tree.materials and methods : a 6-year case record of 35 patients with foreign body inhalation was reviewed from january 2005 to december 2010 at the otorhinolaryngology department of bayero university / aminu kano teaching hospital , kano north - western nigeria .
basic information such as date of birth , age groups , gender , indication for bronchoscopy , type of foreign bodies , sites of foreign body impaction and surgical outcome were extracted and analysed.results:thirty-five patients [ 18 ( 51.4% ) males and 17 ( 48.6% ) females , aged 6 months-10 years ] with a mean age of 5.1 years and median of 5 years ( + /-
3.0 standard deviation ) .
the most affected age group with 31.4% is 3 - 5 years .
groundnuts and whistles were the most frequently inhaled foreign bodies with an equal frequency of 25.7% , respectively .
foreign bodies were preferentially lodged in the left main bronchus in 8 ( 22.9% ) patients compared to the right main bronchus in 6 ( 17.1% ) patients.conclusion:public enlightenment both in the news / print media on the risk of foreign body aspiration especially among the under - five 's is highly advocated .
the left main bronchus may be a common site for impaction as well . | INTRODUCTION
MATERIALS AND METHODS
Data analysis
RESULTS
DISCUSSION
Limitations
CONCLUSIONS |
PMC4437367 | acute kidney injury is a common clinical syndrome that is characterized by a rapid decline in kidney function , often triggered by glomerular disease and/or tubulointerstitial disease and associated with high morbidity and mortality [ 14 ] . in addition to serum creatinine , 2-microglobulin is a biomarker that can be used to determine underlying causes of acute kidney injury .
serum 2-microglobulin derives from cellular membrane turnover , since 2-microglobulin forms the invariant light chain portion of major histocompatibility complex ( mhc ) class i in membranes of all cells [ 710 ] . as a single - chain small polypeptide ( mw = 11.8 kda ) ,
2-microglobulin is filtered almost completely through the glomeruli of the healthy kidney [ 11 , 12 ] and then reabsorbed by the renal proximal tubules .
only a small amount of 2-microglobulin can be detected in the urine under normal physiological conditions .
therefore , levels of serum and urinary 2-microglobulin reflect the functions of glomeruli and proximal tubules . in patients with acute kidney injury , an increase in serum creatinine , together with an increase in urinary 2-microglobulin , strongly suggests proximal tubule injury . on the other hand ,
an increase in serum creatinine with elevated serum 2-microglobulin is seen in patients with decrease in glomerular filtration rate .
elevated urinary 2-microglobulin was associated with tubular injury caused by viral infection , ischemia , and toxicity from medications or heavy metals [ 15 , 16 ] .
measurement of urinary 2-microglobulin is convenient and has been applied in routine clinical practice ( renalvysion bostwick laboratories , uniondale , ny ) to evaluate the tubular function . using immunohistochemical staining for cd133 in kidney biopsies , we demonstrated that urinary 2-microglobulin is a sensitive indicator for tubular injury .
however , a direct correlation between urinary 2-microglobulin and proximal tubule injury has not yet been fully established due to lack of a specific proximal tubule injury marker .
recently , kidney injury molecule-1 ( kim-1 ) was shown to be a biomarker for proximal tubule injury [ 1820 ] .
kim-1 is a type 1 cell membrane glycoprotein comprised of extracellular , transmembrane and intracellular domains [ 21 , 22 ] .
kim-1 is undetectable in the healthy kidney . with different forms of kidney injury , mrna and protein levels of kim-1
are upregulated and expressed [ 20 , 23 , 24 ] in injured tubules [ 23 , 2527 ] .
kim-1 binds to surface - specific epitopes on the apoptotic bodies and cellular debris derived from injured tubular cells , facilitating macrophages to engulf dying cells .
in addition , the extracellular domain of kim-1 can be cleaved by metalloproteinases into a soluble shedded part and short membrane bound fragments that are released into urine .
moreover , the expression of kim-1 in injured tubule cells is limited to the proximal tubular cells .
using immunohistochemistry , kim-1 staining in kidney tissue has been shown to accurately detect proximal tubular injury associated with ischemic and toxic type of tubular injury , allograft rejection in renal transplantation , and acute pyelonephritis [ 20 , 26 , 28 ] .
the present study was designed to examine the correlation between increase in urinary 2-microglobulin and proximal tubule injury assessed by kim-1 staining in kidney biopsies .
between january 2009 and december 2012 , a total of 5494 patients had urinary 2-microglobulin measurements by renalvysion analysis ( bostwick laboratories ) . among these ,
nine renal biopsies either contained less than 10 glomeruli per histological section or had insufficient tissue to perform immunohistochemical staining and were excluded from the present study .
the remaining forty - six biopsies met selection criteria and were included in the present study .
briefly , 10 ml fresh urine was collected from each patient and urine ph was adjusted to ph 68 with 1 m naoh .
the specimen was delivered to lab by overnight shipping and the level of 2-microglobulin was measured next day .
the majority of immunohistochemical staining was performed in archived unstained slides prepared at the time of renal biopsy . in those biopsies
in which unstained slides were not available , additional new slides were prepared from archival tissue from formalin - fixed paraffin - embedded blocks .
immunohistochemical staining for kim-1 was conducted on paraffin slides using dako autostainer ( dakocytomation , carpinteria , ca ) . to remove paraffin ,
sections were heated at 60c for 60 min , placed into xylene baths for 3 min , 3 times , followed by 100% alcohol , for 3 min , 3 times , and then washed by running water for 30 s. antigen retrieval was carried out using a tris - edta buffer at ph 8.0 for 20 min at 99c and cooled down for 20 min at room temperature , raised by tap water .
slides were placed into h2o2 for 15 min and then placed in tris buffer ( ph 7.6 ) .
finally , slides were placed into dako autostainer programmed by thermo scientific ultravision lp detection system ( kalamazoo , mi ) .
staining procedures included 5 min ultra v block , 30 min incubation with primary kim-1 monoclonal antibody ( akg7 clone at 1 : 10 dilution , kindly provided by dr .
joseph v. bonventre from renal division brigham and women 's hospital , boston ) , 8 min primary antibody enhancer , 10 min hrp polymer ( equivalent to secondary antibody ) , and 5 min of chromogen dab to achieve brown staining .
granular staining in the cytoplasm and membrane of proximal tubular epithelium was considered positive . staining intensity of kim-1
was scored from 0 to 3 + as follows : 0 , absence of staining ; 0.5 , focal weak fine granular staining ; 1 + , weak fine granular staining cytoplasm and/or membrane in entire tubular luminal surface ; 2 + , moderate granular staining ; and 3 + , strong coarse granular staining .
parameters related to tubular injury including serum creatinine , granular and cellular casts , and urine cytological findings were also recorded . staining with kim-1
was evaluated by two pathologists who were blinded to the clinical data , final pathological diagnosis and patient outcome .
linear regression analysis was used to correlate values of urinary 2-microglobulin and kim-1 staining scores .
odds ratio analysis was applied to express the association of urinary 2-microglobulin level and intensity of kim-1 staining .
data were also stratified according to urinary 2-microglobulin level as normal ( < 300 g / l ) versus increased to kim-1 staining ( positive or negative ) and evaluated by 2 2 contingency table ( table 2 ) .
the sensitivity , specificity , predictive value for positive result , and predictive value for negative result were calculated as follows : sensitivity = true positive/(true positive + false negative ) ; specificity = true negative/(true negative + false positive ) ; predictive value for positive result = true positive/(true positive + false positive ) ; predictive value for negative result = true negative/(true negative + false negative);the age of the patients was expressed as mean sd . sensitivity = true positive/(true positive + false negative ) ; specificity = true negative/(true negative + false positive ) ; predictive value for positive result = true positive/(true positive + false positive ) ; predictive value for negative result = true negative/(true negative + false negative ) ;
among 5494 patients with urinary 2-microglobulin measurement , renal biopsy was obtained out in 56 patients ( 56/5494 , 1.0% ) . among these ,
thirty patients had increases in urinary 2-microglobulin ; it was in normal range in the remaining 16 patients .
the demographic details for 35 patients ( m : f = 22 : 13 ) with positive kim-1 staining in proximal tubules are shown in table 1 .
the age of patients at the time of biopsy was 54.9 10.7 years ranging from 16.0 to 87.0 years .
glomerular diseases included membranous nephropathy , antineutrophil cytoplasmic antibody- ( anca- ) associated glomerulonephritis , diabetic nephropathy , focal and segmental glomerulosclerosis , iga nephropathy , and lupus nephritis .
in addition to glomerular disease , five patients had tubular injury ( figures 1(c ) and 1(d ) ) , two of which were toxic type of injury with cytoplasmic vacuoles ( figure 1(d ) ) .
of the 26 patients with elevated urinary 2-microglobulin and positive kim-1 staining in proximal tubules , 18 patients had increased serum creatinine , ranging from 1.50 mg / dl to 6.00 mg / dl ( table 1 ) .
since the elevated urinary 2-microglobulin is associated with proximal tubular injury , in these 18 patients it was considered that the proximal tubular injury is the primary cause of increase in serum creatinine .
nine patients with normal urinary 2-microglobulin had positive kim-1 staining in proximal tubules , 6 of which had increase in serum creatinine , ranging from 1.10 mg / dl to 3.20 mg / dl .
granular casts were presented in 2 patients , who also had increase in both urinary 2-microglobulin and serum creatinine .
thirty - five of 46 biopsies showed positive kim-1 staining with a granular pattern in the cytoplasm and/or apical membrane of tubular epithelium , ranging from finely granular to coarsely granular ( figure 1(b ) ) .
the corresponding h&e slides of kidney biopsy showed flattening and simplification of tubular epithelium , ranging from focal to diffuse , overall focal tubular injury with varying degree , and mild tubular injury in the majority of the kidney biopsies . staining
intensity ranged from 0.5 + to 3 + with 1 + staining in 18 patients ( 18/35 , 51.4% ) .
the relation between value of urinary 2-microglobulin and kim-1 staining score is shown in figure 2 .
there was a significant correlation between levels of urinary 2-microglobulin and kim-1 staining ( r value = 0.293 , p < 0.05 ) ( figure 2 ) .
odds ratio was 7.229 , implying that levels of urinary 2-microglobulin are strongly associated with intensity of kim-1 staining .
confidence interval at the 95% range was 2.868 to 12.01 . among 35 biopsies with kim-1 positive staining ,
using positive kim-1 staining as the end point , sensitivity was 86.6% , specificity was 43.7% , positive predictive value was 74.2% , and negative predictive value was 63.6% .
we found that increased urinary 2-microglobulin was significantly correlated with kim-1 staining in proximal tubules , indicating that urinary 2-microglobulin is a sensitive ( sensitivity 86.6% ) assay for detecting tubular injury .
further , because kim-1 is a selective proximal tubule injury marker , the present study also demonstrated that measurement of urinary 2-microglobulin was a reliable assay to detect proximal tubule injury .
therefore , in patients with acute kidney injury with increase in serum creatinine , measurement of urinary 2-microglobulin is also helpful to identify the underlying tubular dysfunction , that is , proximal tubule injury .
acute tubular injury is a common clinical diagnosis . in a patient with classic clinical presentations such as hypotension together with a rapid rising of serum creatinine
, the diagnosis of severe tubular injury ( acute tubular necrosis ) can be made and does not require kidney biopsy to initiate treatment .
however , the diagnosis of glomerular disease accompanied by tubular injury is not easy to establish , as both may contribute to elevated serum creatinine .
tubular injury was the secondary diagnosis in 5/35 patients but other evidence of tubular injury such as tubular casts and an increase in tubular cells by urine analysis was found in 22/35 patients . moreover , 2 of 4 patients with secondary diagnosis of acute interstitial nephritis showed tubulitis , a feature associated with tubular injury .
these findings indicate that glomerular diseases associated with varying degree of tubular injury could be missed in renal biopsies due to sampling . in a prior study , using cd133 staining in injured tubular epithelium , it was demonstrated that urinary 2-microglobulin was a sensitive indicator for tubular injury . as a progenitor marker
in addition , cd133 positive cells are present not only in proximal tubular cells but also in distal tubular cells and/or atrophic tubules .
therefore , a careful morphological correlation is needed to exclude false positive staining . by comparison
, kim-1 is a selective proximal tubule injury marker that only presents in patients with proximal tubule injury [ 1820 ] .
therefore , kim-1 is a more specific marker than cd133 for detecting proximal tubular injury . in the present study ,
kim-1 was able to detect patients with mild , focal tubular injury evidenced by urine analysis as an increase in number of tubular cells ; some patients presented with normal levels of serum creatinine ( table 1 ) .
this was also supported with the finding in the present study that more than half of kim-1 staining score was weakly positive ( 1 + ) .
a good correlation of urinary 2-microglobulin and kim-1 staining ( r value of 0.293 ) indicates that measurement of urinary 2-microglobulin is a reliable assay for proximal tubule injury .
similar to cd133 staining , kim-1 was also able to identify two major types of tubular injury : ischemic and toxic type ( figures 1(c ) and 1(d ) ) .
as shown in table 2 , tubular injury was an additional diagnosis included in 5 of 26 biopsies ( 25% ) ( table 1 ) , and all showed positive kim-1 staining in proximal tubules . using kim-1 staining as a proximal tubule injury marker in the present study ,
nine patients , rather than six patients with normal urinary 2-microglobulin , presented with positive kim-1 staining , which were considered false positive . with low specificity , clinical interpretation of elevated urinary 2-microglobulin should be closely correlated with clinical history , ( e.g. , history of hypotension , recent history of medications use , etc . ) and clinical presentation , including value of serum creatinine , as well as urine cytological findings .
the diagnosis of tubular injury with renalvysion was made not only based on the elevation of urinary 2-microglobulin and serum creatinine but also combined with morphological findings of tubular casts and the number of sloughed tubular epithelial cells in urine analysis .
renalvysion is a urine - based analysis that combines urine chemistry and cytology to detect glomerular disease and tubular injury .
elevation of urinary 2-microglobulin over 300 g / l was considered to be abnormal by renalvysion .
g / l and positive kim-1 staining in biopsies were considered to be false positive .
however , the actual values of urinary 2-microglobulin in 2 patients were marginally high in 280 g / l and 290 g / l .
the remaining 7 patients were found to have levels less than 210 g / l , which is the measurement threshold of the assay .
because the confidence interval at the 95% range in lower end was 2.868 in present study , the normal range of urinary 2-microglobulin applied in renalvysion should be reconsidered to be 2.868 g / l .
g / l should be considered as abnormal and belongs to true positive for kim-1 staining .
therefore , corrected sensitivity is 87.1% , specificity is 46.6% , positive predictive value is 77.1% , and negative predictive value is 63.6% .
our study has multiple limitations . only a small number of patients had follow - up renal biopsies after renalvysion ( 56/5494 , 1.0% ) , raising concern for selection and ascertainment biases .
as shown in table 1 , these patients had glomerular disease and/or tubular - interstitial disease .
the reason for this might be that renal biopsy is usually avoided in patients with a clear clinical diagnosis of severe tubular injury . a larger study cohort involving patients with severe tubular injury is needed to better correlate renal histopathology and immunohistochemistry . owing to limited sampling by biopsies ,
the retrospective nature of the study represents an additional limitation . in summary , measurement of urinary 2-microglobulin is a sensitive assay to screen patients with proximal tubule injury .
our data indicate a correlation of urinary 2-microglobulin with results of immunohistochemical staining of kim-1 in renal biopsy and consequently with proximal tubular injury . |
objective . after filtration through glomeruli , 2-microglobulin is reabsorbed in proximal tubules . increased urinary 2-microglobulin indicates proximal tubule injury and measurement of 2-microglobulin in urine is useful to determine the source of renal injury .
kidney injury molecule-1 ( kim-1 ) has been characterized as a selective proximal tubule injury marker .
this study was designed to evaluate the correlation of urinary 2-microglobulin concentration and kim-1 expression as evidence of proximal tubule injury
. methods . between 2009 and 2012 , 46 patients with urine 2-microglobulin ( renalvysion ) had follow - up kidney biopsy .
diagnoses included glomerular and tubule - interstitial disease .
immunohistochemical staining for kim-1 was performed and the intensity was graded from 0 to 3 + .
linear regression analysis was applied to correlate the values of urinary 2-microglobulin and kim-1 staining scores .
p < 0.05 was considered statistically significant .
results .
thirty patients had elevated urinary 2-microglobulin .
kim-1 staining was positive in 35 kidney biopsies .
there was a significant correlation between urinary 2-microglobulin and kim-1 staining ( p < 0.05 ) .
sensitivity was 86.6% , specificity was 43.7% , positive predictive value was 74.2% , and negative predictive value was 63.6% . conclusion . increased urinary 2-microglobulin is significantly correlated with kim-1 staining in injured proximal tubules .
measurement of urine 2-microglobulin is a sensitive assay for proximal tubule injury . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion |
PMC4345076 | insulin resistance ( ir ) is associated with obesity and characterized by its metabolic consequences , including hyperglycemia , dyslipidemia , and hypertension .
these metabolic consequences are known collectively as metabolic syndrome ( mets ) and are potent risk factors for adverse clinical outcomes .
ir has been found to be associated , directly and indirectly , with cardiovascular complications , including atherosclerosis that may lead to myocardial infarction and stroke .
moreover , obesity is associated with increased production of proinflammatory adipokines , including monocyte chemoattractant protein-1 , interleukin-6 ( il-6 ) , and tumor necrosis factor- ( tnf- ) .
they contribute to chronic , low - grade inflammation and play a pivotal role in the development of insulin resistance [ 4 , 5 ] .
fructose intake has been directly linked to hyperuricemia , which may result in obesity and mets [ 7 , 8 ] .
the mechanism by which uric acid ( ua ) induces the development of mets may be due to ua inhibition of nitric oxide synthase and induction of endothelial dysfunction [ 9 , 10 ] .
recent evidence supports the concept that hyperuricemia itself can be a significant and independent cardiovascular risk factor .
lowering ua in fructose - induced mets has been found to be associated with lowering bp , improving insulin sensitivity , and reducing hypertriglyceridemia , through improving endothelial and adipocyte dysfunction .
therefore , the aim of the current study was to evaluate the effect of allopurinol , as a xanthine oxidase enzyme inhibitor reducing the level of ua , on insulin resistance induced experimentally in rats and its possible protective effect on the cardiovascular complications .
the following drugs and chemicals were used : allopurinol and urethane ( sigma - aldrich , st .
male wistar rats weighing 120140 g ( king fahd center for medical research , king abdulaziz university , saudi arabia ) were housed in clear polypropylene cages ( four rats per cage ) and kept on equal durations of the dark - light cycle , under constant environmental conditions .
experimental protocol was approved by the research ethics committee of the faculty of medicine , king abdulaziz university .
rats were randomly divided into four experimental groups ( eight animals each ) as follows : control , insulin resistant ( ir ) , allopurinol - treated insulin resistance ( ir - allo ) , and allopurinol - treated control ( allo ) . ir was induced by adding fructose ( 10% ) to everyday drinking water and feeding rats on high fat , high salt diet ( 16% crude protein , 28.2% crude fat , 2.8% crude fiber , 4.8% ash , and 3.4% salt ) for 12 weeks , while control animals receive tap water and standard diet ( 20% crude protein , 4% crude fat , 3.5% crude fiber , 6% ash , and 0.5% salt ) . allopurinol ( 20 mgkgday ) was daily administered by dissolving in drinking water ( 90110 mg / l ) depending upon water consumption .
drinking water was measured every week and allopurinol concentration in drinking water was readjusted . at the end of the study
then the rats were anesthetized by intraperitoneal injection of urethane ( 1.5 gkg ) and the invasive bp was recorded .
finally , venous blood was withdrawn and allowed to coagulate for 30 min at 4c and then was centrifuged ( 3000 g , 4c , 20 min ) to separate serum .
serum was divided into aliquots and stored at 20c till being analyzed for insulin , tnf- , adiponectin , and lipid profile .
glucose was determined in tail blood by a glucose meter ( bionime gmbh ) using noble metal electrode strips .
serum insulin level as well as fructosamine was measured by enzyme - linked immunosorbent assay ( elisa , millipore , billerica , ma , usa ) that uses a plate coated with monoclonal anti - rat insulin antibodies .
the homeostasis model assessment of insulin resistance ( homa - ir ) ( ir index ) was calculated using the serum nonfasting glucose and insulin levels according to the equation of matthews et al .
: homa - ir = glucose concentration ( mmol / l ) insulin ( u / l)/22.5 .
serum levels of ua , triglycerides ( tg ) , total cholesterol ( tc ) , and high density lipoprotein cholesterol ( hdl - c ) were determined using the elitech assay kit ( elitech , laindon , essex , france ) .
the low density lipoprotein cholesterol ( ldl - cholesterol ) was computed using the friedewald equation : total cholesterol ( hdl - c + 1/5tg ) .
invasive bp was continuously recorded for 10 minutes by microtip catheter ( millar , bella vista , australia ) inserted in the thoracic aorta through the right carotid artery .
the microtip catheter was connected to power lab data interface module connected to a pc running labchart professional software ( v7.3 , adi instruments , bella vista , australia ) containing bp module .
the bp module detects and calculates different bp parameters like systolic bp , diastolic bp , mean bp , notch pressure , heart rate , cycle duration , ejection duration , and diastolic duration .
serum tnf- and adiponectin levels were determined by elisa using quantikine kit ( r&d systems , minneapolis , mn , usa ) using rat tnf- or rat adiponectin and antibodies raised against the rat tnf- or rat adiponectin , respectively .
immunofluorescence staining of 4-hydroxy-2-nonenal ( 4-hne ) , ang ii , ang r1 , and collagen protein expression in rat paraffin embedded aortic sections ( 5 m ) was carried out according to the method used in our previous works [ 1618 ] .
fixed aortic tissue section slides were deparaffinized in xylene and rehydrated in ethanol and distilled water . then , perforation is carried out by incubation with methanol at 20c for 30 min followed by washing with distilled water .
epitopes were retrieved ( antigen retrieval ) in citrate buffer for 30 min at 95c followed by washing with pbs .
nonspecific binding sites were blocked ( pbs containing 5% ngs , 1% bsa , and 0.1% triton ) at room temperature for 1 h. after the blocking , sections were washed ( 3 5 min ) with pbs .
aortic sections were then incubated with the intended primary antibody diluted in blocking buffer at 4c overnight .
the sections were then washed ( 3 5 min ) with pbs followed by incubation with a fluorescent conjugated secondary antibody ( dilution 1 : 200 in blocking buffer ) for 1 h in dark . then sections were washed ( 3 5 min ) with pbs and slides were dried and mounted with prolong lasting mounting media .
the slides were stored in dark overnight before examination with zeiss lsm 780 confocal microscope ( carl zeiss , gottingen , germany ) at excitations ( 488 and 561 nm ) and filters ( 497542 and 596655 nm ) .
quantitative comparisons of images fluorescence were made with imagej software ( national institute of health , bethesda , md , usa ) .
for printing purposes , the level of the confocal images was equally adjusted after the fluorescence quantifications were carried out on unmanipulated images .
sections treated with the secondary antibody alone did not show specific staining while incubating the primary antibody with the blocking peptide significantly reduced the signal .
the used primary antibodies were rabbit polyclonal anti - ang ii ( 1 : 2000 , phoenix pharmaceuticals inc . , burlingame , ca , usa ) , mouse
monoclonal anti - angiotensin ii type 1 receptor ( 1 : 133 , abcam , cambridge , ma , usa ) , mouse monoclonal anti - collagen type i ( 1 : 1000 , abcam ) , and rabbit polyclonal anti-4hne ( 1 : 250 , millipore , billerica , ma , usa ) .
the used secondary antibodies were alexa fluor ( ex = 488 ) conjugated goat anti - mouse and alexa fluor ( ex = 594 ) conjugated goat anti - rabbit ( 1 : 200 , life technologies , grand island , ny , usa ) .
statistical analysis was performed by analysis of variance ( anova ) followed by newman - keuls ' post hoc test using a computer based fitness program ( prism 5 , graphpad , ca , usa ) .
body weight gain of the ir group was significantly increased ( p < 0.001 ) compared to control group .
this increase was ameliorated in ir - allo group but still significantly higher than the control group ( p < 0.01 ) .
body weight gain was also higher in allo group ( p < 0.01 ) ( table 1 ) .
treatment with allo has decreased the level of ua significantly ( p < 0.01 ) in ir - allo group , while there was no effect in allo group .
fructosamine , fasting blood glucose , and insulin were significantly increased in ir group ( p < 0.01 , p < 0.001 , and p < 0.01 , resp . ) .
allo showed no effect on fructosamine or fasting blood glucose in allo group but showed significant increase ( p < 0.01 ) in insulin level compared to control group ( table 1 ) .
this increase was significant in ir and ir - allo groups ( p < 0.05 ) ( figure 1 ) .
table 2 revealed that ir model in the current study failed to show any significant changes regarding the fasting lipoprotein profile .
however , there was tendency to increase both tg and hdl - c , but this increase was nonsignificant .
allo treatment has decreased nonsignificantly the tg level and increased nonsignificantly the ldl - c level with no effect on tc or hdl - c in either ir - allo group or allo group .
bp tracing showed normal bp values including systolic pressure , diastolic pressure , mean arterial pressure , and dicrotic notch pressure as well as normal heart rate in both control group and allo group ( figures 2 and 3 ) .
moreover , cycle duration in all rats in both control and allo groups was found to be normal ( table 3 ) .
ir has significantly increased systolic ( p < 0.01 ) , diastolic ( p < 0.01 ) , mean ( p < 0.01 ) , pulse ( p < 0.01 ) , and dicrotic notch ( p < 0.01 ) pressures .
treatment of ir with allo has significantly decreased the elevated systolic ( p < 0.05 ) , diastolic ( p < 0.05 ) , mean ( p < 0.05 ) , and dicrotic notch ( p < 0.05 ) pressures with no effect on pulse pressure . moreover , allo has decreased the heart rate compared to control and ir groups ( figures 2 and 3 ) . regarding the cycle duration ir has no significant changes , including total cycle duration , ejection duration , diastolic duration , and time to peak .
in addition , allo has no effect in all cycle durations except for the ejection duration where it has significantly ( p < 0.05 ) increased compared to the control group .
allo treatment was significantly ( p < 0.01 ) able to decrease this increased level to the nearly normal control value , while allo treatment has no effect on allo group ( figure 4(a ) ) .
regarding adiponectin , ir showed no effect on its level and the treatment with allo has no effect also in ir - allo group .
moreover , allo treatment in allo group revealed no effect on the level of adiponectin ( figure 4(b ) ) .
ir group showed significant increase in 4hne ( p < 0.05 ) compared to control group .
allo treatment significantly ( p < 0.01 ) ameliorated this increased level to the normal control values ( figure 5 ) . on the other hand
, ir caused no significant changes in ang ii or ang r1 but allo treatment caused significant decrease ( p < 0.05 ) of both even when compared with normal control values ( figures 6 and 7 ) .
regarding the aortic collagen , no significant changes were observed between all groups ( figure 8) .
in the present study , rats fed on fructose and high fat and high salt diet revealed ir evidenced by an increase in both insulin and glucose levels .
ir is an important predisposing factor for several clinical disorders , including type 2 diabetes , obesity , dyslipidemia , and hypertension .
although dyslipidemia was not observed in the current study , obesity , increased levels of both glucose and insulin , and hypertension were clearly evident .
moreover , fructosamine , which is an indicator of the average blood glucose concentration over a short - medium period , has increased significantly in the ir model .
the prevalence of hyperinsulinemia and ir rise with increasing body mass index and obesity , but whether insulin causes these phenomena or is a compensatory response has remained unsettled for decades .
the current study showed that allo has increased the level of insulin without effect on glucose level and associated with increased body weight in control groups .
this increase in the body weight may be explained by the allopurinol - induced secretion of insulin observed in the current study with subsequent increase in the glucose uptake in muscles and adipose tissue leading to weight gain with no effect on insulin resistance as the level of glucose was comparable to the normal control group .
regarding dyslipidemia , a possible explanation for insignificant change in the lipid profile is that fructose - induced hyperuricemia is maximal in the first few hours following ingestion of fructose and is also dose - dependent .
moreover , the small sample size used in the present study may be another explanation for this insignificant change in the lipid profile .
clinical and experimental studies have assessed the relationship between hyperuricemia and hypertension and found that hyperuricemia might play a double role as a risk factor for hypertension and as a pathological condition enhanced by hypertension itself . moreover
, many studies revealed that ir is a risk factor for cardiovascular disease [ 3 , 23 ] . despite the fact that it was nonsignificant , the results of the present study demonstrated that ir caused an increase of the ua level with significant vascular complications including the systolic , diastolic , mean , pulse , and dicrotic notch pressure with no effect on the cycle durations or heart rate .
previous studies found that xo activity is increased by various cytokines , including tnf- [ 24 , 25 ] , which is significantly increased in the current study .
moreover , xo was found to be significantly elevated in a variety of vascular diseases including limb ischemia , coronary artery disease , and heart failure .
circulating xo binds to glycosaminoglycans on the surface of endothelial cells where it is involved in the pathogenesis of endothelial injury [ 29 , 30 ] .
in addition , our previous study demonstrated that tnf- has an important role in ir and vascular impairment .
in contrast , the nonsignificant elevation of ua in the current study may be attributed to the fact that the present model is not the ideal model representing the hyperuricemia using 2% oxanic acid .
this may pay attention to the possibility that the effect of allo is not dependant on decreasing the ua level , but due to effect on other byproducts of purine metabolism by xo as superoxide .
moreover , hyperglycemia and hyperinsulinemia in ir may reduce arterial wall compliance by promoting plaque growth , vascular smooth muscle cell proliferation , and nonenzymatic glycosylation of vessel wall proteins [ 32 , 33 ] .
it was reported that the vascular endothelium expresses membrane insulin receptors and is a target for the biologic effects of insulin , which stimulate production of nitric oxide ( no ) leading to vasodilatation .
moreover , hyperglycemia itself increases the release of the vasoconstricting peptide endothelin-1 from cultured endothelial cells , offsetting the vasodilating actions of no .
the results of the current study showed that allo has alleviated the vascular complications of ir including systolic , diastolic , mean , and dicrotic notch pressures compared to the untreated ir group .
the elevations in diastolic and notch bp are largely attributed to the increased peripheral arterial resistance , whereas pulse pressure reflects stiffening of large arteries .
pulse pressure was not significantly changed in ir as a result of significant increase in both systolic and diastolic bp in ir group .
the present study may provide an explanation for these palliative effects which may be related to the effect of allo on increasing insulin secretion observed in the allo control group which in turn have a vasodilator effect on blood vessels .
moreover , previous studies found that allo improves endothelial dysfunction and prevents the development of arteriosclerosis .
moreover , inhibition of xo by allo was found to improve endothelium - dependent dilation and reduced superoxide production in isolated coronary arterioles following ischemia reperfusion .
another explanation may be related to the effect of allo on renal function improvement and increasing glomerular filtration rate as shown by neal et al . .
on the other hand , it was found that allo treatment decreased the heart rate significantly even when compared to the normal controlled group which is in accordance with the results of sakabe et al . which revealed that allo has a protective effect on atrial fibrillation in dogs associated with tachycardia - induced cardiomyopathy .
despite the fact that it was nonsignificant , this bradycardia was associated with increase in the total cycle duration . moreover
, the current study showed an increased level of tnf- which is an important inflammatory cytokine that is overexpressed in adipose and other tissues in cases of ir and obesity .
this is in accordance with many studies which revealed the role of tnf- in the induction of ir and type 2 diabetes [ 43 , 44 ] .
although there was no significant change in the lipid profile as mentioned before , this elevated tnf- may be responsible for the mild increase in the tg and hdl - c and decreased level of ldl .
previous studies showed that hepatic tg and vldl production are increased in both human and murine studies after tnf- administration [ 45 , 46 ] .
this may be due to increased hepatic levels of citrate , the rate - limiting enzyme in free fatty acid synthesis , or through inhibition of lipoprotein lipase activity .
whereas tnf--induced changes in tg metabolism are similar in all species , its effect on cholesterol metabolism differs between rodents and primates . whereas the administration of tnf- in rodents is followed by increase in hepatic cholesterol synthesis and ldl [ 45 , 49 ] ,
nonhuman primates and humans show either no change or a decrease in serum cholesterol and ldl levels .
the mechanisms underlying this species difference are not known . regarding the elevated hdl - c ,
it may be related to the inhibitory effect of tnf- on cholesteryl ester transfer protein ( cetp ) which transfers cholesteryl esters from hdl to tg - rich lipoproteins leading to high levels of hdl - c .
allo treatment significantly alleviated this increased tnf- level nearly to the normal control levels which is in accordance with previous studies that showed the reducing effect of allo on tnf- [ 52 , 53 ] .
a novel explanation of this decrease in the tnf- evidenced by the results of the current study is that it may not be due to direct effect , but may be due to secondary to the ability of the allo to increase the level of insulin . beyond its classic metabolic actions , insulin has also anti - inflammatory effect , decreasing the activity of proinflammatory cytokines including tnf- . unlike many studies which revealed that , in ir , the levels of leptin and resistin increase while adiponectin , which has insulin - sensitizing and anti - inflammatory effect , decreases [ 5557 ] , the current study revealed no significant change in adiponectin level
this may be explained by the finding of shapiro et al . who reported that rats fed fructose ad libitum chronically become leptin resistant with a compensatory increase in adiponectin .
in addition , the results of the present study showed that treatment with allo has ameliorated and decreased the increased level of 4hne that was observed in ir group .
it was found that 4hne is a biomarker aldehyde of oxidative stress which readily binds covalently to nucleophilic residues of proteins , peptides , phospholipids , and nucleic acids and thereby exhibits cytotoxic effects .
4hne can also modulate signaling pathways involved in cell proliferation , fibrosis , apoptosis , and inflammation , which are all hallmarks of cardiovascular diseases [ 5961 ] .
moreover , the current study revealed that allo treatment decreased significantly ang ii and ang r1 .
angiotensin ii directly causes vasoconstriction , increases the release of antidiuretic hormone via release of vasopressin , thus causing the reabsorption of water at the level of the kidneys , and triggers release of aldosterone from the adrenal cortex , causing the kidney to reabsorb sodium .
it is assumed that the current study reveals that the ameliorative effect of allo treatment is not related to its effect on ua and may be related to other mechanisms as the ua in the present model of ir was not significantly increased compared to the normal control group . in conclusion , the results of the current study show that allo has a protective effect on vascular complications of ir which may be attributed to the effect of allo on decreasing the level of tnf- , 4hne , ang ii , and ang r1 as well as increasing the level of insulin secretion regardless of the effect of associated dyslipidemia . | the aim of the current study was to evaluate the possible protective effect of allopurinol ( allo ) on experimentally induced insulin resistance ( ir ) and vascular complications .
rats were divided into four groups : control , ir , allopurinol - treated ir ( ir - allo ) , and allopurinol - treated control ( allo ) .
ir was induced by adding fructose and high fat , high salt diet for 12 weeks .
the results showed that allo has alleviated the increased level of tnf- and the systolic , diastolic , mean , and notch pressure observed in ir with no change in pulse pressure .
in addition , allo decreased the heart rate in the treated group compared to ir rats . on the other hand
, it has no effect on increased levels of insulin , glucose , fructosamine , or body weight gain compared to ir group , while it increased significantly the insulin level and body weight without hyperglycemia in the control group .
moreover , allo treatment ameliorated increased level of 4hne , ang ii , and ang r1 . in conclusion , the results of the current study show that allo has a protective effect on vascular complications of ir which may be attributed to the effect of allo on decreasing the tnf- , 4hne , ang ii , and ang r1 as well as increasing the level of insulin secretion . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion |
PMC3498750 | one of the main objectives of dentistry is rehabilitation of the stomatognathic system , restoring form , function , cosmesis , phonation , and self - esteem in individuals who have suffered tissue loss due to trauma , disease , atrophy , surgical intervention , or congenital defects .
when a fracture occurs , an osseointegrated implant is placed , or a graft is used for bone augmentation prior to implant placement .
the purpose is induction of bone regeneration , that is , the formation of new bone that , after undergoing remodeling processes , is identical to preexisting tissue.1 bone is the second most widely transplanted of all tissues ; only blood is transplanted or transfused more often .
over 2.2 million bone grafts are placed worldwide every year , 450,000 in the united states alone.2 autogenous tissue is the ideal substrate for bone augmentation , but harvesting procedures increase operative time and cost and require a second surgical field , which may increase postoperative discomfort . the limited quantity of autogenous tissue that can be obtained from intraoral sites such as the maxillary tuberosity , extraction areas , edentulous alveolar ridge , symphysis menti , ramus , and retromolar space also poses a challenge.3 for these and other reasons , researchers are increasingly searching for alternative approaches or biomaterials that might circumvent these hurdles and reduce surgical morbidity .
the advantages of allograft bone include greater availability , avoidance of a second surgical field , prevention of donor site morbidity , lower treatment costs , and improved patient acceptance.4 during the first hundred years ( 1880 - 1980 ) of allograft use , the main issue was availability , as there was no legislation in place to protect tissue processing and tissue donors . during the two decades that followed ( 1980 - 2000 )
more sensitive blood testing and major efforts on the part of tissue banks to develop better mechanisms for graft cleansing and removal of infectious agents have helped enable safe bone transplantation , and the key concern is now the efficacy of bone grafts.5 incorporation may be defined as the process of union between graft material and host tissue .
bone graft incorporation involves a cascade of events similar to the stages of fracture consolidation.6,7 regardless of graft type , incorporation is preceded by a sequential process , which may be divided for educational purposes into the stages of inflammation , revascularization , osteoinduction , osteoconduction , and remodeling.8 allograft bone is necrotic , and incorporation to adjacent bone occurs through identification of necrotic bone spicules ( lacking cells in the osteocyte lacunae ) bounded by the cementing lines of new bone formation .
this process , known as creeping substitution , mimics the way in which necrotic bone is resorbed and neoformed.9 incorporation of allogeneic bone is slower than that of autologous bone at the graft - host interface , as it involves a stronger initial inflammatory reaction and less revascularization and because allogeneic bone plays no role in the early stages of incorporation due to its absence of osteogenic function , allograft bone is entirely reliant on the host site to provide the vital substrate for this stage.7,10 - 12 allogeneic bone appears to act more as a mineral matrix or scaffold that supports cell migration and proliferation.13 the growing popularity of dental implants increased the possibility of treating dental loss .
dental care professionals also began to seek excellence in cosmetic results , due to increasingly discerning and demanding patients . in case of major bone loss at the alveolar ridge ,
implant placement usually must be preceded by bone grafting to restore function , form , and cosmesis.14 - 18 restorative - driven or backwards planning , in which implant placement is determined by the ideal position of the crown for optimal function and cosmesis , is the mainstay of current dental implant treatment.18 this approach sometimes requires the use of bone grafts to correct alveolar ridge defects and thus enable optimal implant placement.4,17,19 several methods are available to improve bone repair , including the use of grafts and laser therapy.20 use of the latter began empirically in the 1960s , and has since gained credibility and adherents worldwide . over the past two decades , several studies on the theme have been published in scientific journals , enabling evidence - based clinical use of laser therapy.21 the present study thus sought to assess the effect of low - level laser therapy on deep - frozen block allograft incorporation in the rabbit calvaria , comparing irradiated and non - irradiated sites .
this study was approved by the universidade do estado de santa catarina ( udesc ) animal experimentation ethics committee with judgment no .
1.04/08 and by the ethics of animal use committee of the pontifcia universidade catlica do rio grande do sul with registry number ceua / pucrs 09/00112 .
the animal model chosen for this study was the new zealand rabbit ( oryctolagos cuniculus ) .
a total of 14 adult female rabbits ( age 8 - 10 months , weight 3.5 - 4.5 kg ) were used in the study .
twelve were randomly allocated into two groups , l ( experimental ) and c ( control ) , and received allogeneic bone grafts .
after measurement of weight and several clinical parameters ( including respiratory rate , heart rate , and capillary refill ) , animals were premedicated with tiletamine / zolazepam ( zoletil 100 , virbac saude animal , so paulo , brazil ) 20 mg / kg and xylazine ( anasedan , vetbrands saude animal , brazil ) 3 mg / kg i m .
animals were then placed in sternal recumbency on an active warming blanket and the marginal ear vein was cannulated with a 24-gauge catheter for administration of normal saline .
anesthesia was maintained with 1.0 - 1.5 mac isoflurane ( isoforine , cristlia produtos qumicos e farmacuticos ltda .
, so paulo , brazil ) , provided through a non - rebreather mask , diluted in 100% oxygen ( 2 l / min ) in a universal anesthetic vaporizer ( oxygel , so paulo , brazil ) .
the frontoparietal region was further anesthetized with local infiltration of 0.5 ml plain lidocaine ( 2% ) ( xylestesin 2% , cristlia produtos qumicos e farmacuticos ltda , so paulo , brazil ) .
the surgical field was disinfected with povidone - iodine ( povidine ) and isolated with sterile drapes . a 5 cm - long
, full thickness incision was made down to the periosteum overlying the sagittal suture and center of the frontal bone with a # 15 blade .
soft tissues were dissected with a freer elevator ( golgran , so paulo , brazil ) .
ltda ) set to 800 rpm and an 8 mm trephine ( neodent , curitiba , brazil ) , under copious irrigation with saline solution .
six bone blocks were harvested from the calvaria of each donor rabbit , for a total of 12 block allografts .
block allografts were thoroughly washed with saline solution , cleaned of any soft tissues , placed into sterile containers and frozen at -70c .
allografts were thawed in saline at room temperature , drilled through with a 1.6-mm bit ( neodent ) , and fastened to the cranial vault with a 1.5 mm x 6 mm self - tapping screw ( neodent ) on the right , using a conventional philips screwdriver ( neodent ) .
eight - millimeter pieces of bone were trephined from a more anterior region of the skull vault and placed on the left ( opposite the experimental site ) to serve as an autogenous positive control for bone incorporation .
the wound bed was copiously irrigated with saline solution and the incision was closed with simple running sutures ( ethicon 5 - 0 nylon ) .
postoperative analgesia consisted of meloxicam 0.1 mg / kg once daily , with rescue tramadol 2 mg / kg ( tramadon 50mg / ml , cristlia produtos qumicos e farmacuticos ltda , so paulo , brazil ) as needed in case of intense pain .
immediately after conclusion of the procedure , animals in group l ( experimental ) underwent infrared laser irradiation with aluminum gallium arsenide diode laser ( algaas ; wavelength 830 nm ; thera lase , dmc equipamentos ltda . ,
brazil ) , which was subsequently repeated every 48 hours for a total of eight sessions .
the laser was applied to four sites ( anterior , posterior , left lateral and right lateral aspects of the cranial vault ) at an energy density of 4 j / cm , for a total dose of 16 j / cm per session .
animals in group c ( control ) underwent sham irradiation , with the laser unit switched off , to simulate the stress of restraint .
six animals were euthanized at 35 days post - graft implantation , and the remaining six at 70 days .
anesthesia was induced with tiletamine / zolazepam 20 mg / kg and xylazine 3 mg / kg i m , followed by a lethal injection of 300 mg potassium chloride ( cristlia produtos qumicos e farmacuticos ltda .
the surgical site was dissected and a 10-mm trephine bit was used to obtain a sample of bone containing the graft and host tissue margins .
specimens were kept in 10% buffered formalin solution for 48 hours , decalcified in 5% aqueous nitric oxide , and bisected lengthwise with a microtome knife .
specimens then underwent routine processing ; 5-m thick longitudinal slices were obtained and stained with hematoxylin and eosin ( h&e ) .
the other half of each sample was sent for scanning electron microscopy ( sem ) at the pontifcia universidade catlica do rio grande do sul ( pucrs ) microscopy and microanalysis center .
samples were attached to specimen holders with double - stick carbon tape , sputter coated with gold , and placed in a vacuum chamber for observation under a scanning electron microscope .
assessment of the effects of laser therapy was based on descriptive , semiquantitative histological analysis based on the methods proposed by weber et al.20 , torres et al.22 and pinheiro et al.23 fisher 's exact test was used for statistical analyses .
histological assessment showed significant changes , particularly regarding filling of osteocyte lacunae , replacement of graft myeloid tissue with fibrocollagenous tissue , inflammatory reaction ( with a macrophage and neutrophil infiltrate ) , bone remodeling , and neovascularization .
allograft group ( 35 days ) : collagen fiber deposition was visible throughout the medullary spaces in both groups ( moderate in the lllt group and mild to moderate in the control group ) .
bone remodeling was also evident in both groups ( mild to moderate in the lllt group and mild in the control group ) . in both groups ,
osteocyte lacunae were mostly filled at the graft - to - host interface , while the surface , center , and periphery of the trabeculae were empty in most lacunae .
the inflammatory reaction was mild in the irradiated group and mild to moderate in the control group ( table 2 ) .
allograft group ( 70 days ) : collagen fibers were present in moderate to marked density in the medullary spaces of irradiated grafts and in slight to moderate density in the control group . bone remodeling was moderate in the lllt group and mild in the control group .
lacunae at the graft - to - host interface and surface of irradiated grafts were mostly filled with osteocytes , whereas only moderate filling was visible in the center of the graft . in the control group ,
most lacunae were empty in the center and periphery , and partially filled at the graft - to - host interface .
mild inflammatory reaction with a macrophage and neutrophil infiltrate was present in both groups ( table 3 ) . autograft group ( 35 days ) : in both groups , deposition of collagen fibers ranged from mild to moderate in intensity .
bone remodeling was moderate to marked in the experimental group and mild to moderate in controls .
most osteocyte lacunae were filled in the irradiated group , whereas lacunae in control grafts were filled mostly at the graft - host interface and surface , with most lacunae empty at the center .
mild - to - moderate inflammatory infiltrates were present in both groups ( table 4 ) .
autograft group ( 70 days ) : in the irradiated group , deposition of collagen fibers ranged from mild to marked . in the control group
, fibers were seen only in some specimens , and never at a moderate or higher density .
moderate to marked bone remodeling was present in the irradiated group , and moderate remodeling in the control group .
most osteocyte lacunae were filled in irradiated group grafts , whereas lacunae in control grafts were mostly filled at the graft - host interface , at the surface , and , to a lesser extent , in the core area ( table 5 ) .
sem at 45x ( figure 1 a and b ) and 500x magnification and optical microscopy at 40x magnification and 100x magnification ( figures 2 a and b , 3 a and b , 4 a and b and 5 a and b ) clearly showed graft incorporation at the graft - host interface in all speciments . in areas of cortical bone , grafts were in close contact with recipient areas ; filling of osteocyte lacunae was evident , as were medullary degeneration , necrosis and revascularization in the medullary spaces at the graft - host interface , with deposition of collagen fibers and bone remodeling .
assessment showed the osteoconductive properties of block allografts are preserved after deep - freezing , as are their structural characteristics and bone matrix , providing a scaffold for host - to - graft migration of blood vessels and cells .
histological assessment showed significant changes , particularly regarding filling of osteocyte lacunae , replacement of graft myeloid tissue with fibrocollagenous tissue , inflammatory reaction ( with a macrophage and neutrophil infiltrate ) , bone remodeling , and neovascularization .
allograft group ( 35 days ) : collagen fiber deposition was visible throughout the medullary spaces in both groups ( moderate in the lllt group and mild to moderate in the control group ) .
bone remodeling was also evident in both groups ( mild to moderate in the lllt group and mild in the control group ) . in both groups ,
osteocyte lacunae were mostly filled at the graft - to - host interface , while the surface , center , and periphery of the trabeculae were empty in most lacunae .
the inflammatory reaction was mild in the irradiated group and mild to moderate in the control group ( table 2 ) .
allograft group ( 70 days ) : collagen fibers were present in moderate to marked density in the medullary spaces of irradiated grafts and in slight to moderate density in the control group . bone remodeling was moderate in the lllt group and mild in the control group .
lacunae at the graft - to - host interface and surface of irradiated grafts were mostly filled with osteocytes , whereas only moderate filling was visible in the center of the graft . in the control group ,
most lacunae were empty in the center and periphery , and partially filled at the graft - to - host interface .
mild inflammatory reaction with a macrophage and neutrophil infiltrate was present in both groups ( table 3 ) . autograft group ( 35 days ) : in both groups , deposition of collagen fibers ranged from mild to moderate in intensity .
bone remodeling was moderate to marked in the experimental group and mild to moderate in controls .
most osteocyte lacunae were filled in the irradiated group , whereas lacunae in control grafts were filled mostly at the graft - host interface and surface , with most lacunae empty at the center .
mild - to - moderate inflammatory infiltrates were present in both groups ( table 4 ) .
autograft group ( 70 days ) : in the irradiated group , deposition of collagen fibers ranged from mild to marked . in the control group
, fibers were seen only in some specimens , and never at a moderate or higher density .
moderate to marked bone remodeling was present in the irradiated group , and moderate remodeling in the control group .
most osteocyte lacunae were filled in irradiated group grafts , whereas lacunae in control grafts were mostly filled at the graft - host interface , at the surface , and , to a lesser extent , in the core area ( table 5 ) .
sem at 45x ( figure 1 a and b ) and 500x magnification and optical microscopy at 40x magnification and 100x magnification ( figures 2 a and b , 3 a and b , 4 a and b and 5 a and b ) clearly showed graft incorporation at the graft - host interface in all speciments . in areas of cortical bone , grafts were in close contact with recipient areas ; filling of osteocyte lacunae was evident , as were medullary degeneration , necrosis and revascularization in the medullary spaces at the graft - host interface , with deposition of collagen fibers and bone remodeling .
assessment showed the osteoconductive properties of block allografts are preserved after deep - freezing , as are their structural characteristics and bone matrix , providing a scaffold for host - to - graft migration of blood vessels and cells .
histological assessment showed significant changes , particularly regarding filling of osteocyte lacunae , replacement of graft myeloid tissue with fibrocollagenous tissue , inflammatory reaction ( with a macrophage and neutrophil infiltrate ) , bone remodeling , and neovascularization .
allograft group ( 35 days ) : collagen fiber deposition was visible throughout the medullary spaces in both groups ( moderate in the lllt group and mild to moderate in the control group ) .
bone remodeling was also evident in both groups ( mild to moderate in the lllt group and mild in the control group ) . in both groups ,
osteocyte lacunae were mostly filled at the graft - to - host interface , while the surface , center , and periphery of the trabeculae were empty in most lacunae .
the inflammatory reaction was mild in the irradiated group and mild to moderate in the control group ( table 2 ) .
allograft group ( 70 days ) : collagen fibers were present in moderate to marked density in the medullary spaces of irradiated grafts and in slight to moderate density in the control group . bone remodeling was moderate in the lllt group and mild in the control group .
lacunae at the graft - to - host interface and surface of irradiated grafts were mostly filled with osteocytes , whereas only moderate filling was visible in the center of the graft . in the control group ,
most lacunae were empty in the center and periphery , and partially filled at the graft - to - host interface .
mild inflammatory reaction with a macrophage and neutrophil infiltrate was present in both groups ( table 3 ) . autograft group ( 35 days ) : in both groups , deposition of collagen fibers ranged from mild to moderate in intensity .
bone remodeling was moderate to marked in the experimental group and mild to moderate in controls .
most osteocyte lacunae were filled in the irradiated group , whereas lacunae in control grafts were filled mostly at the graft - host interface and surface , with most lacunae empty at the center .
mild - to - moderate inflammatory infiltrates were present in both groups ( table 4 ) .
autograft group ( 70 days ) : in the irradiated group , deposition of collagen fibers ranged from mild to marked . in the control group
, fibers were seen only in some specimens , and never at a moderate or higher density .
moderate to marked bone remodeling was present in the irradiated group , and moderate remodeling in the control group .
most osteocyte lacunae were filled in irradiated group grafts , whereas lacunae in control grafts were mostly filled at the graft - host interface , at the surface , and , to a lesser extent , in the core area ( table 5 ) .
sem at 45x ( figure 1 a and b ) and 500x magnification and optical microscopy at 40x magnification and 100x magnification ( figures 2 a and b , 3 a and b , 4 a and b and 5 a and b ) clearly showed graft incorporation at the graft - host interface in all speciments . in areas of cortical bone , grafts were in close contact with recipient areas ; filling of osteocyte lacunae was evident , as were medullary degeneration , necrosis and revascularization in the medullary spaces at the graft - host interface , with deposition of collagen fibers and bone remodeling .
assessment showed the osteoconductive properties of block allografts are preserved after deep - freezing , as are their structural characteristics and bone matrix , providing a scaffold for host - to - graft migration of blood vessels and cells .
a variety of methods have been studied as potential means of improving the speed and quality of allograft incorporation , including platelet - rich plasma ( prp),3,24,25 bone morphogenetic proteins ( bmps),5 bisphosphonates,26 adjuvant stem cell and gene therapy,27 and processing techniques , such as laser drilling and partial demineralization of block allografts.28 several studies using various graft materials have shown that infrared laser therapy stimulates osteoblast proliferation , collagen deposition , and new bone formation.29,30 in this study , allogeneic bone tissue was processed by deep freezing .
this proved to be an adequate method for allografts , preserving the osteoconductive and structural properties of the graft tissue as a scaffold for vessel and cell proliferation , as demonstrated by partial filling of osteocyte lacunae , early - stage bone remodeling , and incorporation at the graft - host interface .
the use of other methods , such as freeze - drying , demineralization , gamma irradiation , ethylene oxide , or hydrogen peroxide , can jeopardize the structural properties of the donor tissue , which are of the utmost importance in onlay grafting.9,12,13,31 - 37 in the present study , collagen deposition was greater in allogeneic and autogenous blocks exposed to laser irradiation .
this was consistent with the findings of pinheiro et al.38 , lopes et al.39 and lopes et al.40 .
statistical comparisons of all lllt groups versus controls showed a positive effect of lllt on bone remodeling ( p = 0.0269 ) .
bone remodeling was significantly predominant ( p = 0.0151 ) in irradiated allograft groups versus controls at 35 and 70 days .
this finding is consistent with that of several prior studies that have shown greater bone remodeling after laser therapy.38 - 46 filling of osteocyte lacunae was also greater in the laser group , most markedly at 70 days in the allograft group and at 35 and 70 days in the autograft group , providing evidence of partial allograft bone vitality and preserved osteoconduction during this brief experiment .
drtbudak et al.42 and jakse et al.47 both reported a similar effect on autogenous grafts .
statistical analysis showed greater filling of osteocyte lacunae ( p = 0.0009 ) and greater bone remodeling ( p = 0.0267 ) in autogenous grafts ( positive controls ) than in allograft blocks .
comparison of the autograft and allograft + lllt groups showed greater filling of osteocyte lacunae in the autograft group ( p = 0.0375 ) , most likely due to transport across the autogenous graft of cells that remained viable during the incorporation stage10,11,13 and to the biomodulation effects of lllt on host cells and on graft cells that remained viable in the early stages of bone regeneration , during osteoblast proliferation.48,49 inflammatory reaction in the experimental ( irradiated ) allograft group at 35 days was less intense than in the control group .
animal experiments provide a useful means of estimating tissue reaction to bioactive materials ; however , results obtained from animal models should not necessarily be extrapolated to humans.30
optical and scanning electron microscopy showed that the combination of deep - frozen bone allografts and lllt is an adequate alternative for the treatment of bone defects .
after lllt , deep - freeze - processed bone allografts showed incorporation at the graft - host interface , moderate bone remodeling , partial filling of osteocyte lacunae , less inflammatory infiltrate in the early postoperative period , and higher collagen deposition than the control group , over the course of this brief study .
use of the deep - freezing method for processing of bone grafts preserves the structural and osteoconductive characteristics of bone tissue . | objective to assess the effect of low - level laser therapy ( lllt ) on the incorporation of deep - frozen block allografts in a rabbit model.background data studies have shown that lllt has beneficial effects on tissue repair and new bone formation.methods bone tissue was harvested from two rabbits , processed by deep - freezing and grafted into the calvaria of 12 animals , which were then randomly allocated into two groups : experimental ( l ) and control ( c ) .
rabbits in group l were irradiated with an aluminum gallium arsenide diode laser ( algaas ; wavelength 830 nm , 4 j / cm2 ) , applied to four sites on the calvaria , for a total dose of 16 j / cm2 per session .
the total treatment dose after eight sessions was 128 j / cm2 .
animals were euthanized at 35 ( n = 6 ) or 70 days ( n = 6 ) postoperatively.results deep - freeze - processed block allografts followed by lllt showed incorporation at the graft - host interface , moderate bone remodeling , partial filling of osteocyte lacunae , less inflammatory infiltrate in the early postoperative period , and higher collagen deposition than the control group.conclusion optical microscopy and scanning electron microscopy showed that allograft bone processed by deep - freezing plus lllt is suitable as an alternative for the treatment of bone defects .
use of the deep - freezing method for processing of bone grafts preserves the structural and osteoconductive characteristics of bone tissue . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
HISTOLOGICAL TESTING
Descriptive, semiquantitative, light microscopy-based histological assessment
Scanning electron microscopy (SEM) and optical microscopy (OM)-based assessment of incorporation at the graft-host interface
DISCUSSION
CONCLUSIONS |
PMC4215527 | magnetic resonance imaging ( mri ) permits to assess tissue abnormalities in vivo and approximate histopathological changes of the multiple sclerosis ( ms ) disease ( ganiler et al . , 2014 ; kearney et al . , 2014
several studies have shown that the percentage of change in brain atrophy tends to correlate with the progression of the disease ( prez - miralles et al . , 2013 ; sormani et al . , 2014
moreover , changes in gray matter ( gm ) atrophy are observed independently from white matter ( wm ) , and hence atrophy measures based on segmentation - based methods are nowadays employed as they allow classifying brain tissues separately ( prez - miralles et al . , 2013 ) . the performance of different segmentation methods used to quantify brain atrophy or volume estimation has been evaluated deeply in the last 5 years ( klauschen et al . , 2009 ; derakhshan et al . ,
2010 ) . however , it is well known that the presence of wm lesions can induce errors on brain tissue volume measurements ( chard et al . , 2010 ; battaglini et al . , 2012 ; gelineau - morel et al . , 2012 ) and non - rigid registration ( sdika and pelletier , 2009 ; diez et al . , 2014
) , if lesions are processed within the images . for instance , if wm lesion voxels are classified as wm , lesion voxels with hypointense signal intensities are added into the wm tissue distribution , increasing the probability of gm voxels with similar intensity to be misclassified also as wm ( chard et al . , 2010 ) . in the last years
, some authors have proposed different techniques to overcome these issues in ms patients by filling wm lesions with intensities similar to those of wm before performing tissue segmentation and image registration .
these methods can be divided into two groups : methods which use local intensities from the surrounding neighboring voxels of lesions ( sdika and pelletier , 2009 ; battaglini et al .
2013 ) and methods which use global wm intensities from the whole brain ( chard et al . , 2010 ) .
in all cases , the performance of these methods is directly related with their ability to minimize the impact of refilled voxels on original tissue distribution , not only due to the addition of these voxels into the tissue distribution , but also due to the effect on the global tissue distributions of filled images . within local methods , sdika and pelletier ( 2009 )
have proposed to refill each wm lesion voxel with the mean of its three - dimensional neighboring normal appearance white matter ( nawm ) voxels .
( 2012 ) have suggested refilling each wm lesion voxel with intensities derived from a histogram of nawm voxels surrounding the two - dimensional lesions . in a recent study , magon et al .
( 2013 ) have proposed to refill each two - dimensional lesion with the intensity from the mean of the surrounding area of the lesion . regarding global methods ,
( 2010 ) have proposed a different approach by using intensities re - sampled from a global wm distribution to refill wm lesion voxels , based on the mean and standard deviation of the total nawm of the whole image . both chard et al .
fsl - l ( battaglini et al . , 2012 ) runs from a computer command - line and does not provide any graphical interface that aids the process .
this technique has been integrated into the latest fsl package , and therefore it depends on the whole fsl installation . in the case of leap ( chard et al . , 2010 )
, the method runs as a stand - alone script also from the command - line and requires the installation and configuration of several external dependencies , which may be difficult to install for non - computer experts . in this paper
we propose a new technique to refill wm lesions which is a compromise between global and local methods .
hence , for each slice composing the three - dimensional image , we compute the mean and standard deviation of the signal intensity of nawm tissue .
on the one hand , compared to local methods ( battaglini et al . , 2012 ; magon et al . ,
2013 ) which only make use of a limited range of voxel intensities , the fact of using global information from the whole image slice reduces the bias caused by refilled voxels on gm and wm tissue distributions , especially on images with high lesion load . on the other hand , compared to other global methods ( chard et al . , 2010 ) ,
which are based on the mean signal intensity of the nawm of the three - dimensional image , our method re - computes the mean signal intensity of the nawm at each two - dimensional slice with the aim of reproducing more precisely the signal variability between mri slices , especially in low resolution images . in order to easily integrate it into current platforms ,
the proposed method called slf is currently available as a stand - alone program and as spm1 extension at the salem group site ( http://atc.udg.edu/salem/slftoolbox ) . to evaluate the performance of our method
, we estimate the deviation in gm and wm tissue volume between a set of healthy images and the same images where artificial wm lesions have been refilled with the proposed technique .
to do so , we register wm lesion masks from diagnosed ms patients into two sets of 30 1.5 and 3 t t1-weighted ( t1-w ) images of healthy subjects , respectively . afterwards , we simulate realistic lesions on healthy images by replacing the signal intensities of registered lesion voxels with values similar to those of the mean gm / wm interface . brain tissue volume is computed using both fast ( zhang et al . ,
2001 ) and spm8 ( ashburner and friston , 2005 ) segmentation methods , in order to avoid possible correlations between the filling and segmentation processes .
furthermore , we compare our results with the same images where artificial wm lesions have been segmented as normal tissue , masked - out before tissue segmentation , and refilled using also the methods proposed by chard et al .
the first set of images is composed of 30 images of healthy subjects ( matrix size : 176 208 176 , voxel size : 1 1 1.25 mm ) , acquired on a 1.5 t vision scanner ( siemens , erlangen , germany ) and obtained from the open access series of imaging studies ( oasis ) repository2 ( marcus et al . , 2007 ) . only images from young and middle - aged subjects are selected ( age < 50 ) as they have not been diagnosed with any related pathology .
image references included in the study are as follows : 2 , 4 , 5 , 6 , 7 , 9 , 11 , 12 , 14 , 17 , 18 , 20 , 25 , 26 , 27 , 29 , 34 , 37 , 38 , 40 , 43 , 44 , 45 , 47 , 49 , 50 , 51 , 54 , 55 , and 57 .
the second set of images is composed of 30 images of healthy subjects ( matrix size : 256 150 256 , voxel size : 0.92 0.92 1.20 mm ) acquired on a philips 3 t scanner ( philips healthcare , best , nl ) and obtained from the information extraction from images ( ixi ) repository maintained by the imperial college london in london , uk.3 we selected 30 images acquired from the hammersmith hospital .
image references included in the study are as follows : 12 , 13 , 14 , 15 , 33 , 34 , 39 , 48 , 49 , 51 , 52 , 57 , 59 , 72 , 80 , 83 , 92 , 95 , 96 , 97 , 104 , 105 , 126 , 127 , 128 , 131 , 136 , 137 , 146 , and 159 .
skull - stripping is performed using the brain extraction tool ( bet ) ( smith , 2002 ) , following the optimization workflow suggested by popescu et al .
( 2012 ) , with the exception that cerebrospinal fluid tissue has been refilled on skull - stripped images again .
this procedure is preferred over other alternatives as it provides the best performance on some lesion - filling methods such as chard et al .
( 2010 ) , being also the choice in other recent studies ( popescu et al . , 2014 ) .
ixi images are corrected from possible intensity non - uniformities and acquisition artifacts using n4 , the itk ( ibez et al . , 2003 ) implementation of the n3 package ( sled et al . , 1997 ) .
we use a set of 37 patients with clinically confirmed ms , provided with initial and follow - up studies ( diez et al . , 2014 ) . in these patients , lesions have been annotated semi - automatically on proton density - weighted ( pd - w ) images by a trained technician using jim software4 and afterwards co - registered with t1-w images . in order to maintain the independence between the 1.5 and 3 t sets of images , we match randomly 30 patients from the initial study into the oasis images , and we repeat the same procedure with the follow - up study and the ixi image set .
ms lesion masks are registered into healthy images by a non - rigid transformation ( rueckert et al . , 1999 ) . to ensure that resulting lesion masks are placed on wm , we remove registered lesion voxels that have not been segmented as wm by both fast and spm8 on the healthy image .
we computed a wilcoxon rank sum test to analyze the difference in lesion volumes generated between oasis and ixi datasets , obtaining that differences were not statistically significant ( p = 0.162 ) .
the obtained mean lesion volume on oasis images was 21.1 20.8 ml ( range from 0.5 to 65 ml ) , while 15.4 16.2 ml ( range from 0.8 to 62 ml ) on ixi 3 t images .
note that due to the existing anatomical differences between 1.5 and 3 t image subjects and the enforced wm tissue constraint , the effect of registering the same ms lesion mask into a 1.5 and 3 t image results in two different lesion masks .
for instance , the effect of registering lesions from the initial study into the 3 t dataset provided different lesion volumes ( 10.30 12.10 ml ) and reported statistically significantly differences ( p = 0.007 ) on the wilcoxon rank sum tests .
artificial lesions are simulated by replacing registered lesion voxel intensities with ones between the gm and wm interface , following the same strategy shown in battaglini et al .
for each original image , gm and wm tissue distributions are computed using only voxels in agreement between fast and spm8 .
wm lesion voxels are filled with random intensities coming from a newly generated normal distribution , with mean equal to the average of the gm and wm mean values and standard deviation equal to the difference between mean wm and gm , divided by 4 ( battaglini et al . , 2012 ) .
artificial lesions are refilled with the aim of simulating a profile which clearly separates their signal intensity with healthy tissue .
this intensity profile chosen does not reflect the entire scope of possible real lesions , but allows us to visualize the magnitude of the differences in tissue volume between images with artificial lesions and the same images where lesion have been filled with the proposed method .
the intensity profile chosen would not affect any of the methods studied since they do not take into account the artificial lesion intensities .
( 2010 ) with the similarity between refilled voxel intensities and their surrounding voxels of local methods such as battaglini et al .
basically , for each slice composing the three - dimensional image , lesion voxel intensities are replaced by random intensities of a normal distribution generated from the mean nawm intensity of the current slice .
the proposed algorithm requires two input images : a preprocessed t1-w image ( skull - stripped and intensity inhomogeneity corrected ) and its corresponding binary wm lesion mask . after testing the performance of the method with different skull - stripping approaches ( smith , 2002 ; shattuck et al . , 2001 )
, we observed that including this step inside the filling process is not necessary , because the skull - stripping method employed seems to not interfere significantly in the results obtained ( wilcoxon significant rank - sum tests between differences in tissue volume between lesion - filled and original images of both datasets for gm and wm tissue , p > 0.13 ) .
wm lesions are masked out from the t1-w image using the provided lesion mask , in order to avoid the influence of artificial lesions on tissue distributions .
the resulting image is used to estimate the probability of each voxel to be classified as csf , gm , and nawm , by segmenting tissue with a fuzzy - c - means approach ( pham , 2001 ) .
the fuzzy - c - means implementation used here follows the algorithm described in pham ( 2001 ) , with clusters initialized according to bezdek et al .
moreover , input signal intensities are constrained to the mean plus three standard deviations of the signal intensity of the image , in order to avoid outlier signal intensities , such as residual parts of the eyes or neck . from the obtained tissue segmentation output ,
we compute the three - dimensional nawm mask from the image voxels with the highest probability to pertain to the wm cluster .
finally , the lesion - filling process is achieved as follows : for each axial slice composing the three - dimensional image , we compute the mean and standard deviation of the signal intensity of nawm tissue .
axial sampling is motivated because after testing the sampling procedure on the coronal , axial and sagittal planes , we found that the best results were obtained when we sampled the axial plane .
this was due to the fact that using the axial plane reduced the variability of possible existing wm intensities , when compared to coronal and sagittal sampling .
the fuzzy - c - means approach used to estimate the tissue probabilities is a simple method which in fact does not take into account neither spatial nor neighboring information , and hyper - intense signal intensities such as residual parts of the eyes or the neck produced in the skull - stripping process can bias significantly the clusters .
the risk of adding these parts into the wm distribution is minimized in the axial plane because we are reducing it to a certain slice where lesion volume is usually lower than that in central slices .
the computed mean and standard deviation values are used to generate a normal distribution with mean equal to the computed nawm mean intensity and standard deviation equal to half of the computed nawm standard deviation .
standard deviation is always fixed to half of the wm mean independently of the dataset used .
this value was chosen empirically with the aim of balancing the accuracy of the method with both 1.5 and 3 t images .
although a specific tuning of this parameter could provide a better performance on certain cases , we decided to fix it avoiding therefore the number of parameters to tune .
lesion voxel intensities from the current image slice are replaced by random values of the generated distribution .
we compute the absolute percentage % difference in normalized gray matter volume ( ngmv ) and normalized white matter volume ( nwmv ) between each original and its correspondent lesion - filled images .
normalized volumes are obtained as the ratio of voxels outside lesion regions segmented as gm or wm and the total number of segmented voxels , respectively .
for instance , the % difference in ngmv is computed as:%=ngmvfilledngmvorigngmvorig100 where ngmvfilled and ngmvorig values refer to the computed volumes for the lesion - filled and original images , respectively .
the higher the performance of the lesion - filling method , the lower the percentage difference between lesion - filled and original images . in order to analyze possible correlations between the filling process and the segmentation method employed , brain tissue volume
is calculated independently on the same subjects using fast ( zhang et al . , 2001 )
( v.5.0.5 ) and spm8 ( ashburner and friston , 2005 ) ( v.4667 ) approaches .
we compare the performance of our method with respect to other existing techniques such as the ones proposed by chard et al .
we also add two more sets of images into the comparison : images segmented with artificial lesions and images where wm lesions have been masked out before tissue segmentation .
given the small differences in ngmv and nwmv between original and lesion - filled images , the use of a standard analysis of the variance ( anova ) or a classic t - test is impractical here .
instead , we perform a series of permutation tests to determine significant differences in tissue volume between pairs of methods ( menke and martnez , 2004 ; valverde et al . , 2014 ) .
the permutation tests return the mean and standard deviation of the fraction of times that the difference in ngmv and nwmv for a current lesion - filling method is smaller than the rest of methods with p - value 0.05 .
afterwards , methods are presented in 3 ranks determined by the mean and standard deviation of the best method and the distance with respect to the mean of the rest of methods ( valverde et al . , 2014 ) .
in our experiments , we set the number of comparisons between each pair of methods to n = 1000 .
2 depicts the absolute mean % difference in ngmv and nwmv between the 30 original 1.5 t images and the same images with artificial lesions ( none ) , masked - out lesions before segmentation ( masked ) , and lesion - filled using magon et al .
( 2010 ) ( leap ) , and finally our proposed algorithm slf . when fast is used , slf reports the lowest absolute mean difference in ngmv ( 0.16 0.14 ) , followed by leap ( 0.40 0.30 ) and fsl - l ( 0.43 0.58 ) methods .
our proposal also provides the lowest difference in nwmv ( 0.29 0.36 ) , followed by fsl - l ( 0.81 1.28 ) .
maximum values in ngmv are found in none images , with differences up to 2.30 2.62 in ngmv and 3.85 4.81 in nwmv .
when spm8 is used , slf also reports the lowest differences in ngmv ( 0.09 0.14 ) , followed by leap method ( 0.12 0.13 ) .
our proposed method also performs better than the rest of the methods on nwmv ( 0.20 0.24 ) , followed by the leap method ( 0.36 0.40 ) .
again , the highest differences in ngmv ( 1.84 1.97 ) and nwmv ( 4.82 4.58 ) are found in none images .
table 1 shows the absolute mean difference in wm volume for all methods where lesion volume has been ranged by size intervals .
table 2 presents the performance of each filling - method after running all possible pair - wise permutation tests . with a significant p - value of 0.05 ,
all tests run on images segmented with fast show the superiority of slf over the other methods presented . on images segmented with spm8 ,
all tests show a clear superiority of slf over the other methods on nwmv , while a similar performance of slf and leap over the other methods on ngmv .
we also test the performance of our algorithm using 3 t data . as before , fig .
3 shows the absolute mean % difference in ngmv and nwmv between the 30 original 3 t images and the same images with added lesions ( none ) , masked - out lesions before segmentation ( masked ) , and lesion - filled methods magon , fsl - l , and leap , and our proposed approach slf . when fast is used , slf reports the lowest absolute mean % difference in ngmv ( 0.06 0.06 ) , followed by leap ( 0.09 0.10 ) .
our method slf also performs the lowest difference in nwmv ( 0.09 0.09 ) , followed again by leap ( 0.12 0.08 ) .
maximum values in ngmv are found in none images , with differences up to 1.40 1.56 in ngmv and 1.00 1.32 in nwmv . when spm8 is used , both leap ( 0.04 0.06 ) and slf ( 0.05 0.05 ) yield the lowest absolute % mean difference in ngmv .
on nwmv , also leap ( 0.09 0.12 ) and slf ( 0.08 0.09 ) report the lowest absolute mean % difference in volume between original and lesion - filled images . again
, highest differences in ngmv ( 1.84 1.97 ) and nwmv ( 4.82 4.58 ) are found in none images .
table 3 shows the absolute mean difference in wm volume for all methods on ixi images , where lesion volume has been ranged by size intervals .
table 4 shows the performance of each filling - method after running the permutation tests .
tests run on images segmented with fast show a significant superiority of slf over the rest of the methods on nwmv , and a slightly better performance of slf with respect to leap on ngmv , although both methods are clearly superior to the rest of methods presented .
when spm8 is used , tests show a similar performance of slf and leap over the rest of the methods on both nwmv and ngmv .
several studies have proposed to use different filling techniques in order to reduce the effects of wm lesions on brain tissue measurements of t1-w images . up to date , only leap ( chard et al . , 2010)5 and fsl - l ( battaglini et al .
, 2012)6 are publicly available methods that permit to refill t1-w images given a wm lesion mask . the lesion segmentation toolbox ( lst ) proposed by schmidt et al .
( 2012 ) also provides a lesion - filling approach based on the work of chard et al .
( 2010 ) , but it is dependent of a flair image and an internal lesion - probability map obtained during the lesion segmentation step .
in general , deviation in tissue volume between original and lesion - filled images tends to be higher on 1.5 t oasis images than on 3 t ixi images .
the observed deviation is caused by differences in intensity , slice thickness and dimensionality between datasets .
on ixi images , the distance between gm and wm signal intensity distributions is narrower than that of 1.5 t data . applying the lesion generation algorithm ( battaglini et al . , 2012 ) with identical parameters of those used with 1.5 t images
creates simulated lesions whose intensity are noticeably similar to the mean wm , because the standard deviation of the generated lesion distribution is the mean between the gm and wm tissue divided by 4 .
however , this fact only explains the difference found on images segmented with artificial lesions . in the rest of the methods ,
the signal intensity of the generated lesions is not interfering with the obtained results since in all cases lesion voxels are replaced before tissue segmentation . on images where lesions have been masked before segmentation ( masked ) , the lower deviation in tissue volume of 3 t images can be explained by the increase in the resolution of the images when compared to 1.5 t data , which reduces the effect of masked voxels in tissue distributions .
the same reason can be behind the lower deviation found on all four lesion filling methods . by increasing the number of slices , differences produced by the methods on certain slices can be smoothed by tissue segmentation methods .
moreover , the use of a reduced sampling space or a better tuning of the parameters involved in the wm tissue distribution generated to refill lesion voxels could increase the performance of the presented method .
nevertheless , in all our experiments we decided to fix the standard deviation to 2 for simplicity . analyzing the results by dataset , on 1.5 t images from the oasis dataset , our results show that compared to the available methods , the proposed algorithm slf reduces significantly the differences in nwmv between original and filled images , independently of the brain tissue segmentation method used to measure the tissue volume . with the same data ,
although our method reports the lowest mean % difference in ngmv when spm8 method is used , the permutation test clearly shows that differences between slf and leap are not relevant . on 3 t images from the ixi dataset , slf also yields the lowest mean % differences in ngmv and nwmv , when fast is used to measure tissue volume .
these results are clearly significant in nwmv , but not in ngmv , although our method reports also the lowest difference among all methods .
when spm8 is used , slf presents a similar performance of that of leap , and both methods tie on the results of the significance tests .
compared with local methods , our algorithm performs quantitatively better than local methods on images with high lesion load ( > 36 ml ) .
the magon method incorporates all neighbor voxels surrounding a wm lesion region to compute a mean intensity which is used to refill all lesion voxels . on images with high lesion load touching gm tissue , including gm voxels can decrease refilled intensities and modify the tissue distribution of filled images .
fsl - l overpasses this limitation by building an intensity distribution based only on wm voxels surrounding lesions .
however , on large lesion regions , all lesion voxels will be filled with a narrow range of intensities coming from the neighboring voxels that can have a direct incidence on gm and wm tissue distributions . by contrast
, lesion volume appears to affect less global methods . in our case , the intensity distribution generated to refill lesion voxels will be independent of both the size and the position of lesion .
furthermore , the effect of filled voxels on the global wm tissue distribution is smoothed by the addition of intensities which try to reassemble the global nawm of the current slice .
compared with global methods , there are some interesting differences between our method and leap .
contrary to local methods , global methods have to deal with the skull - stripping process before processing images .
leap incorporates the skull - stripping process as part of the processing pipeline . in addition
by contrast , our method does not deal with skull - stripping internally , and the method requires an already skull - stripped image or a brain mask . as noted previously , the skull - stripping method employed seems to not interfere significantly in the results obtained by our method .
while setting up each of the different processes involved in the proposed pipeline , we found that , at least with our data , the performance of leap decreased or failed in 1.5 t scans when the skull - stripping methods bse ( shattuck et al . , 2001 ) and bet ( smith , 2002 ) were used with default options . by contrast
, leap provided the best results when the optimized method proposed by popescu et al .
( 2012 ) was used . this fact motivated the selection of this skull - stripping method for all the experiments of the study .
furthermore , on both datasets , we have also compared the differences between our method and leap estimating the mean nawm intensity used as a basis to fill lesion voxels . in most of the images ,
the global mean nawm intensity does not differ significantly between fityk7 on leap and our fuzzy - c - means approach .
hence , we can reject the hypothesis that observed differences in 1.5 t images can be caused by the approach employed to compute the nawm tissue distribution before filling lesion voxels .
however , on both lesion - filling approaches , tissue segmentation methods tended to increase the apparent mean wm tissue distribution on 1.5 t images with high lesion load ( > 40 ml ) due to the increase of voxels refilled with intensities higher than the actual mean wm signal intensity .
this effect is clearly more visible on leap than in our method , especially when fast is used .
the resolution of the oasis 1.5 t images ( 176 208 176 slices ) is lower than that of ixi 3 t images ( 256 150 256 slices ) . on images with low number of slices ,
after comparing the a priori wm tissue distribution values estimated by both the leap and slf methods with the already computed wm tissue distributions obtained from healthy images , we found that as lesion size increases , global methods such as leap and slf tend to increase the differences in tissue volume with respect to original images . in both methods
, we have observed that the a priori estimated mean intensity of the wm distribution tends to be higher than the actual tissue distribution as computed by fast and spm8 on healthy images . as lesion volume increases , the addition of more filled voxels with intensity higher than the actual mean tissue intensity is more prominent , causing a displacement of the mean intensity of the wm distribution returned by the segmentation methods on filled images .
consequently , more voxels bordering gm / wm are segmented as gm and wm tissue volume decreases . in this scenario ,
the strategy followed by slf , where wm is sampled independently at each slice , is more robust to the increase of lesions size than a global estimation of the wm tissue ( leap ) because possible errors introduced by a particular slice are not propagated into the rest of the slices .
contrary to spm8 , which estimates the tissue distributions based on a gaussian mixture model approach of the whole image , fast builds a network of neighboring relations based on a markov random field approach , more sensible to changes between slices .
the same reason can also be behind the better performance of our method on 3 t when fast is used .
compared with 1.5 t images , the probability of intensity change between slices is less prominent on 3 t images due to a higher resolution between slices . analyzing the possible deviations in tissue volume caused by each tissue segmentation process
, we obtained results which suggest that the chosen tissue segmentation method does not affect significantly the performance of our filling - method .
results between the same filled images segmented with fast and spm8 differ ( < 0.1% ) in the worst case on both datasets and tissues .
by contrast , magon , fsl - l and leap switch their rank on 1.5 t images , depending on the segmentation method used . on 3 t images , only magon and fsl - l appear to switch between ranks when fast or spm8 is used , respectively .
the most important one is the lack of images of ms patients with brain tissue expert annotations .
( 2014 ) have been only provided with lesion annotations delineated by a trained expert , but not brain tissue annotations . to overpass this limitation ,
we have registered wm lesions from ms patients into healthy images as performed in battaglini et al .
( 2012 ) and double - checked that registered lesions have replaced voxels segmented as wm by fast and spm8 .
this strategy has a negligible impact on the performance of the filling - methods analyzed in this study , because we assure a priori that generated lesions are on wm , and moreover none of the methods use information from the artificial lesions generated .
furthermore , although we tested the performance of the proposed method with two datasets with different magnetic field strengths , our results are limited to these two different scanners with particular configurations , and hence it is difficult to generalize the results to all 1.5 and 3 t scanners . in conclusion ,
the results of this study show that regardless of the lesion size , the slf method performs consistently well compared to other existing methods such as leap , especially on 1.5 t images .
furthermore , the results obtained show that the proposed method can be an effective method for low resolution images .
the skull - stripping process does not especially affect the accuracy of the method , which allows integrating it with different preprocessing pipelines .
additionally , volume estimations of lesion filled images processed by our algorithm appear to be not affected by the segmentation method employed .
in contrast to other approaches , slf may be installed by non - computer experts who can easily use it without any parameter tuning .
slf is currently available to researchers as a stand - alone script and as an spm library extension which facilitates to incorporate the lesion filling process into the expert workflow for tissue volume segmentation . | multiple sclerosis white matter ( wm ) lesions can affect brain tissue volume measurements of voxel - wise segmentation methods if these lesions are included in the segmentation process .
several authors have presented different techniques to improve brain tissue volume estimations by filling wm lesions before segmentation with intensities similar to those of wm . here , we propose a new method to refill wm lesions , where contrary to similar approaches , lesion voxel intensities are replaced by random values of a normal distribution generated from the mean wm signal intensity of each two - dimensional slice .
we test the performance of our method by estimating the deviation in tissue volume between a set of 30 t1-w 1.5 t and 30 t1-w 3 t images of healthy subjects and the same images where : wm lesions have been previously registered and afterwards replaced their voxel intensities to those between gray matter ( gm ) and wm tissue .
tissue volume is computed independently using fast and spm8 . when compared with the state - of - the - art methods , on 1.5 t data our method yields the lowest deviation in wm between original and filled images , independently of the segmentation method used .
it also performs the lowest differences in gm when fast is used and equals to the best method when spm8 is employed . on 3 t data , our method also outperforms the state - of - the - art methods when fast is used while performs similar to the best method when spm8 is used .
the proposed technique is currently available to researchers as a stand - alone program and as an spm extension . | Introduction
Materials and methods
Results
Discussion |
PMC4386784 | glial cell line - derived neurotrophic factor ( gdnf ) is a small protein that potently promotes the survival of many types of neurons and is able to prevent apoptosis of motor neurons induced by axotomy .
the detection of gdnf is vital to monitoring the survival and maintenance of sympathetic and sensory neurons.16 several approaches have so far been devised for the detection of gdnf .
conventional antibody - based methods , including western blotting , dot blotting , immunohistochemistry , and enzyme - linked immunosorbent assay , are widely used analytical approaches in both clinical diagnosis and biological research.7 however , these methods always suffer from the limitations of long assay time , complex experimental procedures , high cost , and low sensitivity . therefore , continuing efforts have been made to seek ideal methods for rapid , sensitive , low - cost , and user - friendly detection of gdnf .
a molecular translator can be employed as a signal transducer by highly converting the input target molecules into unique output deoxyribonucleic acid ( dna ) for signal amplification .
for example , picuri et al8 have developed a novel nucleic acid detection method through converting any target nucleic acids to a predesigned output dna .
li et al911 and zhang et al12,13 have designed a molecular translator base on binding induced dna assembly for fluorescence detection of protein , including prostate - specific antigen and platelet - derived growth factor ( pdgf ) .
although these approaches are effective , most of them are time - consuming , laborious , and high cost with low sensitivity .
recently , lateral flow biosensors ( lfbs ) have attracted considerable research interest because of their simple assay procedure , user - friendly platform , short assay time , and cost - effectiveness .
the result of the assay can be observed by naked eyes within 15 minutes , and quantitative analysis can be realized by recording the color intensity of the red band on the test zone with a portable lfb reader.14,15 isothermal strand - displacement polymerization reaction ( isdpr ) is a commonly used approach for dna signal amplification due to its robustness and simplicity.16 in this work , for the first time , we propose an lfb for detecting gdnf with a molecular translator and isdpr .
we employ a molecular translator to convert the protein signal to a dna signal , which can be further amplified by isdpr .
gdnf and rabbit anti - gdnf polyclonal antibody , bovine serum albumin ( bsa ) , streptavidin , and gold nanoparticles ( aunps ) ( 5 nm , 10 nm , and 20 nm ) were purchased from sigma - aldrich ( steinheim , germany ) .
the polymerase klenow fragment exo- and deoxynucleoside triphosphates ( dntps ) were obtained from new england biolabs ( ipswich , ma , usa ) .
all buffer solutions used in this study were prepared in our laboratory with ultrapure water .
the oligonucleotide sequences were synthesized and purified through high - performance liquid chromatography by shanghai sangon biological engineering technology & services co. ltd ( shanghai , people s republic of china ) .
the conjugates were prepared by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and n - hydroxysuccinimide ( edc / nhs ) activity .
briefly , 20.6 mg edc and 11.5 mg nhs were mixed in 1 ml of water for 10 minutes . from this solution
, 10 l was added to 200 l of 500 g / ml rabbit anti - gdnf polyclonal antibody , rabbit antitransforming growth factor [ tgf]- , anti - tgf- , antiepidermal growth factor [ egf ] , anti - insulin - like growth factor [ igf]-i , and antifibroblast growth factor [ fgf ] ) polyclonal antibodies solution in carbonate buffer , ph 11 , and reacted for 2 hours .
the solution was then centrifuged for 5 minutes at 4,000 r / min and washed twice with 200 l of water for 5 minutes at 4,000 r / min before being brought up to a final volume of 200 l with water .
the biosensor consisted of a sample pad , a conjugate pad , a nitrocellulose membrane , and an absorbent pad .
the sample pad ( 1.730 cm ) was prepared by soaking a glass fiber pad in sample pad buffer ( ph 8.0 ) ( 1% triton , 2% bsa , 3% glucose , and 50 mm boric acid ) .
the sample pad was then dried at room temperature . the conjugate pad ( fiberglass : 0.830 cm )
was prepared by dispensing a desired volume of aunp antidigoxin conjugates onto the glass fiber using a dispenser .
the conjugate pad was dried at room temperature for 12 hours and stored at 4c .
streptavidin ( 1 mg / ml ) and rabbit antimouse immunoglobulin g ( igg ) antibody ( 30 l , 1 mg / ml ) were dispensed onto the nitrocellulose membrane to form a test zone and a control zone , respectively , with a lateral flow dispenser .
finally , the four components of the lateral flow dna biosensor were assembled on plastic adhesive backing ( 630 cm ) .
each part overlapped by 2 mm to ensure solution migration through the strip during the assay .
strips were cut into 3 mm width with a paper cutter . a dna probe for the molecular translator
was prepared at a final concentration of 5 m by mixing 20 l 50 m mouse anti - gdnf antibody conjugated dna1 with 13.3 l 50 m output dna3 in 166.7 m tris ethylenediaminetetraacetic acid ( te ) buffer ( containing 10 mm mgcl2 and 0.05% tween 20 ) , heating to 90c for 5 minutes , and allowing solution to cool down to 25c slowly in a period of 3 hours .
strand displacement of molecular translator was performed in 20 l te buffer , 10 nm dna1/dna3 complex , 10 nm dna2 .
isdpr was performed in a 25 l 50 nm tris - hcl ( ph 8.0 ) buffer containing 50 nm short primer , 3u polymerase klenow fragment exo- , 50 m dntps , 6% dimethyl sulfoxide , 0.1% bsa , 1 mm dtt , 5 mm mgcl2 , and 2 l of product of strand displacement of molecular translator .
the red bands were observed within 5 minutes , and the biosensor was scanned with a portable lfb reader .
the optical intensities of the test zone and the control zone were recorded simultaneously by the reader , which automatically located the red bands in a fixed reaction area and then measured strip parameters such as peak height and area integral . for the process of molecular translator and strand - displacement amplification , dna1 conjugated with the gdnf antibody is initially hybridized to dna3 to form a stable dna1/dna3 duplex .
dna2 sequences are designed in such a way that the complementary sequences between dna1 and dna2 are 4-nt shorter than the complementary sequences between dna1 and dna3 . in the absence of gdnf ,
the strand displacement activity is poor between dna2 and dna3 at 25c , and the displacement of output dna3 by competing dna2 is extremely minimal .
however , in the process of gdnf , the binding of the same target protein to its antibodies that are coupled with dna1 and dna2 brings dna1 and dna2 to a close proximity , resulting in an increasing in their local effective concentration .
this process triggers the strand - displacement reaction between dna2 and dna3 . as a result
, the output dna3 is released for the next signal amplification ( figure 1a ) .
the releasing output dna3 recognizes and hybridizes with the loop region of the hairpin probe , which is composed of an eleven base pair stem , a 25-nt loop , and a biotin at the 5 end , causing the hairpin probe to undergo a conformational change and leading to stem separation . the digoxin - coupled primer ( 8-nt ) complementary to the stem region of the hairpin probe at the 3 end
thus anneals with the open stem and triggers a polymerization reaction in the presence of dntps and dna polymerase . in the process of primer extension ,
the output dna3 is released by dna polymerase with strand - displacement activity , after which complementary dna is synthesized , leading to the formation of a hairpin dna complex . to start the next cycle ,
the releasing output dna3 hybridizes with another hairpin probe , triggering another polymerization reaction . through this cyclic reaction comprising release of output dna3 and hybridization of output dna3 with remaining hairpin probe ,
a great amount of duplex dna products with a biotin and digoxin tag at the 5 end are synthesized .
the hairpin probe retains its original stem - loop structure in the absence of gdnf , the primer is unable to anneal to the hairpin to initiate a polymerization , and no duplex dna is produced ( figure 1b ) .
an lfb consists of four components : sample pad , conjugate pad , nitrocellulose membrane , and absorption pad .
streptavidin and goat antimouse igg antibody ( secondary body ) are dispensed on the nitrocellulose membrane , respectively , to form the test zone and the control zone .
the sample solution containing the isdpr product and running buffer is applied on the sample pad of the lfbs .
the solution migrates along the lfb by capillary action to the conjugate pad , where antidigoxin aunp conjugates have been deposited .
antidigoxin antibody couples with digoxin of the isdpr duplex dna product to form a biotin duplex dna
the complexes are captured at the test zone by the specific reaction between preimmobilized streptavidin and biotin .
the accumulation of aunps on the test zone is then visualized as a characteristic red band
aunp conjugates continue to migrate and are captured at the control zone by immunoreactions between antidigoxin antibody and the preimmobilized secondary antibody on the aunp surface , thus forming a second red band . in the absence of gdnf , no isdpr duplex dna product is produced ; therefore , no red band is observed at the test zone . in this case , the red band at the control zone indicates that the lfb is working properly .
visual detection is performed by observing the color change caused by the accumulation of aunps on the test zone , and quantitative detection can be realized by recording the color intensity of the red band on the test zone with a portable lfb reader ( figure 1c ) .
the data in this study were analyzed with spss statistics software version 19.0 ( ibm corporation , armonk , ny , usa ) .
gdnf and rabbit anti - gdnf polyclonal antibody , bovine serum albumin ( bsa ) , streptavidin , and gold nanoparticles ( aunps ) ( 5 nm , 10 nm , and 20 nm ) were purchased from sigma - aldrich ( steinheim , germany ) .
the polymerase klenow fragment exo- and deoxynucleoside triphosphates ( dntps ) were obtained from new england biolabs ( ipswich , ma , usa ) .
all buffer solutions used in this study were prepared in our laboratory with ultrapure water .
the oligonucleotide sequences were synthesized and purified through high - performance liquid chromatography by shanghai sangon biological engineering technology & services co. ltd ( shanghai , people s republic of china ) .
the conjugates were prepared by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and n - hydroxysuccinimide ( edc / nhs ) activity .
briefly , 20.6 mg edc and 11.5 mg nhs were mixed in 1 ml of water for 10 minutes . from this solution ,
10 l was added to 200 l of 500 g / ml rabbit anti - gdnf polyclonal antibody , rabbit antitransforming growth factor [ tgf]- , anti - tgf- , antiepidermal growth factor [ egf ] , anti - insulin - like growth factor [ igf]-i , and antifibroblast growth factor [ fgf ] ) polyclonal antibodies solution in carbonate buffer , ph 11 , and reacted for 2 hours .
the solution was then centrifuged for 5 minutes at 4,000 r / min and washed twice with 200 l of water for 5 minutes at 4,000 r / min before being brought up to a final volume of 200 l with water .
the biosensor consisted of a sample pad , a conjugate pad , a nitrocellulose membrane , and an absorbent pad .
the sample pad ( 1.730 cm ) was prepared by soaking a glass fiber pad in sample pad buffer ( ph 8.0 ) ( 1% triton , 2% bsa , 3% glucose , and 50 mm boric acid ) .
the sample pad was then dried at room temperature . the conjugate pad ( fiberglass : 0.830 cm )
was prepared by dispensing a desired volume of aunp antidigoxin conjugates onto the glass fiber using a dispenser .
the conjugate pad was dried at room temperature for 12 hours and stored at 4c .
streptavidin ( 1 mg / ml ) and rabbit antimouse immunoglobulin g ( igg ) antibody ( 30 l , 1 mg / ml ) were dispensed onto the nitrocellulose membrane to form a test zone and a control zone , respectively , with a lateral flow dispenser .
finally , the four components of the lateral flow dna biosensor were assembled on plastic adhesive backing ( 630 cm ) .
each part overlapped by 2 mm to ensure solution migration through the strip during the assay .
a dna probe for the molecular translator was prepared at a final concentration of 5 m by mixing 20 l 50 m mouse anti - gdnf antibody conjugated dna1 with 13.3 l 50 m output dna3 in 166.7 m tris ethylenediaminetetraacetic acid ( te ) buffer ( containing 10 mm mgcl2 and 0.05% tween 20 ) , heating to 90c for 5 minutes , and allowing solution to cool down to 25c slowly in a period of 3 hours .
strand displacement of molecular translator was performed in 20 l te buffer , 10 nm dna1/dna3 complex , 10 nm dna2 .
isdpr was performed in a 25 l 50 nm tris - hcl ( ph 8.0 ) buffer containing 50 nm short primer , 3u polymerase klenow fragment exo- , 50 m dntps , 6% dimethyl sulfoxide , 0.1% bsa , 1 mm dtt , 5 mm mgcl2 , and 2 l of product of strand displacement of molecular translator .
finally , the isdpr product was loaded onto the sample pad together with 20 l pbs .
the red bands were observed within 5 minutes , and the biosensor was scanned with a portable lfb reader .
the optical intensities of the test zone and the control zone were recorded simultaneously by the reader , which automatically located the red bands in a fixed reaction area and then measured strip parameters such as peak height and area integral .
for the process of molecular translator and strand - displacement amplification , dna1 conjugated with the gdnf antibody is initially hybridized to dna3 to form a stable dna1/dna3 duplex .
dna2 sequences are designed in such a way that the complementary sequences between dna1 and dna2 are 4-nt shorter than the complementary sequences between dna1 and dna3 . in the absence of gdnf ,
the strand displacement activity is poor between dna2 and dna3 at 25c , and the displacement of output dna3 by competing dna2 is extremely minimal .
however , in the process of gdnf , the binding of the same target protein to its antibodies that are coupled with dna1 and dna2 brings dna1 and dna2 to a close proximity , resulting in an increasing in their local effective concentration .
this process triggers the strand - displacement reaction between dna2 and dna3 . as a result
, the output dna3 is released for the next signal amplification ( figure 1a ) .
the releasing output dna3 recognizes and hybridizes with the loop region of the hairpin probe , which is composed of an eleven base pair stem , a 25-nt loop , and a biotin at the 5 end , causing the hairpin probe to undergo a conformational change and leading to stem separation . the digoxin - coupled primer ( 8-nt ) complementary to the stem region of the hairpin probe at the 3 end
thus anneals with the open stem and triggers a polymerization reaction in the presence of dntps and dna polymerase . in the process of primer extension ,
the output dna3 is released by dna polymerase with strand - displacement activity , after which complementary dna is synthesized , leading to the formation of a hairpin dna complex . to start the next cycle
, the releasing output dna3 hybridizes with another hairpin probe , triggering another polymerization reaction . through this cyclic reaction comprising release of output dna3 and hybridization of output dna3 with remaining hairpin probe ,
a great amount of duplex dna products with a biotin and digoxin tag at the 5 end are synthesized .
the hairpin probe retains its original stem - loop structure in the absence of gdnf , the primer is unable to anneal to the hairpin to initiate a polymerization , and no duplex dna is produced ( figure 1b ) .
an lfb consists of four components : sample pad , conjugate pad , nitrocellulose membrane , and absorption pad .
streptavidin and goat antimouse igg antibody ( secondary body ) are dispensed on the nitrocellulose membrane , respectively , to form the test zone and the control zone .
the sample solution containing the isdpr product and running buffer is applied on the sample pad of the lfbs .
the solution migrates along the lfb by capillary action to the conjugate pad , where antidigoxin aunp conjugates have been deposited .
antidigoxin antibody couples with digoxin of the isdpr duplex dna product to form a biotin duplex dna
the complexes are captured at the test zone by the specific reaction between preimmobilized streptavidin and biotin .
the accumulation of aunps on the test zone is then visualized as a characteristic red band
aunp conjugates continue to migrate and are captured at the control zone by immunoreactions between antidigoxin antibody and the preimmobilized secondary antibody on the aunp surface , thus forming a second red band . in the absence of gdnf , no isdpr duplex dna product is produced ; therefore , no red band is observed at the test zone . in this case , the red band at the control zone indicates that the lfb is working properly .
visual detection is performed by observing the color change caused by the accumulation of aunps on the test zone , and quantitative detection can be realized by recording the color intensity of the red band on the test zone with a portable lfb reader ( figure 1c ) .
the data in this study were analyzed with spss statistics software version 19.0 ( ibm corporation , armonk , ny , usa ) .
the molecular translator is composed of target recognition elements ( dna1 and dna2 ) and a signal output element ( dna3 ) , which is illustrated in figure 1a .
the output dna3 triggers isdpr for signal amplification by producing a large amount of duplex dna ( figure 1b ) .
figure 1c illustrates the schematic illustration of the strip biosensor for visual detection of duplex dna .
we investigated the optical response of 10 nm dna3 on the lfb with and without isdpr amplification . in the case without isdpr , there was no polymerase in the reaction buffer .
as shown in figure 2 , the response of the sample with isdpr was 35 times higher compared with that without isdpr or the blank group ( p<0.01 ) .
the experimental parameters , such as isdpr temperature and reaction time that could affect the analytical performance , were optimized systematically .
we compared the signal to noise ( s / n ) ratio of 10 ng ml and 1 ng ml gdnf with four different isdpr temperatures ( 25c , 37c , 42c , and 50c ) .
as shown in figure 3a , the s / n ratios increased with increasing reaction temperature in the range from 25c to 42c .
as shown in figure 3b , the optical response elevated gradually with the augmenting of reaction time from 10 minutes to 30 minutes .
the peak area of the 10 ng / ml gdnf and 1 ng / ml gdnf was significantly higher compared with the blank group ( figure 3b , p<0.05 ) . further increasing the reaction time led to the increasing background signal .
a longer reaction time at 42c may cause stem separation of the hairpin probe , even in the absence of output dna3 , resulting in a high background signal .
the results indicated that 30 minutes is the optimal reaction time ( figure 3b ) .
we have examined the sensitivity and dynamic range of the lfb using different concentrations of gdnf under optimal conditions . as shown in figure 4a , the color intensities of the red bands on the test zone increased with the increasing of gdnf concentrations .
no distinct red band was observed on the test zone in the absence of gdnf . for quantitative detections , the optical responses
were further confirmed by recording the color intensities of the red bands by a portable strip reader .
the peak areas versus the incremental concentrations of gdnf have been plotted in figure 4b , which followed a sigmoid increase .
the resulting calibration curve shows that the peak areas are proportional to the logarithm of gdnf concentrations in the range from 1 pg ml to 10 ng ml ( figure 4c ) .
the specificity of the lfb for gdnf was studied by testing the responses of the assay to other kinds of growth factors , including pdgf , tgf- , tgf- , egf , igf - i , and fgf , which are similar to gdnf .
as shown in figure 5a , 1 ng / ml gdnf produced a visible bright red band on the test zone of lfb , whereas all other growth factors at a concentration of 100 ng / ml did not yield a red band on the test zone of lfb .
the prepared lfbs were sealed and stored at 37c for the following time periods : 12 hours , 24 hours , 14 days , and 1 month . as shown in figure 5b
, there were no significant performance differences among different storage times , indicating that the stability of the assay is acceptable .
the molecular translator is composed of target recognition elements ( dna1 and dna2 ) and a signal output element ( dna3 ) , which is illustrated in figure 1a .
the output dna3 triggers isdpr for signal amplification by producing a large amount of duplex dna ( figure 1b ) .
figure 1c illustrates the schematic illustration of the strip biosensor for visual detection of duplex dna .
we investigated the optical response of 10 nm dna3 on the lfb with and without isdpr amplification . in the case without isdpr , there was no polymerase in the reaction buffer .
as shown in figure 2 , the response of the sample with isdpr was 35 times higher compared with that without isdpr or the blank group ( p<0.01 ) .
the experimental parameters , such as isdpr temperature and reaction time that could affect the analytical performance , were optimized systematically .
we compared the signal to noise ( s / n ) ratio of 10 ng ml and 1 ng ml gdnf with four different isdpr temperatures ( 25c , 37c , 42c , and 50c ) .
as shown in figure 3a , the s / n ratios increased with increasing reaction temperature in the range from 25c to 42c .
as shown in figure 3b , the optical response elevated gradually with the augmenting of reaction time from 10 minutes to 30 minutes .
the peak area of the 10 ng / ml gdnf and 1 ng / ml gdnf was significantly higher compared with the blank group ( figure 3b , p<0.05 ) . further increasing the reaction time led to the increasing background signal .
a longer reaction time at 42c may cause stem separation of the hairpin probe , even in the absence of output dna3 , resulting in a high background signal .
the results indicated that 30 minutes is the optimal reaction time ( figure 3b ) .
we have examined the sensitivity and dynamic range of the lfb using different concentrations of gdnf under optimal conditions . as shown in figure 4a , the color intensities of the red bands on the test zone increased with the increasing of gdnf concentrations .
no distinct red band was observed on the test zone in the absence of gdnf . for quantitative detections , the optical responses
were further confirmed by recording the color intensities of the red bands by a portable strip reader .
the peak areas versus the incremental concentrations of gdnf have been plotted in figure 4b , which followed a sigmoid increase .
the resulting calibration curve shows that the peak areas are proportional to the logarithm of gdnf concentrations in the range from 1 pg ml to 10 ng ml ( figure 4c ) .
the specificity of the lfb for gdnf was studied by testing the responses of the assay to other kinds of growth factors , including pdgf , tgf- , tgf- , egf , igf - i , and fgf , which are similar to gdnf .
as shown in figure 5a , 1 ng / ml gdnf produced a visible bright red band on the test zone of lfb , whereas all other growth factors at a concentration of 100 ng / ml did not yield a red band on the test zone of lfb .
the prepared lfbs were sealed and stored at 37c for the following time periods : 12 hours , 24 hours , 14 days , and 1 month . as shown in figure 5b
, there were no significant performance differences among different storage times , indicating that the stability of the assay is acceptable .
a molecular translator can be employed as a signal transducer by highly converting the input target molecules into a unique output dna for signal amplification.17,18 isdpr is a commonly used approach for the dna signal amplification.1618 therefore , we combined the molecular translator and isdpr method to detect the gdnf in cells . in this work
, isdpr was used to produce a large amount of digoxin- and biotin - coupled duplex dna for visual detection of gdnf .
compared with the solutions without isdpr , the response of the sample with isdpr was 35 times higher .
figure 3a shows that the s / n ratios increased with increasing reaction temperatures in the range from 25c to 42c , however s / n ratios decreased with temperatures over 42 .
these results could be explained by conformational change of the hairpin probe at high temperature , which leads to the stem separation of the hairpin probe .
the primer can anneal with the open hairpin probe and trigger a polymerization reaction , even in the absence of output dna3 , resulting in a high background signal .
usually , the isdpr can be enhanced with increasing the reaction time . in this study , 30 minutes was selected as the reaction time throughout the experiments to obtain the highest signal . as shown in figure 4 , the red band on the test zone was observed with as little as 1 pg ml of gdnf , which can be considered the limit of detection . based on the molecular weight of gdnf ( 13.5 kda ) , the limit of detection was calculated to be 74 fm .
the detection limit of our proposed approach is two orders of magnitude better than the antibody - based immunoassay.7 generally , human basal serum gdnf concentrations are between 20 pg / ml and 30 pg / ml .
this lfb holds great promise in monitoring gdnf by naked eyes with its superior detection sensitivity and large dynamic range . from the results of figure 5a , 1
ng / ml gdnf produced a visible bright red band , while the other growth factors ( such as tgf- , tgf- , egf , igf - i , and fgf ) did not yield a red band even at a concentration of 100 ng / ml . these results indicated that our constructed lfb exhibited excellent selective response to gdnf .
our results also showed that this method is very stable after different periods of storage .
in summary , we have successfully fabricated an lfb for amplified detection of gdnf using a molecular translator for converting the input protein to output dna signal and isdpr for signal amplification .
compared with other reported antibody - based immunoassays , the sensitivity of the lfb developed here is higher . by using this assay , gdnf as low as 1 pg ml
the response of the optimized assay is highly linear over a range of 1 pg / ml to 10 ng / ml . this new gdnf detection method is expected to be a great potential platform for the detection of protein , which would be valuable for clinical therapy for some diseases related to gdnf . | backgroundglial cell line - derived neurotrophic factor ( gdnf ) is a small protein that potently promotes the survival of many types of neurons .
detection of gdnf is vital to monitoring the survival of sympathetic and sensory neurons .
however , the specific method for gdnf detection is also un - discovered .
the purpose of this study is to explore the method for protein detection of gdnf.methodsa novel visual detection method based on a molecular translator and isothermal strand - displacement polymerization reaction ( isdpr ) has been proposed for the detection of gdnf . in this study ,
a molecular translator was employed to convert the input protein to output deoxyribonucleic acid signal , which was further amplified by isdpr .
the product of isdpr was detected by a lateral flow biosensor within 30 minutes.resultsthis novel visual detection method based on a molecular translator and isdpr has very high sensitivity and selectivity , with a dynamic response ranging from 1 pg / ml to 10 ng / ml , and the detection limit was 1 pg / ml of gdnf.conclusionthis novel visual detection method exhibits high sensitivity and selectivity , which is very simple and universal for gdnf detection to help disease therapy in clinical practice . | Introduction
Materials and methods
Materials
Preparation of anti-GDNF antibody DNA1/DNA2 conjugates
Preparation of the LFB
Detection of GDNF by LFB
Process of molecular translator and ISDPR
Statistical analysis
Results
Principle of visual detection of GDNF
Verification of the signal amplification of ISDPR
Optimization of ISDPR
Analytical performances
Discussion
Conclusion |
PMC3742112 | the imaging was performed using a 1300 nm sd - oct system shown in fig .
s1 . a superluminescent diode ( sld ) with a bandwidth of 105 nm was used as the light source , giving an axial resolution of 9 m .
an identical pair of achromatic doublets with focal lengths of 30 mm was used to image the fiber core to the sample , resulting in a numerical aperture ( na ) of 0.1 .
the rayleigh range was calculated to be 50 m ( using 4.7 m as the radius of the beam at the beam waist ) while the imaging depth from the spectrometer ( bayspec , inc . )
the beam was raster scanned along the fast - scanning and slow - scanning axes over the sample .
customized driving waveforms ( 85% linear , 15% fly - back ) for the x - y galvanometer pair ( scanlab , scancube 7 ) were generated by a daq board ( national instruments , ni - pcie-6353 ) , which also generated a control signal to synchronize the scanning with the acquisition from the ingaas line - scan camera ( goodrich , su - ldh2 ) .
the camera was operated at the maximum a - scan rate of 91.912 khz and was interfaced through an image acquisition board ( national instruments , ni - pcie-1427 ) .
the software for data acquisition and graphical user interface was developed in labview and the data was processed in real - time through dynamic link library ( dll ) function calls implemented in c ( microsoft visual studio 2008 environment ) .
a computer with an intel core i7 processor ( 975 @ 3.3 ghz , 12 gb ddr3 ram ) was used for running the system and the compute unified device architecture ( cuda ) extension v4.1 from nvidia was used for gpu kernel calls on the nvidia geforce gtx 580 gpu .
the ex vivo samples were placed on a three - axis stage separate from the beam collimator and scanning galvos while the in vivo data was taken by gently pressing the tissue of interest onto a custom mount attached to the scancube and beam collimator . by attaching a coverslip mount directly to the lens tube containing the objective lens , which was attached to the scanning optics and fiber collimator , the phase stability requirements for isam
acquired volumes consisted of 1024 pixels per a - scan and 810 a - scans along each transverse dimension resulting in an acquisition time of approximately 8.5 seconds .
a multi - streamed gpu program was developed for real - time 3-d isam ( see supplementary fig .
the streams executed concurrently , performing two orthogonal 2-d isam reconstructions where one stream processed the fast - axis frames while the other stream processed the orthogonal dimension of the previously acquired , 2-d isam processed data .
this decomposition required only a single volume buffer in gpu memory at any given time because each previously acquired slow - axis frame can be replaced by the currently acquired fast - axis frame .
all the processing on the gpu was done with single precision floating point operations and gamma correction was applied on the processed data for display purposes . in this manuscript , the term real - time means that data acquisition was never paused for processing , and that data is continuously being updated for the user , albeit , the data being displayed is at a small latency of one volume .
( 8.5 seconds for the dimensions mentioned in experimental setup ) . if we were to allow the camera to pause between volume acquisitions to complete the isam reconstruction , the latency would be reduced even further ( to 1.5 seconds for the same volume ) .
see supplementary methods for further details . to remove any bias in displaying comparisons between oct and isam
, all comparisons were viewed on the same histogram scale using the same upper and lower saturation values . in real time , our system has the ability to both process and display cross - sectional and user - selected en face planes in the 2-d and 3-d isam reconstructions . in post - processing for further visualization ,
the calculated energy in each isam tomogram ( en face plane by en face plane ) was normalized to that of the oct tomogram .
after power - normalization , the noise floors in the oct and the isam tomograms were in excellent agreement . to further visualize complete oct and isam datasets ,
the volume was first segmented into two regions , the stratum corneum / stratum lucidum ( containing the sweat - ducts ) and the dermis , then different color maps were used for each .
image metrics such as anisotropy , signal - to - noise ratio , and contrast were computed to quantitatively evaluate the improvement in reconstruction quality .
the anisotropy image quality metric was originally devised to be sensitive to additive gaussian noise in incoherent imaging systems .
further testing on various tissue phantoms and tissue data ( data not shown ) confirmed that this metric is reliable for noise present in oct data as well .
signal - to - noise and contrast were computed by using the 20% ( noise ) and 90% ( signal ) quantiles of the intensity histograms .
the imaging was performed using a 1300 nm sd - oct system shown in fig .
s1 . a superluminescent diode ( sld ) with a bandwidth of 105 nm was used as the light source , giving an axial resolution of 9 m .
an identical pair of achromatic doublets with focal lengths of 30 mm was used to image the fiber core to the sample , resulting in a numerical aperture ( na ) of 0.1 .
the rayleigh range was calculated to be 50 m ( using 4.7 m as the radius of the beam at the beam waist ) while the imaging depth from the spectrometer ( bayspec , inc . )
the beam was raster scanned along the fast - scanning and slow - scanning axes over the sample .
customized driving waveforms ( 85% linear , 15% fly - back ) for the x - y galvanometer pair ( scanlab , scancube 7 ) were generated by a daq board ( national instruments , ni - pcie-6353 ) , which also generated a control signal to synchronize the scanning with the acquisition from the ingaas line - scan camera ( goodrich , su - ldh2 ) .
the camera was operated at the maximum a - scan rate of 91.912 khz and was interfaced through an image acquisition board ( national instruments , ni - pcie-1427 ) .
the software for data acquisition and graphical user interface was developed in labview and the data was processed in real - time through dynamic link library ( dll ) function calls implemented in c ( microsoft visual studio 2008 environment ) .
a computer with an intel core i7 processor ( 975 @ 3.3 ghz , 12 gb ddr3 ram ) was used for running the system and the compute unified device architecture ( cuda ) extension v4.1 from nvidia was used for gpu kernel calls on the nvidia geforce gtx 580 gpu .
the ex vivo samples were placed on a three - axis stage separate from the beam collimator and scanning galvos while the in vivo data was taken by gently pressing the tissue of interest onto a custom mount attached to the scancube and beam collimator . by attaching a coverslip mount directly to the lens tube containing the objective lens , which was attached to the scanning optics and fiber collimator , the phase stability requirements for isam
acquired volumes consisted of 1024 pixels per a - scan and 810 a - scans along each transverse dimension resulting in an acquisition time of approximately 8.5 seconds .
a multi - streamed gpu program was developed for real - time 3-d isam ( see supplementary fig .
the streams executed concurrently , performing two orthogonal 2-d isam reconstructions where one stream processed the fast - axis frames while the other stream processed the orthogonal dimension of the previously acquired , 2-d isam processed data .
this decomposition required only a single volume buffer in gpu memory at any given time because each previously acquired slow - axis frame can be replaced by the currently acquired fast - axis frame .
all the processing on the gpu was done with single precision floating point operations and gamma correction was applied on the processed data for display purposes . in this manuscript , the term real - time means that data acquisition was never paused for processing , and that data is continuously being updated for the user , albeit , the data being displayed is at a small latency of one volume .
( 8.5 seconds for the dimensions mentioned in experimental setup ) . if we were to allow the camera to pause between volume acquisitions to complete the isam reconstruction , the latency would be reduced even further ( to 1.5 seconds for the same volume ) .
to remove any bias in displaying comparisons between oct and isam , all comparisons were viewed on the same histogram scale using the same upper and lower saturation values . in real time , our system has the ability to both process and display cross - sectional and user - selected en face planes in the 2-d and 3-d isam reconstructions . in post - processing for further visualization ,
the calculated energy in each isam tomogram ( en face plane by en face plane ) was normalized to that of the oct tomogram .
after power - normalization , the noise floors in the oct and the isam tomograms were in excellent agreement . to further visualize complete oct and isam datasets , the tomograms were depth - normalized to a common histogram scale .
the volume was first segmented into two regions , the stratum corneum / stratum lucidum ( containing the sweat - ducts ) and the dermis , then different color maps were used for each .
image metrics such as anisotropy , signal - to - noise ratio , and contrast were computed to quantitatively evaluate the improvement in reconstruction quality .
the anisotropy image quality metric was originally devised to be sensitive to additive gaussian noise in incoherent imaging systems .
further testing on various tissue phantoms and tissue data ( data not shown ) confirmed that this metric is reliable for noise present in oct data as well .
signal - to - noise and contrast were computed by using the 20% ( noise ) and 90% ( signal ) quantiles of the intensity histograms . | high - resolution real - time tomography of scattering tissues is important for many areas of medicine and biology16 .
however , the compromise between transverse resolution and depth - of - field in addition to low sensitivity deep in tissue continue to impede progress towards cellular - level volumetric tomography .
computed imaging has the potential to solve these long - standing limitations .
interferometric synthetic aperture microscopy ( isam)79 is a computed imaging technique enabling high - resolution volumetric tomography with spatially invariant resolution . however , its potential for clinical diagnostics remains largely untapped since full volume reconstructions required lengthy postprocessing , and the phase - stability requirements have been difficult to satisfy in vivo .
here we demonstrate how 3-d fourier - domain resampling , in combination with high - speed optical coherence tomography ( oct ) , can achieve high - resolution in vivo tomography .
enhanced depth sensitivity was achieved over a depth - of - field extended in real time by more than an order of magnitude .
this work lays the foundation for high - speed volumetric cellular - level tomography . | Methods
Experimental setup
GPU implementation
Image display and analysis
Supplementary Material |
PMC2952819 | nitric oxide ( no ) plays multiple roles in the lung in both injury and repair ; it is an airway and vascular smooth muscle cell signaling molecule , an inhibitory nonadrenergic noncholinergic ( inanc ) signaling molecule , a modulator of apoptosis , and a component of the bactericidal arsenal of lung inflammatory cells .
the primary molecular sources of no are the nos enzymes , including the inducible nos2 isoform , which is upregulated in the ovalbumin- ( ova- ) induced allergic airway inflammation model [ 13 ] and the constitutively expressed nos1 ( neuronal nos ) and nos3 ( endothelial nos ) isoforms , which contribute to the generation of no in the murine airway epithelium [ 4 , 5 ] .
no can be further metabolized to produce the more stable products , nitrate and nitrite .
both of these products are considered bioactive , capable of enzyme - dependent and enzyme - independent reconversion into no [ 6 , 7 ] . in asthma , increased no concentration in exhaled breath is considered a disease biomarker and is supported by current guidelines for use in clinical settings .
the conventional treatment of asthmatics using inhaled steroids decreases exhaled no , but whether alleviation of the asthmatic symptoms is a result of decreasing exhaled no or if the decreased exhaled no is a byproduct of decreasing overall lung inflammation is still debated .
in the cell , maintenance of a stable no concentration , or no homeostasis , is essential , as fluctuations in the concentration of no can alter intra and intercellular signaling and affect survivability .
, for example , detected an increase in the concentration of no in exhaled breath correlating to an increase in nos2 expression in nos3 mice , thus indicating a compensatory mechanism for the regulation of baseline no production . also , exposure of astrocytes to no - scavenging hemoglobin increased nos2 expression and was dependent on nf-b activation .
in addition to maintaining a baseline of no production , it is necessary to upregulate no production under specific conditions such as during oxidative stress , release of heme - containing compounds [ 13 , 14 ] , or the launching of an inflammatory response .
thus , the ability to further regulate nos2 expression in constitutive nos isoform knockout strains may be necessary for the organism to modulate its reaction to an insult and also resolve the response .
a study by connelly et al . , using lps stimulation , determined that nf-b translocation was necessary for nos2 expression under inflammatory conditions and the study by gobeil jr .
we previously identified nos2 mice as being more susceptible to severe allergic inflammation and subepithelial fibrosis than their c57bl/6 wild - type counterparts indicating that nos2 expression and activity are necessary for regulating the intensity of the inflammatory response .
thus , we chose to examine no homeostatic dysregulation in the development of allergic airway disease . in this paper , we hypothesized that under normal conditions , deleting the nos1 or nos3 gene would upregulate nos2 protein expression .
thus , the lung function and total cellular population of filtered air - exposed nos1 and nos3 mice should be comparable to filtered air - exposed c57bl/6 wt mice , while the nos2 mice should have a heightened inflammatory cell profile as observed in our previous study .
furthermore , ten broeke and colleagues showed that nos2 expression was nos3 dependent , and we therefore hypothesized that exposure of mice to ovalbumin ( ova ) would inhibit this increase in nos2 protein expression in the nos3 mice resulting in lower nos2 protein concentrations compared to nos3 filtered air controls .
we also hypothesized that this would result in increased lung inflammation in the nos3 mice compared to both ova - exposed c57bl/6 wt and nos1 mice . to this end , we performed lung function analysis with a methacholine challenge protocol and collected bronchoalveolar lavage for the calculation of lung inflammatory cell influx and inflammatory cell profile
. we also examined tissue - specific nos2 expression patterns in the lungs and changes in nitric oxide production of both filtered air - exposed and ova - exposed mice from nos1 , nos2 , nos3 , and c57bl/6 wt genotype .
all procedures were performed as per our iacuc - approved protocol , following their standards and regulations .
all animals were maintained in an hepa - filtered laminar flow cage rack with a 12-hour light / dark cycle and allowed free access to food and water .
animals were housed and cared for by the veterinary staff of animal resource services at ucd in aalac- accredited facilities , in plastic cages over autoclaved bedding in hepa - filtered cage racks .
animals were routinely screened for health status by serology and histology by our veterinary animal resources facility .
nos1 , nos3 , and the wild type strain c57bl/6 mice were purchased from jackson laboratories .
the nos2 mice were initially purchased from taconic laboratories , germantown , ny and were rederived by embryo transfer to establish a breeding colony in the targeted genomics laboratory of the mouse biology barrier facility at uc davis .
mice were sensitized by intraperitoneal ( ip ) injection of chicken egg albumin ( ovalbumin , grade v , 98% pure , sigma , st .
louis , mo , 2 10 g/0.1 ml , 2 weeks apart ) with alum as an adjuvant .
exposure to ova aerosols was performed using chambers and generators we have described elsewhere .
exposures to ova aerosol , 10 ml of a 10 mg / ml ( 1% ) solution , were begun on day 28 .
mice were exposed for 30 minutes , three times per week for the duration of a given experiment .
age - matched control animals were injected ip with ova ( sensitized ) but were then exposed to filtered air .
histological evaluations of c57bl/6 wt mice ova - exposed and filtered air - exposed control mice demonstrated that we were able to induce airway inflammation , epithelial cell sloughing , and goblet cell hyperplasia in the exposed mice .
dynamic compliance and resistance of the respiratory system were measured using a plethysmograph for restrained animals .
mice were deeply anesthetized and sedated with medetomidine , 0.5 mg / kg ( domitor , orion pharma , finland ) , and tiletamine / zolpidem , 50 mg / kg ( telazol , fort dodge laboratories , fort dodge , ia ) and ventilated at 7 - 8 cc / kg with a mouse ventilator ( minivent , harvard apparatus , cambridge , ma ) for the duration of the procedure .
compliance and resistance measurements were made at baseline and immediately following serial 3-minute nebulizations of saline and methacholine ( 0 , 0.5 , 1.0 , and 2.0 mg / ml ) .
after the physiological measurements , animals were euthanized with an overdose of phenobarbital and dilantin administered via intraperitoneal injection .
animals were placed on a restraining board and their lungs lavaged with two 1 ml aliquots of phosphate - buffered saline ( pbs ) , ph 7.4 , and each aliquot was passaged twice through the lungs .
cells were pelleted at 2500 rpm for 10 minutes , and the acellular supernatant was removed and stored at 20c for nitrate and nitrite ( nox ) analysis .
the remaining cell pellet was treated with a lysis buffer ( 0.15 m nh4cl , 1 mm khco3 , 0.1 mm edta , and ph 7.3 ) , repelleted , and resuspended in 0.5 ml pbs .
total lavage cell number was determined using a hemocytometer and 100 l aliquots of the remaining cell suspension processed onto slides using a cytocentrifuge at 1650 rpm for 15 minutes .
slides were air dried and stained with a hema3 stain set as described in the manufacturer 's instructions ( biochemical sciences , swedesboro , nj ) and sealed using cytoseal ( stephens scientific , kalamazoo , mi ) .
cells were classified as alveolar macrophage , neutrophil , eosinophil , lymphocyte , or other based upon morphological characteristics and staining profile .
five - minute samples of exhaled gases were collected into a specially constructed mylar bag from the cannulated mice via the ventilator exhalation port immediately after insertion of a mouse into the plethysmograph . this 5-minute sample was adequate for the measurement of no concentration in the expired air , using a sievers nitric oxide analyzer ( sievers inst . , boulder , co ) .
nitrate and nitrite ( nox ) was measured in lavage fluid with this analyzer as previously described .
the isolated airways were prepared by microdissecting the left lung lobe to obtain a preparation of the larger airways distal to the carina from the left bronchus through the third generation of conducting airway , separate from adhering parenchyma .
samples containing 20 g total protein were electroporated under reducing conditions and transferred to a polyvinylidene difluoride ( pvdf ) membrane .
membranes were incubated in 0.6 g / ml rabbit antimouse nos1 , 0.8 g / ml nos2 , 0.6 g / ml nos3 , or 0.4 g / ml -actinin igg ( used as a gell loading control ) in 5% dry milk in pbs overnight at 4c and incubated in 40 ng / ml horseradish peroxidase- ( hrp- ) conjugated goat antirabbit igg ( pierce biotechnology , rockford , il ) in 5% milk in pbs .
bands were visualized using immobilon western chemiluminescent hrp substrate kit ( millipore , billerica , massachusetts ) and band intensity - assessed using the kodak 1d version 3.5.4 scientific imaging system ( eastman kodak co , ct ) .
half of the animals had their lungs fixed for histological evaluation at 30 cm pressure using 1% paraformaldehyde in pbs ( ph 7.5 ) .
after 24 hours of fixation , the left lung was placed in 70% ethanol and embedded in paraffin .
lung sections of 5-um thickness were made with special attention to cutting through the larger lobar bronchi in parallel then dried at 37c overnight .
lung sections were deparaffinized and processed for hematoxylin and eosin ( h&e ) staining for evaluation of total inflammation , or nos2-specific immunohistochemistry for semiquantitative assessment of tissue - specific nos2 expression or alcian blue - periodic acid - schiff ( pas ) staining for quantitation of mucus - containing goblet cells . left lung sections were prepared as described .
slides were incubated in 1 mm etda , ph 7.5 at 100c for 20 minutes to decloak antigen .
sections were processed using the r&d systems cell and tissue staining kit hrp - dab system ( minneapolis , mn ) for rabbit antibodies .
sections were incubated overnight at 4c in 0.2 g / ml rabbit anti - mouse nos2 igg diluted in 5% goat serum in 1% bsa , 0.2 g / ml rabbit igg ( isotype control ) , or serum + bsa only ( negative control ) .
incubation times were as follows : 1 hour in biotinylated goat antirabbit secondary antibody , 30 minutes in hss - conjugated hrp , and 15 minutes in diaminobenzidine .
a grading system of 010 was established prior to the grading based upon a series of prestained slide standards .
the lung tissue compartments were divided into airway epithelium , smooth muscle , and macrophages and were scored under 200x power .
the linear intensity grading scale used was 0no nos2 stain as compared to a primary antibody negative control to 10dramatically increased nos2 staining comparable to a lung section from an lps - treated mouse
. left lung sections , prepared as described above , were immersed in a 1% alcian blue , 3% glacial acetic acid solution ( ph2.5 ) for 30 minutes , rinsed in tap water , and then immersed in a 1% periodic acid solution for 7 minutes .
slides were then rinsed again in distilled water and immersed in schiff reagent solution consisting in 0.45% basic fuchsin , 10% hcl , and 0.45% sodium bisulfite for 15 minutes .
slides were cleared in running tap water , counterstained using harris ' hematoxylin , and then dehydrated and mounted in cytoseal .
each animal was represented by a single section of lung selected for maximal visualization of the main airway .
each section had 5 randomly selected regions evaluated ( two segments of the 1 conducting airway , two segments from separate 2 conducting airways , and one segment from a 3 conducting airway ) .
a minimum of 100 sequential airway epithelial cells were counted from each region and the total number of pas positive cells per total epithelial cells was determined for each region .
means were compared by t - test or by anova , with tukey 's correction for multiple comparisons applied where appropriate .
analysis of the compliance and resistance changes was done by both two - way anova with bonferroni correction and by linear regression analysis , using the prism software package ( graphpad prism 5.0 , san diego , ca ) .
we believe that the combination of these methods allowed for better understanding of the interaction between the effect of ova exposure and methacholine aerosol challenge .
r , a common open source statistical computing package ( url : http://www.r-project.org ) , was used to perform this analysis .
nos2 mice exposed only to filtered air contained 8.65 6.90 10 total cells per lavage sample ( figure 1(a ) ) , of which 92 2% were pulmonary alveolar macrophages , significantly more total cells than were found in the nos1 , nos3 , and c57bl/6 mouse strains exposed only to filtered air ( p < .01 ) .
after two weeks of ova exposure , the total inflammatory cell number recovered by lavage increased significantly in all strains of mice evaluated .
nos2 mice exposed to ova for two weeks had significantly more lung lavage cells than the nos1 , nos3 , or c57bl/6 mice exposed to ova ( figure 1(b ) , p < .05 ) .
the normal lung lavage from a healthy mouse contains more than 90% alveolar macrophages , and our observations in this study were consistent with this finding . there were < 1% eosinophils ( 88 230 eosinophils ) in all of the strains of mice tested after exposure to filtered air ( figure 1(c ) ) .
nos2 mice exposed to ova had 11.90 1.76 10 eosinophils in their lavage fluid , which was significantly more than were observed in nos1 , nos3 , or c57bl/6 mice exposed to ova ( p < .01 ) .
there was a small ( < 17,000 cells ) but significant increase in the number of lymphocytes in all groups of ova - exposed mice compared to filtered air - exposed mice ( data not shown ) , but no significant differences between the four different strains exposed to ova . in addition , the percentage of neutrophils was < 1% in all mouse strains exposed to ova .
these results extend our previous finding that mice lacking the inducible nos2 gene are more susceptible to allergic airway inflammation than wild - type or constitutive nos knockout mice strains . to determine which nos isoform is responsible for the production of no in the expired breath , we compared the concentration of exhaled no between c57bl/6 mice and the three nos mouse strains exposed to filtered air .
all nos strains exposed to filtered air had similar exhaled no concentrations ( figure 2(a ) ) ; nos1 ( 5.1 1.6 ppb ) , nos2 ( 5.0 0.5 ppb ) , and nos3 mice ( 7.5 1.8 ppb ) , respectively .
c57bl/6 mice exhaled a significantly greater no concentration than nos2 animals ( 9.7 0.5 versus 5.0 0.5 ppb , p < .001 ) .
after exposure for two weeks to ova aerosol , there was no significant change in exhaled no concentrations in any of the knockout mice strains compared to their respective filtered air - exposed groups ( figure 2(a ) ) .
however , the nos2 animals exposed to ova had significantly lower no concentrations in exhaled breath than the nos3 mice exposed to ova ( 4.9 0.5 versus 7.7 1.1 ppb , resp . , p < .05 ) .
in contrast , there was a decrease in exhaled no in the c57bl/6 mice exposed to ova compared to their controls exposed to filtered air ( 9.7 0.5 versus 7.2 0.6 ppb , p < .05 ) .
lung lavage nox concentrations of filtered air - exposed mice ( figure 2(b ) ) from mice of all four strains were not significantly different from each other .
after exposure to ova , both nos3 and c57bl/6 mice had significant increases in nox levels compared to their respective filtered air groups .
c57bl/6 mice exposed to ova also had significantly higher nox levels compared to nos1 and nos2 mice exposed to ova .
we examined whether nos isoform protein expression , no levels in exhaled breath , or nox concentrations in bronchoalveolar lavage were correlated by linear regression analysis .
we found that exhaled no levels were lower in c57bl/6 mice after exposure to ovalbumin compared to their matched controls exposed only to filtered air .
total nitrate / nitrite , the more stable products of no metabolism , was increased in the c57bl/6 mice exposed to ovalbumin .
it is unclear to us why this might be a strain - related result .
there is no obvious reason to believe that no consumption is greater in lungs of inflamed wild type mice compared to others .
overall , we have some evidence that total lung no content was increased in the mice after ovalbumin exposure .
there was no correlation between nos2 protein expression in the isolated airways and exhaled no in any of the strains examined ( data not shown ) .
however , there was a significant correlation between nos2 protein expression in the isolated airways and bronchoalveolar lavage nitrate / nitrite concentration in the filtered air and ova - treated groups of the nos3 strain ( band intensity versus nox concentration m = 18.02 6.17 , r = 0.36 , and p < .05 , figure 3 ) .
there were no significant correlations between nox concentration and nos protein expression in the other mouse strains ( data not shown ) . to measure the development of airway hyperreactivity ( ahr ) in our model , we compared the total lung resistance and dynamic compliance at baseline and after inhalation of nebulized methacholine using serial doses of methacholine ( 0.5 , 1.0 , and 2.0 mg / ml ) .
lung compliance decreased significantly in nos2 mice exposed to ova compared to filtered air ( figure 4(c ) ) . the difference in lung compliance between
the air and ova - exposed nos2 mice was significant at each dose of methacholine ( p < .001 for 02.0 mg / ml methacholine ) .
the slope of the mch response by linear regression analysis for nos2 mice exposed to ova ( 2.6 0.27 10 , f = 96.35 ) was significantly different from that of the nos2 mice exposed to air ( 1.8 0.12 10 , f = 226.9 ; p < .05 )
. measured lung compliance after exposure to methacholine also significantly differed for nos3 mice exposed to ova versus air ( figure 4(d ) ) .
the slopes of the best fit lines by linear regression analysis for the methacholine dose - response curve for ova - exposed nos3 mice , 0.5 0.2 10 , f = 4.627 , and for filtered air - exposed nos3 mice was 3.0 0.3 10 , f = 95.07 ( p = .004 ) .
neither the c57bl/6 nor the nos1 mice demonstrated significant decreases in lung compliance with methacholine challenge after exposure to ova ( figures 4(a ) and 4(b ) ) .
lung resistance measurements from the four different strains of mice examined did not show significant differences when analyzed by two - way anova ( figures 5(a)5(d ) ) but differed significantly compared to their corresponding filtered air exposures when analyzed by linear regression analysis . upon challenge with increasing doses of mch , nos1 mice exposed to ova ( slope = 0.266 0.027 , f = 94.09 ) had a significantly greater increase in lung resistance compared to the same strain of mice exposed to filtered air ( slope = 0.079 0.008 , f = 93.15
air- and ova - exposed nos2 mice had similar increases in lung resistance upon mch challenge testing ( slopes = 0.12 0.01 and 0.15 0.02 , resp . ) .
these results suggest that these strains of mice behaved differently than the nos1 or nos3 mice after their respective exposures , which could reflect differences in their inflammatory responses , differences in airway remodeling , or both factors .
the relative amount of the different nos isoforms in airways isolated from each of the mouse strains was assessed using western blot band intensity analysis .
protein blots of airways from both the nos1 ( figure 6(a ) ) and nos3 ( figure 6(b ) ) mice exposed to filtered air showed a higher nos2 protein expression than the c57bl/6 controls ( nos1 60.2 10.7 versus wt c57 bl/6 6.5 3.6 , p < .01 ) and ( nos395.6 28.1 versus wt c57 bl/6 26.6 5.1 , p < .05 ) . upon ova exposure ,
the nos2 expression in nos1 animals was further upregulated ( 96.5 30.1 ( ova ) versus 60.2 10.7 ( fa ) , p = .019 ) , while nos3 animals ( figure 6(b ) ) showed no change in nos2 with ova treatment compared to their filtered air controls .
in contrast , the nos2 mice ( figures 7(a ) and 7(b ) ) showed no significant change in the protein levels of either nos1 or nos3 in the airway samples from mice that were exposed to filtered air or ova .
we analyzed smooth muscle tissue , airway epithelium , and macrophage populations to evaluate differences in nos2 protein content between these three lung tissue compartments .
consistent with the western blot data derived from the isolated airways , nos1 and nos3 mice exposed to filtered air had a greater nos2 protein content than c57bl/6 mice exposed to filtered air ( figures 8 and 9 ) .
quantitative assessment by immunohistochemistry of these air - exposed mice showed that increases in nos2 protein content were limited to the airway epithelium ( nos1 versus c57bl/6 : p < .05 and nos3 versus c57bl/6 : p < .05 ) and the smooth muscle of the airways and vasculature ( nos1 versus c57bl/6 : p < .01 and nos3 versus c57bl/6 : p < .01 ) , but no change in nos2 content was detected in the tissue macrophage population ( figure 9 ) .
ova exposure significantly reduced total nos2 protein in the airway epithelium and smooth muscle tissue compared to the filtered air - exposed animals in the nos3 mice ( figures 9(a ) and 9(b ) , p < .01 ) . in contrast to the nos3 mice exposed to ova , the nos1 mice exposed to ova maintained the nos2 protein content in the airway epithelium and also showed significant increases in nos2 protein content in the macrophage population ( figures 9(a ) and 9(c ) , p < .05 compared to ova - exposed nos3 and filtered air - exposed nos1 mice ) . to determine if the decrease in nos2 protein content in the airway epithelium was due to the replacement of ciliated epithelium with mucus - producing goblet cells in the nos3 animals exposed to ova , we used the periodic acid - schiff ( pas ) to stain for mucus - containing cells in the airway epithelium and determined the overall ratio of mucus - containing cells to total cell population by cell counting .
all of the filtered air - exposed groups contained essentially no mucus - producing cells in the airways ( see , e.g. , c57bl/6 animals ; figure 10(a ) ) .
in contrast , exposure to ova significantly increased the number of mucus - containing cells in all three strains of mice tested ( figures 10(b)10(d ) ) .
we observed that 937% of the total cell population of the upper airway epithelium was mucus - producing cells , and that the observed value depended on the genotype ( figure 11 ) with nos1 mice exposed to ova having significantly fewer pas positive - stained cells compared to the ova - exposed c57bl/6 ( p < .01 ) or nos3 ( p < .05 ) mice .
no modulates pulmonary vascular tone , non - adrenergic non - cholinergic mediated bronchodilation , the lung inflammatory response , and apoptosis [ 2629 ] . in previous work using an nos2 strain , we observed increased airway inflammation as compared to wild - type mice exposed only to air , and especially upon exposure to ova .
we concluded that the ability to upregulate active nos2 enzyme in response to ova exposure is necessary for the anti - inflammatory effect imparted by no .
however , these earlier findings could not define the mechanistic basis for these observations , for example , whether nos2 was itself the required source for no in allergen - induced airway inflammation or whether the effects were caused by increased l - arginine turnover by the other nos isoforms or the reconversion of oxidization products , nitrate and nitrite , into no . to attempt to answer these questions ,
we examined the effects of deleting individual nos isoforms on the overall dynamic of total lung no balance in allergic airway disease . measurements of exhaled no in the various mouse strains tested focused our attention on nos2 as the most likely source of no in exhaled air from mice exposed to ovalbumin ( figure 3 ) .
examination of the correlation between nos2 protein expression in isolated airways and total concentration of nox in the lung lavage fluid showed that the nos3 strain was the only strain studied , where nos2 protein expression was tightly coupled to nox production , and such coupling was observed in the nos3 mice exposed to either air or ovalbumin .
in contrast , the nos1 and nos3 mice actually exhaled more no than their wild - type counterparts .
the upregulation of nos2 we observed in the two constitutive nos knockout strains may explain this finding . in previous work using the nos2 strain , we observed increased numbers of cells in the lavage fluid ( predominantly macrophages ) , which raised the question of whether deletion of either of the other individual nos isoforms ( nos1 and nos3 ) would have similar effects on the lavagable alveolar macrophage population .
because we hypothesized that there would be an upregulation of nos2 in the constitutive knockouts , we expected to find no significant increase in total lung cells in lavage fluid from the nos1 and nos3 animals exposed to filtered air .
our results were consistent with this hypothesis , as we found similar bal total cell counts in the constitutive knockout strains and the c57bl/6 mice . we conclude that the deletion of individual nos isoforms affects no output by specific isoforms , but that the inability to normalize the total lung no concentration by upregulating nos2 results in an increase in the recruitment of bone marrow - derived cells as illustrated by an increase in the population of lavagable macrophages only in the nos2 mice . in this study , nos1 animals exposed to filtered air demonstrated a significant change in lung compliance and resistance upon challenge with methacholine . with increased nos2 protein expression induced by ova exposure
we also saw a significant decrease in the respiratory system compliance of nos2 mice exposed to ova compared to their counterparts exposed to filtered air .
we interpret these findings to suggest that changes in lung compliance in this model directly reflect lung inflammation .
only the nos1 animals exposed to ova demonstrated a significant decrease in lung compliance associated with a concomitant increase in lung resistance that reflects a bronchoconstrictive response .
these data suggest that no from a specific enzyme source in this case from nos1 in the subepithelial smooth muscle and/or the epithelium is responsible for the bronchodilatory properties of no , but that the induction of nos2 may be sufficient to mitigate some aspects of ahr .
attribute decreased nos1 activity in pulmonary and tracheal smooth muscle after ova challenge in guinea pigs to airway hyperresponsiveness , and our data are consistent with these results .
de sanctis et al . also examined changes in inflammatory response in the nos2 strain compared to the c57bl/6 strain but observed no significant increase in total inflammatory cell number .
although our current and previous observations appear to conflict with the findings of de sanctis , substantial variations in the exposure protocol may have caused the differing outcomes .
nos2 is capable of producing one thousand times more no than either of the constitutive isoforms and has multiple levels of regulation that are dependent upon no concentration [ 10 , 3541 ] .
we hypothesized that under normal noninflammatory conditions , deletion of the nos1 or nos3 gene would upregulate nos2 protein expression .
knocking out individual constitutive nos isoforms would lower overall cellular no concentration , resulting in increased nos2 expression in order to maintain a minimal total lung no concentration . to test this hypothesis
, we measured lung no and nox concentrations , total airway nos protein content , and localized the expression of nos2 protein in the four strains of mice ( c57bl/6 wild - type strain and nos1 , nos2 , and nos3 ) exposed to filtered air only . the constitutive nos knockout strains , nos1 and nos3 , exposed to filtered air showed significant upregulation of nos2 protein expression compared to the c57bl/6 wild - type control . as predicted , the nos1 mice showed no change in nos3 expression and nos3 mice showed no change in nos1 expression .
these results support our hypothesis that no homeostasis is established by maintaining a concentration of available total no independent of nos isoform origin .
the nos2 strain showed no significant increase in protein expression of either of the two constitutively expressed nos isoforms ( nos1 or nos3 ) .
these results indicate different upstream regulatory mechanisms inducing gene expression and potential translational control of the two constitutive isoforms and indicate that the control of constitutive nos expression is not no dependent .
thus , we conclude that at baseline , nos2 enzyme production can be induced to maintain total lung no homeostasis via a mechanism that is capable of detecting decreased concentrations of cellular no and activating nos2 expression .
this conclusion is in accordance with observations by cook et al . , who noted increased nos2 expression and exhaled no concentrations in nos3 mice .
immunohistochemical analysis of whole lung sections identified the cellular origins of nos2 expression in the filtered air - exposed genotypes .
consistent with the western blot results , the c57bl/6 mice exposed to filtered air showed low expression of nos2 protein , with only light staining present in the smooth muscle and airway epithelium .
in contrast , both the nos1 and nos3 animals displayed a significant increase in nos2 protein expression compared to the c57bl/6 mice by western blot analysis , and these increases were limited to the airway epithelium and smooth muscle tissue of the airway and vasculature ( see figures 8 - 9 ) . as the airway epithelium constitutively expresses both nos1 and nos3 and is capable of upregulating nos2 under inflammatory conditions , this result implies a tight regulatory control of no homeostasis in these particular cell types .
the smooth muscle tissue of the lung expresses low levels of nos2 protein in the c57bl/6 mice and may benefit from vectorial production of no derived from adjacent epithelial and endothelial cells . as localized no concentrations decrease , nos2 expression
et al , which suggest that induction of nos2 gene expression by nf-b is dependent upon nos3 activity ( figure 8) , we hypothesized that exposure to ovalbumin would inhibit the increase in nos2 protein expression in the nos3 mice . we examined the upregulation of nos2 in nos1 and nos3 mice exposed to ova to determine if the no - dependent mechanism to induce nos2 expression is isoform specific , that is , derived solely from the nos3 isoform .
if the ova - induced increase in nos2 expression is driven by the nos3 , we would expect no significant increase in nos2 protein expression in the nos3 animals while we would expect an increase in nos2 protein expression in the nos1 animals . in our study , the patterns of nos2 expression in the ova - exposed nos1 and nos3 animals were consistent with this mechanism being nos3 specific .
in fact , we observed a surprising lack of nos2 expression in the lungs of nos3 animals overall by immunohistochemistry .
analysis of nos2 expression in the nos1 and nos3 lung compartments of mice exposed to ova indicated a significant reduction of nos2 in the airway epithelium and smooth muscle compartments of the nos3 mice only , with nos1 mice maintaining nos2 protein expression in both compartments , including upregulation of macrophage nos2 expression . while examining nos2 protein content in the airway epithelium , we observed a difference in the cellular population of the airway epithelium between the different mouse genotypes with exposure to ova that may have contributed to changes in nos2 protein content .
one limitation of our study is that we used nos gene knockout strains only and did not use overexpressing mice to confirm our findings .
however , we are encouraged by the complimentary data of ten broeke and colleagues .
they found that transgenic mice overexpressing nos3 increased no production in the lungs , decreased lung lavage inflammatory cell counts , and improved airway hyperresponsiveness compared to littermate controls after exposure to ovalbumin .
one possible explanation for the seeming overlap between ten broeke 's results and ours is the interrelationship among the noss in the lung .
this suggests that important feedback mechanisms to nos3 regulate much of the measurable no that is produced in the lung in response to allergen .
the nos 1 mice had a significantly reduced number of goblet cells present compared to the other three strains .
nos1 mice exposed to ova also had reduced airway reactivity and airway inflammation compared to the other strains , which may indicate roles for nos1 in inflammatory cell recruitment signaling and , potentially , airway epithelial cell apoptosis .
in addition to its effects on airway hyperreactivity , no has pro- and antiapoptotic effects that are concentration and cellular compartment dependent .
the nos1 isoform is also localized to the mitochondria ( mtnos ) and despite the ubiquitous nature of no in the lung during inflammation , deletion of the nos1/mtnos isoform may contribute to the reversal of airway goblet cell metaplasia seen in the other isoform knockout and c57bl/6 strains exposed to ovalbumin .
although we were able to identify increased goblet cell metaplasia in the airways of nos3 mice exposed to ovalbumin as compared to nos1 mice , this change in epithelial cell content did not account for the lack of nos2 protein in the remaining intact epithelial cells .
the cells that were pas - negative still comprised 50% of the airway cell population .
in addition , the lack of nos2 protein in the intact smooth muscle of the airways and vasculature of the nos3 mice was also noteworthy .
though our hypothesis indicates that knockout of nos3 results in an inability to signal for increased nos2 expression upon allergen exposure , an alternative explanation exists for these results that we could not address within the scope of our studies in intact animals .
nf-b - dependent transcription can also be affected by nitrosation of the rel and p50 components of the active nf-b complex . in high no environments such as cell culture medium with high concentrations of no donor compounds added ,
cysteine residues of both p65 and p50 have been shown to be nitrosated , resulting in reduced dna - binding capacity [ 35 , 3941 ] ( figure 9 ) . increased
no production by nos2 under inflammatory conditions may be sufficient to nitrosate these residues resulting in the activation of this negative feedback loop . despite
the lack of nos2 in the lungs of nos3 animals exposed to ova , increases in bal nox concentrations in the nos3 mice may indicate an accumulation of no metabolites in the lung from another source .
there is increasing evidence suggesting that nitrite serves as a bioavailable pool of no to act as a vasodilator , as deoxyhemoglobin may have nitrite reductase activity , promoting this conversion . while we did not measure the arterial oxygen saturation of our mice during this experiment , it is possible that the ventilation - perfusion mismatching as a result of dense inflammatory infiltrate and/or methacholine challenge could lead to regional hypoxemia and have an effect on nox to no conversion .
however , we did not see changes in either no or nox concentrations in nos2 animals after exposure to ova that would be consistent with this pathway .
in addition , it has been theorized that alterations in the ph of the airway lining fluid as a result of inflammatory cell influx and oxidative damage may result in nitrite conversion to no .
both the nos3 and nos2 mice had more inflammatory cells in their bal fluid but their exhaled no concentrations were relatively unaffected .
examination of individual nos isoform knockout and c56bl/6 strains exposed to filtered air supports the hypothesis that there is an nos - dependent mechanism in the cell that maintains a
baseline no production in both airway epithelium and airway and vascular smooth muscle and is unable to distinguish between nos isoforms of origin . under inflammatory conditions ,
the expression of the nos2 isoform is essential for reducing lung inflammation and may also contribute to the normalization of airway reactivity .
nos3 isoform activity is essential for the upregulation of nos2 in response to ovalbumin exposure .
in contrast , nos2 can be expressed independently of nos1 activity in the inflamed lung , but nos1 activity contributes to goblet cell metaplasia in the airways of ovalbumin - exposed animals . |
objectives and design . the function of the airway nitric oxide synthase ( nos ) isoforms and the lung cell types responsible for its production are not fully understood .
we hypothesized that no homeostasis in the airway is important to control inflammation , which requires upregulation , of nos2 protein expression by an nos3-dependent mechanism .
materials or subjects .
mice from a c57bl/6 wild - type , nos1/ , nos2/ , and nos3/
genotypes were used .
all mice strains were systemically sensitized and exposed to filtered air or ovalbumin ( ova ) aerosol for two weeks to create a subchronic model of allergen - induced airway inflammation .
methods .
we measured lung function , lung lavage inflammatory and airway epithelial goblet cell count , exhaled no , nitrate and nitrite concentration , and airway nos1 , nos2 , and nos3 protein content .
results .
deletion of nos1 or nos3 increases nos2 protein present in the airway epithelium and smooth muscle of air - exposed animals .
exposure to allergen significantly reduced the expression of nos2 protein in the airway epithelium and smooth muscle of the nos3/ strain only .
this reduction in nos2 expression was not due to the replacement of epithelial cells with goblet cells as remaining epithelial cells did not express nos2 .
nos1/ animals had significantly reduced goblet cell metaplasia compared to c57bl/6 wt , nos2/ , and nos3/ allergen - exposed mice . conclusion .
the airway epithelial and smooth muscle cells maintain a stable airway no concentration under noninflammatory conditions .
this homeostatic
mechanism is unable to distinguish between nos derived from the different constitutive nos isoforms .
nos3 is essential for the expression of nos2 under inflammatory conditions , while nos1 expression contributes to allergen - induced goblet cell metaplasia . | 1. Introduction
2. Experimental Methods
3. Results
4. Discussion
5. Conclusions |
PMC4458484 | a 54-year - old male presented with painful , swollen bilateral knees following a minor motorcycle accident where he fell from a motorcycle traveling at a relatively low speed .
the accident occurred when a driver backed into the street in front of him riding a motorcycle in an alleyway .
radiographs revealed schatzker type iv tibial plateau fractures involving medial condyles in both knees ( fig .
two years and four months before the accident , the patient had undergone an arthroscopic acl reconstruction using double - bundle technique with tibialis posterior allograft and medial collateral ligament ( mcl ) repair on his left knee at a hospital . in spite of our efforts to obtain more information , such as the type of the screw used and
tunnel size created in the previous operation , additional information from that hospital was not available .
he had the same surgery on his right knee about eight months ago at another hospital .
an arthroscopic acl reconstruction was performed using single - bundle technique with a 25 mm femur endobutton ( smith & nephew endoscopy , andover , ma , usa ) , a 9 mm in length and 25 mm in diameter bio - absorbable interference screw ( conmed linvatec , largo , fl , usa ) , and tibialis posterior allograft .
mcl femoral tensioning using an 11 mm bone staple was performed during the operation with removal of a spiked washer and a screw on the left tibia that had been implanted in the previous acl reconstruction .
due to severe pain , knee joint stability and range of motion could not be assessed .
computed tomography was performed to evaluate the fracture pattern in relation to the acl tibial tunnel , which demonstrated intra - articular involvement of split fractures on the bilateral medial tibial plateaus .
1 ) . considering the unique nature of the fractures , we decided to attempt treatment and confirm the state of the previously reconstructed acl later by second - look arthroscopy .
fracture reduction on the left side was achieved intraoperatively under fluoroscopic control , which was followed by fixation using a six - hole medial locking compression plate ( lcp medial proximal tibial plate ; depuy synthes , oberdorf , switzerland ) with a 6.5 cancellous screw and a washer ( depuy synthes ) for anatomical reduction of the intra - articular surface .
subsequently , removal of the spiked washer and screw on the right tibia was done . in the same way , utilizing a four - hole medial locking compression plate ( lcp medial proximal tibial plate ) with a 6.5 cancellous screw and a washer ( depuy synthes ) , fracture reduction and fixation on the right side was performed . the planned second - look arthroscopic examination for confirming the previously reconstructed acl state
was achieved three weeks afterwards because the patient had suffered from fever and urinary tract infection since immediately after surgery .
examination under general anesthesia revealed negative anterior lachmann test and pivot shift test with feeling of firm as the endpoint .
however , we could not identify the degree of screw resorption or tunnel widening . physical therapy with continuous passive motion was started immediately postoperatively , and protected weight bearing was maintained for two postoperative months . at five months follow - up ,
follow - up radiographs of the patient showed callus formations with healed fractures in both tibias ( fig .
tibial plateau fractures presenting as a serious complication after acl reconstruction are rare , and only isolated cases have been reported .
interestingly , in most of these cases , the tibial plateau fractures occurred through the tibial tunnel .
authors of these reports suggested that the presence of the tibial tunnel could be the main factor in predisposing the patient to the fracture acting as a stress riser3,4,5,6,7 ) .
more recently , a case described by a. gobbi et al.6 ) had some interesting features .
tibial plateau fractures had occurred after primary anatomic double - bundle acl reconstruction following minimal trauma of running backwards during a soccer game in their case .
they suggested that two tunnels made in the proximal tibia might have caused a greater increase in local stresses around the tunnels with less bone available to withstand the applied load compared with a single tunnel .
we could not find any studies that specifically measured bone strength of the tibia following tibial tunnel drilling or compared the mechanical effect of two tunnel creation to one tunnel creation .
however , it has been documented that bone defects , such as screw holes , can concentrate stress and significantly decrease bone resistance to bending and torsional forces8,9 ) .
for instance , diaphyseal drill holes with a diameter greater than 20% of the bone have been shown to reduce bone strength by 55%-90%8,9,10 ) . in our case , the fracture pattern had some unique and distinct features . although the patient fell from a motorcycle while riding it at a relative low speed , the fractures involved bilateral tibias and occurred through tibial tunnels that had been formed during previous acl reconstruction .
furthermore , we could not identify other injuries to the articular structures in our patient .
he had undergone an arthroscopic acl reconstruction using double - bundle technique on his left knee and the same surgery using single - bundle technique on his right knee .
fortunately , the acls on both knees were found to be intact when examined by second - look arthroscopy .
although we were not able to find differences between the effect of single and double - bundle reconstructions on this unique fracture in our patient , it was obvious that the fracture lines on the patient 's tibias extended into the tibial tunnel .
our study suggests that a transosseous tibial tunnel alone can somehow act as a stress - riser that decreases strength at the level of proximal tibial metaphysis .
we would like to emphasize that the surgeon must be very careful in tunnel placement and should not perform extensive bone removal during acl reconstruction .
also , it is crucial to be aware of the possible occurrence of this , albeit rare , postoperative complication considering most cases required surgical fixation of the fractures . | tibial plateau fractures after arthroscopic anterior cruciate ligament ( acl ) reconstruction are rare , and only isolated cases have been reported .
the authors describe a case of bilateral medial tibial plateau fracture following a minor motorcycle accident in a patient who underwent arthroscopic acl reconstruction in the past .
two years and four months before the accident , the patient underwent an arthroscopically assisted acl reconstruction using double - bundle technique on his left knee at a hospital .
he had the same surgery using single - bundle technique on his right knee about eight months ago at another hospital .
the fractures in his both involved knees occurred through the tibial tunnel and required open reduction with internal fixation . at three weeks after fixation , a second - look arthroscopy revealed intact acls in both knees . at five months
follow - up , he was able to walk without instability on physical examination .
follow - up radiographs of the patient showed callus formations with healed fractures . | Case Report
Discussion |
PMC3948198 | literature has identified and defined evening - fever syndrome ( efs ) as the thermal capacitance effect of overheated buildings that manifests in neonatal hyperthermia usually during the mid afternoon and early evening periods of sunny days .
efs affects all newborns , especially the premature escalating morbidity , prolonging hospitalisation period and impoverishing overall outcome .
the effect of efs has been reported from many parts of nigeria with extreme cases occurring within the savannah regions of the middle - belt and the desert northern regions of the country . at the federal medical centre nguru ( fmc - nguru ) located within this zone , efs is a significant contributor to neonatal morbidity resulting from soaring local ambient temperature that goes as high as 47c at times . in a recent publication amadi et al .
this implicated the climate , environment , and civil structures among other reasons for excessive room warming that instigates efs .
this suggested that efs is a condition of time - dependent neonatal hyperthermia that can be clinically diagnosed , typically being associated with the following constituents : it notably occurs on high ambient days , often in excess of 37cit readily affects neonates within room / nursery building of which walls , windows , and/or ceiling are directly exposed to high intensity of rays of sunlightneonates acquire high body temperature during early postmeridiem ( p.m. ) perhaps due to heat transfer from the energised surroundings but regain thermal stability much later in the evening when the surroundings have cooled down .
it notably occurs on high ambient days , often in excess of 37c it readily affects neonates within room / nursery building of which walls , windows , and/or ceiling are directly exposed to high intensity of rays of sunlight neonates acquire high body temperature during early postmeridiem ( p.m. ) perhaps due to heat transfer from the energised surroundings but regain thermal stability much later in the evening when the surroundings have cooled down .
apart from occasional planting of trees within residential compounds in nguru town and the raising of concrete flooring by up to 1 ft above ground level as seen from the structures at fmc - nguru , there are no other building standards that technically suggest adaptation to this extreme high ambient temperature . as a result
the interiors of buildings including the special care baby unit ( scbu ) of this hospital often get higher than 40c of room temperature .
this is significantly high enough to trigger hyperthermal crisis for the immature neonate even for a short period of exposure [ 4 , 5 ] . neonatal thermal stability , defined as the condition of maintaining the body temperature of a given neonate within the physiologically allowable range of 36.5c to 37.4c , is a crucial factor for the survivability of any newborn .
the scbus of nigerian hospitals often respond to this extreme condition of environmental high temperature with all kinds of desperate clinically inefficient measures .
some clinicians often suspect a disease process responding with doses of unnecessary antibiotics and complicating situations and from which many neonates might have lost their lives .
in addition , nurses resort to the desperate practice of opening incubator access windows / doors , turning - on fans , turning - on air conditioners or entirely switching - off the incubator . at the fmc - nguru babies
there are down - sides to these desperate measures : opening port - holes compromises the microenvironment as a mini - isolation unit for the delicate premature baby [ 6 , 7 ] ; fans and air conditioners are subject to adequacy of power supply , frequent breakdown , and threat of hypothermia for cot babies ; incubator switch - off often results in sudden hypothermia during environmental thermal recovery at sunset ; baby bathing has momentary effect only and often demands unaffordable high volume of nursing time as this has to be repeated several times during the day .
this hence results in frustration for the few nurses that would attempt to thermally stabilise any neonate .
we hypothesise that any measures that can achieve naturally cooled nursery environment would eliminate the uncontrollable excessive warming of neonates due to efs .
the relatively poor setting of tropical africa would constitute complications due to factors that are naturally difficult to change or be sustainably managed .
these involve the sun and sunlight intensity as the source of heat generation , the relocation of the hospital complex to a more appropriate site , provision of uninterruptable air conditioning units , or supply of specially designed adaptable incubators to replace a host of the climate - stricken standard models . in the face of these seeming difficulties it became imperative to seek to investigate how design or building structural factors could be optimized for a probable permanent solution [ 9 , 10 ] .
therefore , the aims and objectives of this study were ( 1 ) to develop miniaturised laboratories with distinct unconventional building factors that are capable of ameliorating the warming effect of the sun in a nursery and ( 2 ) to validate these by carrying out a comparative analysis of room warming and neonatal thermal stability of babies being nursed in the laboratories .
the project was divided into two segments involving ( 1 ) design and construction of two different nursery laboratories and ( 2 ) validation of the new constructs through controlled comparative analyses of outcomes .
ethical approval for the execution of the project was obtained from the medical ethics committee of the federal medical centre nguru , nigeria .
patient - informed consents for the validation aspect of the research were also obtained from carers , in most cases the mothers , after carefully explaining the project and how their babies might be minimally involved in the use of data collected during normal nursing / clinical procedures whilst treating them .
it was more difficult recruiting from illiterate carers who were more in number than the educated ones .
a total of 97 neonates were recruited , 33 for the wet season months ( september - october 2012 ) analyses and 64 for the dry season months ( march - april 2013 ) analyses .
average admission - weight was 1966 g ( range : 1050 g2500 g ) .
average gestation age of the sample population could not be accurately quantified as most cases lacked antenatal information .
literature reveals that nursery warming due to extreme sunlight intensity comes through capacitance effect of nursery walls that are directly heated by the sun amongst other mediums .
the laboratory design therefore incorporated a double - wall with in - between air space for all sides that were exposed to direct rays of sunlight .
these were flooring , roof / ceiling height and materials , window size and positioning , window blind materials , and room lighting .
the first ( lab-1 ) represented the construction of a purpose built nursery structure that targeted the total elimination of efs .
this integrated all possible constraints that were capable of minimising the transmission and concentration of sun - heat within the interior of the structure .
the second laboratory ( lab-2 ) represented a possible renovation that could be done to an existing nursery structure so that the warming of the interior by the sun would be considerably reduced .
lab-2 was designed to avoid expensive or extensive constructions that could be either unaffordable or lengthily disruptive to the activities of an existing nursery .
this was implemented by the renovation of an existing office space at the special care baby unit ( scbu ) complex of fmc - nguru .
the double - wall for the side exposed to sunlight was provided by adding an inner wall on that side .
an arbitrary air space of 6 cm was provided in - between the walls and vented into the roof space through the ceiling .
this created escape route for heated air within the space and was expected to enhance the reduction of heat transfer to the inner wall .
lab-2 was finished to a rectangular floor area of 5.9 m , the original ceiling height of 3 m , and one door and one window .
the design of lab-1 extensively integrated other measures that could contribute to further heat reduction on the inner wall and the interior of the structure as explained below.prior to design , a simple thermometric investigation revealed that a significant temperature difference existed between the sandy ground level surface of the site and the bottom of an excavation 90 cm below the surface .
it was therefore considered a good idea to take advantage of this and tap the coldness for the room .
the floor was therefore designed as a 5 cm thick concrete mixture that was set at 120 cm below ground level .
the surrounding single wall continued until ground level where this transformed externally into the double wall as described for lab-2 .
inner walls and floor were finished in smooth ceramic tiles.the floor - to - ceiling height was 380 cm , that is , 80 cm more than the standard height of rooms in the hospital complex .
this was to achieve a good height for warmer air that tends to raise the room temperature at the height of the cots and incubators .
a roof extractor fan that automatically covered its outlet whilst switched - off was provided to maintain the escape of heated air from the room and vented air within the roof space as shown in figure 1.a controlled valve - operated heat exchanger was integrated in the design .
this was a system of flowing water made to traverse the interior surface of the inner wall , behind the tiling .
the copper - piped water entered building from the left end via a manual valve , crisscrossed the entire upper half of the inner wall above ground level , and exited through the right end ( figure 2(a ) ) .
water was supplied from an insulated overhead tank to ensure adequate pressure head and emptied into the same underground tank from which the overhead tank received supply ( figure 2(b ) ) .
the underground water storage served a purpose of recooling the water before recycling to the overhead tank .
heat exchanger was operated during data collection on few prescribed days only for the investigation of any possible contribution such application might offer towards the cooling of lab-1 .
the coolness pattern of lab-1 relative to heat exchanger days was evaluated by the quantification of average temperature rise of the water from the system outlet as a measure of heat extraction from the lab interior.two opposite wall windows were integrated such that these were indirectly facing each other to ensure better air circulation .
cotton material window blinds were affixed and operated when sun - heat intensity through the windows became very intense .
electric lighting of the interior and medical examination lamps were provided by low wattage led system installations to minimise heat generation by these applications . prior to design ,
a simple thermometric investigation revealed that a significant temperature difference existed between the sandy ground level surface of the site and the bottom of an excavation 90 cm below the surface .
it was therefore considered a good idea to take advantage of this and tap the coldness for the room .
the floor was therefore designed as a 5 cm thick concrete mixture that was set at 120 cm below ground level .
the surrounding single wall continued until ground level where this transformed externally into the double wall as described for lab-2 .
the floor - to - ceiling height was 380 cm , that is , 80 cm more than the standard height of rooms in the hospital complex .
this was to achieve a good height for warmer air that tends to raise the room temperature at the height of the cots and incubators .
a roof extractor fan that automatically covered its outlet whilst switched - off was provided to maintain the escape of heated air from the room and vented air within the roof space as shown in figure 1 .
this was a system of flowing water made to traverse the interior surface of the inner wall , behind the tiling .
the copper - piped water entered building from the left end via a manual valve , crisscrossed the entire upper half of the inner wall above ground level , and exited through the right end ( figure 2(a ) ) .
water was supplied from an insulated overhead tank to ensure adequate pressure head and emptied into the same underground tank from which the overhead tank received supply ( figure 2(b ) ) .
the underground water storage served a purpose of recooling the water before recycling to the overhead tank .
heat exchanger was operated during data collection on few prescribed days only for the investigation of any possible contribution such application might offer towards the cooling of lab-1 .
the coolness pattern of lab-1 relative to heat exchanger days was evaluated by the quantification of average temperature rise of the water from the system outlet as a measure of heat extraction from the lab interior .
two opposite wall windows were integrated such that these were indirectly facing each other to ensure better air circulation .
cotton material window blinds were affixed and operated when sun - heat intensity through the windows became very intense .
electric lighting of the interior and medical examination lamps were provided by low wattage led system installations to minimise heat generation by these applications .
lab-1 was finished to a rectangular floor area of 8.3 m , roof height of 3.8 m , and one door and two windows .
the existing nursery section for babies born within the fmc - nguru facility ( in - born unit ) was applied in the study as the control ward while lab-1 and lab-2 were the test - wards .
this has a rectangular floor area of 70.5 m , room height of 3 m , and two opposite french doors and six windows arranged for effective crossventilation with window - blinding shutters .
electric power supply to all three wards was provided via two assembles of 3.5 kva inverter
the power - banks ensured uninterrupted power supply to the experimental wards all through the periods of data collection .
these acted as ready backups during unexpected power failures as normally witnessed in nigerian cities .
environmental temperature , humidity , and other meteorological data within all three wards and the outside of the complex were automatically collected and stored every 30 minutes .
this was achieved by the application of three assembles of watson 's w-8681 weather station .
babies from consenting carers / parents were randomly assigned to available cots and incubators in all three wards .
neonates were nursed and vital signs data were collected by nurses at the usual practice times of the day .
nurses were posted to the wards on rota basis and the fmc - nguru standard neonatal nursing techniques and procedures particularly remained unaltered in the three wards .
this also involved the practice that required any hyperthermic baby to be water - bathed ( sponged ) if body temperature climbed up to 37.9c .
exclusion / inclusion criteria were carried out as follows : it included all preterm neonates during special care nursing.it included all full - term neonates below 2500 g birth weight but was limited to half of the neonatal period ( first 2 weeks of life ) as healthy older neonates might be capable of significant auto thermal regulation .it excluded any neonates undergoing treatment for a well - established disease or disease process .
it included all full - term neonates below 2500 g birth weight but was limited to half of the neonatal period ( first 2 weeks of life ) as healthy older neonates might be capable of significant auto thermal regulation .
it excluded any neonates undergoing treatment for a well - established disease or disease process .
experimental setup and data collection continued for over 24 months , covering every month of the year for up to two times .
data for the latest 12 months were applied for the comparative analyses of outcomes from the three wards in relation to the impacting environmental conditions of the outside surrounding .
the collected data was applied to study impact of extreme hot days in the laboratories and the control ward .
this was treated as an extreme hot day if the day 's peak temperature exceeded 36c .
the entire data covering the 12 months of the year was carefully examined , extreme hot days were identified , and their data were extracted into a file and were applied for further processing as described below.plots of temperature against time - of - the - day were produced in superimposition for all three wards and outdoor temperature .
these were produced for each extreme hot day to study the daily pattern of heat gain or loss in each ward in relation to changes in out - wind temperature.records of relative - humidity ( rh ) against time - of - the - day were studied to assess the suitability of the wards for effective neonatal nursing without extra room humidification .
this was also used to determine how the wards responded to changes in outdoor rh.the daily peak temperatures for each of the four environments ( i.e. , the 2 labs , the control ward and outdoor environments ) for all extreme hot days were identified and their averages for each environment quantified.the averages of the daily relative humidity were quantified for each of the four environments.the behaviour of incubators in all three wards on the extreme hot days under assessment was evaluated from an incubator data collection chart to determine how environmental overheat affected them.all the patients ' temperature chart folders for the period of study were recalled .
patients admitted during the two hottest months of the dry season ( march and april ) and the two relatively hot months of the wet season ( september and october ) were extracted and applied to obtain the total number of baby water - sponging events for each of the three wards being studied.the effect of the heat exchanger was evaluated by activating this on other specific days primarily to test the exchanger only .
temperature of water at laboratory inlet and outlet were measured and noted at 12 noon , 3 pm , and 6 pm on each day .
data was applied to quantify the averages of inlet and outlet water temperatures during the specified times of the day .
this would enable the evaluation of the most cost - effective time of operating such a device to improve heat extraction from the ward .
plots of temperature against time - of - the - day were produced in superimposition for all three wards and outdoor temperature .
these were produced for each extreme hot day to study the daily pattern of heat gain or loss in each ward in relation to changes in out - wind temperature .
records of relative - humidity ( rh ) against time - of - the - day were studied to assess the suitability of the wards for effective neonatal nursing without extra room humidification .
this was also used to determine how the wards responded to changes in outdoor rh .
the daily peak temperatures for each of the four environments ( i.e. , the 2 labs , the control ward and outdoor environments ) for all extreme hot days were identified and their averages for each environment quantified .
the averages of the daily relative humidity were quantified for each of the four environments .
the behaviour of incubators in all three wards on the extreme hot days under assessment was evaluated from an incubator data collection chart to determine how environmental overheat affected them .
patients admitted during the two hottest months of the dry season ( march and april ) and the two relatively hot months of the wet season ( september and october ) were extracted and applied to obtain the total number of baby water - sponging events for each of the three wards being studied .
the effect of the heat exchanger was evaluated by activating this on other specific days primarily to test the exchanger only .
temperature of water at laboratory inlet and outlet were measured and noted at 12 noon , 3 pm , and 6 pm on each day .
data was applied to quantify the averages of inlet and outlet water temperatures during the specified times of the day .
this would enable the evaluation of the most cost - effective time of operating such a device to improve heat extraction from the ward .
the working floor areas of the control ward and labs were not equal in dimension to enable a direct building cost comparison .
however , engineers were commissioned to design and produce bill - of - quantities for 2 equal size structures of floor area of 16.6 m , each based on the construction details of the control ward and lab-1 , respectively .
they also separately quantified the cost of adding double - wall to the two sides of the control ward patterned design .
all quantifications were based on the prevailing costs of building materials and labour within and around the town of nguru nigeria .
this revealed that the sum of 4.69 million naira ( about us$29,000 ) was required to complete the project based on the control , including all fittings and air conditioning .
lab-1 patterned counterpart was estimated to cost 4.93 million naira ( about us$31,000 ) including entire heat exchanger assembly .
extra double wall on two sides for lab-2 pattern would require ngn317,000 ( about us$2,000 ) .
superimposed daily plots of environmental warming for 24 hours of the day typically showed that ward peak temperatures were always lower than the out - wind peak temperature during the day times .
these also revealed that lab-1 peak temperatures were always the lowest and rarely exceeded 33.5c even when out - wind peak soars up to 46c ( figures 3 and 4 ) .
on the contrary , the control ward always registered the hottest peak temperatures of the day amongst the three tested wards .
this was typically lower than the out - wind peak temperatures , often within 3c . on the average ,
lab-1 was constantly much cooler than out - wind peak - temperature often with margins of up to 11c ( figure 4 ) . when the four overall hottest days of the study period ( each registering over 44c peak temperature ) were separately examined as shown in figure 5 , lab-1 remained stably cool at 32.9 0.5c ; range : 32.1c to 33.2c across the four days .
figure 6 shows a chart of the overall average peak temperatures of the entire extreme hot days for the four environments during the extreme hot days of dry season months of march / april and wet season months of september / october with their average relative - humidity ( rh ) . typically , all the incubators in lab-1 maintained set - point values at all times without overheating during the extreme hot days .
the incubators of lab-2 overshot set - point on few occasions while those of the control ward were constantly overheated beyond set - point values .
figure 7 shows the typical performance of three incubators set at the same operational set - point value of 32.2c in the three respective wards as captured on october 17 , 2012 .
figure 8 shows the outcome of baby water - sponging events as a result of efs - induced hyperthermia in the 3 wards for the months of march , april , september , and october . on the overall impact on the wellness of neonates ,
70% ( representing 28 ) of babies in the control ward were water - sponged 63 times during the wet season months of september and october . during the dry season months of march and april , 100% ( representing all 16 )
lab-1 folders revealed only 2 water - sponging events on 1 of 13 participating babies during the dry season months of march and april .
there was no reported case of water sponging in lab-1 all through the wet season study period .
we observed that preterm and low - birth - weight ( lbw ) babies were quicker to succumb to efs attack as compared to full term and bigger neonates .
in addition to excessive high body temperatures , attack was found to be associated with relative increase in heart - rate ( hr ) most of the time .
respiration - rate ( rr ) was also affected as this was faster than normal in some patient but slower than normal in other patients during efs attack .
water sponging would bring about sudden quench of fever for a short period of time .
this improved rr in some patients for the short time but this did not necessarily slow down hr during this period .
figure 9 is a graphical display of the signs of skin - temperature , hr , and rr in a typical case of comparing physiological events in the control ward and lab-1 during a 5-day period from the very hot month of april 2013 .
the two babies being compared were water - sponged several times during the day due to efs attack whilst they remained in the control ward .
however when one of them was transferred to lab-1 on april 11 just before midnight , this baby recovered and never had any need for water sponging again for the rest of the comparing period unlike its counterpart that remained in the control ward ( figure 9 ) .
there was observable difference in the relative coolness of lab-1 with the heat exchanger operation , especially just before the sun began to set at 6 pm .
average inlet / outlet temperatures of water through the exchanger are shown in table 1 .
the present study has sought to create and validate a method of building a sustainable weather - proof neonatal nursing environment that can curb morbidity due to tropical evening - fever syndrome ( efs ) . at study inception
we hypothesised that a naturally cooled nursing environment with minimised heat - impact of the sun would guarantee effective incubator performance in maintaining desired incubation set - points without externally influenced overheating .
we also hoped that such thermal - friendly environment would guarantee a more stable nursing of open - cot babies within the environment with lower frequency of hyperthermia due to efs .
an earlier study demonstrated that neonates readily tend to assume body temperatures similar to their environmental room temperatures when exposed to these .
this is consequential to the classical procedures of maintaining effective neonatal warming in a cold environment using appropriately designed wrapping suits for full - term neonates and adequately controlled incubator microenvironments for preterm neonates [ 12 , 13 ] .
preterm neonates exhibit higher ratio of skin - surface - area to body - mass as compared to full - term neonates .
this delimits the preterm neonate 's ability to autoregulate its body temperature when exposed to a room temperature outside the physiological range .
therefore , efs can fundamentally set in when environmental room temperature exceeds the acceptable physiological body upper limit of 37.4c .
the present study created lab-1 and lab-2 , testing their respective abilities to maintain room temperatures that were well below this physiological limit even with prevailing extreme heat intensity from the sun .
our lab-1 results have shown that by carefully integrating some factors as considered in this study , a naturally cooled environment could be guaranteed .
lab-2 was relatively warmer than lab-1 because this lacked the additional cooling effect the underground floor / walls provided in lab-1 .
the control ward 's room temperatures readily exceeded this physiological upper limit as can be seen from figure 6 .
this consequently led to a very frequent manifestation of efs , incubator overheating , and general un - wellness of babies in this ward .
in general , the room temperatures of all the wards during the early hours of the day were far below the hypothermic threshold of 36.5c and at times as low as 26c
. this condition would normally require the incubators to operate at high set - points to supply extra heating for the preterm neonates while cot - babies were given adequate extra wrapping .
the room temperatures of our control ward changed very rapidly with the appearance of the sun and its rising intensity as the day progressed into the afternoon .
this rapid increase of room temperature resulted in additional uncontrollable heating that would soon overheat the cot - babies , the incubator , and its occupant . in most cases
the attending nurses were unaware of the hyperthermic babies until the next vital - signs check that happened only once every 4 hours ( 6 am , 10 am , 2 pm , 6 pm , and 10 pm ) , with 2 am being verily inconsistent .
the only exception to this was in cases where the uncomfortable babies reserved any energy to cry or show signs of unrest or the incubators produce overheat alarm .
in extreme cases complete switch - off of incubator heating would not remove the problem as the sun singularly supplied enough energy to warm the nursery beyond 38c .
this results in more and more babies being given the ultimate solution of temporary comfort of water sponging .
we do not yet know of the consequences of such sudden quench of high fever on the neonates as this has not been studied .
however , such practice of sudden temperature crash from hyperthermia to hypothermia and a quick buildup back to hyperthermia without any obvious thermal stability creates a condition of neonatal thermal shock that could readily initiate apnoeic attacks .
this extreme complication was however not observed in the test wards due to their relative coolness and more sluggish rate of temperature increases as compared to the control ward .
lab-1 maintained an average temperature - gain rate of 0.75c / hr to average maximum peak of 33 1c as compared to the control ward at average rate of 1.63c / hr to average maximum peak of 39 1c .
this situation guaranteed a more stable and relatively cooled lab-1 enabling the incubators to solely control their neonatal microenvironments , maintaining their operational set - points for the entire day .
the operational effectiveness of the new lab designs in maintaining such coolness in a life - size nursery unit becomes more believable when lab-1 relative disadvantages are considered .
lab-1 , for example , was only a small room less than 12% of the area size of the control , with only 2 windows and 1 door .
hence , this lacked the airy advantage that could possibly aid the cooling of the control . during the period of this experimentation
, nurses reported instances when incessant hyperthermia would have led doctors to suspect a disease process and hence commenced antibiotic treatment as a matter of procedural practice .
instead , some of the nonparticipating patients were temporarily moved to lab-1 ( one at a time due to lack of cot space ) and the patients quickly recovered , similar to the example in figure 9 .
this clearly demonstrated no presence of disease for which the use of antibiotic would have led to complications that could be capable of claiming the life of such baby .
lab-1 clearly eliminated efs but still required well - trained nurses for effective dynamic regulation of the incubator to determine the appropriate set - point that could stabilise baby within the safe zone of 36.5c37.4c
. baby could become hypothermic if lower - than - required incubator set - point was maintained .
this explained why baby control - lab-1 showed signs of hypothermia during the first few hours in lab-1 ( figure 9 ) .
this revealed that the control ward was overheated far above the physiologically acceptable neonatal upper limit temperature of 37.4c .
this explains why so many water - sponging events were recorded in the control on the one hand ( figure 8) . on
the other hand , the temperature peak values of the 2 labs never rose up to this physiological limit , resulting in the relative neonatal stability as depicted by figure 8 .
we can therefore conclude that neonatal efs can be reduced by correcting an existing scbu building using the specifications of lab-2 or where the funds could be afforded , eradicated by the construction of entire new structure with the specifications of lab-1 .
the cost of erecting lab-1 patterned structure might be slightly higher than that of the control ward based on projected bill - of - quantity ; however , this could be quickly offset by the running cost of the air - conditioning units in control ward during operation . at experimented heat extraction factor of 2.2c / unit - time
the heat exchanger time of operation would be between 3 pm and 4 pm for best result .
further study would investigate a possible link of disease appearance to the presence of efs .
we do not understand why efs resulted in extremely low rr sign in some patients and extremely high in others whilst hr remained slightly high .
( 2009 ) noted that tachycardia is a sign of fever ; this is consistent with the high rr in our observation .
however , the cases of lower rr might as well be associated with the thermal - shock effect explained earlier and hence a gradual approach of apnoea . a foreseeable limitation to the application of the present proposals ( i.e. , lab-1 ) is in the event of flooding .
since such nursery design is likely going to be the lowest floor in the hospital complex , adequate allowance to prevent any possible occurrence of flooding must be incorporated in the design .
adequate precautions should be taken to avoid development of wall cavities that could lead to stagnation of leaking water as this could become breeding grounds for mosquitos and dampness with possible consequences of malaria or other infections . | neonatal thermal stabilisation can become challenging when uncontrollable factors result in excessive body temperature .
hyperthermia can rapidly slow down baby 's progress and response to treatment .
high sunlight intensity in tropical countries such as nigeria manifests in incessant high neonatal temperatures towards early evenings .
the ugly consequences of this neonatal evening - fever syndrome ( efs ) can only be eradicated by the development of a controlled weatherproof nursery environment .
two laboratories and a control ward ' were applied .
lab-2 was a renovation of an existing room in a manner that could correct an existing nursery .
lab-1 was an entirely new building idea .
the laboratories were assessed based on comparative ability to maintain environmental coolness and neonatal thermal stability during hot days .
data collection continued for 12 full calendar months . on average ,
at evaluated out - wind peak temperature of 43c ( range : 41c46c ) , the control - ward peak was at 39c , lab-2 peak at 36c , and lab-1 peak at 33c .
all incubators in the control overheated during the hot periods but there was no overheating in lab-1 .
forty - four ( 86% ) of sampled babies were fever - quenched by water sponging 131 times in the control whilst only one baby received same treatment in lab-1 .
nursery designs patterned after lab-1 can significantly reduce efs - induced neonatal morbidity . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion |
PMC3015299 | laparoscopic inguinal hernioplasty is being performed more frequently everywhere , now that several well - designed prospective and randomized studies have shown that it produces results that compare favorably to the open conventional approaches with the added and well - known advantages of this type of mini - invasive surgery . however , like any other surgical procedure , it has complications , some of which appear to be unique to this technical modality like intestinal obstruction related to tapp .
a 65-year - old white male was seen in our office with the main complaint of bilateral inguinal hernias diagnosed elsewhere . he had had an appendectomy 15 years before .
he recently complained of pain in the right side and was referred to our office by his brother , who had been operated with the same diagnosis using laparoscopy in 1993 .
the rest of the clinical examination was unremarkable , and a chest film and electrocardiogram and pre - operative laboratory workup were within normal limits .
accordingly , on january 22 , he was admitted to the spanish hospital in mxico city and , under general endotracheal anesthesia , a 2 cm lower umbilical incision was made . under direct vision
the anterior fascia of the rectus muscle was incised and its fibers retracted . the preperitoneal space was dissected and a balloon trocar was inserted and advanced to the level of the pubis . during the dissection of the preperitoneal space , it was observed that the peritoneum was torn at the site of the previous appendectomy incision , and the procedure was converted to a transperitoneal approach .
two 10 mm trocars were placed in each flank under direct vision and transillumination to avoid injury to the epigastric vessels .
the right peritoneal incision was extended and a large indirect hernia with an accompanying direct defect were seen .
a lipoma was excised , and the peritoneal sac was dissected free up to the level of the umbilicus .
a 15 7 cm polypropylene mesh was introduced , unrolled and properly placed and anchored using a 5 mm tacker ( origin medical systems , inc .
, menlo park , california ) in cooper 's ligament , the upper medial corner of the hesselbach 's triangle , and in the lateral upper aspect above the level of the anterior and superior iliac spine .
the peritoneal defect was closed with a running suture of 00 pds ( polydioxanone ethicon , ltd , u.k . ) , ( figure 1 ) .
a similar procedure was carried out in the left side for a smaller indirect hernia .
the patient was discharged eight hours later with normal and stable vital signs , tolerating well a liquid diet .
the next morning he requested pain medication at home and took the analgesic prescribed ( ketorolac p.o .
10 mg ) , and soon after that he presented nausea and several episodes of forceful vomiting .
he was seen and examined at the emergency room at which time he was found to have normal vital signs , mild dehydration and abdominal distention with hyperactive bowel sounds without rebound .
he was hospitalized , a nasogastric tube was inserted , intravenous fluids and a second generation cephalosporin was given iv .
he failed to improve overnight , having a large ng aspirate , and he developed inguino - scrotal swelling for which reason he was taken to the operating room and re - operated using general endotracheal anesthesia . upon introduction of the 10 mm laparoscope , it was obvious that 30 - 35 cm of small intestine loops were herniated through a right peritoneal suture defect ( figure 2 ) . with the aid of two 10 mm babcock - type of forceps ,
all the loops of small bowel were carefully extracted from the defect and none of them showed signs of severe ischemia nor perforation .
the preperitoneal defect was irrigated and 10 ml of normal saline containing 1 g of kanamycin were left in .
the peritoneal defect was closed using interrupted figure of x stitches of polydioxanone until a water - tight closure was achieved ( figure 3 ) .
the postoperative period was uneventful ; he passed flatus in the second postoperative day , tolerated a liquid diet and was discharged on the 3rd postoperative day on oral antibiotics for an additional 10 days . he has remained asymptomatic to this date , having assisted to the office several times and is satisfied with the result of the operation .
closure of the defect with interrupted figure of x stitches using 00 polydioxanone .
at least nine prospective studies have compared the frequency of complications between laparoscopic and open inguinal hernia repair .
overall , the complication index is higher for the open procedures ( 22% vs 18% ) , though there is not statistical significance .
, in their multicentric prospective and randomized study , also found a higher morbidity for the open techniques ( 20.3% vs 20.1% ) even though in this report the use of extra trocars , rupture of instruments , and conversion from tapp to tep techniques were considered complications .
fortunately , most of the complications following laparoscopic hernia repair are minor ( seromas , hematomas , pneumoscrotum , orchitis , etc . ) although there are some serious complications of the operation like nerve entrapment and intestinal obstruction .
the latter is rare and may be caused by herniation of an intestinal loop through a port incision ( usually bigger that 5 mm ) when the wound has not been closed correctly .
we found at least 15 cases reported in the literature of intestinal obstruction due to herniation of the intestines or adhesions at the site of peritoneal closure after a transabdominal preperitoneal inguinal repair ( tapp ) . in five of these patients reoperation
was done laparoscopically , whereas in three it was done by laparotomy and in seven cases the approach was not specified .
one patient with a staple peritoneal closure developed an internal herniation , was re - operated by laparoscopy and presented a second intestinal obstruction in the same place that had been closed again with the staples and had to be re - operated for the second time by laparotomy .
herein reported - the 420th consecutive laparoscopic inguinal repair in our group since 1992 - it is possible to speculate that the vomiting crisis that our patient suffered soon after the analgesic ingestion could have caused a sudden increase of the intra - abdominal pressure leading to herniation of intestinal loops through the peritoneal inguinal closure . in general , the authors agree that staple closure can be defective if a large gap is left between them and that this may lead to the shower - curtain effect , which also occurred in this patient who had a running stitch type of closure .
our group previously repaired peritoneal defects with a running - suture or figure of x interrupted stitches with 00 pds ( polydioxanone ) and , as shown in figure 1 , the closure seemed to be adequate , initially .
after the presentation of this complication , our current opinion is that when tapp is indicated , a safer closure should be done with interrupted figure of x stitches , leaving a space of not more than 0.5 cm between each .
the procedure of choice at the present time is to carry out a tep procedure whenever it is possible , in which this complication is avoided .
there are , though , indications for a tapp procedure with advantages , and a reduced number of tep procedures will be converted .
so , it is necessary to keep in mind the possible complications of the former method to prevent them and treat them correctly once they appear .
finally , we think that the approach of choice in a reoperation of this type of case is laparoscopy because the lysis of adhesions and the internal herniation can be solved readily with this method .
if needed , the umbilical trocar incision can be lengthened 3 or 4 cms to assist the procedure in order to exteriorize necrotic intestinal loops to perform the resection / anastomosis procedure and introduce them back into the abdominal cavity . alternatively ,
if deemed necessary , a formal laparotomy can be done once the diagnosis has been established .
even though laparoscopic inguinal repair has gained popularity due to its results and advantages , it should not be forgotten that , as in any other surgical procedure , complications may appear and some of them can be serious or even lethal .
a water - tight peritoneal closure in the tapp procedure , using interrupted stitches with an adequate closure of the rest of trocar wounds , should reduce the risk of postoperative intestinal obstruction , considerably . | a case is presented of a male patient that presented with intestinal obstruction in the early postoperative period of a transabdominal preperitoneal inguinal repair ( tapp ) that was diagnosed and repaired successfully using laparoscopy .
whenever a total extraperitoneal procedure ( tep ) can not be performed , the peritoneal closure of the tapp should be done water - tight using interrupted stitches of absorbable monofilament sutures . | INTRODUCTION
CASE REPORT
DISCUSSION
CONCLUSIONS |
PMC4454767 | thyroid nodules and thyroid cancer are more common in women [ 1 , 2 ] .
the incidence rate of thyroid cancer ( tc ) is increasing , which could be due to better surveillance techniques .
nevertheless , other factors are probably involved because the incidence rates of tumors of all sizes increased .
as the increase in incidence of tc appears to be higher in women , sex hormones might have an etiological role .
reproductive factors , as history of uterine fibroids , greater number of live births , greater number of reproductive years , and greater estimated number of ovulatory cycles , were associated with an increased risk of tc . also , during pregnancy , thyroid nodules were shown to increase in number and size [ 7 , 8 ] , and tc has been shown to affect one in a thousand pregnant women , being the second most common type of cancer in pregnancy .
several studies have shown thyroid cells growth in response to estrogen [ 1013 ] , as reviewed recently , and some data suggest a decrease in differentiated function by this hormone in thyroid cells [ 11 , 15 ] .
progesterone might have a protective effect on the thyroid because the use of oral contraceptives and menopausal hormone therapy , with the combination of estrogen and progesterone , were not associated with thyroid cancer risk , but irregular menstrual bleeding , usually due to anovulatory cycles , has been associated with it .
although progesterone could exert its effects through genomic or nongenomic mechanisms [ 17 , 18 ] , its primary action is to regulate the expression of target genes through binding directly to its cognate sequence in the genes , called progesterone responsive elements .
as pr have been variably described in thyroid follicular cells [ 2023 ] , the aim of the present study was to evaluate the effects of progesterone on gene expression in a model of normal human thyroid cells in primary culture .
tissue samples were obtained from patients submitted to thyroidectomy as part of treatment for differentiated tc . after evaluation by two pathologists , normal tissue that would have been discharged
the project was submitted to , and approved by , the research ethics committee of the hospital de clnicas de porto alegre , porto alegre , rs , brazil ( cep : 09 - 454 ) , which waived a written informed consent . in accordance with the resolution hcpa 02/97 , based on the resolution cns 196/96 of the national health council , brazil , and the guideline 9 of the international ethical guidelines for biomedical research involving human subjects ( cioms , who , geneva , 1993 ) , there was no need to obtain informed consent of the patients , because only after the surgical procedure the researchers would know if a tissue sample would be available , they would not know the identity nor had access to the files of the patients , and the tissue samples included would have been discharged by the pathologists .
the authors signed the statement of commitment to use of data , which states that no data that could identify patients would be used and also that their anonymity would be preserved in any publication of the results .
briefly , thyroid tissue was cut in fragments of about 1 mm and digested by 3 mg / ml type i collagenase ( gibco , grand island , ny , usa ) . the suspension of cells was sequentially filtered through nylon meshes ( 250 , 150 , and 60 m pore size ) and the filtered fraction , containing epithelial thyroid cells , was resuspended and seeded in a 35 mm petri dish at a density of 1 10 cells / cm .
thyroid cells were cultured initially in ham 's f-12 coon 's modification medium supplemented with 10% fetal bovine serum , 10 g / ml insulin , 5 g / ml transferrin , 1 mu / ml tsh , and 100 u / ml kanamycin at 37c with 5% co2 ; after two changes , the concentration of serum was decreased to 5% ( 3h medium ) .
five or 7 days later , cells were deprived of tsh for 48 hours ( 3h medium minus tsh : 2h medium ) ; subsequently , they were treated with 2h medium and progesterone , mifepristone , or vehicle ethanol , as follows : group 1 : 2h medium ; group 2 : 2h medium + 20 u / ml tsh ; group 3 : 2h medium + 20 u / ml tsh + 10 nm progesterone , and group 4 : 2h medium + 20 u / ml tsh + 10 nm progesterone + 100 nm mifepristone , for 4 h or 48 h , before total rna isolation .
total rna was isolated using the trizol reagent ( invitrogen life technologies , carlsbad , ca , usa ) .
concentration and purity were assessed by the nanodrop nd-1000 spectrophotometer ( nanodrop technologies , rockland , de , usa ) and stored at 80c .
1 g total rna was transcribed into cdna by superscript iii reverse transcriptase ( invitrogen life technologies ) and stored at 20c .
expression of ki-67 , thyroglobulin tg , thyroperoxidase tpo , and sodium - iodide symporter nis genes was assessed by quantitative reverse transcriptase polymerase chain reaction ( rt - qpcr ) on applied biosystems stepone real - time pcr system using kit platinum sybr green qpcr supermix - udg ( invitrogen life technologies ) .
the primers used for the specific amplification of tg , tpo , and nis were described previously .
the sequences of the primers for progesterone receptor were 5-acccgccctatctcaactacc-3 and 5-aggacaccataatgacagcct-3. annealing temperature of 60c was used for amplification ; dissociation curves were performed by running a gradient of 6095c to confirm the specificity of the pcr amplification .
all samples were amplified in triplicate , and cdna standard curves were constructed using the threshold cycles with five successive 10-fold dilution points of a pool of cdna samples .
rt - qpcr experiments were performed in duplicate and repeated in three ( nis gene ) or four ( ki-67 , tg , and tpo genes ) independent experiments , with cells isolated from different patients .
differences in the means of the treatment groups were evaluated with the generalized estimating equation ( gee ) .
after gee , post hoc bonferroni test was used to identify which means were different .
all statistical analyses were performed with spss software , version 18.0 ( spss , chicago , il ) , and considered significant when p was less than 0.05 .
thyroid follicular cells viability and differentiation were assessed by characteristic morphology and by staining tpo protein in immunocytochemistry . as showed in figure 1(a ) , the thyroid follicular cells isolated in our model present a cuboid shape , with the presence of many vacuoles around the nucleus as observed in previous studies of primary culture of follicular cells thyroid . also , as other epithelioid cells , these cells grow as islands and form a monolayer with extensive cell - to - cell contact . in figure 1(b ) , tpo protein staining , characteristic of thyroid follicular cells , is shown . the specific thyroid genes expression ( tg , tpo , and nis )
tsh increased significantly the nis gene expression ( 2.08 times ) after 4 h ; it also increased significantly the tg ( 2.39 times ) , tpo ( 1.58 times ) , and ki-67 ( 1.87 times ) genes expression , after 48 h , as shown in figure 2 . to evaluate the effect of progesterone treatment on the expression of nis ,
gene expression of progesterone receptor was present in all thyroid cells cultures used in this study ( data not shown ) .
when thyroid cells were treated with 20 ui / ml tsh plus 10 nm progesterone , the mean expression of nis mrna after 4 h , and the mean expression of tg , tpo , and ki-67 mrna after 48 h , increased , respectively , by 1.75 , 1.77 , 1.68 , and 1.95 times , when compared to the group treated with tsh ( figure 3 ) .
in the present study , for the first time , an effect of progesterone on thyroid cells was demonstrated .
genes involved in the differentiated protein expression of thyroid cells were upregulated by progesterone , as well as ki-67 , a marker for growth ; these effects were abolished by mifepristone .
isolated human follicular cells derived from normal thyroid tissue have been studied previously [ 27 , 28 ] , with high functional correspondence with the original follicular cells . in our model ,
tg , tpo , and nis mrna were detected even when the cells were deprived of tsh for 48 h , which could be due to other components of the medium and its supplements , such as insulin , or constitutive activation of thyroid cells .
it is known that thyroid cell regulation varies in different species , and , sometimes , the cells can have different phenotypes and responses to tsh [ 29 , 30 ] . in our model , normal human thyroid follicular cells in primary culture were shown to be differentiated and responsive to tsh .
the response of gene expression to tsh has been shown to vary with the gene and time after treatment .
as no direct effect of progesterone on thyroid cells has been described until now , we have no other studies to compare our results with . as progesterone increased the expression of genes involved in growth and differentiated function of thyroid cells , its effect on the thyroid gland could be protective .
observed an increase in free t4 levels in progesterone - treated women , and a tendency towards lower tsh levels during progesterone treatment , in a randomized controlled progesterone trial .
our results suggest that progesterone effects on human thyroid cell occur via a transcriptional level through its nuclear receptor , since a specific antagonist , mifepristone , abolished it .
the ability of mifepristone to abolish progesterone effect in thyroid cells suggests that these receptors are functional in these cells , although its concentration in normal thyroid has been described as low or undetectable [ 22 , 23 ] . in a recent study
pr expression was seen in 75.8% of patients with papillary thyroid cancer . in our study , pr gene expression was identified in all cell cultures . as mifepristone blocks also the action of glucocorticoids and some synthetic progestogens , as medroxyprogesterone , are ligands for the glucocorticoids receptor ( gr ) ,
the effects described in the present study could have been mediated by these receptors . nevertheless , the ability of progesterone to bind to gr is disputable .
although the model used in the present study has contributed to the understanding of thyroid cell growth regulation , the increase in gene expression does not always result in a proportional increase in functional proteins .
besides , our monolayer model does not reproduce the complex organization of thyrocytes in the follicle , so functional and growth studies are necessary to better understand a possible role of progesterone in thyroid cells .
this work provides evidences that progesterone has a direct effect on thyroid cells , through its receptor , upregulating genes involved in thyroid function and growth . | thyroid cancer and thyroid nodules are more prevalent in women than men , so female sex hormones may have an etiological role in these conditions .
there are no data about direct effects of progesterone on thyroid cells , so the aim of the present study was to evaluate progesterone effects in the sodium - iodide symporter nis , thyroglobulin tg , thyroperoxidase tpo , and ki-67 genes expression , in normal thyroid follicular cells , derived from human tissue .
nis , tg , tpo , and ki-67 mrna expression increased significantly after tsh 20 ui / ml , respectively : 2.08 times , p <
0.0001 ; 2.39 times , p = 0.01 ; 1.58 times , p = 0.0003 ; and 1.87 times , p <
0.0001 . in thyroid cells treated with 20 ui / ml tsh plus 10 nm progesterone , rna expression of nis , tg , and ki-67 genes increased , respectively : 1.78 times , p < 0.0001 ; 1.75 times , p = 0.037 ; and 1.95 times , p < 0.0001 , and tpo mrna expression also increased , though not significantly ( 1.77 times , p = 0.069 ) .
these effects were abolished by mifepristone , an antagonist of progesterone receptor , suggesting that genes involved in thyroid cell function and proliferation are upregulated by progesterone .
this work provides evidence that progesterone has a direct effect on thyroid cells , upregulating genes involved in thyroid function and growth . | 1. Introduction
2. Methods
3. Results
4. Discussion
5. Conclusion |
PMC5323018 | roughly
2.7 billion people depend on the burning of biomass and
other solid fuels in three stone fires ( tsf ) and other traditional
cookstoves for their day - to - day cooking purposes .
cookstoves have environmental and health impacts on an
enormous scale , due in large part to emissions
of products of incomplete combustion ( pic ) such as co , pm2.5 , methane ( ch4 ) , and polycyclic aromatic hydrocarbons
( pahs ) .
black carbon ( bc ) , commonly known as soot , is an aerosol component
formed during combustion that is estimated to have the second highest
global warming impact after co2 .
approximately
25% of global annual bc emissions and 6080% of africa s
and asia s bc emissions that are not from open burning ( e.g. ,
wildfires ) are from domestic solid fuel combustion .
bc is coemitted with organic carbon ( oc ) , a component which
is often regarded to have a cooling impact on climate , although recent studies suggest that the oc fraction ( generally
called brown carbon or brc ) that absorbs radiation at short wavelengths
contributes significantly to warming .
the net climate impacts
of cooking - related aerosol emissions are uncertain , though likely
warming .
replacement of traditional cookstoves with alternative technologies
thus has the potential to provide considerable climate and health
benefits by reducing emissions and human exposures .
efforts to reduce these impacts have spurred the development
of
a range of alternative cookstoves with varying configurations , levels
of sophistication , and performance .
models range from rudimentary
low - cost cookstoves often built from local materials to mass produced
state - of - the - art forced draft cookstoves ( fdcs ) which use electrically
driven fans for improved combustion efficiency . using laboratory emission
factors ( ef ; g ( kg wood ) ) , grieshop et al .
estimated that health ( quantified as daily intake
of pm2.5 for users ) and climate impacts ( quantified as
global warming commitment or gwc ) of various cookstove - fuel combinations
can each span 2 orders of magnitude , with all biomass - burning cookstoves
having greater impacts than modern fuel stoves such
as lpg and kerosene .
this variation across cookstoves types
and the need to benchmark
performance has led to the development of a tier framework , with emissions and other parameters quantified
during standardized laboratory testing ( e.g. , the water boiling test ;
wbt ) . while laboratory testing is required
for benchmarking , field data indicate that laboratory tests typically
greatly overestimate performance relative to in - home use .
for example ,
field pm efs are 25 times higher than those measured during
wbt tests .
this is important because benefit estimates for alternatives
rely on accurate estimates of real - world performance .
fdcss have the potential to greatly reduce emissions via improved
combustion efficiency ; their laboratory pm2.5 and elemental
carbon ( ec ) efs are an order of magnitude lower than those for traditional
stoves , putting fdcss in the highest tiers ( 3 or
4 ) for indoor pm emissions .
however , in - field measurements of emissions
from fdcs are limited , report real - time bc concentrations ( not efs ) , and neglect other species ( e.g. , co2 , oc ) .
carbon finance has been held up for its potential to yield
cobenefits
by enabling access to improved cookstove technologies by poor households .
while flagging carbon markets and evidence from early efforts call
the near - term practicality of this into question , it remains an important
possible source of finance .
current carbon finance methodologies include
greenhouse gases ( ghgs ) but fail to account for the climate impact
of pics such as bc , co , oc , and non - methane hydrocarbons ( nmhc ) , mainly
because of the high uncertainty and variability in their emissions and the differing spatial and temporal scales of their impacts relative
to ghgs .
bc dominates cookstove pic climate impacts and has become a focus for mitigating near - term climate change .
in response
, the gold standard foundation recently developed a simplified
methodology , which relies on either laboratory
or ( preferred ) field - based emission measurements , with the latter
collected using the kitchen testing protocol ( kpt ) , to incentivize bc mitigation efforts .
however , the gold
standard does not require field emission measurements
to be made ( only measurements of fuel use reductions ) , and as noted above few cookstoves have actually
been measured in the field .
therefore , an important missing piece
is a rigorous understanding of in situ emissions from cookstove technologies
and the extent to which the emission reductions indicated by laboratory
testing are achieved under real - world conditions .
to address
this gap , we conducted an evaluation of in - field emissions
in malawi , africa focusing on two pre - existing cookstove programs .
the specific objectives of this work were : 1 ) to measure fuel use
and emission factors from in - home use of alternative and baseline
household and institutional cooking technologies ; 2 ) compare emission
factors and rates with existing measurements and emission tiers ;
3 ) analyze real - time optical properties ( absorption and scattering )
of aerosols during in - home use ; and 4 ) estimate and compare health
and climate impacts / benefits suggested by lab and field - based measurements .
malawi
is a small , landlocked country in southeastern africa in which over
90% of the population uses biomass as their main source of domestic
energy .
it is one of the most densely populated
countries in sub - saharan africa and among the poorest in the world ,
ranking 173 among 188 countries in human development index .
high poverty rates , dependence on unsustainably
harvested firewood , and a predominantly rural population means there
is a great need for improvements in household energy systems and makes
malawi an ideal location to study the impacts of alternative cookstove
technologies .
household emission measurements of uncontrolled
in - home cookstove use ( following the kpt protocol ) took place during routine cooking activities in september - october
2015 ( the hot and dry season ) .
emission tests were completed in two
communities on cookstoves at opposite ends of the technology spectrum
discussed above . in one ,
the cooking and pneumonia study ( caps ; www.capstudy.org ) , led by the
liverpool school of tropical medicine ( lstm ) , distributed limited
quantities of two fdcs models ( philips hd4012ls and ace-1 ; cost 90
usd ) as part of pilot activities for this community - level randomized
controlled trial of the effects of the philips hd4012ls cookstove
on the incidence of pneumonia in children under the age of 5 .
fdcs use was not widespread in this community ;
on the order of 1020 cookstoves had been distributed . in the
other ,
the nongovernmental organization ( ngo ) concern universal ( cu ; www.concern-universal.org ) helped establish nearly universal distribution of the chitetezo
mbaula ( cm ) cookstove , a low - cost ( 12 usd ) , locally
produced , natural - draft clay cookstove , with the main objective of
reducing fuel use by users and with funding from the sale of carbon
credits . in both communities , traditional
three stone fires or simple mud stoves
cookstoves are
shown in figure s1 in the supporting information ( si ) . in - home cookstove use
included cooking of traditional foods
such as nsima ( a corn - flour porridge , the staple
food of malawi ) or rice , preparing vegetables or meat , and heating
water for bathing and took place inside , in semicovered verandas and
outside .
in addition to in - home testing , controlled cooking
tests ( cct ) were conducted on several
larger , wood - burning
institutional cookstoves being piloted at an orphanage .
institutional
cookstoves are used where a large number of people are fed ; they are
much larger than household cookstoves to accommodate a dedicated ,
large cooking pot ( 80100 l ) that sits inside the stove body ,
enabling more efficient heat transfer .
institutional cookstoves tested
included a large institutional three stone fire ( i - tsf ) , aleva ( al ) ,
mayankho ( ma ) , and the jumbozama ( jz ) .
the jumbozama is a scaled - up
version of the zama zama rocket gasifier cookstove ( rocket works ,
durban , south africa ) built inside a masonry housing .
figure s2 ( si ) shows the institutional cookstoves tested ,
and table s1 summarizes tests conducted during the campaign . in -
home emission
measurements were performed using the portable stove emissions measurement
system ( stems ; figure s3 in the si ) , which
utilizes the sensor board from a portable emission
measurement system ( aprovecho research , cottage grove , or ) .
the stems
runs on a 12 v battery and measures real - time ( 2 s ) concentrations
of carbon dioxide ( co2 ) , carbon monoxide ( co ) , temperature ,
relative humidity ( rh ) , and particle light scattering ( bsp ; also used as a proxy for real - time pm2.5 mass concentration )
with a laser photometer ( optical wavelength , = 635 nm ) .
integrated filter samples were
collected on two 47 mm diameter filter trains with equal flows for
gravimetric and thermo - optical oc / ec analysis ( see the si for details ) .
one of the filter trains contained
a quartz filter , and the other contained a teflon filter followed
by a backup quartz filter downstream to correct for gas phase absorption
artifacts .
additional details on the
stems sensors , filter analysis and associated uncertainties , and quality
assurance are provided in section s1 in the si .
real - time pm light absorption at = 880 nm was measured
using an ae-51 microaeth ( aethlabs ) incorporated within stems . to
avoid excessive filter loadings and frequent filter ticket changes
in the field , an external flow meter ( honeywell awm3150v ) and vacuum
source were used in place of the internal pump and flow rate set at
1025 cm min .
the microaeth
filter loading artifact was corrected via the algorithm described
by park et al .
a six - armed
stainless - steel probe with sampling ports radially
centered in equal areas was used to capture a representative sample
of naturally diluted emissions approximately 11.5 m above
the cookstove . from the probe
, emissions
passed through conductive sampling tubing to the stems via a 2.5 m
cut - point cyclone ( bgi inc . ) .
wood fuel was set aside
before the start of cooking and wood moisture and weight recorded
as per the kpt protocol .
wood moisture
content was measured with an electronic moisture meter ( lignomat mini - ligno
s / dc ) .
the fire was started using matches or hot charcoal
left from a previous cooking session ; the latter practice was more
common .
one kg of wood can result in up to 161 g of char being
formed .
neglecting starting char likely
biases high the estimates of wood consumed , whereas neglecting left - over
char results in a low bias to wood consumed . to account for this ,
we assume a conservative 20% uncertainty in wood consumed .
a brief ,
anonymous survey was conducted after testing to collect user feedback
on performance and perception of alternative cookstoves .
fuel
based emissions factors were calculated using the carbon balance
method , assuming that carbon comprises 50% of dry wood by weight and
all gaseous carbon in the wood is emitted as co and co2 . since the summed carbon mass obtained from background - corrected
co and co2 concentrations serves as a tracer for the fuel
consumed , the carbon balance does not require all emissions to be
captured .
other carbonaceous species ( e.g. , gaseous hydrocarbons )
contribute a relatively small fraction ( < 5% ) of carbon in emissions
and are neglected in this calculation .
additional details
on ef and er calculations and associated uncertainties are described
in si section s3 .
45 household cooking sessions were measured ,
with durations from
19 to 233 min ( median = 49 min ) across 4 cookstove technologies ( traditional ,
philips , ace-1 and cm ) in 22 households , with two tests ( in some cases
with different cookstoves ) conducted in each household wherever possible .
axis labels in figure 1 indicate the number of tests of individual cookstove types ; cookstove - type ,
household identifier , and emissions data from all tests are listed
in table s5 .
wood moisture ranged from
6 to 26% for all sessions and was not significantly different for
tests of different cookstoves .
traditional cookstoves had the highest pm ef of 7.8
2.9 g kg ( average sd ) , similar to
field observations in honduras , while
philips had the lowest ( 4.1 0.6 g kg ) ,
with 47% lower mean emissions than the traditional stove , a statistically
significant reduction ( p < 0.005 from two - sample t test ) .
mean cm and ace-1 efs were lower than that from
traditional stoves but not significantly so ( p =
0.347 and 0.158 , respectively ) .
linear regressions showed no relationship
between fuel moisture content and pm ef for individual cookstove types
( r < 0.1 for all ) .
box plots of pollutant
emission factors ( in g kg ) and emission rates
( in mg min ) , with the number
of tests of each type indicated in the axis label .
boxes represent
interquartile range , whiskers indicate 5th and 95th percentiles , and
the horizontal line in the box is the median .
95th percentile whiskers
for some of the cookstoves were out of scale on the y axis and are indicated by numbers on the top axis .
panel a : pm2.5 ef ; panel b : co ef ; panel c : pm er , panel d : co er ; panel
e : ec ef ; panel f : ec er ; panel g : ec / tc ratio ; panel h : single scattering
albedo at 880 nm .
tier values for panels c and d were taken from iso
iwa 11:2012 guidelines .
the legend for
all panels is in panel a. an asterisk before the stove name on the
lower axis indicates a statistically significant difference with respect
to traditional stoves ( p < 0.05 ) .
data from shen
et al . is for a movable metal cookstove
used in china .
co efs ( figure 1b ) measured ranged from 28 to 198 g kg , with
traditional stoves the highest ( 98 26 g kg ) , comparable to field measurements from traditional open fires in
honduras ( 116 55 g kg ) .
both fdcs models had statistically significant reductions
of 45% ( p < 0.005 ) .
the mean co ef for cm was
106 g kg , not significantly different from traditional
stoves ( p = 0.507 ) .
although these efs are
similar to those from other field studies ,
they are considerably higher than those observed in laboratory tests .
mean pm2.5 ef for traditional stoves is roughly 3.7 times
higher than laboratory tests .
for the
philips , laboratory mean pm2.5 and co efs were 80 and 65%
lower than our mean values , respectively .
estimated uncertainties in our pm ef values are 1030% , with
an average uncertainty of 15% ( si section
s3 ) , smaller than variability in ef values within groups ( e.g. , pm
ef coefficient of variation ranges from 14% for philips to 44% for
cm ) .
fuel based efs do not account for two parameters important
for
understanding total emissions : cooking time and wood consumed , which
depend on cooking activity and cookstove efficiency . here
figure
s5 in the supporting information shows
a box - whisker plot of wet - basis ( as measured ) wood consumption rate
in kg h. traditional stoves had the highest fuel
consumption rate ; mean reductions from the cm , ace , and philips were
26% , 27% , and 51% , respectively .
observed reductions are consistent
with , but slightly smaller than , reductions in wbt fuel consumption
observed during tests of the cm ( 33% ) and philips ( 61% ) in malawi .
figure 1c and 1d show the pm and co
ers estimated using measured
fuel values ( on a dry fuel basis ) ; also shown are tier boundaries
for indoor emission rates .
the philips shows reductions
of both pm2.5 and co ers by 70% compared to traditional
stoves
. however , pm and co ers based on laboratory testing of philips
cookstove ( for wet wood ) are 76 and 61% , respectively , lower than
our mean values .
this indicates that
laboratory tests may substantially underestimate real - world emissions
of even the most advanced wood - burning cookstoves when fuel properties
and cookstove operation are variable .
figures 1e to 1 g show box plots
of ec efs , ers , and ec / tc , respectively ,
where tc is total carbon ( oc+ec ) .
all quantities show high variability
due to the uncontrolled nature of this combustion and varying usage
and fueling of the cookstoves and skill of the cook .
ec efs and ec / tc
ratios for intervention cookstoves are generally higher than those
for traditional stoves , though only the philips shows a significant
difference for ec / tc .
increases are moderated for ers due to reduced
fuel use , especially for the philips cookstove .
ec efs for cm were
similar to observations for basic improved cookstoves
in other field studies .
fdcss had the highest
ec / tc ratios ( 0.48 ) followed by cm ( 0.42 ) and tsf ( 0.28 ) .
ec / tc is
typically 0.1 for open biomass
burning , significantly lower than values observed in this study .
this
highlights that combustion under these relatively controlled conditions
emits particles with distinct properties compared to those from open
biomass burning .
additional discussion on ec / tc ratios and ssa is
provided in section 3.3 .
a summary of efs and optical properties for each test is provided
in si table s5 .
food- and fuel - based efs
for institutional cookstoves are reported in table 1 .
efs for pm and co followed a consistent
trend , with the i - tsf the highest , followed by the al , ma , and jz .
co and pm emissions were reduced by similar amounts , with co ( pm )
emissions reduced by 67% ( 68% ) and 73% ( 75% ) for ma and jz , respectively .
averaged wood consumption rate was highest for the aleva ( 5.2 kg h ) , followed by i - tsf ( 4.18 kg h ) , mayankho ( 3.85 kg h ) , and jumbozama ( 3.30
kg h ) .
food based pm and co efs follow the
same trend as their fuel based counterparts , with jz exhibiting > 80%
reductions for both .
the jz cookstove used a slightly smaller , different
cooking pot ( 80 l vs 100 l for other cookstoves ) , which may have contributed
to mean pm ef values ( e.g. , due to a quicker warm - up phase ) and cooking
times that were 61% and 32% lower , respectively , than those for other
cookstoves .
further extensive comparisons of these data are complicated
by the small sample size and lack of emission data in the literature .
figure
s6 in the si shows real - time ( 2 s and 1
min average ) concentrations of co , co2 , particle absorption ,
and scattering coefficients ( bap ; bsp ) for representative
fdcs and traditional household stove tests .
gravimetric pm2.5 concentrations correlated well ( r =
0.87 ) with averaged bsp , suggesting that real - time scattering
was a reasonable proxy for real - time pm2.5 mass concentrations
under these test conditions ( si figure
s7 ) .
figure s6 also shows single
scattering albedo ( ssa ; fraction of scattering to total extinction ;
( bsp/(bap+bsp ) , here at =
880 nm ) and modified combustion efficiency ( mce ; co2/(co+co2 ) , where indicates background - corrected
concentrations in ppm ) .
a lower ssa signifies a greater contribution
from absorption to total aerosol light extinction , while higher mces
indicate more efficient combustion .
all cooking events were characterized
by a scattering spike at startup ( evident
in figure s6 ) .
observations of cooking
activity showed that addition and adjustment of fuel typically resulted
in spikes in bap for fdcs and in bsp for traditional
stove tests .
fdcs tests typically had a large scattering peak only
at startup and overall particle extinction was dominated by absorption ,
while extinction from traditional stove tests was dominated by scattering .
as a result , traditional stove tests had comparatively higher ssa
( shown in figure 1h )
and lower mce than fdcs tests .
test - average ssa was highest for traditional
stoves ( 0.36 ) , followed by cm ( 0.28 ) and fdcs ( 0.25 ) ; particles from
all alternative cookstoves are more absorbing than
those from traditional stoves and thus have greater specific warming .
our optical measurements are at = 880 nm , which influences
the quantities ( e.g. , ssa ) presented here , but likely not the relative
trends discussed here and below . for example , while ssa for pure bc
aerosol is typically 0.150.3 at = 530 nm , we observe periods with ssa ranging from 00.2
for fdcs , despite the fact that the particles are not pure bc .
section
s5 and figure s8 in the si present mie
theory modeling indicating that both the longer wavelength and smaller
particle diameter in fdcs emissions likely strongly reduces the scattering
efficiency of these particles . the average mass scattering cross section
( msc ; ratio of scattering to gravimetric pm2.5 concentration )
over all tests was 0.87 0.31 m g , substantially lower than mscs of 3.64.3 m g ( = 550 nm ) and 2.2 0.6 m g ( = 530 nm ) reported for emissions
from dry biomass burning and in - field cookstoves , respectively .
lower msc values in our study are consistent
with the wavelength dependence of scattering . averaged mass absorption
cross - section ( mac ; ratio of bap to ec concentration ) for
all tests was 13.2 4.8 m g ,
overlapping with the mac of 12.5 m g assumed in the microaethalometer .
the data show a general trend of decreasing ssa with increased
mce and ec / tc .
si figure s9 shows the relationships
between mce and ec / tc with ssa for in - home cookstove testing .
greater
specific absorption correlates somewhat with higher mce ( r = 0.29 ) and more strongly with higher ec / tc ( r = 0.57 ) .
these relations are consistent with greater bc
production in more efficient , contained combustion where mce is highest ,
while more scattering oc is produced during less efficient ( perhaps
lower temperature ) combustion . relationships between mce , ec / tc , and
ssa have been proposed based on measurements of open biomass burning
for a range of fuels .
while the general trends we observe
are consistent with the published parametrizations , a direct comparison
is not possible due to differences in measurement wavelength .
however ,
it is likely that the relationships would be different because combustion
technology strongly influences aerosol properties .
test - average
quantities do not reflect the contribution of distinct
combustion phases to total emissions .
analysis of real - time data can
give insight into the variation of parameters such as mce and particle
properties throughout a burn .
patterns
of real - time emissions data ( parted ) analysis approach of
chen et al . to evaluate quantities and
optical characteristics of emissions based on real - time data . in this
analysis , mce and ssa are calculated for each minute of data ( termed
a combustion event ) .
a bivariate histogram of mce and ssa , weighted
by instantaneous scattering emission factor ( iefscat , particle
light scattering normalized by mass of fuel consumed ) and normalized
by total scattering emissions , is then constructed .
the resulting
plot shows the fractional contribution of combustion at specific conditions
( mce ; ssa ) toward total scattering emissions .
this weighting is chosen
to represent the distribution of total particle emissions , as scattering
shows a strong correlation ( r = 0.87 )
with pm2.5 from gravimetric analysis ( figure s5 ) .
figure 2 shows parted
plots for traditional , cm , and fdcs ( ace-1 and philips combined ) emission
tests , with each panel representing all test data from that cookstove
type .
all display a cluster at mce > 0.9 and ssa < 0.4 ,
suggesting
that all produce more absorbing particles during more efficient flaming
combustion .
however , all test types also had combustion events and
particle emissions at lower mce and higher ssa , with noticeably more
spread in this direction among the traditional cookstove and cm tests .
the fdcs plot shows a more distinct and concentrated cluster at high
mce , low ssa .
figure s10 in the si shows
histograms weighted with fuel consumption ( calculated as co+co2 ) rather than particle scattering and shows that the vast
majority of fuel consumption occurs at high mce / low
ssa ( 88% at mce > 0.9 and ssa < 0.4 for fdcs ) , whereas only
12%
of scattering emissions took place under these conditions .
the corresponding
fractions ( fuel consumption / scattering ) in this mce - ssa range are
57%/15% and 58%/25% for the cm and traditional tests , respectively .
this reinforces that the relative distribution of combustion conditions
varies between stoves and has substantial impacts on particle emissions
and properties .
( though less so for traditional / cm tests )
but emit a large fraction of scattering particles / particle mass .
time
series data ( figure s6 ) show that all tests
had sharp peaks in scattering emissions during cookstove startup ,
consistent with other field measurements .
the parted plots also suggest that these startup emissions may make
outsized contributions to overall pm emissions .
we examine the contribution
of startup emissions in si figure s11 ,
which plots the running average of iefscat against normalized
time for each test of a cookstove type . in all tests , the average
peaks strongly during the startup phase , confirming the importance
of start emissions . although the test averaged iefscat ( at
the right edge of the graph )
is highest for traditional and lowest
for fdcs tests , we see a much higher peak for gasifiers and in nearly
all cases a monotonic decrease in iefscat during the test ,
reinforcing the dominance of startup emissions for these cookstoves .
the outsized contribution of startup to pm emissions from fdcs has
important implications for exposure as during startup the cook is
assured to be in close proximity to the cookstove .
bivariate histogram of
mce and ssa weighted by particle emissions
( parted plots ) for a. traditional ; b. chititezo mbaula , and c. fdcs .
the bottom axis delineates bins of single scattering albedo ( ssa )
at 880 nm , and the left axis shows modified combustion efficiency
( mce ) bins .
each location on the plot represents an ssa and
mce at which a combustion event ( 1 min ) may occur ; the color scale
indicates the percent contribution of emissions at that condition
to the total scattering ( pm mass ) emitted .
three of the traditional
stove tests were excluded from this analysis due to lack of either
co2 ( 1 test ) or absorption data ( 2 tests ) .
100- and 20-year gwc ( tons
of co2-equivalent per year of cookstove use ) were estimated
based on measured pollutant emission factors ( co2 , co ,
oc , and ec ) for household and institutional cookstoves following the
approach of previous work , as briefly
discussed in si section s6 .
gwcs associated
with the use of a modern fuel , lpg , are also included as a benchmark
( based on laboratory efs ) .
the intergovernmental panel on climate
change ( ipcc ) default value of 0.81 for the fraction of nonrenewable
biomass ( fnrb ) in malawi was assumed
for all calculations , though this is highly uncertain due to factors
including spatial heterogeneity in fnrb and uncertainties in data
on fuel demand and its dynamics .
a fixed
energy demand was assumed and annual fuel use for each cookstove estimated
based on fuel use rate reductions observed in this study ( figure s5 ) relative to the baseline ( traditional
cookstoves ) .
gwc calculations used global warming potential ( gwp )
values recommended by the gold standard foundation and ipcc and account only for emissions during fuel combustion .
gwc associated with upstream processes ( e.g. , fuel production and
transport ) related to cookstove fuel use have been found to be negligible
for woodfuel and relatively small ( 1020% of combustion stage )
for lpg , though they may be considerable
for other fuels such as coal or charcoal .
ch4 makes a substantial contribution to the gwc associated
with cookstove use but was not measured here . to
approximate ch4 gwc
, ch4:co ratios from the
literature were used to estimate ch4 efs ; ch4:co ratios of 0.05 for ace-1 and philips
and 0.08 for the traditional , cm , and institutional cookstoves were
used in calculations .
gwc contributions from other hydrocarbons and
n2o are small for biomass emissions and are neglected here . also not included
in this accounting is brown carbon ( brc ) , the component of oc that
absorbs energy across visible and ultraviolet wavelengths .
we did
not measure absorption at multiple wavelengths in this work but expect
that the brc absorption would make a relatively small contribution
to short - wavelength absorption considering the very high ec : oc ratios
( mean for all tests was 1 ) observed in emissions from all
cookstoves .
brc is expected to have little additional impact on climate
forcing for emissions with ec : oc ratios above 0.1 , though this merits further study .
figure 3 shows the gwc values estimated
across a 100-year horizon ( figure s12 shows
20 year gwcs ) .
as expected , traditional cookstoves show the highest
gwc , followed by the cm , ace , and philips . across a 100 year horizon
for household cookstoves , cm , ace , and philips show overall reductions
of 13% , 23% , and 55% , respectively , from the household traditional
( hh - trad ) stoves . across household stove models ,
the highest relative
contribution by species is from co2 ( 5169% ) followed
by ec ( 2638% ) with other species contributing less than 10% .
calculations using laboratory efs suggest much larger reductions ( 59%
and 93% for improved and fdcs , respectively ) relative to traditional
stoves , highlighting again the implications
of the lab - field discrepancy in emissions . due to high combustion
efficiency for lpg
, pics contribute minimally to its gwc , and the
vast majority is contributed by co2 .
the gwc of lpg is
a factor of 4 lower than the cleanest cookstove tested in the field
( philips ) . among institutional cookstoves ,
only the jz yielded substantial
fuel use and emission reductions during our limited testing , leading
to an 50% reduction in gwc relative to baseline . across a
20-year horizon ( figure s12 ) ec contributes
the most to biomass cookstove gwc , contributing 5365% across
all cookstoves .
100 year gwc values for one year of use of in - home cookstoves
( l )
and institutional cookstoves ( r ) from major short- and long - lived
climate forcing species emitted by cookstoves .
the ch4 component
is estimated based on the ch4:co ratio from other studies .
note that the daily energy use is different for household and institutional
cookstoves . in - home cookstoves
institutional cookstoves are assumed to be used for twice
a day for 5 days a week for 40 weeks a year with an energy basis based
on average fuel use measured in this study .
figure 4 combines
gwc estimates with those for human exposure to pm2.5 to
examine the cookstoves measured in a
the exposure estimation applies an individual intake fraction of 1300
ppm ( 1 ppm = 1 mg inhaled per kg emitted ) to link emissions to human
exposure .
the figure shows estimated
daily pm intake ( horizontal axis ) and gwc ( vertical axis ) of several
cookstove technologies evaluated in that study , with added estimates made based on data from in - home testing
during this study .
it should be noted these calculations assume complete
adoption of the cookstove in question , while field trials have shown
that new technologies are rarely used exclusively and
the exposure - response relationship for all - age mortality risk from
ischemic heart disease ( ihd ) from burnett et al . is used to estimate adjusted relative risk of mortality
due to ihd ( dose response for chronic obstructive pulmonary
disease mortality is similar ) , shown on the lower horizontal axis .
this extrapolation of our field emissions data suggests that the philips
cookstove reduces pm emissions and intake by approximately 75% relative
to the traditional stove ; this is associated with a smaller reduction
in estimated mortality relative risk ( from 2.2 to 1.9 ) due to the
nonlinear dose
this figure dramatically demonstrates the implications of the performance
decrement observed in field measurements .
field - tested biomass stoves
do not meet expectations based on laboratory tests in terms of emissions / exposure .
for example ,
relative to estimates based on laboratory measurements ,
the field - measurement - based daily pm intake and gwc for the philips
are a factor of 8.6 and 2.8 times higher , respectively .
the philips
and jz have the lowest estimated intake and gwc among in - home and
institutional cookstoves , respectively , but are still associated with
far greater impacts than the benchmark lpg cookstoves .
for example , compared to the estimated impacts of lpg cookstove use ,
in - home use of a philips cookstove results in 4.9 times higher gwc
and around 66 times higher daily exposure , corresponding to increase
in adjusted relative risk for ihd mortality from 1.25 to 1.95 .
impact
estimates for hh - trad , cm , and three of the institutional cookstoves
( i - tsf , ma , and al ) are within the bounds of impact estimates for
laboratory - tested traditional stoves , whereas the best performing
fdcs is in the range estimated based on laboratory performance of
a basic improved biomass cookstove ( w - im - u ) . field observations of
emissions from a range of cookstoves used in interventions give important
insights into the potential for these technologies to mitigate the
health and climate impacts associated with traditional cookstoves .
health
and climate impacts of various cookstove - fuel combinations
based on laboratory emission test data ( shown with circles ; adapted
from grieshop et al . with fuel renewability
and energy demand values described in text ) along with the estimates
from this study are shown , marked with diamonds and squares with error
bars for in - home and institutional cookstoves , respectively .
abbreviations
for laboratory - based calculations : w - tr - u : wood - burning traditional
unvented cookstoves ; w - im - u : wood - burning improved unvented cookstoves ;
w - gas - u : wood - burning gasifier unvented cookstoves ; w - fan - u : wood - burning
fan unvented cookstove .
household traditional ; cm : chitetezo mbaula ; philips : philips
hd4012ls ; ace : ace-1 ; i - tsf : institutional three stone fire ; al : aleva ;
ma : mayankho ; jz : jumbozama .
our results suggest that
both simple improved cookstoves
and more advanced biomass cookstoves provide some benefits but fall
short of those indicated by laboratory testing or that may be possible
through the use of modern fuels / devices .
impact estimates shown here
( figure 4 ) are rough
approximations but are consistent with field trials that have seen
less than expected benefits from cookstove interventions .
for example , the fact that no effect on childhood pneumonia incidence
was observed during caps may be partly
due to poorer - than - expected performance of these cookstoves under
real - world conditions ; other factors such as continued use of traditional
stoves and exposure to air pollution from other sources likely also
contributed to this outcome .
forthcoming findings from other intervention
trials will report on the health effects associated with other fuel / cookstove
technologies including lpg and give insights into whether or not they
provide the benefits that might be predicted .
a range of factors contribute to reduced performance observed
in
the field , all related to the difficulty of controlling combustion
of heterogeneous fuels under widely ranging conditions .
part of the
performance decrement observed is due to cookstoves not being used
in accordance with manufacturer recommendations .
for example , wood
pieces sticking out of the top of fdcs were commonly observed in the
field ( figure s1 in the si ) and lead to
suboptimal combustion of the volatiles emitted from pyrolysis of wood .
however , this practice is unsurprising when one considers that processing
larger logs and branches to the size recommended for the fdcs models
considered here ( 1 5 cm ) represents additional work
for the household .
this source of variability may be addressed via
a cookstove that is highly robust to changing fuel type / configuration
or a situation in which a homogenized fuel source ( e.g. , pellets )
is provided or readily available .
the former is only likely possible
in a more advanced combustion device ( e.g. , the enclosed heating stoves ,
often with catalytic after treatment of exhaust used in developed
countries ) that are beyond the budget of the target population , while
the latter requires a close look at the broader system , beyond the
cookstove .
these observations highlight the need to expand the
view beyond
clean cookstoves to clean and controlled cooking systems ,
which could provide considerable health and climate benefits and perform
consistently and reproducibly under both laboratory and field conditions .
one approach advocated is to focus efforts on a switch to modern appliances
( e.g. , electrical induction cookers and lpg ) rather than promoting
improved biomass cookstoves , though such technologies would be out of reach in the short- to
medium - term for the poorest of the world s poor ( e.g. , rural
malawians ) .
improvements using biomass can likely be made by improving
the cookstove / fuel system in tandem .
our findings emphasize
that laboratory protocols do not fully anticipate
real - world emissions when fuel properties and cookstove operation
are variable and highlight the need for testing approaches that more
accurately represent real - world cookstove use .
field evaluation of
emissions performance early in product development would be one way
to achieve this ; another way would be to develop testing protocols
that
simulate the range of ways a cookstove may be used , including low - efficiency
emissions such as smoldering , often observed in field emissions .
these data were collected during
a dry season , and fuel moisture may also have important impacts on
stove performance ; future field studies
should assess stove performance across an annually representative
period if possible . | emissions
from traditional cooking practices in low- and middle - income
countries have detrimental health and climate effects ; cleaner - burning
cookstoves may provide co - benefits . here
we assess
this potential via in - home measurements of fuel - use and emissions
and real - time optical properties of pollutants from traditional and
alternative cookstoves in rural malawi .
alternative cookstove models
were distributed by existing initiatives and include a low - cost ceramic
model , two forced - draft cookstoves ( fdcs ; philips hd4012ls and ace-1 ) ,
and three institutional cookstoves . among household cookstoves , emission
factors ( ef ; g ( kg wood)1 ) were lowest for the
philips , with statistically significant reductions relative to baseline
of 45% and 47% for fine particulate matter ( pm2.5 ) and
carbon monoxide ( co ) , respectively .
the philips was the only cookstove
tested that showed significant reductions in elemental carbon ( ec )
emission rate .
estimated health and climate cobenefits of alternative
cookstoves were smaller than predicted from laboratory tests due to
the effects of real - world conditions including fuel variability and
nonideal operation .
for example , estimated daily pm intake and field - measurement - based
global warming commitment ( gwc ) for the philips fdcs were a factor
of 8.6 and 2.8 times higher , respectively , than those based on lab
measurements .
in - field measurements provide an assessment of alternative
cookstoves under real - world conditions and as such likely provide
more realistic estimates of their potential health and climate benefits
than laboratory tests . | Introduction
Methods
Results and Discussion
Implications |
PMC3747626 | childhood obesity remains one of the most serious threats to the public 's health , with 1 in 3 children and adolescents overweight or obese ( body mass index ( bmi ) 85th percentile ) .
there is limited evidence for effective behavioral prevention interventions . to fill this gap , the institute of medicine , the strategic plan for nih obesity research , shaping america 's youth , and the white house task force on childhood obesity
have called for community - engaged , family - centered approaches to pediatric obesity prevention .
these approaches are thought to have the greatest potential for sustained efforts and effects in our obesogenic environment . in parallel , comparative effectiveness research
is being discussed within the national health reform debate as a mechanism for improving healthcare quality and decreasing healthcare spending .
clinical research typically examines the effectiveness of one prevention or treatment method at a time .
comparative effectiveness research compares multiple methods to determine the effectiveness of an intervention relative to alternatives .
identifying the most effective and efficient interventions has the potential to reduce unnecessary treatments , which should lower costs .
it is estimated that 8.4 million children attend after - school programs ( asp ) , and an additional 18.5 million would do so if a program was available .
the purpose of this study was to evaluate the comparative effectiveness of a community - driven asp designed to combat physical inactivity versus a standard - of - care school - based asp available to working parents .
the community asp was derived directly from local input and sustained through the collaboration and sharing of resources by the parks department and the public school system ( davidson county , tn , usa ) .
this community - engaged effort has the potential to serve as a new model for youth obesity prevention because ( 1 ) it systematically addresses the top four barriers , identified by shaping america 's youth , that prevent children from being active ( lack of access to safe and appropriate places to be active , parental time constraints , cost of programs , and lack of parental motivation ) and ( 2 ) it engages multiple sectors of society to support program attendance and sustainability .
systematic reviews suggest that it is possible to improve activity levels , physical fitness , body composition , and blood lipids in the after - school setting and that limitations in study design , lack of statistical power , and problems with implementation have hindered the evaluation of most asps to date . to assess the comparative effectiveness of this community - driven asp as a pediatric obesity prevention intervention , we compared it to the routine aftercare available to working parents in the community and asked two research questions : ( 1 ) are children in the alternative asp more physically active than children in the standard asp ? ( 2 )
this study was guided by principles of community - based participatory research ( cbpr ) .
cbpr is an important research approach that equitably involves community members who are affected by the issue being studied in all phases of the research process .
the community asp was developed by the parks department to address the community 's need for an affordable asp .
families provided input about their needs and preferences ( e.g. , transportation from school to the community center , flexibility in pick - up times , homework time , reduction of screen time , and increased physical activity ) .
the city 's public school system changed its policy around permissible bus stops allowing buses to deliver students to the community recreation center to support program attendance .
the leadership and staff of the parks department were involved in all aspects of this research project : grant acquisition , study design , implementation , interpretation of results , and dissemination .
the study design was an observational prospective cohort study and a natural experiment in nashville , tn , usa . the naturally occurring event , the parks department 's new asp , was the intervention , and children attending this community asp formed the intervention group ( n = 54 ) .
comparison participants ( n = 37 ) were recruited from an asp located in the same ( low income ) school district and operated by a national company that operates a high proportion of school - based asps in the city , making it de facto standard - of - care for the majority of the city 's school - aged children with working parents .
children were eligible for the study if the following was true : ( 1 ) age 5 and < 13 years ; ( 2 ) attended one of the glencliff [ neighborhood ] cluster of public elementary or middle schools ; ( 3 ) enrolled in the community or school - based asp .
parents of eligible children underwent a 15-minute oral consent process before providing written consent for their child .
children provided assent . the consent / assent process was conducted in the preferred language of english or spanish .
the study was approved by the vanderbilt university institutional review board ( # 090986 ) .
the two asps followed similar formats , and operated from 36 pm every day public schools were open .
both asps included time for snack , homework , and play and did not focus on a single activity ( e.g. , tutoring , chess , and team sport ) .
the community asp was set in a community recreation center and involved staff - led games .
the school - based asp was set in a school cafeteria and involved opportunities for arts and crafts and playing on the playground .
the main differences between the two asps were ( 1 ) format of active play time ( adult - led versus unstructured ) and ( 2 ) location ( community recreation center versus public school ) .
all measures were collected at the asps at three time points over approximately 12 weeks ( february may 2010 ) , with six weeks separating each wave of measurement .
the measurement period was selected based on the cochrane review that states that obesity prevention interventions should last at least 12 weeks for behavior change to be observed .
physical activity was assessed using actigraph gt1 m accelerometers ( actigraph , pensacola , fl , usa ) only during asp programming time .
accelerometry is considered an objective measure of physical activity and has been used with children [ 15 , 16 ] , including latino and african - american children , with high reliability : r = 0.93 .
the actigraph is a small monitor that is worn on an elastic waist belt and measures the intensity of physical activity associated with locomotion .
monitors were programmed to record in continuous 10-second epochs to capture the short , spurt - like activity characteristic of children . at each measurement period , the children wore monitors for five consecutive days , from the time they signed into the asp until they were picked up .
measurement start and stop times were recorded by study staff at each site ; these were used as precise wearing cut - off points , eliminating the need for wearing / nonwearing time analysis .
data were retained in analysis if the child wore the accelerometer a minimum of 3 days of the given measurement period [ 18 , 19 ] .
freedson 's age - dependent cut points were used to determine time spent in sedentary , light , moderate , and vigorous activity .
trost 's validation study comparing various accelerometer cut points for predicting physical activity in children supports the application of freedson equations in field - based studies of school - aged children .
in particular , trost found that , for classification of mvpa ( moderate - vigorous physical activity ) , freedson cut points exhibited excellent classification accuracy .
the analyses described below were also conducted using pate 's cut points and resulted in similar findings ( not reported here ) .
daily percentage of time spent in each level of physical activity ( i.e. , sedentary , light , moderate , and vigorous ) was determined by dividing the minutes spent in each activity level by the sum of minutes the actigraph was worn in a day ( i.e. , time in attendance at the asp ) .
thus , the continuous outcome measures were the proportion of time spent in lmvpa ( light - moderate - vigorous physical activity ) or mvpa ( moderate - vigorous physical activity ) out of total time in attendance , rather than the number of minutes the program was open , to allow for a meaningful comparison within individuals and across groups .
daily percentages were averaged across days to create individual participants ' physical activity ( pa ) scores at each measurement period . body weight was measured after voiding while children wore light clothing without shoes .
calibrated digital scales ( detecto , webb city , mo , usa , model#758c ) were accurate to the nearest 0.1 kg .
body height without shoes was measured to the nearest 0.1 cm with the scale 's stadiometer .
weight categories were defined by bmi percentile , according to centers for disease control growth charts : underweight : < 5th percentile ; healthy weight : 5th to < 85th percentile ; overweight : 85th to < 95th percentile ; obese : 95th percentile .
body composition was measured by the rjl systems bia quantum ii ( rjl systems , clinton , mi , usa ) after voiding .
standard procedures for whole body bioelectrical impedance measurement were used , along with the vendor - provided child - specific regression equation to estimate percent fat mass from total body water .
children were asked to complete a 1/2 mile run as fast as possible on a running track .
parents completed a survey asking about child 's date of birth ( used to calculate age ) , gender , race / ethnicity , and name of school . because children were not randomly assigned , preexisting differences between groups were potential confounders .
therefore , we compared children enrolled in the asps to test for differences on basic demographic and process variables , using bootstrap t - tests that controlled for the familywise false discovery rate . to assess change in pa over time ,
a conditional linear latent growth model was used with random intercepts and slopes that were free to covary and time varying error variances .
this approach offers important advantages over older analysis of variance ( anova ) models , such as ( a ) better accuracy in assessing change over time , ( b ) graceful handling of missing values and unequal time intervals between waves and participants , and ( c ) repeated measurements that increase statistical power .
the key result is a group by time interaction , which shows whether groups differ in their slopes / rates of change in pa .
centering time zero at the first measurement let us answer two questions : ( 1 ) did the groups start out equally ? and ( 2 ) did their time slopes differ ?
the analysis assumed data were missing at random and used full information maximum likelihood to maximize sample size by including all participants with at least one wave of data .
using the ingredient method , we estimated the implementation costs , without estimating indirect costs or externalities associated with the programs , to indicate how much it would cost to replicate each asp . instead of accounting expenditures paid during the implementation , we valued resources using standard costs to society .
all personnel time ( including volunteer time ) was valued by using the median earning per hour of a comparable worker published by the bureau of labor statistics 2010 ; thus , differences in human capital endowment did not affect our estimates of implementation costs .
of the 91 children who attended the asps , baseline demographics were obtained from parents of 83 children .
the analytic sample included the 82 children with pa data from at least one time point ; one child in the school - based asp did not provide at least 3 days of pa data in any measurement period and was not included in the analyses .
of the 82 participants , 62 had data for all three time points , 16 had data for two time points , and 4 had data for only one time point .
the baseline sample was 65% female and 7.9 years of age ( sd = 1.7 ) on average ; 57% were healthy weight , 23% overweight , and 20% obese ; 40% were african - american , 40% white , and 20% latino . on average at baseline , children spent 77.4% ( sd 10.3% ) of the asp in lmvpa and 27.5% ( sd 14.3% ) in mvpa . at baseline , children in the two asps did not differ in gender , age , bmi , percent body fat , fitness ( table 2 ) , or physical activity level ( table 3 ) .
however , children in the community asp were less likely to be white than children in the school - based asp ( p = .027 , table 2 ) . at baseline , children spent approximately 30% of their asp time in mvpa ( sd = 15.6 ) .
the linear latent growth model implied that , on average , children in the community asp became more active over time ( average change between baseline - week 6 and week 6-week 12 ) , compared to the children in the school - based asp ( table 3 ) .
children in the school - based asp reduced their total physical activity ( lmvpa ) by an average of 3.4 percentage points over each measurement period ( p = .002 ) , for a total 6.8 percentage point decrease over the 12-week study period .
in contrast , children in the community asp increased their total physical activity ( lmvpa ) by an average of 3.0 percentage points over each measurement period ( p = .006 ) , for a total 6 percentage point increase over the 12-week study period ( figure 1 ) .
children in the school - based asp did not significantly change their mvpa on average ( p = .12 ) .
however , children in the community asp increased their mvpa by an average of 2.8 percentage points over each measurement period ( p = .006 ) , for a total 5.6 percentage point increase over the 12-week study period ( figure 2 ) .
taken together , the model - implied average difference between the two groups of children at week 12 was 15.4 percentage points in lmvpa ( p < .001 ) and 14.7 percentage points in mvpa ( p < .001 ) , favoring the activity - enhanced community asp .
however , as a more conservative indicator , the observed average difference between the two groups of children who had data at week 12 was 10.8 ( p = .001 ) percentage points in lmvpa and 13 percentage points in mvpa ( p < .001 ) .
the community asp served 54 children ; the school - based asp served 37 children .
total implementation costs ( valued in 2010 dollars ) for the 12-week study period were $ 1,184 per child ( $ 19.25 daily per child ) for the community asp , compared to $ 1,087 per child ( $ 17.67 daily per child ) for the school - based asp ( 9% difference ; table 4 ) .
the facility cost represented 66% and 65% of the total implementation costs to run the community asp and school - based asp , respectively .
the main source of cost differential between programs was the child to staff ratio ( 6 : 1 at the community asp , 12 : 1 at the school - based asp ) . to run the asps for 12 weeks , the personnel cost was $ 380 per child for the community asp compared to $ 314 per child for the school - based asp ( 21% difference ) . with more than 23 million parents of school - aged children employed full - time ,
this study demonstrated that , compared to a standard - of - care school - based asp , an asp set in a community recreation center with activities directed by recreation staff significantly increased total physical activity in a multiethnic sample of public school children by 6 percentage points over 12 weeks .
most ( 5.6% ) of this increase was in mvpa , which is the type of physical activity that has the greatest health benefits [ 3335 ] .
the incremental cost of implementing the activity - enhancing asp compared to the traditional asp was $ 1.59 per day per child .
the main source of cost differential between programs was their child to staff ratios . assuming the improvement in activity was solely due to the intervention ; these findings suggest that children attending traditional school - based asps , already costing an average of $ 17.67 per day , would need an additional daily investment of $ 1.59 per child over 12 weeks to increase their lmvpa by 15.4 percentage points or their mvpa by a model - implied 14.7 percentage points .
the annual cost of childhood obesity - related health expenses in the us is $ 14.1 billion for outpatient care and $ 237.6 million for inpatient care which translates to about $ 5 in healthcare expenses per day per child , without including other relevant long - term costs related to school performance , labor market involvement , quality of life , welfare needs , and so forth . given this , providing structured pa programming by qualified staff in a community recreation center in the after - school hours could be a reasonable low - cost investment .
asps have long played a critical role in supporting academic achievement , safety , discipline , and avoidance of risky behaviors .
they could now be leveraged as part of a broader approach to address physical inactivity .
community centers operated by local parks and recreation departments ( 20,000 nationally ) provide an ideal venue for structured pa programming for children in large part because these centers , in conjunction with school transportation departments , can address community - based barriers to increasing children 's activity levels . it is noteworthy that the parks staff initiated and led this program on their own .
their intimate knowledge of the community and the respect they commanded from both the children and adults in the community likely contributed to the program 's success .
we speculate that the combination of the built environment that supported activity , low child to staff ratio , and intentional activity leadership resulted in the increased pa levels .
the school - based asp could have let children play in the school 's gymnasium if there had been additional staff available to supervise .
thus , we speculate that importing adult - led activities with lower child to staff ratios into school - based asps might be a cost - effective approach to increasing activity in that setting as well
first , accelerometers do not adequately measure body movements of upper and lower extremities , but they are considered the gold standard for measuring pa under free - living conditions .
this should not have biased our results since the limitation of accelerometry was the same across groups .
second , our sample was small but having three waves of data increased statistical power and was sufficient for detecting a significant increase in pa under free - living conditions .
third , despite efforts to select a comparable comparison group and measure potential confounders , we can not rule out all systematic differences between the two groups .
we did rule out the most important possible confounds in the literature : body composition , fitness , age , and gender .
it is possible that the difference in racial composition of the groups could explain baseline variance but is unlikely to explain change over time in activity levels .
fourth , for the community asp , there were significant differences between the observed and model - implied averages at weeks 6 and 12 .
these discrepancies highlight the fact that the final specified model did not perfectly recreate the observed data .
this could have been partially due to missing data for this group at either time point .
the discrepancies could also have arisen because the community asp 's growth rate was not linear ; yet , a model with only three time points does not have the degrees of freedom to investigate more sophisticated growth parameter specifications ( e.g. , quadratic ) . nonetheless , applying latent growth models has provided further insight into how asps might impact children 's pa over time ( e.g. , what effect does asp type have on pa change over time ? what is the typical growth rate of pa for children over time ?
do some programs increase the growth rate of certain types of pa ( e.g. , light , moderate , or vigorous ) more than others ?
an asp set in a community recreation center and led by recreation staff incorporating structured physical activity opportunities was associated with significant increases to physical activity during asp time in a multiethnic sample of public school children in 12 weeks , compared to a standard school - based asp .
utilizing community recreation centers ' built environment and staff could be a promising low - cost proposition to improve health trajectories among school - aged children . |
background .
we conducted a comparative effectiveness analysis to evaluate the difference in the amount of physical activity children engaged in when enrolled in a physical activity - enhanced after - school program based in a community recreation center versus a standard school - based after - school program .
methods .
the study was a natural experiment with 54 elementary school children attending the community asp and 37 attending the school - based asp .
accelerometry was used to measure physical activity .
data were collected at baseline , 6 weeks , and 12 weeks , with 91% retention . results . at baseline ,
43% of the multiethnic sample was overweight / obese , and the mean age was 7.9 years ( sd = 1.7 ) .
linear latent growth models suggested that the average difference between the two groups of children at week 12 was 14.7 percentage points in moderate - vigorous physical activity ( p < .001 ) .
cost analysis suggested that children attending traditional school - based asps at an average cost of $ 17.67 per day would need an additional daily investment of $ 1.59 per child for 12 weeks to increase their moderate - vigorous physical activity by a model - implied 14.7 percentage points .
conclusions . a low - cost
, alternative after - school program featuring adult - led physical activities in a community recreation center was associated with increased physical activity compared to standard - of - care school - based after - school program . | 1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion |
PMC3523546 | the proprotein convertases ( pcs ) are members
of a mammalian family
of endoproteases related to the bacterial subtilisin and the yeast
kexin .
there are seven pcs that cleave proteins at paired basic
amino acid residues , namely furin , pc2 , pc1/3 , pc4 , pace4 , pc5/6 ,
and pc7 .
the optimal pc recognition sequence
is r - x - k / r - r , while the minimal consensus sequence is r - x - x - r.
a variety of substrates have been described including precursors of
hormones , enzymes , growth factors , receptors , cell membrane proteins ,
and plasma proteins but also a number of pathogenic proteins such
as viral glycoproteins and bacterial toxins .
our previous work showed that pace4 has a role in prostate cancer
cellular proliferation .
pace4 has a wide
expression pattern and is constitutively secreted into the extracellular
media .
it has been suggested from immunohistochemical
observations that in addition to its localization within the secretory
pathway , pace4 is also localized at the cell surface through interactions
between its cysteine - rich domain ( crd ) and heparan sulfate proteoglycan
( hspg ) or tissue inhibitors of metalloproteinases
( timps ) .
recently , two independent studies
( including one from our group ) showed a specific overexpression of
pace4 mrna in prostate cancer tissues .
this overexpression
is correlated with higher circulating protein levels in some patients . using a molecular inhibition approach ,
the relevance
of pace4 in a prostate cancer model has been demonstrated . as the expression levels of other
pcs remains
unchanged , it was suggested that a selective pace4 inhibitor , with
limited inhibition toward furin , might provide a useful tool against
prostate cancer . to our knowledge
, no such inhibitor has been yet
reported ( for complete review see ref . ) . designing
specific pc inhibitors represent an important challenge .
the high homology level deep within the catalytic cleft suggests that
small - molecule inhibitors acting as competitive inhibitors will be
unlikely to produce any specificity . indeed
, structural evidence indicates that the pc
active sites are nearly identical in their s1s4 subsites.a however , there are notable
differences found at the s5 subsite and beyond .
this suggests that peptide - based inhibitors could
be designed to achieve the desired specificity , although they would
require a minimum of six residues .
there is some proof for this concept
based on discovered endogenous peptide inhibitors , such as the 7b2
ct - peptide , which is a highly potent ( nm range ) and specific pc2 inhibitor .
of course , each pc also has an endogenous inhibitor within its structure ,
namely their prodomains , of which the c - terminal
region provides the critical positions for inhibition at the catalytic
sites .
pc prodomains first act in the er as intramolecular chaperones
and then as activity delayers through interactions in cis with the
active site of their cognate pc .
the derived prodomains have been
shown to be potent pc inhibitors in trans but display minimal levels
of specificity .
last , the screening of peptide combinatorial libraries
has led to the identification of polyarginine peptides as furin inhibitors , however , these are also not highly specific .
the present study
reports the development of a new pace4 inhibitor ,
named the multi - leucine ( ml)-peptide .
our focus remains primarily
on discriminating between our target pace4 and furin , which is the
only ubiquitously pc enzyme in normal tissues .
since it is known that
furin inhibition can be lethal ( i.e. , as demonstrated in furin knockout
mice ) , it appears logical to design an inhibitor
that primarily avoids furin as a target .
we also present some of the
characteristics of the ml - peptide as a potent inhibitor of proliferation
in prostate cancer cell lines .
pc prodomains
act as cis regulatory inhibitors during the maturation process and
have been considered as lead compounds for pc inhibition .
our previous
work assessed the inhibitory potency of the prodomains as trans competitive
inhibitors .
however , their inhibitory
potency has never been carried out for pace4 until now ( figure 1a ) .
we observed that most pc prodomains displayed
similar inhibition potencies for pace4 and furin , with ki values ranging from low nanomolar ( 12.4 nm ) to subnanomolar
levels ( 0.34 nm ) .
the inhibition ratios ( furin / pace4 ) were always
in the range of 15 for all prodomains , with the exception
of the pc7 prodomain , which displayed a 36-fold inhibition preference
for pace4 over furin .
the ki obtained
with the pc7 prodomain on pace4 ( 0.34 nm ) is lower than the ki previously reported for inhibition of pc7
by its own prodomain .
( a ) pcs prodomains were produced
and purified to perform inhibition assays toward pace4 and furin .
the ratio between ki for furin and pace4 ,
namely the specificity ratio , point out the selectivity of pc7 prodomain
toward pace4 .
kis in the table
are means and standard deviations of three independent experiments .
( b ) pcs prodomain sequence alignment was performed for the region
p7p1 downstream primary cleavage site .
dark background indicates
conserved residues , while light - gray background indicates residues
of same type than consensus .
uniprotkb accession numbers are the following : hfurin ( p09958 ) , mpc1/3
( p63239 ) , hpc2 ( p16519 ) , mpc4 ( p2921 ) , hpc5/6 ( q92824 ) , hpc7 ( q16549 ) ,
and hpace4 ( p29122 ) . to further investigate the selectivity of pc7 prodomain
inhibition
toward pace4
, a sequence alignment was performed , focusing on the c - terminal regions ( figure 1b ) .
although
prodomains are approximately 100 amino acids in length , our analysis
focused on the dissimilarities in the p1p7 region of the primary
cleavage site . because those residues are directly implicated in molecular
recognition by pcs , we hypothesized that the 36-fold difference might
be related to this c - terminal region .
the sequence
alignment ( figure 1b ) reveals that the pc7
prodomain differs from the consensus in position p6 , as it does not
exhibit a basic amino acid at this position .
apart from the pc2 prodomain ,
a poor inhibitor of both furin and pace4 , only pc7 is dissimilar from
consensus in this position .
it is well - known that the presence of
a basic residue in position p6 promotes increased inhibition potency
for furin .
however , the observation of leu residues
at p6 and p7 positions of the pc7 prodomain suggests that pace4 may
have a preference for hydrophobic residues .
the use
of combinatorial libraries has proven its relevance for the rational
design of inhibitors .
this method has also been used for pc recognition
pattern studies . an 8-mer positional scanning - synthetic peptide combinatorial libraries
( ps - spcl ) approach was used to further study differences in furin
and pace4 inhibition . because it is hypothesized that specific pc
inhibition will be reached by varying the p5p8 positions ,
this library was synthesized by fixing the p1p4 positions
with the core consensus motif rvkr while using the combinatorial method
for the p5p8 positions .
this pattern ensures that each peptide
in the library has the ability to be recognized and bind to the pc s
active site .
the inhibition profiles for pace4 and furin at the p6
position are shown in ( figure 2 ) .
as expected ,
peptides with arg and lys at the p6 position were the most potent
pace4 inhibitors ( ki = 40 and 37 nm , respectively ) .
it is interesting to note that peptides with hydrophobic leu residue
displayed equally strong pace4 inhibitory potency ( ki = 49 nm ) ( figure 2 ) .
in contrast ,
when testing furin inhibition , peptides containing leu in p6 had a
much higher inhibition constant ( 900 nm ) whereas peptides with lys
and arg at this position maintained mid - nm range inhibition constants
( 230 and 300 nm , respectively ) . for this purpose ,
the specificity
ratios were most important and led to the observation that peptides
containing a leu residue showed a 18-fold ratio ( furin / pace4 ) .
the
second highest ratio was obtained with peptide containing his residue
with a 11-fold specificity ratio .
it is relevant to mention that ph
differences in the enzymatic assays might influence this result ( ph
6.5 for pace4 and ph 7.5 for furin ) given that the histidine side - chain
pka is 6.08 .
use of sp - spcl to profile
pc - inhibitor recognition . to better understand
the recognition patterns of pace4 and furin , sp - spcl was used toward
both enzymes .
for each sample in this table , the general recognition
pattern rvkr is present in positions p1p4 and p6 position
are occupied by a unique amino acid .
the other positions of those
peptides are occupied by an equimolar mixture ( x ) of the 19 natural
amino acids , cysteine excluded .
ki was
calculated from ic50 using the cheng and prussof equation
for competitive inhibitors .
thus , this partial combinatorial library approach
confirmed that
pace4 tolerates the presence of a hydrophobic amino acid in the p6
position whereas furin did not and presents a clear preference for
basic amino acids in this position . on the basis of these results ,
peptides containing leu at the p6 position
as
a leu containing peptide could offer a selective inhibition toward
pace4 , the effects of leu n - terminal extensions of
the core sequence ac - rvkr - nh2 , a poor micromolar inhibitor of pcs , was investigated in vitro
( figure 3 ) .
the peptide ac - llrvkr - nh2 was a midnanomolar inhibitor of pace4 , but the progressive
addition of n - terminal leu decreased the inhibition
constant to the low nanomolar range ( 1822 nm ) for both ac - lllrvkr - nh2 and ac - llllrvkr - nh2 .
however , the subsequent
addition of leucine residues increased ki values , reaching higher nanomolar values ( 300 nm ) for the decapeptide
ac - llllllrvkr - nh2 .
peptides
containing three to four leu residues were the most potent inhibitors
of pace4 evaluated in this study and were significantly more effective
on pace4 than furin ( 2022-fold ) .
the peptide ac - llllrvkr - nh2 and now designated as the
ml - peptide was chosen as lead inhibitor for further characterization
on pace4 inhibition .
the inhibitory potency of the ml - peptide was
also assayed with other members of the pc family and also showed high
levels of specificity ( supporting information
table s1 ) . multi - leucine peptides . to stabilize pc
inhibitor
interaction , n - terminal leucine extensions were added
to the core rvkr
sequence .
( a , b ) each peptide was assayed with pace4 and furin in an
inhibition assay .
( c ) kis in this table
are means and standard deviations of three independent experiments .
peptides ac - lllrvkr - nh2 and ac - llllrvkr - nh2 were
the most potent and the most selective inhibitors of pace4 of this
library .
the peptide ac - llllrvkr - nh2 , named multi - leu peptide ( ml ) , was selected as lead compound
for further studies . in a previous study ,
pace4 was proposed as a new therapeutic target
in prostate cancer based on a molecular
inhibition approach using the prostate cancer cell line du145 . because
the molecular inhibition of pace4 in du145 cells had dramatic effects
on cell proliferation in vitro and in vivo
, we decided to test the
ml - peptide as a pharmacological inhibitor to achieve identical results .
in the present study , two additional prostate cancer cell lines
thus , pace4 expression level
was first evaluated in lncap and pc3 cell lines in comparison to du145
cells using a rt - qpcr approach ( figure 4a ) .
pace4 was most highly expressed in lncap cells , with nearly 6-fold
higher levels than du145 cells , but was almost absent in pc3 cells .
du145 and lncap cells also exhibited higher levels of furin mrna than
pc3 cells .
similar expression levels were observed for pc5/6 and pc7
within all cell lines investigated and pc1/3 and pc2 were undetectable .
the effect of the multi - leu peptide on cellular proliferation of each
cell line was evaluated using mtt assays ( figure 4b ) .
the ml - peptide showed a very poor inhibition of pc3 cells ,
whereas the half - maximal inhibitory concentrations ( ic50 ) were in the micromolar range for du145 and lncap cells ( 100
10 and 180 60 m , respectively ) .
thus , the ml - peptide
inhibited the proliferation of du145 and lncap cells , but not pc3
cells , showing a strong correlation with cellular pace4 expression .
in previous work , our research team proposed pace4 as a therapeutic
target against prostate cancer progression .
( a ) du145 , lncap , and pc3 prostate - cancer derived cell lines were
first screened to compare their pcs expression levels using rt - qpcr .
( b ) to assess the efficiency of our new pace4 inhibitor in such
context , mtt assays were performed on those cell lines .
ml is efficient
to inhibit proliferation and metabolic activity in du145 and lncap ,
two pace4-expressing cell lines , indicating a possible role for ml
as a prostate cancer therapeutic .
( c ) to prove that the inhibition
observed is a pcs dependent mechanism , the peptide ac - llllrvk[darg]-nh2 was used as negative control .
because the p1 position is a key residue of the recognition pattern ,
the replacement of p1 arg by darg significantly affected the ki for this peptide .
( d , e ) as expected from inhibition
constant values , the peptide ac - llllrvk[darg]-nh2 is a poor proliferation inhibitor in a mtt assays with
du145 and lncap .
an additional control experiment was performed
to test the pc - specific
interaction of the ml - peptide resulting in cell proliferation inhibition
by designing a ml - peptide substituted at the p1 position with a darg .
as the p1 arg position is critical for pc recognition
, this modification
should strongly abrogate the observed effects unless they are not
pc - mediated .
as expected , the peptide ac - llllrvk-[darg]-nh2 showed a substantial loss of affinity
in vitro going from a nm to a m inhibitor ( figure 4c ) ( kis = 1380 and 2600 nm for
pace4 and furin , respectively ) .
consistent with this affinity loss ,
this peptide also showed a significant loss of potency in both du145
( figure 4d ) and lncap ( figure 4e ) cell - based assays ( ic50 440 80 and 390
10 m , respectively ) .
these data make a strong case that
decreased cell proliferation is mediated by pc inhibition .
pace4 is localized both at the cell surface
and within the intracellular secretory pathway .
thus , it was relevant to determine whether
the inhibitory effect of the ml - peptide is mediated by cell surface
and/or intracellular pace4 . to elucidate this question , ml - peptide
analogues bearing n - terminal modifications with strikingly
different cell penetration properties
was coupled with an n - terminal amino - poly(ethylene
glycol)-8 ( -amine--propionic acid octaethylene glycol ,
known as peg8 ) group that should render the peptide much less hydrophobic ,
whereas a n - terminal hydrophobic 8-amino - octanoyl
( c8 ) chain was used in order to increase its hydrophobicity . in vitro
enzymatic activity assays demonstrated that pegylation and alkylation
of the ml - peptide did not alter its potency or selectivity for pace4
inhibition ( figure 5a ) .
the uptake of fitc - labeled
versions of these ml - peptide analogues were tested on du145 cells
and analyzed using a fluorescence - activated cell sorter ( facs ) ( figure 5b ) .
the fitc--ala - ml - peptide had excellent
cell penetration properties , considering it is relatively unmodified ,
suggesting that the hydrophobic content of this peptide is sufficient
to penetrate cell membranes ( see also confocal microscopy in supporting information figure s1 ) .
however , cells
treated with fitc-[peg8]ml - peptide showed a geometric mean of the
distribution ( geomean ) of about 1 log unit lower than the one measured
for the fitc--ala - ml - peptide ( 5.437.84 vs 48.9052.82 ) .
these results indicate that the fitc-[peg8]-ml - peptide penetrates
the cell membrane very poorly . as expected , the fitc - c8-ml - peptide
displayed greater cell penetration properties with 5-fold greater
geomean compared to the control fitc--ala - ml - peptide ( 260.08261.95
vs 48.9052.82 , respectively ) .
when all three peptides were
tested on du145 cells , the [ c8]-ml - peptide gave very similar results
to the ml - peptide by maintaining antiproliferative effects ( ic50s 80100 m ) while the [ peg8]-ml - peptide could
no longer reduce cell proliferation ( ic50 was estimated
to be > 500 m ) ( figure 5c , d ) .
this
result
provides strong evidence that the antiproliferative actions of the
ml - peptide were largely mediated by intracellular pace4 .
a second
conclusion is that the cell penetration properties of the unmodified
ml - peptide are sufficient to achieve a near maximal effect .
ml - peptide
targets intracellular pace4 to inhibit prostate cancer
cell lines proliferation .
( a ) to determine whether the cell proliferation
effects are mediated by cell surface or intracellular pace4 , ml n - terminal extensions were design to modify cell penetration
properties of our inhibitor . neither pegylation ( peg8 ) nor alkylation
( c8 ) modified the affinity of multi - leucine inhibitor toward pace4
and furin , as determined in a kinetic assay .
( b ) the uptake of fitc - labeled
peptides was tested on du145 cells and analyzed by facs .
comparison of geomeans
( numbers adjacent the peaks ) obtained with these three peptides indicates
that pegylation prevents cell entry , whereas alkylation increases
the uptake of the inhibitor .
( c ) using a mtt metabolic assay , it was
clearly demonstrated that alkylation increases inhibitory potency
of ml peptide , whereas pegylation leads to a poor proliferation inhibitor
in du145 cell line .
( d ) ic50 in the table are means of
five independent experiments . n.c .
means the curve did not converged
to 50% with doses up to 500 m .
this assay demonstrates that
ml peptide must enter the cell in order to inhibit du145 proliferation . to obtain
further support for the antiproliferation effects observed , a dna
content analysis on lncap treated cells
was performed with 100 or
200 m of ml - peptide ( figure 6 ) . as these
assays are performed over a 96 h period , which could result in peptide
degradation , a modified analogue ac-[dleu]lllrvkr - nh2 was also used .
the substitution of
the n - terminal leu by a dleu provided improved stability
of the peptide to amino - peptidases ( unpublished data ) . a dose - dependent
g0/g1 accumulation and s phase decrease were
observed following exposure to ml - peptide and ml - peptide analogue .
following a 200 m treatment with the ac-[dleu]lllrvkr - nh2 , a 10% increase in the g0/g1 population
was observed along with an increase
in cells with hypodiploid dna content ( sub - g1 ) proportions ,
which represent apoptotic cells .
the effect of pace4 inhibition on cell cycle distribution was observed
by flow cytometry .
lncap cells were treated for 96 h with 100 or 200
m of peptide .
( a ) ac - llllrvkr - nh2 or ( b ) ac-[dleu]lllrvkr - nh2 .
cell cycle distribution was assessed from
cellular dna content analysis of cells treated with propidium iodine .
the significance of the results were established from
an unpaired two - tailed t test .
( * p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 ) .
( c ) data in the table are mean and standard deviation
of a representative experiment .
furin was the first discovered
pc , and ever since the discovery
of additional pcs , issues of distinct and redundant functions have
been debated for this family of enzymes .
while this remains
a fundamental interrogation , it can also be envisaged that an answer
to this question could have repercussions on the druggability of pcs
in various pathologies .
structural data have been very clear that
within the deep subsites of the catalytic cleft ( s1s4 ) , the
pcs appear to be virtually identical .
this has an immediate consequence
from a medicinal chemistry point of view , that small - molecule inhibitors
( i.e , small organic compounds ) will not be specific if they are competitive
inhibitors .
it can not be excluded that small molecules could specifically
inhibit pcs if they have an allosteric mechanism of action , but to
date no such inhibitors have been reported nor have druggable allosteric
pc sites been established . therefore , the most probable option presently
is to use peptides as lead compounds to design specific pc inhibitors .
this is plausible based on the observed structural differences within
the s4s7 subsites , as deduced by homology modeling of the
furin structure .
peptide - based inhibitors afford a number of advantages ,
the principal one being a great variety of structures that could provide
an optimal fit , providing the sought specificity .
the disadvantages
of peptide - based inhibitors are in their further use as in vivo drug
compounds because they can be readily degraded , may be excreted rapidly ,
and are said not to be very cell penetrable .
there is mounting
evidence of the potential benefits of inhibiting pace4 in prostate
cancer cells .
should a therapeutic application be possible , it would
have to be through the development of a relatively specific pace4
inhibitor .
thus , we have focused our studies on a peptide - based approach
to target pace4 , with the full knowledge of the obstacles that may
arise . as the starting point to design a specific pace4 inhibitor ,
the
potency and the selectivity of purified pc prodomains toward pace4
and furin were evaluated ( figure 1a ) .
as furin
is the only basic amino acids cleaving pc ubiquitously expressed ( i.e. ,
defined as expressed in every single normal cell ) and furin full knockouts
in mice have proven to be lethal , it
seemed appropriate to establish an inhibitor that favors pace4 as
much as possible over furin .
this objective may appear almost insurmountable
due to the stated structural similarities of the two enzymes and multiple
data suggesting very similar substrates . nonetheless , the peptide
designs focus on obtaining the best specificity ratio of furin / pace4
inhibitory potency ( furin ki /pace4 ki ) . as previously reported in various studies , including
our own previous
work , pcs prodomains are highly potent inhibitors of pcs
( nm and sub - nm range ) , except for pc2 prodomain , which displays no
inhibition at concentrations up to 1 m
. however , one observation
stands out , namely the specificity ratio of the pc7 prodomain , which
was 36-fold ( furin ki / pace4 ki ) .
the alignment of pc prodomains was performed for region
p7 to p1 , as this region contains key residues for substrate - pcs recognition
( figure 1b ) and highlights the higher density
of hydrophobic residues in this region .
the pc7 prodomain clearly
stands out as it differs from the consensus in position p6 .
this suggests
that basic residue in p6 position could be crucial for potent furin
inhibition , whereas pace4 may tolerate a broad range of residues for
this position .
the combinatorial peptide library provided additional
support for
this hypothesis ( figure 2 ) .
this library was
anchored at the c - terminal with the consensus motif
rvkr to allow the study of p5p8 positions .
it was first observed
that kis were higher for furin than pace4
for every peptide mixture . as positions p8 , p7 , and p5 of those samples
are occupied by an equimolar mix of the 19 natural amino acids ( cysteine
excluded ) , this could indicate that pace4 can accommodate a more diversified
selection of amino acids .
thus , when looking at kis for position p6 for furin , it can be concluded that
furin has a marked preference for basic residues , as lys and arg peptides
are the only midnanomolar inhibitors of this enzyme in this library .
on the other hand , although p6 lys and p6 arg peptides are mid - nm
inhibitors of pace4 , similar levels of inhibition can be reached with
p6 leu peptides .
the highest specificity ratios for p6 position were
obtained from leu , his , val , ile , and ala containing peptides .
on
the basis of the hydrophobic nature of leu , val , ile , and ala , this
suggests that pace4 tolerates the presence of hydrophobic residues
in position p6 and this opens possibilities to design specific inhibitors .
the synthesized series of ml - peptides validated the differences
between furin and pace4 in the p5p8 region . to date , the ml - peptide
is the best pace4 specific inhibitor described .
although furin and
pace4 share very identical catalytic domains and an almost identical
catalytic cleft , our design of the ml - peptide selective
pace4 inhibitor shows that even with these similarities , both enzymes
have different binding affinities .
the results highlight the fact
that furin does not provide nonpolar peptide stabilization in its
catalytic cleft while pace4 has good binding affinity with both cationic
and nonpolar peptides . to hypothesize on the origin of partial selectivity
between differently charged substrates , a homology model of pace4
containing peptide ac - rvkr - cmk in the binding cleft
was built using modeler 9v8 from crystalline furin 1p8j(31 ) ( figure 7 ) .
stereoscopic views of ( a ) mouse furin
crystal catalytic cleft ( 1p8j ) and ( b ) pace4 homology model .
asp and glu negative
charges are shown in red , whereas arg and lys positive charges are
shown in blue .
green spheres represent dicationic calcium ions and
decanoyl - rvkr - cmk inhibitor has been modified to
ac - rvkr - cmk for clarity .
a close examination of the whole catalytic domain
of pcs shows
various degrees of charge excess , as previously reported for other
subtilisins . by looking at the number
of positively charged arg and lys ( i.e. , his being mostly neutral
at cytoplasmic ph
is left out ) and negatively charged asp and glu ,
the overall total charge calculated for both convertase catalytic
domains are 7 for human furin and 0 for human pace4 ( i.e. ,
the catalytic domain of furin goes from n - terminal
prodomain primary cleavage site asp108 to val444 and from gln150 to
ala496 in pace4 ) .
the active site of both furin and pace4 also reveals
that the s1 , s2 , and s4 sites are formed by residues with similar
properties , e.g. , glu257 and glu230 in furin replace asp309 and asp282
in pace4 , respectively ( figure7 ) . on the other
hand ,
the differences in affinity observed with multiple leu extension
in the ml - peptide propose the existence of notable differences in
subsite s5 and beyond . to gain a deeper insight of the phenomenon ,
it is necessary to dissect the binding cleft of both pcs .
one common
feature among all the subtilases is the presence of a groove running
from the catalytic site to helixes 3 and 4 that stabilizes
a peptidic substrate in an antiparallel -sheet conformation
through an induced fit process .
this interaction can also be seen
in the noncatalytic pcsk9 structure ( 2pmw ) between the prodomain and the region
homologous to the catalytic cleft of the other pcs ( ser147 to gln152 ) . despite the fact that other residues might be
involved ,
the same trend remains for furin and pace4 : a way of hosting
a peptidic substrate or inhibitor adopting an antiparallel -sheet . on the basis of these data
, we propose that
the linear conformation of the peptide brings the p6p8 residues
of the inhibitor in the vicinity of the residues located at the tip
of helix 4 , and this region of the enzyme would represent the
subsites s6 , s7 , and s8 .
we also focused our analysis on the disparities
in this region to better understand the specificity observed for ml - peptide
because the solvent accessible residues of helix 4 differ in
pace4 and furin . from the homology model of pace4
, it appears that
the most critical dissimilarity in the helix 4 comes from the
replacement of acidic residues in furin by either basic or neutral
residues in pace4 ( glu271 and glu272 in furin exchanged for lys323
and gln324 in pace4 ) .
consequently , the n - terminal
end of helix 4 has a charge of 1 in furin and + 2 in
pace4 .
finally , an intramolecular quench of charges in pace4 may explain
its potential to complex neutral ligands .
such self - quenching is unlikely
for furin in the n - terminal region of the helix 4 .
in sum , the fact that furin catalytic clef is more negatively charged
than pace4 might very well account for much of the selectivity showed
by the two pcs .
the cellular effects of the ml - peptide are remarkable ,
as they
phenocopy the antiproliferation effects observed in our previous molecular
studies knocking down pace4 in du145 cells .
this study also provided additional evidence for the pace4 targeted
antiproliferation using lncap cells .
however , the effects of the ml - peptide
are largely ineffective in the pc3 cell lines , as it expresses very
little if any pace4 . as a control ,
the polybasic peptide ac - rarrrkkrt - nh2 , which is a potent furin
inhibitor of similar length , was tested
in the mtt cell - based assays ( with lncap and du145 cells ) and no antiproliferative
effects could be observed with this peptide ( supporting
information figure s2 ) .
we are confident that
the furin target was reached by the peptide , as in a previous study
we showed that this potent furin inhibitor has cell penetration properties .
additional control peptides provide evidence
that the ml - peptide indeed targets pace4 because the ac - llllrvk[darg]-nh2 shows a substantial loss
of affinity in vitro and is also a poor inhibitor of du145 and lncap
cell proliferation ( figures 4d , e ) .
pace4
is localized at the cell surface and in the extracellular
matrix due to interactions between its cycteine - rich domain ( crd ) ,
heparin sulfate proteoglycans ( hspgs ) , and tissue
inhibitors of metalloproteinases ( timps ) . to assess the importance of cell surface pace4 in tumor progression ,
the ml - peptide was modified with n - terminal pegylation ,
significantly reducing its cell penetration property while not affecting
its inhibitory potency ( i.e , pegylated ml - peptide has a similar ki to the ml - peptide ) .
because the pegylated
ml - peptide significantly losses its ability to inhibit du145 proliferation ,
it leads to the conclusion that intracellular pace4 is required for
this function .
a reduction of cellular proliferation could occur
by a number of
mechanisms downstream of pace4 inhibition , but most likely a number
of important growth factors which are substrates of pace4 have lost
their activity due to lack of processing .
this general lack of growth
factor activity would eventually have effects on the cell cycle .
therefore ,
a cell cycle analysis was performed to evaluate if cell cycle arrest
or slow - down was possible .
a dose - dependent accumulation of cells
in g0/g1 phase was observed , thus preventing
cells entry into s phase .
the transition between g1 and
s phase is a finely regulated mechanism controlled by a combination
of environmental considerations mostly influenced by the presence
of growth signals and the discontinuation of extracellular inhibitory
signals .
this could suggest that the ml - peptide inhibits the processing
of growth factors required to go beyond the restriction point and
therefore triggers a cell cycle arrest at this point .
however , while
outside of the scope of the present manuscript , further studies identifying
these substrates and signaling pathways involved will be required .
nonetheless , various potential substrates have been suggested in various
other studies . of interest was the observation
of apoptosis following an exposure to 200 m ac-[dleu]-lllrvkr - nh2 in lncap cells .
this phenomenon
may be easily explained by the fact that cell cycle arrest is usually
poorly tolerated and prolonged cytostasis must be escaped by cell
death .
if this is true , then more potent
and stable versions of the ml - peptide inhibitor could result in exceptional
drugs that reduce prostate cancer cell proliferation as well as inducing
specific cell death . in the context of the metastasis phase of prostate
cancer
synthesis of sp - spcl peptide library
was performed as described previously ( torrey pines institute for
molecular studies ) .
all other peptides were obtained
by solid - phase peptide synthesis ( 50 mol scale ) on a polystyrene
resin , tentagel s ram ( rapp polymere , capacity 0.23 mmol / g ) , with
a pioneer peptide synthesizer ( applied biosystems ) , according to standard
coupling procedures and fmoc strategy .
the protected amino acids were coupled at 3-fold excess using hatu
as coupling agent in the presence of dipea in dmf .
after the final
fmoc deprotection , with the exception of fitc - labeled peptides , n - terminal acetylation was carried out in dmf with acetic
anhydride ( 0.5% ) and 2.6-lutidine ( 0.6% ) .
the fitc - peptides were labeled
with fluorescein isothiocyanate isomer i ( fitc ) through their n - terminus ( -ala was used as a spacer for the peptide :
fitc-[-ala]-llllrvkr - nh2 ) .
dcm ( 1:4 ) was added
to the resin and allowed to couple overnight .
after completion of
the synthesis , the protected peptidyl resins were treated with tfa h2o
tis ( 95:2.5:2.5 ) and stirred for 3 h. the solutions
with the released peptides were filtered and evaporated in vacuo to
a volume of about 1 ml . then the peptides were precipitated with diethyl
ether to afford crude products .
the crude compounds were purified
by semipreparative hplc ( agilent technologies , 1100 series hplc equipped
with a diode array detector ( dad ) ) on reversed - phase support agilent
c18 column ( 15 m , 100 , 7.8 mm 300 mm ) .
the purity of the peptides was controlled using analytical hplc . a
seldi - tof mass spectrometer ( bio - rad laboratories )
was used to confirm
the identity of the pure products ( molecular ion ) . according to both
hplc and mass spectrometry ,
the peptide content analyses were performed
in vacuo with a beckman 120c autoanalyzer following 24 h hydrolysis
in 5.7 n hcl at 110 c and analyzed using a varian ds604 system
integrator .
recombinant
soluble human pcs were produced from s2 insect cells and purified
as previously described .
briefly , s2
conditioned medium was purified using ultrafiltration , anion exchange
chromatography , hydrophobic interaction chromatography , and gel filtration .
the prodomains of pcs were produced in one shot top 10 cells ( invitrogen )
transformed with cdna construct of full - length prosegment .
the recombinant
prodomains were purified from bacterial lysate by nickel chromatography
and reverse - phase hplc as previously described .
enzyme inhibition assays for
furin were performed in 100 mm hepes ph 7.5 , 1 mm cacl2 , 1 mm -mercaptoethanol , and 1.8 mg / ml bsa , while assays for
pace4 were performed in 20 mm bis - tis ph 6.5 , 1 mm cacl2 , and 1.8 mg / ml bsa . all assays were performed with the substrate
pyroglu - arg - val - lys - arg - methyl - coumaryl-7-amide ( bachem , ca ) at 100
m .
assays were carried out at 37 c for 60 min , and real - time
fluorescence was measured using a gemini em 96-well spectrofluorometer
( molecular devices , ca ) ( em , 370 nm ; ex .
inhibitory peptides and prodomains
were added to the assays at decreasing concentrations to perform a
competitive inhibition assays .
kinetics assays were analyzed using
softmaxpro5 , and ki values were determined
from ic50 using cheng and prusoff s equation with km values of
4.61 m for furin and 3.5 m for pace4 .
the quality of the total rna sample
was assessed using an agilent bioanalyzer with the rna nano chip ( agilent
technologies ) .
briefly , 1 g rna was reverse transcribed and
the obtained cdna was used to carry out qpcr analysis .
relative expression were calculated using -actin
as a reference gene and the formula ( 1 + amplification efficiency )
(ct ) for each cell line , as described previously . to perform mtt assays ,
both du145 and pc3 cell lines were seeded at a density of 1500 cells
per well in 96-wells plates .
lncap cells were seeded at a density
of 2500 cells per well in a poly lysine coated 96-well plate .
after
24 h , media was changed and inhibitory peptides were added to the
cells . the peptides were incubated with the cells for 72 h prior to
addition of mtt reagent at a final concentration of 1 mg / ml . mtt reagent
was incubated 4 h with du145 and lncap and 6 h with pc3 cells , and
then media was removed and formazan was solubilized with 100 l
of 2-propanol hcl ( 24:1n ) .
the total metabolic activity was
normalized relatively to vehicle treated cells ( sterile bidistilled
water 0.1% dmso ) .
each step of mtt , as well as maintenance of du145 ,
lncap , and pc3 cells , were carried in rpmi 1640 5% fbs for du145 and
10% fbs for lncap and pc3 .
the uptake of fitc peptides
was tested on du145 cells and analyzed with a facscan cytometer ( becton
dickinson , mountain view , ca ) .
first , 4 10 cells
were incubated 1 h in serum - free rpmi media with 10 m of fitc--ala - ml ,
fitc-[peg8]-ml , or fitc-[c8]-ml and collected by centrifugation .
then
the cell pellets were washed twice with trypsin ( 0.05% v / v ) during
5 min at 37 c to remove nonspecific interactions with the membrane .
cells were incubated 2 min with propidium iodine ( 10 g / ml )
in order to exclude cells with altered membrane .
a minimum of 10000
events per sample was acquired , excluding cell clumps and debris .
geomeans were determined using cellquest software ( becton dickinson ,
mountain view , ca ) . to perform cell cycle analyses
on lncap , 4
10 cells were seeded in 10 cm culture
dishes and grown for 24 h without treatment .
cells were then treated
with vehicle ( 0.1% dmso ) or with 100 m or 200 m of peptides
ac - llllrvkr - nh2 or ac-[dleu]lllrvkr - nh2 .
treatments were carried
out in complete medium ( 10% fbs ) for a period of 96 h , and cell media
were changed every 24 h to offset peptide degradation .
cells were
harvested using trypsin , washed once with pbs , resuspended in 0.5
ml of pbs , and fixed by dropwise addition of 1.5 ml of ice - cold ethanol .
after a 30 min incubation at room temperature , cells were washed with
pbs and dna staining was performed in 20 mm hepes ph 7.2 , 0.16 m nacl ,
and 1 mm egta buffer containing 10 g / ml of rnasea and 10 g / ml
of propidium iodine .
flow cytometry was performed using a facscan
cytometer ( becton dickinson , mountain view , ca ) equipped with a 15
mw argon ion laser tuned at 488 nm .
forward and side scatter signals were used
to establish the live gate to exclude debris and cell clumps and a
second live gate was set using the fl3-a and fl3-w parameters of the
doublet discrimination module ( ddm ) , allowing single cell measurements .
the percentages of cells in different phases of cell cycle were calculated
by modfit software ( verity software house , topsham , me ) . | the proprotein convertases ( pcs ) play an important role
in protein precursor activation
through processing at paired basic residues .
however , significant
substrate cleavage redundancy has been reported between pcs .
the question
remains whether specific pc inhibitors can be designed .
this study
describes the identification of the sequence llllrvkr , named multi - leu
( ml)-peptide , that displayed a 20-fold selectivity on pace4 over furin ,
two enzymes with similar structural characteristics .
we have previously
demonstrated that pace4 plays an important role in prostate cancer
and could be a druggable target .
the present study demonstrates that
the ml - peptide significantly reduced the proliferation of du145 and
lncap prostate cancer - derived cell lines and induced g0/g1 cell cycle arrest .
however , the ml - peptide must enter
the cell to inhibit proliferation .
it is concluded that peptide - based
inhibitors can yield specific pc inhibitors and that the ml - peptide
is an important lead compound that could potentially have applications
in prostate cancer . | Introduction
Results
Discussion and Conclusions
Experimental Section |
PMC3163818 | the american demographer and sociologist valerie oppenheimer wrote a series of influential articles in which she emphasized the role of men s socioeconomic position in demographic change , in particular in the declining rates of marriage and the underlying tendency to increasingly postpone and perhaps even forego marriage ( oppenheimer 1988 , 2000 , 2003 ; oppenheimer et al .
i review oppenheimer s original theoretical study , i discuss how her study was held up in empirical research in europe , and i provide a new test of the theory for the european setting . in doing so , i try to resolve some remaining gaps in the empirical literature , and i evaluate whether the theory is equally valid in different countries that make up the european context . given the recent economic crisis in the united states and in europe , and the growing concerns about economic inequality , the influence of men s economic position on marriage and family formation remains a vital concern . at the time oppenheimer began writing her articles on
how men s economic position influenced marriage formation in the late 1980s and early 1990s this was generally not a popular idea .
the declining rates of marriage and increasing rates of divorce were typically conceptualized in terms of an " erosion of marriage . "
one theory looked for the culprit in the growing economic role of women in society .
this theory was voiced by demographers and economists working from a micro - economic perspective ( becker 1981 ; espenshade 1985 ; farley 1988 ) , although , as oppenheimer noted ( 1988 , p. 575 ) , it bore a strong resemblance to classic sociological theories formulated by functionalists like talcot parsons ( parsons 1949 ) .
the explanation basically argued that more symmetrical economic roles of men and women would lead to a decline in the gains to marriage , or to put it in parsonian terms , would undermine marital solidarity .
the second explanation argued that the decline of marriage was related to value change , and in particular to the increasing need for individual autonomy on the one hand , and the ideological condemnation of traditional institutions like marriage on the other .
this second perspective was expressed more strongly by european demographers like lesthaeghe and van de kaa although it was also used by the influential american demographers at the time ( bumpass 1990 ; rindfuss and van den heuvel 1990 ) . in their second demographic transition theory , lesthaeghe and van de kaa argued that ideological change in combination with secularization was driving not only the postponement of marriage , but also the increase in cohabitation , the rise in divorce , and the decline of fertility ( lesthaeghe 1983 ; lesthaeghe and meekers 1986 ; lesthaeghe and surkuyn 1988 ; van de kaa 1987 ) .
while the first explanation saw the engine of the demographic transition in economic change , the second emphasized the primacy of cultural change .
both theories , however , were pessimistic about the future of marriage : the economic perspective saw marriage as incompatible with symmetrical gender roles , the second saw it as incompatible with individualistic values . while there was a considerable debate between the proponents of economic and cultural explanations , oppenheimer criticized both perspectives .
for example , she noted that there were no signs of a so - called independence effect .
women with attractive economic resources were not less likely to enter marriage , as would be predicted from the micro - economic perspective ( oppenheimer and lew 1995 ) .
although women s employment and education had an effect on fertility and divorce , this did not appear to be the case for marriage timing ( oppenheimer 1997 ) .
oppenheimer also had empirical critique on the cultural perspective . when looking at simple descriptive statistics on what people want for themselves on people s hopes and desires she noted that the majority of both single men and women still wanted to be married ( oppenheimer 1994 ) .
the anti - marriage ideology may have existed in feminist circles or in the pop culture of the sixties , but it had not spread to a larger audience in the way that , for example , egalitarian gender norms had done .
oppenheimer also had theoretical criticisms of the two explanations ( oppenheimer 1994 , 1997 ) .
first , she believed that the theories were basically about nonmarriage and not about delays in marriage .
as other demographers also had observed , the declining marriage rate was primarily driven by increases in the age at marriage , and not so much by a decline in the proportion of persons who marry eventually , although the latter could of course not yet be observed in the late 1980s .
this seems by and large correct now , although the proportion of the marrying persons among the lower educated in the united states did appear to decline ( goldstein and kenney 2001 ) .
a second part of her theoretical critique was against the micro - economic model of specialization . quoting historical demographic work , oppenheimer noted that wives in the past had always worked for pay when circumstances required this .
wives worked to make ends meet when the husband was not making enough money , when he was unemployed , or when household costs were temporarily pressing ( oppenheimer 1982 ) .
oppenheimer argued that specialization in marriage is an inflexible and risky strategy in many different societal contexts .
if marriage was not based on a model of full specialization in the more distant past , oppenheimer argued , why would it then cease to exist in the modern era in which wives began to work ?
oppenheimer not only criticized the then dominant perspectives on demographic change , she also presented an alternative .
her explanation can be placed in the economic rather than the cultural camp , but it was different in that it focused on men rather than women . during the 1980s and 1990s , young men
s economic position in the united states had deteriorated quickly , especially for those with little schooling . in the poor and uncertain economic prospects of young men
, oppenheimer saw an important potential for understanding the decline of marriage . because the earlier explanation had focused more on women especially through arguments about women s economic independence
one could say that oppenheimer was in fact " bringing men back into the debate . "
first , she reinstated older malthusian ideas about the economic costs of marriage ( hajnal 1965 ; easterlin 1980 ) . as setting up and running a household costs money , men who are unable to fulfill
oppenheimer recognized that this traditional male - breadwinner hypothesis may have lost some of its force when gender roles become more symmetrical .
nonetheless , she argued that it would also be naive to expect men s economic resources to become unimportant in influencing marriage prospects : this would be " throwing out the baby with the bathwater .
" the second way in which she brought men back in the debate was through her uncertainty hypothesis ( oppenheimer 1988 ) .
the argument is that unstable careers , as indicated by low - status jobs , nonemployment , and irregular and temporary employment , signal uncertainty .
this uncertainty applies not only to whether the husband will be able to provide in the future , but also to the type of life he will lead . work structures the lifestyle a person will develop , and when men have not yet settled in their career it is difficult to predict what married life will be like . in this way
. an important difference between the breadwinner and the uncertainty hypotheses is that the former focuses primarily on the financial aspects of employment whereas the latter is also concerned with its social consequences .
an implication is that the neo - malthusian argument would be fully covered by effects of income , whereas the uncertainty argument would also be reflected in indicators like irregular attachment to the labor market , the amount of work experience , career trajectories , and temporary employment . in a separate article
, oppenheimer also developed and operationalized the concept of a stopgap job , i.e. , a job that is not a reflection of an employee s educational credentials and that is meant as temporary by both employer and employee ( oppenheimer and kalmijn 1995 ) .
men in such stopgap jobs would postpone marriage because they are not settled in their career and therefore can not yet make a suitable match in the marriage market .
compared to the other two perspectives , oppenheimer s theory has a more optimistic implication for the future of marriage .
after all , female labor force participation was unlikely to decline in the future and individualism did not appear to be receding . in oppenheimer
s theory , the economic position of young men largely depends on macro - economic conditions . because unemployment rates tend to have cyclical rather than linear trend patterns , the economic position of young men could improve and this would then have positive repercussions for marriage .
moreover , the theory only implies the postponement of marriage until men accumulate more work experience and become settled in their career , and not an erosion of the institution of marriage , as the other theories seem to imply .
oppenheimer 's explanation had a second attractive feature : it could also explain another important demographic trend , namely the rise in cohabitation ( oppenheimer 2003 ) .
oppenheimer argued that a man s failure to provide economically would be less of a problem for cohabitation than for marriage .
for many couples , cohabitation is a trial stage before marriage , and it may be that uncertainty about a young man s position is more tolerable during the cohabiting stage than it would be for a long - term commitment to marriage .
assuming that the costs of breaking up a cohabiting union are lower , cohabitation can therefore provide a way for couples to reduce uncertainty about future career prospects . in a sense , oppenheimer argued that a cultural innovation like cohabitation before marriage ( on a massive scale ) was the outcome of economic needs rather than the result of ideological change . in line with this , other authors even argued that cohabitation is a rational response to uncertainty : a flexible partnership well - suited for a flexible labor market ( mills et al .
american research generally supports the view that poor economic prospects for men are associated with a delay in marriage .
this has been demonstrated for a range of indicators , including employment per se , unstable employment , low earnings , and other indicators of career " immaturity " ( clarkberg 1999 ; lichter et al .
1992 ; lloyd and south 1996 ; mare and winship 1991 ; oppenheimer 2003 ; oppenheimer et al .
1997 ; sassler and schoen 1999 ; smock and manning 1997 ; sweeney 2002 ; xie et al .
there is also evidence in the united states that cohabitation is less strongly influenced by men s economic position than marriage , although there is no clear reverse income effect , i.e. , that the poor are being selected into cohabiting unions .
furthermore , in the united states , the income effect on marriage timing appears to be stable over time . sweeney ( 2002 )
compared two cohorts in the united states and found that in the cohort marrying during the 1980s and 1990s , men s income had an equally strong positive effect on the entry into marriage as in the cohort marrying during the 1960s and 1970s ( sweeney 2002 ) .
in this article , i develop a new test of oppenheimer s theory for the european context .
first , the demographic trends that occurred in europe were similar , although sometimes less dramatic and sometimes occurring later .
the age at marriage has risen , the rate of marriage has declined , and cohabitation has increased as well ( kiernan 2002 ; lesthaeghe 1983 ; van de kaa 1987 ) .
second , many european countries experienced economic problems that were similar to those in the united states .
they were especially salient for outsiders on the labor market , such as young adults , ethnic minorities , and women .
several authors argued that partly in response to economic globalization , young european men ( and women ) faced increasing levels of economic uncertainty in their transition from school to work ( blossfeld et al . 2005 ) . in many european countries , especially in southern europe ,
levels of youth unemployment were even higher than in the united states , a phenomenon which has often been linked to the higher degrees of employment protection in europe ( mller and gangl 2003 ; nickell 1997 ) .
there are also reasons to believe that the theory would be less applicable to europe .
one counter argument lies in the role of the welfare state . in several european societies , and particularly in social - democratic welfare states like sweden , denmark , and the netherlands , social security is more generous and more universally provided than it is in the united states ( arts and gelissen 2002 ; esping - andersen 1993 ) .
this means that in many european countries , young men receive unemployment benefits when they are out of work .
moreover , for those who have never worked , basic welfare is provided , albeit at a minimum level . as a result
following the neo - malthusian argument , it could thus be argued that employment problems do not per se lead to marriage postponement in europe .
a rebuttal of this point is that oppenheimer s argument about uncertainty and assortative mating , which is not only about money , but also about stability and predictability , could still apply to europe .
a young man who is on unemployment benefits remains an uncertain candidate on the marriage market even if he has the financial means to support a household at that point in time .
another important difference between the american and the european case lies in the degree of heterogeneity .
although the united states is certainly not a homogeneous country there are important ethnic , racial , and regional differences it is fair to say that europe is more heterogeneous ( at least regionally ) than the united states . in comparative studies , it has often been argued that european countries can be rated on a continuum from more traditional societies such as spain , greece , and italy on the one hand , to more modern and more economically developed societies such as sweden and the netherlands on the other ( hagenaars et al .
these differences are expressed in a number of social and cultural domains , including differences in marriage and family living .
for example , in more traditional european societies , cohabitation and divorce are less common and less accepted , marriage has a higher social status , gender roles in marriage are more unequal , female labor force participation is lower , and extended family ties are stronger ( hans - peter blossfeld and hakim 1997 ; gelissen 2003 ; kalmijn 2003 ; knudsen and waerness 2008 ; reher 1998 ) .
these indicators are strongly correlated , both with each other and with the level of economic development in a country ( gdp ) .
this degree of heterogeneity suggests that oppenheimer s theory may not apply equally to all european countries .
for example , in contexts where gender roles are more egalitarian , men s economic situation could be less important for the entry into marriage and cohabitation . in these settings , men are not the only breadwinners and women s economic resources should be of growing importance , making men s economic resources less important . what has the evidence in europe shown so far ?
perhaps the most important source of evidence comes from a large multi - nation project initiated by the german sociologist blossfeld and his colleagues ( blossfeld et al .
in this project , blossfeld brought together a number of demographers and sociologists from different parts of the world ( with an overrepresentation of european countries ) , with the aim of examining the effect of men and women s individual economic resources on the timing of marriage and parenthood .
while the authors used their own country - specific longitudinal data , they used similar methods and variables , leading to a reasonably uniform and comparable set of outcomes .
the project s goal was to test the exact same set of hypotheses in each country .
the hypotheses were borrowed in part from oppenheimer s work but they were translated by mills and blossfeld to make them fit for a broader societal setting ( mills and blossfeld 2005 ) .
the articles were combined in a volume for which oppenheimer wrote the foreword ( blossfeld et al .
the articles in blossfeld s volume provide generally positive evidence for the theory in the european countries studied ( germany , the netherlands , france , sweden , hungary , great britain , italy , and spain ) . in
virtually all countries , school enrollment one of the indicators of uncertainty negatively affected the entry into marriage .
more importantly , men s unemployment appeared to lower the chances of entering marriage in most countries ( bernardi and nazio 2005 ; kieffer et al .
2005 ; liefbroer 2005 ; noguera et al . 2005 ; robert and bukodi 2005 ) . in britain ,
an effect of unemployment was observed only on the transition from cohabitation to marriage , and not on the transition from being single to living together ( francesconi and golsch 2005 ) . in sweden , only unemployment after leaving school appeared to delay marriage formation , not unemployment after a period of employment ( bygren et al . 2005 ) .
some evidence was also found for the effect of temporary contracts . in italy , spain ,
france , and the netherlands , it was shown that men who were employed temporarily were less likely to enter marriage than men who had permanent employment . in germany and hungary
, there was no effect of temporary work , however , and in several other countries , the effect could not be studied .
a recent analyses of fertility in europe has also pointed to the delaying effect of temporary contracts ( adsera 2011 ) . outside the blossfeld project
, there were a number of important individual articles in which aspects of oppenheimer s theory were tested .
for example , in sweden , it was found that men s employment increased the chances of union formation while it did not affect the chances of marriage after cohabitation ( bracher and santow 1998 ) . in norway , men s employment increased the chances of marrying after being single and the chance of marrying after living together ( kravdal 1999 ) . in the netherlands ,
men s employment had a stronger effect on direct marriage than on cohabitation but there was no effect of employment on marriage after cohabitation ( kalmijn and luijkx 2005 ; cf .
the evidence outside europe ( i.e. , israel ) has been supportive as well ( raz - yurovich 2010 ) , as has been the evidence in central and eastern europe , a region not included in the present article ( gerber and berman 2010 ) . while the role of employment has often been studied in europe , less
is known about how men s income and earnings affect union formation . many of the studies discussed above were based on retrospective rather than prospective longitudinal data .
the panel data that exist have been collected by economists and labor market researchers and do not always have the desirable demographic indicators . because income can not be measured well in a retrospective fashion , this has also meant that we know little about the income effects on marriage and divorce in europe . this is unfortunate because employment and income need to be examined simultaneously , especially if one wants to make a distinction between the neo - malthusian breadwinner hypothesis on the one hand , and oppenheimer s uncertainty hypothesis on the other . because in many european welfare states nonemployment does not , per se ,
another drawback of the prevailing evidence is that most studies are based on single countries .
blossfeld s multi - nation project is clearly a major step forward in trying to summarize the evidence for europe as a whole , but the analyses are not pooled so the results can only be summarized verbally .
moreover , possible differences that exist between countries can be described but they can not be compared or tested in a more rigorous fashion . for these reasons ,
in answering this question , i not only look at employment , but also i look at men s income , work experience , and type of labor contract . by looking at income and employment patterns simultaneously
, i obtain more direct evidence on the underlying mechanisms . the period for which i answer
unemployment rates increased substantially in the early 1990s before declining again in the mid to late 1990s ( oecd 2009 ) .
in italy , greece , and belgium , unemployment remained high in the late 1990s but began to decline later , in the early 2000s .
in other words , for most countries , the period that i examine covers a recovery stage of the economy , a stage which should have been positive for marriage and family formation .
second , are the effects of men s economic position similar or different for the chances of entering marriage and the chances of entering a cohabiting union ? with this part of the study , i replicate the last influential study of oppenheimer ( 2003 ) , in which she studied this issue for the united states
. we would expect effects to be weaker for cohabitation than for marriage : marriage would require a stronger economic underpinning than cohabitation ( kravdal 1999 ; oppenheimer 2003 ) .
in addition , i examine the chances that cohabiting unions turn into marriage . here too , oppenheimer expects men s economic position to have an effect , but because those who cohabit already have an independent household , the effects of income will probably be weaker .
third , to what extent are the effects of men s economic position on union formation different across societal contexts ? in this part , i focus on differences between traditional and egalitarian societies .
the expectation is that men s economic characteristics remain important in traditional societies but are less important in more modern , egalitarian ones . by looking at differences among societal contexts
, i try to generalize the cross - cohort comparison that sweeney ( 2002 ) made for the united states . answering this question is also of more general importance because if we find conditions under which the theory is ( not ) true , this could in principle lead to theoretical progress in the field .
i use panel data that are collected in the same format for a number of european countries , i.e. , the european community household panel ( echp ) .
the echp was an annual panel survey held between 1994 and 2001 ( behr et al .
samples are large and representative , and ( almost ) the same questionnaire was used each year . for the analyses in this article , i use data from 13 countries
: denmark , finland , germany , austria , the netherlands , belgium , france , the united kingdom , ireland , portugal , spain , italy , and greece .
my sample is limited to men who were never married in the first wave of the panel .
although i am able to exclude previously married men , i can not exclude men who ended a cohabiting union before the panel began .
never married men who were cohabiting in the first wave are also included because these men can make the transition from cohabitation to marriage . the first wave of data from the netherlands is excluded because no information is available on cohabitation status
there have been previous demographic analyses of the echp most notably by adsera ( 2011 ) who analyzes the effect of individual and aggregate labor market characteristics on fertility .
although adsera focused more on women than on men , her general conclusion is that labor market uncertainty is very influential in delaying fertility , in line with the perspective suggested above . in the current article , we go back one step by analyzing how labor market uncertainty affects union formation , a transition which probably remains the most important necessary condition for family formation .
i use discrete time event - history analysis by estimating logistic regression models on a person - year file .
the person - year file begins at the first wave and ends in the last wave or when a transition is made . as is the case with all event - history analyses of panel data ,
such left truncation problems can be solved in principle , but not without losing our time - varying independent variables ( guo 1993 ) .
the first logit model is estimated for person - years in which men are single and never married .
the dependent variable is whether a man is living without a partner in wave t and living with a partner in wave t + 1 ( union formation ) . in the second logit model ,
i estimated which choice was made , cohabitation or marriage , using only the person - years in which the event occurred .
this sequential approach is slightly different from the approach taken by oppenheimer , who estimated multinomial logit ( i.e. , competing risk ) models ( 2003 ) .
the sequential approach does not need the assumption implicit in a competing risk model that the two choices
this assumption is problematic because it is plausible that unmeasured factors like personality , wealth , attractiveness , and the like , influence marriage and cohabitation to the same extent ( hill et al .
if a person is single in year t , missing in year t + 1 , and married or cohabiting in year t + 2 , i also regarded this as an event .
duration dependency is modeled with two age effects , according to the approach developed by blossfeld and huinink ( 1991 ) .
blossfeld used log ( age a1 ) and log ( a2age ) to capture the nonlinear age - dependency of union formation , where a1 is the lowest and a2 the highest age in the sample .
there were 4,492 transitions to a first union of which 2,499 were to cohabitation and 1,993 to marriage .
the second logit model is estimated for person - years in which men were living with a partner without being married .
the dependent variable is defined as living with a partner unmarried in wave t and being married in wave t + 1 ( marriage after cohabitation ) . respondents who were living alone in wave t + 1 are truncated .
hence , separation is treated as a competing risk . if a person is cohabiting in year t , not in the panel in year t + 1 , and married in year t + 2 , i also regarded this as an event .
duration dependency could not be modeled directly because for those who are in a cohabitating union in the first wave of the panel , no data on the start of the union is available . as an alternative
age is obviously less ideal than duration since persons enter a cohabiting union at different ages .
it is noted that there was no question in the interview about whether the partner in one wave was the same partner as in the previous wave .
hence , i could not check if a man changed ( cohabiting ) partners between the subsequent waves , nor could i check if the married partner was the same person as the cohabiting partner in the previous wave .
all independent variables , except where noted , refer to time t. as the dependent variables refer to whether or not a transition was made between the time t and t + 1 , the independent variables precede the dependent variable in time .
the first indicates a man is working on a paid job or is self - employed at the time of the interview for at least 15 h a week .
the second variable indicates other less common forms of employment , such as military service , apprenticeships , unpaid family work , and working less than 15 h. given that a man is employed , i also considered the type of contract he has .
temporary contracts include fixed - term or short - term contracts , casual work with no contract , and " some other working arrangement " that is not a permanent contract ( all included within the 15 + hours category ) . the coding is cumulative so that the effect of temporary work refers to the difference between the men with a temporary contract and men with a permanent contract .
i also include whether a person is enrolled in full - time schooling in the interview week .
data on work history are obtained from a monthly calendar that all respondents had to fill out . for the year
t 1 , i counted the number of months that a man was employed , self - employed , or in school ( hereafter called " active " ) .
i checked whether the effects of this variable were different when excluding the netherlands but this was not the case .
i consider personal income and not only income from work since social security income may also provide a stable source of income .
ideally , we would like to measure the income a man had in the interview month or in the 12 months before the interview .
unfortunately , incomes are measured for ( full ) calendar years only . to solve this
, i took a weighted average of the income in calendar year t 1 and the income in calendar year t , using information on the month of interview .
for example , if the interview was in september 1994 , income for that person - year is 9/12 of the income earned in 1994 plus 3/12 of the income earned in 1993 .
it is noted that some of the income in year t may be earned after a man marries .
marrying in the year of the interview is more likely when the interview took place early in the year , but then , this income receives a lower weight .
i corrected all incomes for changes in the consumer price index and converted them to pounds sterling . to estimate effects of income in a comparable way across countries
i first calculated income quintiles in each country using data from all men in the first wave of the echp .
next , i used these quintiles to categorize the men in the person - year file .
i also use a linear income variable which is coded 15 for the five quintiles .
three control variables are used which may affect union formation : general health ( time varying ) , the highest level of educational attainment , and the year of the interview ( using dummies ) .
health is controlled because having a poor health may be detrimental for finding a partner and is also related to job and income opportunities ( waldron et al .
descriptive findings for the independent variables for each country are presented in table 1.table 1means of independent variables in person - year file by countryyear ( 18)agegeneral health ( 15)secondary educationtertiary educationworking 15 + hoursother worktemporary contractenrolled in schoolmonths active ( 012)relative income ( 15)income in poundsuk ( n = 7,585)4.0731.883.910.080.680.750.030.050.059.943.0410546ireland ( n = 7,741)3.4032.314.370.440.230.620.050.080.129.522.707184germany (
n = 9,581)4.0630.123.790.640.260.680.090.070.1210.412.559536austria ( n = 5,782)4.4829.124.400.790.080.700.080.060.1310.672.508995france ( n = 11,385)3.9031.993.870.420.320.600.060.090.149.492.528537belgium ( n = 3,842)3.8132.254.170.440.440.630.010.080.199.962.469182netherlands ( n = 5,248)4.5232.664.110.540.220.730.030.100.149.002.449841italy ( n = 18,316)4.0229.884.110.520.140.530.060.070.178.552.494696greece ( n = 7,942)3.8929.644.690.470.280.610.110.120.119.572.574220spain ( n = 12,764)3.8330.834.080.250.390.550.040.200.168.682.544553portugal ( n = 10,097)4.0929.423.690.220.110.680.040.150.1210.062.883062finland ( n = 5,360)5.0132.954.060.560.260.610.040.090.179.452.5511526denmark ( n = 5,381)3.8833.384.420.450.400.710.060.080.1110.202.9912814total (
n = 111,024)4.0330.974.090.430.280.630.060.100.139.502.627227source : echp ( own calculations ) means of independent variables in person - year file by country source : echp ( own calculations ) to explore differences across societies , i constructed two measures at the macro level .
first , i considered a measure of the division of household labor in marriage in a society .
this measure was obtained from a article by knudsen and waerness ( 2008 ) and measures the extent to which husbands and wives share four household tasks ( i.e. , laundry , grocery shopping , meal preparation , caring for sick family members ) .
the second measure is the female labor force participation rate in the 1990s , which is defined as the percentage of women aged 2049 who are active in the labor force ( obtained from ilo , geneva ) .
. countries in which wives participate more often in the labor force are also countries in which the household tasks are divided more equally ( r = .74 ) .
the most traditional societies are southern european countries , the most egalitarian are northern european countries .
i construct a single macro - level indicator , which is the means of the two standardized items .
i use discrete time event - history analysis by estimating logistic regression models on a person - year file .
the person - year file begins at the first wave and ends in the last wave or when a transition is made . as is the case with all event - history analyses of panel data ,
such left truncation problems can be solved in principle , but not without losing our time - varying independent variables ( guo 1993 ) .
the first logit model is estimated for person - years in which men are single and never married .
the dependent variable is whether a man is living without a partner in wave t and living with a partner in wave t + 1 ( union formation ) . in the second logit model ,
i estimated which choice was made , cohabitation or marriage , using only the person - years in which the event occurred .
this sequential approach is slightly different from the approach taken by oppenheimer , who estimated multinomial logit ( i.e. , competing risk ) models ( 2003 ) .
the sequential approach does not need the assumption implicit in a competing risk model that the two choices
this assumption is problematic because it is plausible that unmeasured factors like personality , wealth , attractiveness , and the like , influence marriage and cohabitation to the same extent ( hill et al .
if a person is single in year t , missing in year t + 1 , and married or cohabiting in year t + 2 , i also regarded this as an event .
duration dependency is modeled with two age effects , according to the approach developed by blossfeld and huinink ( 1991 ) .
blossfeld used log ( age a1 ) and log ( a2age ) to capture the nonlinear age - dependency of union formation , where a1 is the lowest and a2 the highest age in the sample .
there were 4,492 transitions to a first union of which 2,499 were to cohabitation and 1,993 to marriage .
the second logit model is estimated for person - years in which men were living with a partner without being married .
the dependent variable is defined as living with a partner unmarried in wave t and being married in wave t + 1 ( marriage after cohabitation ) . respondents who were living alone in wave t + 1 are truncated .
hence , separation is treated as a competing risk . if a person is cohabiting in year t , not in the panel in year t + 1 , and married in year t + 2 , i also regarded this as an event .
duration dependency could not be modeled directly because for those who are in a cohabitating union in the first wave of the panel , no data on the start of the union is available . as an alternative ,
age is obviously less ideal than duration since persons enter a cohabiting union at different ages .
it is noted that there was no question in the interview about whether the partner in one wave was the same partner as in the previous wave .
hence , i could not check if a man changed ( cohabiting ) partners between the subsequent waves , nor could i check if the married partner was the same person as the cohabiting partner in the previous wave .
all independent variables , except where noted , refer to time t. as the dependent variables refer to whether or not a transition was made between the time t and t + 1 , the independent variables precede the dependent variable in time .
the first indicates a man is working on a paid job or is self - employed at the time of the interview for at least 15 h a week .
the second variable indicates other less common forms of employment , such as military service , apprenticeships , unpaid family work , and working less than 15 h. given that a man is employed , i also considered the type of contract he has .
temporary contracts include fixed - term or short - term contracts , casual work with no contract , and " some other working arrangement " that is not a permanent contract ( all included within the 15 + hours category ) .
the coding is cumulative so that the effect of temporary work refers to the difference between the men with a temporary contract and men with a permanent contract .
i also include whether a person is enrolled in full - time schooling in the interview week .
data on work history are obtained from a monthly calendar that all respondents had to fill out .
for the year t 1 , i counted the number of months that a man was employed , self - employed , or in school ( hereafter called " active " ) .
i checked whether the effects of this variable were different when excluding the netherlands but this was not the case .
i consider personal income and not only income from work since social security income may also provide a stable source of income .
ideally , we would like to measure the income a man had in the interview month or in the 12 months before the interview .
unfortunately , incomes are measured for ( full ) calendar years only . to solve this
, i took a weighted average of the income in calendar year t 1 and the income in calendar year t , using information on the month of interview .
for example , if the interview was in september 1994 , income for that person - year is 9/12 of the income earned in 1994 plus 3/12 of the income earned in 1993 .
it is noted that some of the income in year t may be earned after a man marries .
marrying in the year of the interview is more likely when the interview took place early in the year , but then , this income receives a lower weight .
i corrected all incomes for changes in the consumer price index and converted them to pounds sterling . to estimate effects of income in a comparable way across countries ,
i first calculated income quintiles in each country using data from all men in the first wave of the echp .
next , i used these quintiles to categorize the men in the person - year file .
i also use a linear income variable which is coded 15 for the five quintiles .
three control variables are used which may affect union formation : general health ( time varying ) , the highest level of educational attainment , and the year of the interview ( using dummies ) .
health is controlled because having a poor health may be detrimental for finding a partner and is also related to job and income opportunities ( waldron et al .
descriptive findings for the independent variables for each country are presented in table 1.table 1means of independent variables in person - year file by countryyear ( 18)agegeneral health ( 15)secondary educationtertiary educationworking 15 + hoursother worktemporary contractenrolled in schoolmonths active ( 012)relative income ( 15)income in poundsuk ( n = 7,585)4.0731.883.910.080.680.750.030.050.059.943.0410546ireland ( n = 7,741)3.4032.314.370.440.230.620.050.080.129.522.707184germany ( n = 9,581)4.0630.123.790.640.260.680.090.070.1210.412.559536austria ( n = 5,782)4.4829.124.400.790.080.700.080.060.1310.672.508995france ( n = 11,385)3.9031.993.870.420.320.600.060.090.149.492.528537belgium ( n = 3,842)3.8132.254.170.440.440.630.010.080.199.962.469182netherlands ( n = 5,248)4.5232.664.110.540.220.730.030.100.149.002.449841italy ( n = 18,316)4.0229.884.110.520.140.530.060.070.178.552.494696greece ( n = 7,942)3.8929.644.690.470.280.610.110.120.119.572.574220spain ( n = 12,764)3.8330.834.080.250.390.550.040.200.168.682.544553portugal ( n = 10,097)4.0929.423.690.220.110.680.040.150.1210.062.883062finland ( n = 5,360)5.0132.954.060.560.260.610.040.090.179.452.5511526denmark (
n = 5,381)3.8833.384.420.450.400.710.060.080.1110.202.9912814total ( n = 111,024)4.0330.974.090.430.280.630.060.100.139.502.627227source : echp ( own calculations ) means of independent variables in person - year file by country source : echp ( own calculations ) to explore differences across societies , i constructed two measures at the macro level .
first , i considered a measure of the division of household labor in marriage in a society .
this measure was obtained from a article by knudsen and waerness ( 2008 ) and measures the extent to which husbands and wives share four household tasks ( i.e. , laundry , grocery shopping , meal preparation , caring for sick family members ) .
the second measure is the female labor force participation rate in the 1990s , which is defined as the percentage of women aged 2049 who are active in the labor force ( obtained from ilo , geneva ) .
. countries in which wives participate more often in the labor force are also countries in which the household tasks are divided more equally ( r = .74 ) .
the most traditional societies are southern european countries , the most egalitarian are northern european countries .
i construct a single macro - level indicator , which is the means of the two standardized items
figure 1 shows that there are small differences within europe in the age at first union entry . in most countries ,
male age at entry is low in portugal and austria , and high in the netherlands and italy .
for example , the age at union formation is quite high in italy and greece where the family is a strong institution .
europe , youth unemployment is high . when we look at the type of first union chosen by men , presented in the left side of fig . 1
even more in northern europe , direct marriage is a minority experience . in many countries , more than 80% of men cohabit first . in southern europe , the proportions are almost reversed : 6080% of the men in these countries marry directly .
the united kingdom resembles western europe but ireland resembles the south , with most men choosing marriage as their first way to enter a union .
1men s first union formation in 13 countries men s first union formation in 13 countries the effects of men s economic characteristics on union entry are presented in table 2 .
the odds of entering a union are 58% higher for employed men than for men who are not employed and not in school ( e ) .
the type of labor contract also matters for the choice between marriage and cohabitation . compared to men with a permanent job , men with a temporary job who enter a union are 23% less likely to choose marriage than cohabitation .
we also note that school enrollment has a negative effect on union entry while it does not affect the type of union.table 2discrete time event history models of union formation : logit regression coefficients and p - values in parentheses union formationmarriage vs. cohabitationmarriage after cohabitationmodel amodel bmodel cmodel dmodel emodel fmodel gmodel hmodel iln ( age15)0.841 * * * ( 0.000)0.661 * * * ( 0.000)0.676 * * * ( 0.000)0.746 * * * ( 0.000)0.745 * * * ( 0.000)0.741 * * * ( 0.000)0.230 * ( 0.040)0.317 * * ( 0.006)0.316 * * ( 0.006)ln ( 65age)1.872 * * * ( 0.000)1.816 * * * ( 0.000)1.847 * * * ( 0.000)0.810 * * * ( 0.000)0.813 * * * ( 0.000)0.809 * * * ( 0.000)0.296 * ( 0.038)0.264 ( 0.061)0.268 ( 0.058)general health0.106 * * * ( 0.000)0.100 * * * ( 0.000)0.098 * * * ( 0.000)0.135 * ( 0.012)0.135 * ( 0.013)0.135 * ( 0.012)0.107 * * ( 0.008)0.104 * ( 0.010)0.103 * ( 0.011)education secondary versus primary0.029 ( 0.491)0.002 ( 0.955)0.007 ( 0.867)0.046 ( 0.655)0.035 ( 0.731)0.047 ( 0.648)0.005 ( 0.951)0.012 ( 0.890)0.014 ( 0.873)education tertiary versus primary0.225 * * * ( 0.000)0.141 * * ( 0.003)0.122 * * ( 0.010)0.113 ( 0.311)0.149 ( 0.194)0.111 ( 0.326)0.408 * * * ( 0.000)0.361 * * * ( 0.000)0.349 * * * ( 0.000)working 15 + hours versus no work / school0.457 * * * ( 0.000)0.193 * * ( 0.003)0.220 * * * ( 0.001)0.450 * * ( 0.003)0.419 * * ( 0.007)0.445 * * ( 0.004)0.178 ( 0.166)0.090 ( 0.495)0.102 ( 0.434)other employed versus no work / school0.144 ( 0.088)0.204 * ( 0.016)0.188 * ( 0.026)0.279 ( 0.176)0.350 ( 0.096)0.282 ( 0.173)0.237 ( 0.277)0.250 ( 0.254)0.245 ( 0.263)temporary versus fixed contract0.067 ( 0.182)0.026 ( 0.609)0.031 ( 0.544)0.253 * ( 0.033)0.262 * ( 0.028)0.251 * ( 0.035)0.120 ( 0.249)0.086 ( 0.410)0.090 ( 0.386)in school versus no versus no work / school0.623 * * * ( 0.000)0.431***(0.000)0.468 * * * ( 0.000)0.180 ( 0.388)0.089 ( 0.679)0.173 ( 0.412)0.515 * ( 0.015)0.410 ( 0.059)0.424 * ( 0.046)months worked last 2 years0.022 * * * ( 0.000)0.006 ( 0.243)0.007 ( 0.178)0.008 ( 0.518)0.011 ( 0.405)0.009 ( 0.504)0.031 * ( 0.010)0.021 ( 0.089)0.023 ( 0.066)first income quintile versus third quintile0.604 * * * ( 0.000)0.272 ( 0.054)0.256 * ( 0.044)second income quintile versus third quintile0.253 * * * ( 0.000)0.039 ( 0.735)0.182 * ( 0.037)fourth income quintile versus third quintile0.232 * * * ( 0.000)0.136 ( 0.210)0.094 ( 0.224)fifth income quintile versus third quintile0.366 * * * ( 0.000)0.127 ( 0.328)0.109 ( 0.233)linear income ( 15)0.239 * * * ( 0.000)0.007 ( 0.861)0.098 * * * ( 0.001)constant11.814 * * * ( 0.000)10.677 * * * ( 0.000)11.595 * * * ( 0.000)7.903 * * * ( 0.000)7.789 * * * ( 0.000)7.895 * * * ( 0.000)3.346 * * * ( 0.000)2.771 * * * ( 0.000)3.138 * * * ( 0.000)n776217762177621447244724472116781167811678224024602451162516301625335348346note : controlled for country and year dummies*p < .05 , * * p < .01 , * * * p < .001 discrete time event history models of union formation : logit regression coefficients and p - values in parentheses note : controlled for country and year dummies * p < .05 , * * p < .01 , * * * p < .001 next to the effects of current employment characteristics , we see effects of men s work history .
the more months men were employed or otherwise active in the past calendar year , the more likely they are to enter a union in the next two calendar years .
men who were active for an entire year were e = 30% more likely to enter a union in the next 2 years than men who were inactive the entire year .
this effect comes on top of the effect of a man s current employment situation .
there is no effect of work history on the choice between marriage and cohabitation . in sum
, the findings from model a and model d confirm that men who are not yet settled in their career postpone union formation .
the findings also confirm that cohabitation is less sensitive to economic insecurity than marriage , although this applies more to employment per se and the type of employment than to a man s work history . in model
the poorest quintile of men are 45% less likely to enter a union than the middle quintile , while the richest quintile are 44% more likely to do so .
the income effects increase monotonically but the differences between the quintiles become smaller at higher income levels . a formal test shows
that the effect is not strictly linear : the nonlinear model for union formation ( model b ) has a better fit than the linear model ( model c , = 9.3 , p = .03 ) .
when we look at the income effect on choice between the marriage and cohabitation , we only see one marginally significant effect .
men in the lowest quintile who enter a union are 31% more likely than the middle quintile to choose cohabitation rather than marriage ( p = .054 ) .
this result seems to confirm the notion of cohabitation as the poor man s marriage , although the poor are still more likely not to enter a union in the first place .
when we compare the two models for union entry ( model a and b ) , the employment effects that we observed in model a are reduced by more than half once income is added to the model . in other words , the effects of employment and work history run in part via income .
nevertheless , the employment effect remains statistically significant even when income is included , which means that the influence of employment on union formation also has a non - financial element .
it is also interesting to observe that the effects of employment and temporary jobs on the choice between the marriage and cohabitation are not affected by whether or not income is added ( compare model d and e ) .
hence , the non - financial aspects of work are more important for the type of union than for the chance of entering a union in the first place . in this sense ,
oppenheimer s uncertainty theory seems to work better for the type of union than for union formation per se .
this confirms theories about selection into marriage and suggests that screening or selection may be less strong for cohabitation .
education , finally , has a positive effect on union entry but these effects are reduced when income is controlled for . in the last columns of table 2 , we focus on marriage formation after cohabitation ( model g , h , and i ) .
we see that it is not affected by employment , but being enrolled in school does reduce the chance of marrying .
we also find that men who worked more months in the past are more likely to marry .
the higher the men s income during cohabitation , the more likely they are to change from cohabitation to marriage .
when comparing the linear income effect on marriage after cohabitation with the linear effect on initial union formation , it appears that income is less important for marriage after cohabitation ( b = .10 ) than it is for union formation after being single ( b = .21 ) .
this is in line with the neo - malthusian hypothesis , which suggests that it is primarily for initial union entry that the costs of setting up a household play a role ( although the wedding is a cost factor which applies specifically to marriage ) .
men in good health are more likely to experience a transition from cohabitation to marriage , suggesting that the selection effects also play a role in the decision to marry , not only in the decision to live together .
we also see that higher educated cohabiting men are more likely to marry , hence , for the higher educated , cohabitation is less often seen as a long - term option .
do the effects we found vary across societal contexts ? to assess this , i present interaction effects of key independent variables with the macro - level indicator of traditional versus egalitarian societies .
the p - values are based on standard errors that are corrected for the clustering of cases within countries .
this yields a more conservative test for macro - level effects and is a good alternative to multilevel models when the number of units at the macro level is limited . to check for outliers at the country level ,
i calculated dfbetas for the interaction effects of modernization and employment , and of modernization and income , and re - estimated the models while leaving out countries for which dfbetas exceeded the critical value ( kalmijn 2010 ; ruiter and de graaf 2006 ) . dfbeta is calculated as the difference in an effect with and without the outlier divided by the standard error of the effect .
these outlier analyses are presented in the third and fourth columns of table 3.table 3discrete time event history models of union formation with interactions : logit regression coefficients and p - values in parenthesesmodel a all casesmodel b all casesmodel a w / o outliersmodel b w / o outliersln ( age15)0.953 * * * ( 0.000)0.781 * * ( 0.002)0.943 * * * ( 0.001)0.686 * ( 0.036)ln ( 65age)1.970 * * * ( 0.000)1.956 * * * ( 0.000)1.934 * * * ( 0.001)2.040 * ( 0.010)general health0.121 * * ( 0.001)0.107 * * ( 0.002)0.102 * * ( 0.006)0.119 * ( 0.016)education secondary versus primary0.071 ( 0.287)0.023 ( 0.732)0.031 ( 0.638)0.115 ( 0.079)education tertiary versus primary0.271 * * * ( 0.000)0.152 * ( 0.014)0.248 * * * ( 0.000)0.224 * * * ( 0.001)working 15 + hours versus no work / school0.501 * * * ( 0.000)0.194 * * * ( 0.000)0.549 * * * ( 0.000)0.175 * * * ( 0.000)in school versus no versus no work / school0.672 * * * ( 0.000)0.604 * * * ( 0.000)0.614 * * * ( 0.000)0.673 * * * ( 0.000)income linear0.242 * * * ( 0.000)0.243 * * * ( 0.000)index ( traditional egalitarian)1.916 ( 0.177)2.075 ( 0.144)2.092 ( 0.262)2.039 ( 0.327)index * ln ( age15)0.278 ( 0.058)0.314 * ( 0.037)0.311 ( 0.110)0.217 ( 0.272)index * ln ( 65age)0.233 ( 0.442)0.262 ( 0.386)0.288 ( 0.471)0.309 ( 0.485)index * health0.003 ( 0.914)0.004 ( 0.872)0.007 ( 0.718)0.005 ( 0.897)index * secondary education0.079 ( 0.333)0.087 ( 0.285)0.086 ( 0.368)0.121 * ( 0.041)index * tertiary education0.119 ( 0.106)0.130 ( 0.079)0.139 ( 0.127)0.205 * * * ( 0.000)index * working0.158 * * ( 0.001)0.122 * * ( 0.001)0.152 * * * ( 0.000)0.098 * ( 0.043)index * enrolled0.236 * * ( 0.001)0.210 * * ( 0.002)0.215 * * ( 0.004)0.288 * * * ( 0.000)index * income0.002 ( 0.935)0.037 * * ( 0.008)constant13.107 * * * ( 0.000)12.953 * * * ( 0.000)12.853***(0.000)13.112 * * * ( 0.000)n77621776216883758354note : controlled for country and year dummies .
outliers for model a are denmark and greece , for model b these are italy and the united kingdom * p < .05 , * * p < .01 , * * * p < .001 discrete time event history models of union formation with interactions : logit regression coefficients and p - values in parentheses note : controlled for country and year dummies .
outliers for model a are denmark and greece , for model b these are italy and the united kingdom * p < .05 , * *
p < .01 , * * * p < .001 in table 3 , we see a negative interaction of the societal index with men s employment ( b = .16 , p < .01 ) .
hence , the average effect of employment ( b = .50 ) is reduced by 32% for each standard deviation increase in the egalitarian context ( .16/.50 ) .
this shows that the effect of men s employment on the entry into a union is considerably weaker in more egalitarian countries than in more traditional countries .
i also present the effects for each country separately in a graph ( fig .
, the effect of selected independent variables is plotted against the macro - level index .
although the graph does not provide a test , like the interaction effect , it does give a good intuitive feel of the importance of the interaction effect .
in line with expectations , the graph shows that in more traditional societies like spain , italy , and ireland , the effect of men s employment is quite strong . in more egalitarian countries like the netherlands , denmark , and finland ,
men s enrollment in school deters union formation but this effect is weaker in more egalitarian societies .
s economic characteristics on union entry effects of men s economic characteristics on union entry table 3 further shows no negative interaction of the societal index with men s income effect on union formation . after deleting two outliers ( italy and the united kingdom ) ,
the interaction becomes negative and significant ( b = .037 , p = .01 ) .
hence , for these 11 countries , the effect of men s income on union formation is weaker in more egalitarian countries than in more traditional countries .
the magnitude of the effect is a 15% reduction in the income effect per standard deviation increase in the egalitarian context ( .037/.234 ) .
when we look at the graph , the pattern appears to be weaker than it was for employment .
spain and portugal reveal strong effects of men s income and denmark and the netherlands have weak effects . however , there are also outliers like the united kingdom ( stronger effect than expected ) and italy and belgium ( weaker effect than expected ) .
so far , these results apply to union formation regardless of the type of union . traditional and egalitarian societies also differ in the extent to which cohabitation occurs . the more egalitarian countries like denmark , finland , and
the netherlands also have the highest levels of cohabitation ( soons and kalmijn 2009 ) .
because the effects of men s economic position differ depending on whether marriage or cohabitation is the outcome , the results could in part be due to such compositional differences . for this reason
, i also look at the entry into marriage only ( either after being single or after cohabitation ) .
the interaction with employment is .066 ( p = .11 ) , the interaction with enrollment is .326 ( p <
.01 ) , and the interaction with income is .073 ( p < .01 ) . hence , the employment interaction effect is weaker in this model , although still negative , while the other interaction effects are not affected .
we therefore conclude that part of the reason why the employment effects depend on the context lies in the fact that unions are more often unmarried unions in more egalitarian societies . even apart from that
, however , there is evidence that men s economic status matters more for marriage in more traditional societies .
this article has re - examined the importance of oppenheimer s theory on marriage timing in the european context . by and large
unemployment , little work experience , low income , and temporary employment on the part of men deter union formation . by analyzing income and employment effects simultaneously in a panel perspective , it was possible to obtain more direct evidence for the two contrasting hypotheses suggested by oppenheimer , i.e. , the neo - malthusian male breadwinner hypothesis and the career uncertainty hypothesis ( sometimes also called the career instability or immaturity hypothesis ) .
many previous european analyses have not been able to take income into account and have therefore not been able to separate these two mechanisms empirically .
my analyses first show that income effects are strong and significant , which supports the male breadwinner hypothesis .
second , the income effects explain about half of the effects of employment and work experience , suggesting that employment effects by themselves are not sufficient evidence for the uncertainty hypothesis .
this suggests that employment effects on union formation are more than just a matter of financial resources , in line with the uncertainty hypothesis . by analyzing the choice between cohabitation and marriage ,
marriage in europe appears to be more sensitive to men s economic position than cohabitation .
men who are not employed or who have temporary jobs are more likely to choose cohabitation rather than marriage .
this finding provides additional evidence for the uncertainty hypothesis since cohabitation is more like a trial marriage and hence , more compatible with uncertainty in other life domains ( i.e. , employment ) .
we do not , however , see a clear income effect on the choice between marriage and cohabitation .
hence , the choice between marriage and cohabitation has more to do with employment uncertainty than with income .
there is a small effect of the lowest income group , however . among men who enter a union ,
hence , there is some evidence that cohabitation is the " poor man s marriage . "
following oppenheimer s last study , i also examined the transition from cohabitation to marriage .
we would expect economic uncertainty to also reduce the chances of a transition from cohabitation to marriage , but in general , the effects we find are weaker for this transition
. the weaker income effect could be due to the fact that the cost of setting up a household plays no role for this transition .
while this is in line with the neo - malthusian argument , the finding that the employment effects on the transition from cohabitation to marriage are also weaker is unexpected .
other authors have found this too ( e.g. , bracher and santow 1998 ; liefbroer 2005 ) .
if couples buy a house or have a child , they may decide to marry .
although such transitions may be partly governed by economic uncertainty , they may also be exogenous and hence reduce the effects of other determinants like employment .
another speculation has to do with the lack of information on the ( female ) partner .
the partner may have become economically more secure during the cohabitation stage , thereby promoting the transition to marriage , but we do not observe such changes .
european countries differ considerably in terms of their economic , social , and cultural characteristics , and so , it is important to also examine country variation in the degree to which the theory applies .
we hypothesized that men s economic position would be less influential in more developed countries where gender roles are more symmetrical .
the effects of men s employment and school enrollment on union formation are stronger in more traditional societies than in more egalitarian ones .
income effects are also weaker in egalitarian societies but the evidence for this pattern is weaker .
our interpretation is that in egalitarian settings , the costs of setting up a household are more often shared between men and women .
hence , men can afford to experiment with their career if they have a partner who has a ( more ) stable career .
similarly , women may attach less weight to the career options of men than they did in more traditional circumstances and for instance , pay more attention to other traits , such as men s willingness to participate in child rearing .
my finding is in contrast to a previous cohort comparison for the united states which suggested that men s economic effects on marriage timing did not change over time ( sweeney 2002 ) .
perhaps this difference is due to the limited time period that was examined in sweeney s trend study , a design which may have reduced the variance in contextual gender roles . at the same time
, however , it needs to be investigated whether the patterns that i found still hold when a larger number of countries is considered .
the income interaction effects , for example , are sensitive to outliers and therefore less convincing . more units at the macro - level will be needed to confirm this finding . in closing , it is important to re - emphasize on the role of women .
although oppenheimer was justifiably " bringing men back " into the debate at a time when there was too little attention on the changing economic fate of young ( american ) men , the growing egalitarian model that is now supported by many couples in europe and the united states , suggests that men and women should be examined simultaneously for a better understanding of trends and differentials in marriage and cohabitation .
traditionally , studies of women were largely done from a beckerian perspective in which it was argued that women s status would lead to a decline of marriage ( and fertility ) .
currently , we can speculate that a strong economic position and career stability on the part of women might foster marriage .
it could be that certainity on just one side either the man or the woman
alternatively , it could be that both the man s and the woman s career need to have been settled before couples enter a more committed union . in this respect
, it is somewhat unfortunate that analyses of marriage ( timing or formation ) have often been one - sided , focusing either on men or on women .
this design is unavoidable given that the partner of a respondent is typically observed too late , i.e. , when the respondent is married or living with the partner .
empirically , this problem does not exist when we observe cohabiting couples chances of marrying .
some authors in the past have regarded the transition from cohabitation to marriage as a two - sided problem and have analyzed economic characteristics of both partners in one model ( lichter et al .
these studies have not been able to examine the more important entry into a first union , however . for this transition ,
the problem can , in principle , be solved by examining dating couples in a more systematic fashion .
for example , prospective surveys could collect data on the school and work careers of both members of a dating couple and subsequently analyze whether or not ( and when ) they begin to live together , while using the dissolution of the dating relationship as a competing risk . in this way
, the analysis of marriage and cohabitation can become truly two - sided and the economic characteristics of men and women can be included in one model .
there are some innovative sociological studies of transitions from dating to cohabitation and marriage , but so far , they have not focused on the partners economic characteristics ( joyner and kao 2005 ) . | this article discusses oppenheimer s theory on marriage timing , reviews the way this theory was received in european demography and family sociology , and develops a new test of the theory using annual panel data from 13 european countries for the period 19942001 .
several indicators of men s economic status are used , including school enrollment , employment , type of labor contract , work experience , income , and education .
effects of these indicators are estimated for the transition to marriage and cohabitation , as well as for the transition from cohabitation to marriage .
country differences in these effects are examined as well .
the evidence provides strong support for the male breadwinner hypothesis on the one hand , and for oppenheimer s career uncertainty hypothesis on the other .
however , the relevance of these hypotheses also depends on the national context , and especially on the way gender roles are divided in a society . | Bringing Men Back in
Testing the Theory in Europe
Data, Methods, and Variables
Dependent Variables and Models
Independent Variables
Results
Conclusion and Discussion |
PMC5098508 | the danish head and neck cancer ( dahanca ) group was established in 1976 as a working group by the danish society for head and neck oncology with the primary aim to develop national guidelines for the treatment of head and neck cancer in denmark .
dahanca group s activities have included creating and running a national database , development of treatment guidelines , performing numerous national clinical protocol studies ( > 30 ) , and providing quality assurance programs .
the group is multidisciplinary and consists of representatives from all the oncology centers in denmark treating head and neck cancer , including oncologists , otolaryngologists , pathologists , imaging diagnosticians , experimental researchers , epidemiologists , and other expert support , eg , medical physicists .
the dahanca model was behind the establishment of the structure of the danish multi disciplinary cancer groups ( dmcg ) , and the dahanca group is thus the oldest of the still active dmcg .
traditionally , these include cancer of the larynx , pharynx , oral cavity , sinuses , salivary glands , thyroid glands , and neck nodes from unknown primary type of cancer ( corresponding to the international classification of diseases , tenth revision , classifications c.01c.11 , c.30c.32 , c.73 , and c.80 ) , but did not include cancer of the lip .
the most significant types are cancer of the pharynx , larynx , and oral cavity .
almost all of these are squamous cell carcinomas , which etiologically are strongly linked to tobacco and alcohol use and increasingly also to infections with especially human papilloma virus , since human papilloma virus - related oropharyngeal cancer has become the most rapidly increasing cancer type in denmark .
the therapeutic strategy is therefore aimed toward eradicating the disease at the site of the primary tumor and the regional lymph nodes .
such a treatment is typically surgery or radiotherapy , and over the past years in denmark , focus has deliberately been on radiotherapy as the primary modality , as this ensures the best possibility of organ conservation .
dahanca group has been an active and well - established structure for 40 years and has provided a solid foundation for the treatment of head and neck cancer in denmark and has become recognized internationally as one of the major head and neck cancer groups . in this aspect , it should be noted that the treatment of head and neck cancer in denmark has rather a good outcome seen with international eyes .
the registration of patients with head and neck cancer is the essential backbone of the dahanca structure , and the associated database includes currently > 33,000 patients , with an annual increase of 1,400 patients ( table 1 ) .
historically , the database started as a local home - designed database at the department of oncology , aarhus university hospital , aarhus , denmark , where the medical physicist mogens hjelm hansen in the early 1970s created an elaborate program for the registration of head and neck cancer ( the radiation physics department was among the first places where computers were introduced in health care ) .
this program later formed the basis for equivalent databases in copenhagen and odense and a subsequent retrospectively registration of , especially , laryngeal cancer back to the early 1960s . the registration of cases later expanded to include the new oncology centers in aalborg and herlev , and a full national registration of larynx cancer has taken place since 1971 . in the same time period , there have also been regional and more sporadic registrations of pharynx and oral cavity cancers . at a national level ,
the three initial databases included all patients with larynx cancer between 1971 and 1991 . in connection with a large clinical trial,1
the national database was reconstructed and expanded to include all cancers of the larynx , pharynx , and oral cavity .
each of the five oncology centers had their own databases ( named da 15 ) , which were all copies of a central master .
thus , data were gathered locally and were immediately available for daily use and research purposes at the individual institutions .
the da 15 data files were regularly merged into the joint database at the secretariat in aarhus and thus formed the national dahanca clinical database .
also , the initial three databases named ( akh , hlm , and hl3 ) were merged and restructured using the same program .
the registration in da 15 covered the period 19921999 . in 1995 , a special dathyrca database for registering thyroid cancer2 and more lately databases for unknown primary neck tumor,3 salivary gland tumor,4 and sinonasal cancer were established.5 in 2000 , the dahanca database was changed into a central web - based database ( ocx ) , which allowed interactive registration directly on a central server with the immediate possibility for quality checks of the data and with instant correction of errors .
this increased the quality assurance and gave direct access to information about the current status of the patients .
the ocx database is based on the development tool clarion and uses hypertext transfer protocol secure certificate with an hypertext transfer protocol secure connection ; all communications are securely encrypted .
the operating system is on a windows server 2012 platform , and the server is situated at aarhus university .
the system is flexible and can be modified in connection with specific protocols and projects .
all projects are carried out in accordance with the applicable legislation and regulations , including the european union s clinical trials directive , and corresponding regulations for the use of good clinical practice and quality assurance , and the database fulfills the danish data protection agency s description of standard specification and requirements for security .
the ocx database has been active since 2000 , and the structure has formed the basis for several other clinical quality cancer databases ( eg , melanoma , sarcoma , ocular cancer , childhood cancer databases ) . moreover , in 2011 , the dahanca database was transformed from being a private research database to a clinical quality database in public auspices under the danish clinical registries ( rkkp ) , while still allowing the continued use for research purposes .
this simplified the correlation with other public databases , such as the danish cancer registry ( dcr ) , and allowed the database to be used as an indicator of quality .
approximately 33,000 patients are included in the database , which has ~300 basic variables.6,7 additionally , there is the possibility of specially registered parameters in connection with specific projects .
the aim is that established variables remain in the database , and to have , to a large extent , continuous variables that remain unchanged .
international standardized classifications are used in this context where such classifications exist ( eg , union for international cancer control tumor node metastasis classification ) . with regard to side effects ,
international principles are also applied , but the principles that we initially developed have now been adopted by other organizations , and thus , the dahanca database has contributed to the international standard with regard to registration of side effects from radiotherapy . as not all variables are in use at any time , the daily registration probably involves 100150 different parameters .
the basic registration includes the following ( refer forms at www.dahanca.dk ) :
on study : symptoms and the duration of the symptoms , etiological factors , pretreatment and diagnostic evaluation , including tumor
metastasis classification , imaging , histopathology , and laboratory tests , primary treatment : semi - detailed information of radiotherapy , surgery , and medical treatment.follow-up : registration of vital and tumor status and acute and late side effects.recurrence : registration of relapse and treatment thereof.death form : registration of death and cause of death . on study : symptoms and the duration of the symptoms , etiological factors , pretreatment and diagnostic evaluation , including tumor node metastasis classification , imaging , histopathology , and laboratory tests , primary treatment : semi - detailed information of radiotherapy , surgery , and medical treatment .
follow - up : registration of vital and tumor status and acute and late side effects .
besides comparison with the dcr , vital status is checked regularly through updates from the civil registration number register . in certain situations ,
there have been correlations with specific extracts from the national patient discharge registry and the cause of death register .
the principle of the dahanca database has throughout the years been a uniform structure of data description and a local registration .
for the first many years , no actual clinical record forms existed , but the patient s journal was considered to be the paper - related data documentation and was used as a basis for data entry . for practical reasons , paper registration forms have been used since 1991 ( refer forms at www.dahanca.dk ) .
the gathering and recording of data has always been done by a few knowledgeable people , mainly senior specialist doctors .
owing to the limited amount of such people , who have been involved over many years and who know the problems in detail , both communication and collection of data are done in a very uncomplicated and pragmatic fashion .
this dependency on a few highly specialized persons is both the strength and weakness of the database , because it not only gives it extremely high quality but also makes it vulnerable to changes .
the database has a number of error indicators built in , which safeguards against logical errors and at the same time has the option of reminders if information is missing .
furthermore , lists of irregularities are instantly produced in a continuous attempt to collect missing data or correct any errors .
the database is verified through the numerous related research projects.6,7 simultaneously , a specific comparison to the dcr is performed at least annually , where all cases in the dcr have been sought to be verified through the dahanca database , and in case of errors ( in the dcr ) , these are explored when possible .
thus , there is currently a full national registration of dahanca database ranging back to 1971 ( table 1 ) .
this has shown that the dahanca database contains 99.7% of the originally known cases in the dcr , and an additional number of patients , currently 2% , have been identified , who were not in the dcr . furthermore , a number of erroneous registrations in the dcr have been corrected ( in the dahanca database ) .
the complete interaction between the two databases clearly shows that , in principle , there exists a total nationwide coverage in the dahanca database and further ascertains that the dahanca database has the potential of increasing the quality of the dcr s data .
this investigative high - quality work has been performed in great detail , and in cases of doubt , relevant additional hospital records and/or other material have been obtained .
thus , all patients in the database have had their hospital records scrutinized at least once , and all updates are verified by a specialist doctor . with the dcr s well - known almost complete coverage of cancer cases in denmark as a starting point , it is our impression that with the dahanca database we have a near - perfect clinical register , which probably is the world s most accurate recording of head and neck cancer treatment .
the initial aarhus - derived database was approved with register regulations at vejle county . later ,
the national database was registered and approved as a research database by the danish data protection agency ( record nos 1995 - 1200 - 282 and 2004 - 41 - 4802 ) . since 2011 , it has been a public clinical quality database ( record no 1 - 16 - 02 - 249 - 14 ) .
jens overgaard is the overall data responsible head , and regionally , the responsibility lies with the consultant responsible for the head and neck cancer treatment at the respective oncological centers .
as with other research - based databases , the project has initially been based on con amore
gradually , the need for organized help has increased and the database has throughout the years been partly financed in connection with research projects , where among others , phd students were assigned to the activities mainly sponsored through the danish cancer society and the medical research council ( we have constantly tried to avoid any commercial influence and sponsoring ) .
however , the activities have throughout benefited from being part of the infrastructure at the department of experimental clinical oncology at aarhus university hospital and from the numerous dedicated persons who have used their spare time to strengthen the database .
the value of any database is linked with its use , and the quality data are accessible through access to the rkkp ( http://www.rkkp.dk/forskningsadgang/ ) . throughout the years , the dahanca database has been extensively explored and has served as an active research database that has formed the basis for numerous scientific projects and publications , including 15 phd and doctoral theses ( for full list , refer the dahanca homepage [ www.dahanca.dk ] ) .
it has been the supporting database for > 30 prospective clinical trials and has acted as a baseline for large retrospective studies describing the natural history and treatment outcome in head and neck cancer .
examples of studies6,811 are given in figure 1 , and more can be found on the dahanca web site .
this means that the database is constantly revised and updated and , consequently , is extremely well verified .
since many of the dahanca protocols have focused on biological - based improvement of head and neck cancer treatment , these translational studies have been based on a large amount of clinical material.1013 this dahanca - related biobank contains biological material ( tumor , normal tissue cell lines , blood samples , dna , rna , etc ) from > 4,000 patients with detailed clinical data and follow - up .
currently , we are systematically collecting material from new patients in the national danish cancer biobank .
the database has not only been strengthened by linkage to other registries6,7 but has also shown its value by being a supportive basis for studies of comorbidity,6 value of follow - up,14 or socioeconomical relationship in patients with head and neck cancer.15,16 in the foreseen future , it will be part of international comparisons of treatment strategies , and for such purpose , a clinical database is far superior than using cancer registry comparisons ( such as the nordic nordcan study ) , since such databases are incomplete and inaccurate regarding clinical information .
thus , it is important to use the right tools / databases for the right purposes , and when conclusions about therapeutic strategies are the aim , the data must be obtained from a well - verified clinical quality database . | aim of the databasethe danish head and neck cancer database is a nationwide clinical quality database that contains prospective data collected since the early 1960s .
the overall aim of this study was to describe the outcome of the national strategy for multidisciplinary treatment of head and neck cancer in denmark and to create a basis for clinical trials.study populationthe study population consisted of all danish patients referred for treatment of squamous cell carcinoma of the larynx , pharynx , oral cavity , or neck nodes from unknown primary or any histopathological type ( except lymphoma ) of cancer in the nasal sinuses , salivary glands , or thyroid gland ( corresponding to the international classification of diseases , tenth revision , classifications c.01c.11 , c.30c.32 , c.73 , and c.80).main variablesthe main variables used in the study were symptoms and the duration of the symptoms ; etiological factors ; pretreatment and diagnostic evaluation , including tumor
node
metastasis classification , imaging , histopathology , and laboratory tests ; primary treatment with semidetailed information of radiotherapy , surgery , and medical treatment ; follow - up registration of tumor status and side effects ; registration of relapse and treatment thereof ; and registration of death and cause of death.main resultsdata from > 33,000 patients have been recorded during a period of > 45 years . in this period , the outcome of treatment improved substantially , partly due to better treatment as a result of a series of continuous clinical trials and subsequent implementation in national guidelines . the database has furthermore been used to describe the effect of reduced waiting time , changed epidemiology , and influence of comorbidity and socioeconomic parameters.conclusionhalf a century of registration of head and neck cancer treatment and outcome has created the basis for understanding and has substantially contributed to improve the treatment of head and neck cancer at both national and international levels . | About the Danish Head and Neck Cancer group
DAHANCA database
Structure of the database
How do we register data?
Quality assurance and correlation with other registers
Approval of the database
Economy of the database
Use of the database |
PMC4033809 | anterior cruciate ligament ( acl ) reconstruction is one of the most common orthopedic procedures . however , despite very satisfactory clinical outcomes [ 1 , 2 ] , the surgeon often detects the persistence of a certain rotatory instability , even in cases of reconstructions with no detectable intra- or post - operative complications , and regardless of the type of graft used .
the cause of this phenomenon is not completely understood yet , and the goal of many researchers is to be able to finally reconstruct a knee with the original rotational stability . in this paper
the main function of the acl is to control tibial anterior translation with a secondary effect on knee internal rotation [ 4 , 5 ] .
both acl functions are possible due to the presence of other articular structures which are linked to the acl in maintaining knee stability .
it is well documented how the acl is made of two different bundles , the antero - medial ( am ) and the postero - lateral ( pl ) , with different specific functions , but working synergically so that they can not be considered as separate structures .
one of the main aspects of acl anatomy , in regard to surgical reconstruction , is the exact position of its femoral and tibial insertion .
the femoral insertion has been the most commonly studied since the first attempts at open intra - articular reconstructions , because it is thought to be the most difficult to reproduce due to its very posterior positioning on the medial surface of the external femoral condyle .
it was common opinion that , despite the graft choice , the positioning of the graft could never be posterior enough , so many surgeons tried to perform an over the top technique ; however , it is now appreciated that the native acl femoral insertion site is located along osseous landmarks on the posterior aspect of the medial wall of the lateral femoral condyle , termed the lateral intercondylar and bifurcate ridges .
the lateral intercondylar ridge corresponds to the feature termed the resident ridge reported by clancy et al . .
identification of this ridge when present has been shown to be an accurate and reliable method of locating the native acl insertion site and the true entry point of the femoral tunnel .
diagnosis of acl rupture is basically clinical , and is usually performed with the lachman and the pivot - shift tests , whose sensibility and specificity have been widely documented .
the pivot - shift test assesses both tibial translation and rotation , reproducing the most common traumatic mechanism of rupture of the ligament .
moreover , the pivot - shift test better correlates with a patient s disability . for this reason it is thought to be the most reliable test for detecting rotatory instability of the knee following an acl tear .
the pivot - shift is usually graded in three degrees ( grade i : glide ; grade ii : jerk ; grade iii : subluxation ) , but it is highly dependent on the ability and experience of the operator . since the importance of this test is in evaluating both acl deficiency and the effects of different surgical techniques of reconstruction , many authors have proposed various methods of objectively evaluating and quantitatively measuring the test , such as the use of a navigation device ( fig . 1 ) ,
mechanical or electromagnetic tool , or conventional radiological or magnetic resonance dynamic methods [ 1117 ] .
however , none of these methods have become widespread among surgeons and many of them remain operator - dependent . as a matter of fact
the pivot - shift test is still the most popular test for assessing the rotatory instability of the knee after an acl rupture and reconstruction.fig .
1the pivot - shift phenomenon as detected by a navigator : tibial antero - posterior diagram and rotation in a knee with intact acl ( a ) and in an acl - deficient knee ( b ) the pivot - shift phenomenon as detected by a navigator : tibial antero - posterior diagram and rotation in a knee with intact acl ( a ) and in an acl - deficient knee ( b )
the relationship between acl lesions and the resulting rotatory instability of the knee has mainly been studied on cadaver knees .
in fact , in in vivo studies where the instability was compared to the contralateral healthy knee , we can never assume that the lesion of the acl was the only damage that occurred in the knee as a consequence of the initial trauma or as a result of progressive stretching of other structures due to the lack of the torn acl . in a recent study we performed on ten cadaver knees , we evaluated internal rotator instability after sectioning either component of the acl , showing that an isolated section of the pl bundle did not produce an increase in internal rotator instability ; on the contrary , the section of the entire acl did produce an increase in internal instability in nine cases . in our trials , only after a complete section of the entire acl
did we detect a + pivot - shift ( glide ) in three cases and a + + pivot - shift ( jerk ) in seven cases ; a + + + pivot - shift ( subluxation ) was never detected after isolated complete section of the acl .
however , in regard to rotatory instability , acl lesions , and the pivot - shift test , we should remember that the pivot - shift test assesses rotatory instability , and in particular , according to hughston , the antero - lateral instability , whose origin is more complicated than a simple result of acl lesion .
and feagin et al . stated that even though the pivot - shift is related to an acl tear , it is significantly increased by concomitant lesions of the external compartment and in particular of the middle one - third of the lateral capsular ligament
actually , in our study cited above on cadaver knees we detected a significant increase in rotatory instability along with a + + + pivot - shift in all the cases in which , after tearing the entire acl , even a partial lesion ( 1 cm ) of the alftl was performed .
even more recently , mushal et al . showed , also with the use of a navigation system , a significant increase of the anterior tibial translation in the external compartment during the pivot - shift maneuver in acl deficient knees after external meniscectomy .
the same group of researchers had previously shown that the anterior translation of the external part of the tibia was strictly correlated to the pivot - shift score .
using a robotic / ufs testing system . in conclusion , we can state that , while an isolated lesion of the pl bundle does not seem to increase internal rotation , lesion of the entire acl produces a slight increase of internal rotation but , most of all , a different pattern of rotation whose fulcrum moves from the central pivot of the joint to the medial collateral ligament ( mcl ) , with following increase of the anterior translation of the external tibial plateau .
the pivot - shift test , in assessing this particular biomechanical pattern of instability , is related not only to the lesion of the acl , but it is strongly influenced by associated lesions , in particular those of the lateral compartment ( alftl , lcl , lateral meniscus ) .
even though acl reconstruction is a very widely used operation with a very satisfactory success rate which allows the majority of active patients involved in sports activities to resume their pre - operative levels , the persistence of a certain rotatory joint laxity might produce subsequent meniscal or chondral damage leading to a clear degenerative arthritic disease .
even though the pivot - shift assessment has always been part of the evaluation scoring scales used , it has often been underestimated ; only within the last few years has this topic come back to scientific attention thanks to freddi fu s studies on the functional anatomy of the acl , on its two distinct bundles and , most of all , on failure of the single - bundle reconstruction to restore exact joint stability especially in regard to internal rotation . as a result
, many surgeons , with the aim of improving their success rates , have in turn started using the double - bundle reconstruction of the acl . in recent years , and in particular in the first years of the third millennium , many clinical and laboratory studies have been published on the advantages of reconstruction of the two bundles , thus supporting fu s theory [ 2527 ] . in particular , clinical studies with a minimum follow - up of 2 years in which double - bundle reconstructions were used showed better results than the single - bundle , both in terms of knee stability and in terms of recurrence rate .
moreover , other studies , such as the one by robinson et al . on cadaver knees with the use of a navigation system , further provided objective data about how , in acl reconstructions , the am and pl bundles act differently in stabilizing the knee , particularly during the pivot - shift test , where the pl bundle is important in controlling not only anterior laxity toward knee extension , but also the rotational component . in net contrast with
what was reported by the above mentioned authors , in a similar study we performed on cadaver knees with the use of a navigation system , we found that the further addition of the pl bundle to an am single - bundle reconstruction did not provide any additional stability to the knee in regard to internal rotation .
how might these different results be explained since the methodologies of the studies were similar ?
the explanation may likely be in the different surgical way of reconstructing the am bundle : in fact , in robinson s study , the am bundle was positioned slightly more anterior and vertical than in our study , whereas we tried to place the femoral insertion , approached with an out - in technique , more horizontal and as posterior as possible , thus in accordance with the actual anatomy of the acl .
this is a very important topic which deserves deeper examination . since the beginning of the reconstruction of the acl with open techniques , it was mandatory for the surgeon to scrupulously respect the anatomy of the acl with a very posterior positioning of the femoral insertion . since this goal
could not be reached by drilling the femoral condyle from the articular joint , surgeons started using the out - in technique .
after a few years , with the advent of arthroscopic techniques , the majority of surgeons preferred to perform less invasive techniques ( single - incision techniques ) , performing trans - tibial femoral tunnel drilling , often resulting in a non - anatomical positioning of the acl
. it seems as if , despite the advantages provided by the scope , the arthroscopists preferred mini - invasiveness over respect of the anatomy and function . however , since rotatory instability is a complex phenomenon not simply dependent on the acl rupture or the anatomical reconstruction , other hypotheses have been proposed to correct this type of instability . among these
, an important aspect is represented by the peripheral plasties [ 30 , 31 ] , whose biomechanical role in controlling rotational instability and the pivot - shift has been widely proven , even in recent studies published by our group : internal rotation was better restored in cases in which the anatomic acl reconstruction was performed along with a peripheral plasty than in cases treated with a double - bundle technique .
did not reach the same conclusions as us : however , they did not put any tension on the lateral sling , thus losing a big part of the efficacy of the technique itself .
even more controversial is the clinical effectiveness of the peripheral plasties in long - term follow - up , even though zaffagnini et al .
, in assessing three groups of patients treated with hamstrings ( stg ) , bone - patellar - tendon - bone ( bptb ) and st plus peripheral plasty , obtained the best results in the third group . in our experience
we found peripheral plasty useful in patients with severe rotatory instability ( + + + pivot - shift ) , in women and in cases of revision .
a complete lesion of the acl often causes a rotatory instability of the knee detectable with the pivot - shift test . during this complex phenomenon
the tibia tends to internally rotate with respect to the femur , thus changing its rotational fulcrum which moves medially closer to the mcl , with following anterior translation of the external tibial plateau .
the severity of the pivot - shift , commonly scored in three degrees , essentially depends on the amount of constitutional tibial rotation and on the presence of concomitant associated lesions , such as the alftl and the external meniscus . in order to obtain the best rotatory stability ,
the acl reconstruction must be performed accurately , reproducing its anatomical positions ( either single- or double - bundle ) .
peripheral plasties may contribute to better control of rotator instability and may be indicated in selected types of patients . | although acl reconstructions provide satisfactory clinical results nowadays , regardless of the type of graft or the surgical technique used ( out - in vs in - out or single- vs double - bundle ) , the residual rotatory instability which is often detected at clinical follow - ups is still a matter of concern among surgeons . in this paper
we try to analyze all the aspects which might contribute to this phenomenon by summarizing the biomechanical functions of the two bundles of the acl , and by evaluating all the other factors strictly related to the rotatory instability of a reconstructed knee , such as the anatomical positioning of the single- or double - bundle new acl , or the importance of a valid lateral compartment ( lcl , altfl ) .
clinical , biomechanical and cadaver studies are discussed in order to contribute to better understanding of the origin of post - operative residual rotatory instability . | Introduction
Anatomy and function
Diagnosis
ACL tear and rotatory instability
Effects of ACL reconstruction on rotatory instability |
PMC4211206 | balance dysfunction is one of the main factors leading to impaired mobility and postural control , influences walking after discharge and in the community - dwelling elderly , and also impacts balance control in daily life ( brosseau et al . , 1996 ; wee et al . , 1999
previous studies have shown that more than 33% of people with chronic balance dysfunction have an increased risk of falling in their daily lives ( colledge et al . , 2002 ) , and
the cause of more than half of older people 's accidental death was significantly correlated with falling and balance dysfunction ( agrawal et al . , 2009 ) .
some balance ability training methods have demonstrated postural control and mobility improvement ( yavuzer et al . , 2006 ; lo et al . , 2012 ) , but the effectiveness of existing interventions is limited because patients tend not to be fully involved and adhere to the training protocol . with the development of technology , rehabilitation facilities and technology that can study the simulated real environment
are increasingly used to restore motor function and address balance dysfunction ( bower et al . , 2014 ; mcewen et al . , 2014 )
virtual reality technology is developed on a computer hardware and software environment that enables immersed users to generate visual , audio , and haptic feedback , and obtain an interactive experience in the three - dimensional visual space , which strives to give users the impression of a real environment .
the virtual real environment is created by virtual reality technology with a focus on three characteristics : autonomy , interaction and sense of being .
patients who have perceived deficiencies and motor dysfunction can use virtual reality technology during their rehabilitation training , to produce the greatest recovery in their impaired motor function .
consequently , following this treatment , patients might be able to achieve full self - care , and virtual reality training might allow some of them to work independently , improving their quality of life ( berra , 2004 ) .
balance control relies on the central nervous system at multiple levels ; some studies have shown that exploring the virtual real environment activates the cortical and sub - cortical regions ( nancy et al . , 1980 ; bolton et al . ,
2012 ) . in this current review , the possible central control mechanism of virtual reality technology as well as its clinical application will be introduced .
generally , balancing the body is thought to be achieved by the coordination of three major systems , including visual , vestibular and proprioceptive sensation .
previous studies have shown that the prefrontal cortex is one of the most important brain areas in controlling human balance ( nancy , 1980 ; virk et al . , 2006 ; walker et al .
they provided information about the position and motion of the head with respect to the surroundings , and based on information in the visual surroundings provided by visual cues .
the main role of the proprioceptive and somatosensory system is to sense the distributed tactile input stimuli at the neural level , and to provide a relationship between limb position and the central nervous system ( figure 1 ) .
the vestibular system , located in the inner ear , is used to control and perceive the motion and position of the head in space ( virk et al . , 2006 ) . the function of these systems reduced after the brain is impaired .
when one or two of these sensory input systems are dysfunctional , the allocation of different gains to the sensory inputs can be exploited to compensate for the impaired systems ( nancy , 1980 ; walker et al . , 2010 ) .
( 2006 ) proposed that such an adaptation can take place in everyday life or in a virtual environment . by using a virtual environment to train the eye - head movement , balance in older people
can be improved and occupational falls are minimized . a simplified representation of the human motion control loop .
a desired body state directs the controller within the central nervous system generating motor commands that drive the muscles of the body .
external sources like electric current ( d ) can stimulate the body ( a ) .
the actual state of the body is registered by ( among other things ) the vestibular apparatus and the visual system ( b ) .
the signals from these sensors are processed by the central nervous system ( c ) for comparison with the desired body state . adapted from bos et al .
behavioral experiences including an enriched environment , physical activity and spatial learning substantially improve sensorimotor function outcome after brain injury following ischemia ( johansson et al .
virtual reality systems have been shown by many studies to provide similar real - world training effects , such as in the intensity and repetition referred to as constraint - induced movement therapy , body weight support treadmill training and allow patients with stroke to modify their neural organization and promote neuroplastic changes ( viau et al . , 2004 ;
therefore , we hope to explore the possible mechanism of how virtual reality promotes balance rehabilitation after brain injury in terms of visual , vestibular and proprioceptive feedback .
recent studies have shown that balance control in standing is a complex sensorimotor action based on automatic and reflexive spinal programs under the influence of several distinct and separate supra - spinal centers in the brainstem , cerebellum and cortex ( drew et al . , 2004 ) .
multimodal dance training over 4 weeks could activate the region of the prefrontal cortex ( tachibana et al . , 2011 ) .
some experiments have allowed patients to play and learn spatial navigation in mazes ( ayaz et al . , 2011 ) , and activation
could always be found in brain regions in our preliminary study of virtual reality training of stroke patients in sysu using an xbox 360 ( figure 2 ) .
although the mechanism is not clear , these results suggest a corresponding relationship between the prefrontal cortex and spatial orientation ability .
a further study from miller suggests that during the incremental swing balance task , when unpredictable and external postural perturbations were applied to healthy standing participants , the prefrontal cortex would bilaterally recruit both visual and proprioceptive information to adaptively modify postural instability in response to changes in the virtual environment to complete the action goal ( miller et al . , 2001 ) .
another study by basso moro also demonstrated that , when healthy participants performed an incremental swing balance task in a semi - immersive virtual reality environment , the effect of oxygenation increased in the prefrontal cortex of both hemispheres ( basso et al . , 2014 ) .
( 2005 ) recorded the ankle movement of twelve young healthy participants using force plates , eeg and emg as they performed oscillatory and discrete postural movements in the anterior and posterior directions .
a statistically significant difference in the amplitude of all three components of movement - related cortical potentials , with respect to the baseline at the front - central electrode sites was found in the eeg data .
they also demonstrated that the frontal region may play an important role in postural equilibrium , likes moving continuously in different directions , or only in the forward direction .
areas activated during an active foot movement task by a patient with stroke at the first affiliated hospital , sun yat - sen university , china .
the two functional mri images of preliminary experiments are supplied by the motor recovery laboratory in the department of rehabilitation , showing active tasks of the right paretic foot before ( a ) and after ( b ) virtual reality training . during walking
some studies show that when adjusting walking speed on the treadmill ( suzuki et al . , 2004 ) and during the recovery process following ataxic stroke ( mihara et al . , 2007 ) , the prefrontal cortex participates in the control of locomotion .
basso used functional near - infrared spectroscopy to study changes in regional activation in the frontal cortices in terms of hemoglobin oxygenation in the brain 's vascular system during walking at 3 and 5 km / h and running at 9 km / h on a treadmill .
the density of oxygenated hemoglobin increased , but that of deoxygenated hemoglobin was not found in the frontal cortices .
moreover , other studies found a positive correlation between presence and parietal brain activation , and a negative correlation between presence and frontal brain activation during interactive virtual reality training ( mihara et al . , 2008 ) .
we can see that alongside the prefrontal cortex , the parietal cortex makes a contribution to the control of balance .
through investigating the hemodynamics of patients who had cortical activation induced by postural perturbation , mihara found that there was a strong relationship between continuous activation during ataxic gait and the compensatory mechanisms for ataxic gait after infarct stroke . in conclusion , there are widespread cortical network activations , including the prefrontal , premotor , supplementary motor , and parietal cortical areas in both hemispheres , which have a significant impact on postural control in post - stroke hemiplegic patients ( kober et al .
the simulated real - world scenarios give balance dysfunction patients more and stronger information input than the real world to ensure that they relearn their coordination and sense of balance .
the fidelity of virtual reality may play an important role in the effectiveness of recruiting neural circuits and the delivery of desirable outcomes at the functional level .
the structure of the brain can be enhanced using visual feedback in virtual reality to augment interconnected , distributed cortical regions .
it is suggested that visual information can provide a potent signal for the reorganization of sensorimotor circuits ( lewis et al . , 2000 ; lewis et al . , 2005 ; stepniewska et al . , 2005 ) .
patients with stroke can use visual feedback to adjust the body 's center of gravity , and to achieve the goal of controlling the body state ( srivastava et al . ,
, the multi - sensory virtual reality feedback loop can strengthen the control of balance .
( 1998 ) conducted some animal experiments on monkeys , cats and other animals , and showed that visual experience and action seem to be the keys to functional recovery of vestibular function deficiency ( lacour et al .
a large number of clinical trials support this view . in a study by webster et al .
( 2010 ) , a virtual environment was created to help people control the mobility of their wheelchairs , and participants had to navigate through a virtual obstacle course .
after treatment , compared with participants who did not have training on the virtual course , the experimental group showed a decreased rate of wheelchair accidents and falls and a better performance on obstacle courses ( webster et al . , 2001 ) . to study the influence of virtual reality technology on the recovery of balance function following chronic stroke , belinda and cho used different kinds of video games to stimulate patients balance , coordination , strength and upper limb coordination , and the results showed that all patients had significantly improvement in balance function ( cho et al . , 2012 ; michalski et al . , 2012 ) .
some other studies have suggested that middle cerebral artery infarcts in the right hemisphere might have a relationship with increased dependence on vision , and non - infarct brain regions are recruited to control postural sway .
it is also suggested that the control of lateral posture following a right hemisphere infarct requires more visual input than in a healthy participant ( manor et al . , 2010 ) .
at present , virtual reality use in vestibular rehabilitation is a relatively new concept , and there are not many studies on the relationship between virtual reality and balance in the vestibular field .
common vestibular function disorders may be caused by abnormalities in the vestibulo - ocular reflex .
viirre suggested that the vestibulo - ocular reflex could be adapted in virtual reality simulations with an increase in vestibulo - ocular reflex gain , and virtual reality could be used to increase the rate of adaptation by specifically adapting scenes to a person 's capabilities , thereby facilitating their recovery ( viirre et al . , 2000 ) .
miles study suggested that the key to adjusting the vestibular system is the retinal slip , the movement of a visual image across the retina , which is a powerful signal that induces the adaptation of vestibular responses .
the primary rationale for using virtual reality for vestibular rehabilitation is that realistic visual environments may enhance adaptation by causing retinal slip ( miles et al . , 1980 ) .
( 2001 ) studied people with uncompensated unilateral peripheral vestibular dysfunction using a customized exercise program or a machine - base optokinetic stimulation exercise program .
preliminary findings suggest that both exercise programs could improve vestibular dysfunction , yet the machine - base group demonstrated that physical therapy involving conflicting visual environments might be more effective than a customized program alone or a program that includes only cawthorne - cooksey exercises .
virtual reality scenes may promote rehabilitation more effectively than optokinetic - based therapies , since there is an ability to finely control the virtual scene ( pavlou et al . , 2001 ) . to discover the potential of virtual reality for vestibular disorders , in a small sample investigation , wbitney asked two vestibular disorder patients and three healthy people to stand while viewing a sinusoidal waveform on a force plate .
the results suggested that virtual reality was a valid way to perform vestibular rehabilitation ( whitney et al . , 2002 ) .
nevertheless , because of the small number of participants , the accuracy of the study needs to be validated with further data collection .
another theory to explain the effect of virtual reality on the vestibular system is habituation therapy .
the patients may feel uncomfortable at the beginning , and then a sensory mismatch is sent to the brain and promotes compensation for and adaptation to labyrinthine disorders ( norre et al . , 1989 ) .
ninety consecutive patients with hemiplegic involvement following a single cerebrovascular accident were recruited to assess the relative importance of factors affecting balance , and it was found that balance function and mobility post - stroke were both strongly influenced by proprioception ( keenan et al . , 1984 ) .
proprioceptive training affects mobility and balance function by altering how proprioceptive receptors input information , and improves the control of the musculoskeletal motion system and balance function in stroke patients .
some studies have demonstrated that proprioceptive neuromuscular facilitation promotes neuromuscular control and motor function recovery by stimulating proprioceptive stimuli , such as stretching the limbs , joint compression , traction and so on .
in addition , the use of spiral diagonal movement patterns can stimulate the joint and muscle proprioceptors . by training the proprioceptive system ,
the responsiveness of muscles can be enhanced and the recovery of neuromuscular and motor function can be stimulated ( pan et al . , 2011 ) .
furthermore , virtual reality training could improve standing balance in stroke patients , including normal and abnormal proprioceptive function ( song et al . , 2014 ) . but compared with proprioceptive neuromuscular facilitation technology , virtual reality training exercises can not only train the limbs , but also train the body by enriching proprioceptive information input .
( 2011 ) trained 31 patients using the pro - kin training system and found that the score on the berg balance scale in these patients was significantly increased after training , which indicates that proprioception training played an active role in improving balance function in stroke patients .
compared with conventional therapies ( physiotherapy , occupational therapy , or other exercises ) , virtual reality technology has unique advantages in the field of rehabilitation .
first , it makes treatment more interesting and brings patients more enthusiasm , and the sports favored by the patients in daily life can be used as a training program in a virtual reality system ( figure 3 ) .
second , patients can undergo familiar training in a wheelchair or sitting in a chair as well as standing or walking , so there is no need for stabilized posture control , rehabilitation difficulty is reduced , and the safety of the rehabilitation exercise is increased .
third , the game system is cheap and easy to carry , and can be easily operated in the hospital , at home or in the community .
virtual training with bilateral limb movement in the motor recovery laboratory , department of rehabilitation , the first affiliated hospital , sun yat - sen university , china . ding et al .
( 2013 ) used a simple computer game involving virtual reality - based constraint - induced movement therapy training for balance dysfunction in three chronic stroke survivors , which improved dynamic stability and weight symmetry by encouraging them to use their paretic leg with more effort .
the patients received motion signals from virtual avatars in virtual reality as well as augmenting the control gain .
measured by functional gait assessment , the berg balance scale and a walking speed test , horlings and coworkers confirmed that virtual reality balance training could provide more realistic proprioceptive and visual input , and improved the patient 's reaction time , postural stability , balance and walking function effectively ( bisson , 2007 ; horlings et al . , 2009 ; singh et al . , 2012 ) .
therefore , virtual reality balance games can be used as a potential and useful tool to train adults with balance dysfunction ( schwesig et al . , 2011 ) .
virtual environments are used in many areas , such as in entertainment , training situations and medicine , including the rehabilitation of behavioral and neurological dysfunction .
the use of virtual reality technology in the functional rehabilitation of various debilitating diseases not only provides patients with a very authentic virtual environment and an immersive experience , but also greatly improves their participation enthusiasm and initiation in rehabilitation . compared with conventional rehabilitation therapy , there is a more significant improvement in motor function and activities of daily living when treated using virtual reality ( bryanton et al .
virtual reality therapy is an effective therapy in stroke , spinal cord injury , cerebral palsy and other motor function deficient diseases .
wii games combined with virtual reality technology have been used to treat balance dysfunction . in clinical trials , deutsch and
colleagues randomly allocated stroke patients into a wii exercise training group and a conventional training group , and the experiment demonstrated that the wii group exhibited a higher degree of functional recovery of balance and the patients had higher enthusiasm when they performed the virtual reality exercises ( deutsch et al . , 2009 ) . in another study ,
children with spastic cp completed ankle selective motor control exercises using a virtual reality exercise system and conventional exercises .
the results showed that greater fun and enjoyment were expressed during the virtual reality exercises , and children completed more repetitions of conventional exercises .
furthermore , the range of motion and hold time in the stretched position were greater during virtual reality exercises .
these data suggest that using virtual reality to train or guide exercises may improve exercise compliance and raise exercise effectiveness ( bryanton et al . , 2006 ) . however , a randomized controlled trial by eser et al .
( 2008 ) did not find a statistically significant difference between virtual reality therapy and conventional therapy in terms of lower extremity motor recovery , mobility or activity level .
there is no doubt that the intervention time for equilibrium dysfunction rehabilitation is one of the most critical factors in functional recovery after stroke , which has important research value . at present
, many studies about virtual reality training mainly focus on the clinical effects ; the mechanisms , especially of neural reorganization and neuroplasticity , and which regions are mainly activated at cortical and sub - cortical levels , are still not clear . in addition
, how virtual reality training influences the individual involves the central nervous system at multiple levels .
virtual reality therapy is mostly used in the chronic phase of stroke rehabilitation ( lacour et al . , 1976 ;
however , the early stages are the prime time of exercise for balance recovery in patients with balance dysfunction . according to the literature
, sufficient and early rehabilitation training can greatly improve the prognosis of patients with stroke , and reduce complications ( langhorne et al .
2012 ) , improve motor function of the hemiplegic limb ( li et al . , 2011 ) , promote their skills ( craig et al . , 2010 ) and improve their quality of life ( tyedin et al . , 2010 ) .
therefore , the early phase of brain injury rehabilitation is the most important time in the recovery of patients with stroke .
however , during this phase the patients are often too paretic to do training , an important reason for which is that they have a lack of cortical stimulation .
therefore , using virtual reality in the early phase of brain injury could be feasible .
in addition , to explore the safety and therapeutic effects and search for the most suitable intervention time point for balance recovery , virtual reality exercise training should be used for acute and subacute patients , which is a necessary and valuable research direction . meanwhile
many studies ( flynn et al . , 2007 ; cikajlo et al . , 2012 ; pluchino et al . ,
2012 ) have shown that virtual reality exercise training is likely to be an effective therapy for patients with balance dysfunction at home in the future .
but there are still many problems to solve , such as the evaluation criteria for the treatment and the interactive performance of the virtual reality system .
in addition , what kind of model of virtual reality should be chosen , how long the use should be and what precautions should be taken need to be carefully considered when performing virtual reality training . there is still no clear reference index at present .
the next stage of the study of virtual reality technology applied to the rehabilitation of balance function needs to explore the mechanism of the integrated central and peripheral nervous system , visual , vestibular and proprioceptive sensation , and more detailed clinical techniques . establishing a quantitative standard of virtual reality therapy
furthermore , the most appropriate way of applying virtual reality intervention and suitable intervention intensity according to different patients brain function mechanisms should be determined . in these ways , functional recovery of stroke patients
will be promoted and the operational feasibility of virtual reality will be improved by formulating better plans , and the technology can be widely used in the family in the future . in conclusion ,
virtual reality technology based on the study of the mechanisms of functional rehabilitation and its application following stroke has wide space to develop . | virtual reality is a new technology that simulates a three - dimensional virtual world on a computer and enables the generation of visual , audio , and haptic feedback for the full immersion of users .
users can interact with and observe objects in three - dimensional visual space without limitation . at present ,
virtual reality training has been widely used in rehabilitation therapy for balance dysfunction .
this paper summarizes related articles and other articles suggesting that virtual reality training can improve balance dysfunction in patients after neurological diseases . when patients perform virtual reality training , the prefrontal , parietal cortical areas and other motor cortical networks are activated .
these activations may be involved in the reconstruction of neurons in the cerebral cortex .
growing evidence from clinical studies reveals that virtual reality training improves the neurological function of patients with spinal cord injury , cerebral palsy and other neurological impairments .
these findings suggest that virtual reality training can activate the cerebral cortex and improve the spatial orientation capacity of patients , thus facilitating the cortex to control balance and increase motion function . | Introduction
Neural reorganization mechanism of virtual reality exercise in the balance control of the body
Cortical mechanisms of balance control
Visual input mechanisms of balance control
Vestibular sensory pathway mechanism of balance control
Role of proprioceptive rehabilitation
The feasibility and effect of virtual reality technology applied to balance dysfunction recovery
Discussion |
PMC4462484 | traumatic achilles tendon tears are frequent occurrences in lower - limb trauma , involving young , active patients . to better understand the etiology and etiopathogenesis of tendon tears , a comprehensive model of the achilles tendon behavior during rupture - risky daily situations and motions
analyzing the biomechanics of the achilles tendon requires in fact a dissection of the main kinematic events during a running sequence .
the achilles tendon structure allows it to transmit significant forces from muscle to bone , behaving like a hyperelastic material past a certain longitudinal strain , but shows a limited resistance to shearing and compression loads . at rest , tendon fibers appear wavy , whilst during strain they become taut , behaving like an elastic material up to 2% elongation ; past that , they behave like a hyperelastic material .
partial tears develop at 4% elongation and beyond , while complete tears usually occur at 8% elongation and beyond .
the forces acting on the foot can be measured experimentally with the aid of a force plate , a device that utilises newton s third law . by recording the ground reaction force ( grf ) , we can determine the forces that act on the achilles tendon while running , in particular those acting on the tendon - calcaneus jonction .
a running sequence is much more complex compared to a walking one , completely lacking in bilateral support and having an intermediate phase of flight , when the body is completely airborne .
the achilles tendon behaves like an interface between muscle and bone during such a sequence , establishing the so called calcaneus - tendon - sural triceps system . if we consider the bony surface as completely inelastic , the forces developed by the triceps surae input a maximum load on the tendon s insertion .
understanding and predicting these forces during the ground contact phase of a running step is paramount to better understanding the impact of running on the achilles tendon .
to record the ground reaction force ( grf ) , a kistler force plate was used , with a recording frequency of 2.5 khz ; a 23-years - old human subject with a weight of 77 kg , a height of 182 cm and with no prior tendon injuries and no prior specific physical training was asked to run over the force plate 30 times .
the collected data were filtered and statistically analysed , ignoring any force past 5 sds ( standard deviations ) , rendering a force cloud that was sorted in regards to time : it became readily apparent that the median ( red line ) is a rough , intuitive image of the running function model .
a median regression function applied to the force cloud yields a clearer image : in order to elaborate and compare a mathematical model to an empirical one , we chose the running sequence that was closest to the median regression curve .
although one could easily use the regression function as a model itself , we felt that a mathematical inference model was much more useful to appreciate the biomechanics of the achilles tendon .
given that f = m , where f stands for force , m for mass and is the gravitational pull ( 9.81 m / s ) , f for our subject amounts to 755.3 n. the area below the curve is in fact impulse ( i ) , which can be calculated using the riemann sums : where fk is the force recorded at the moment tk , with t=.384 and t = tktk1 being constant ( 0.004 s ) .
an impulse of i=360.31 ns results . this can be used to compare the accuracy of our mathematical model to the empirical data .
a first modeling attempt is to try and approximate the function with the help of a second degree polynomial , f(t ) = at+bt+c , the first term being negative because the function is concave .
the impulse is obtained through imposing limits f(0)=o and f(t)=0 , thus the initial equation becoming f(t ) = a(ttt ) = at(tt ) , with only a unknown .
impulse becomes : empirical data shows that the maximal value of the function is satisfied at the middle of the interval ,
f(t2)=fmax and
f(t2)=0 , which is actually obvious as we purposedly chose to model using a 2 degree function . and
impulse
i=2fmax t3 , we can calculate the impulse using maximal force ( known empirically ) .
thus , t=0.384 and maximal force is 1,711.66 n , the resulting impulse i=438.18 ns , which is close , but not sufficiently so the empirically devised impulse .
using body weight to model the function can be useful , as in mathematical terms , body weight ( bw ) can be described with the median of the function ( during a sprinting step , the entire body weight is shifted from one foot to another , thus during a whole step the entirety of the weight is supported ) : where t1 and t2 represent the crossing of the curve by the line that expresses the median of the function .
thus , bw can be described as : an obvious relation between weight and impulse is described , but as we can see by using previous results ,
bw=2fmax3 , which in turn means
fmax=32bw . using a fourth order polynomial :
f(t ) = at + bt + ct + dt + f , where t[0 , t ] , with the limits f(0 ) = 0 , f(t)=0 , f(0)=0 , f(t)=0 and
f(t2)=fmax , leads us to :
f(t)=16fmaxt4t2(t - t)2 , which results in
fmax=158bw , a rather accurate description . in the end , by comparing the different methods of approximation , we can observe that a known maximal force approximation seems to be the most accurate :
obtaining a perfectly accurate model of the step in a running sequence is a daunting task if attempted through mathematical means alone ; the higher the order of the polynomial , the more accurate the modeling gets , but this continues ad infinitum , because it is done by approximation .
it needs not to be perfect to serve a predictive purpose though : having established a satisfactory model and developing a quick , repeatable and easy way to provide it is a sufficient goal if one seeks to understand the behaviour of the foot in a running sequence and through it , the strain and loads incurred on the achilles tendon calcaneus joint .
strictly from a mathematical point of view , a fourth order polynomial seems to be sufficient to model running ; a further increase in order will only slim the curve of the approximation , making the transition to the peak force steeper while useful to extract a more accurate impulse and therefore load , it diminishes the inequality of force distribution , opposite to the empirical data gathered that reveals the fact that the running step is in fact a walking step in a much shorter time span .
a high order polynomial approximation function suggests that the step is a sprinting step , where ground contact is achieved only on the forefront of the foot , toe region and distal metatarsal region , foregoing the calcaneus landing altogether .
it is probably more accurate to divide running into light running ( which was the object of the study ) and sprinting , where the foot behaves differently . as for inaccuracy sources , recording noise when collecting data can probably be considered the main source of error , while the presence of medio - lateral and anterio - posterior forces unaccounted for during data collection and filtering could come in second place . when modeling a foot that has a distinctly altered biomechanics due to malformations , recent surgery , pain , neuromotor injuries etc .
, care should be taken in ignoring the concurrent forces ; in a healthy foot these forces are negligible in a running sequence .
building an accurate model of running allows to identify key kinematic moments and events that impact the foot and specifically , the ankle with its main muscle / bone system that is the triceps surae achilles tendon
the heel region in general and the achilles tendon in particular bears loads equivalent to twice and sometimes more than twice the body weight during running .
moreover , the impulse generated by these loads is distributed unequally throughout the running sequence , creating high - strain events for the achilles tendon . the most important and impacting force during running is the vertical traction force ( as described , through newton s third law , by the ground reaction force ) . by establishing and describing the relationship between body weight , ground reaction force and impulse in each step of the running sequence , we can model the entire kinematic chain . a polynomial model ,
although rather intricate , can provide a useful tool in predicting and assessing the behavior of everyday movements , in this particular case during sprinting and running . by recording the movement , one can simply extrapolate and compare between results from healthy individuals or between similar tendon healing rates in traumatic tendon injuries ; pathological motions can be detected as well as abnormal loads or prolonged loading times which can suggest unfit tendons or slow functional healing .
one must remember that while the vertical traction force is the main actor while running , medio - lateral and anterio - posterior forces can be detected , even though minimal , they can impact the achilles tendon and the overall motion , if for some reason their value is greatly increased through pathological movement or antalgic motions .
the achilles tendon is poorly equipped to deal with shear and compression forces , which can be cause for re - rupture . evaluating these forces | the following study attempts to elaborate a model of the achilles tendon while in the process of running , specifically during a step that is part of a running sequence .
data are collected with the help of a force plate and then is processed and modeled to serve as a starting point and comparison to a mathematical model using polynomial functions.the data collected were filtered to diminish recording of noise and an empirical model was established .
mathematical models using second order and fourth order polynomials were employed , as well as an approximation using known maximal force . the increase in the accuracy of modeling was determined as the order of the polynomial function increased.achieving an accurate predictor function is essential in understanding the biomechanics of the achilles tendon . | Introduction
Materials and Methods
Results
Discussion
Conclusions |
PMC5172552 | the structure types consist of the overall and the local models . as an effective tool to analyze gene expression data ,
the clustering analysis is comprehensively applied to many fields , such as gene expression spectrum analysis ( 1 ) , genome study , biological regulatory networks ( 2 ) , medicine filtering , new medicine development , clinical disease diagnosis 3 . , 4 . , and so on .
the basic hypothesis included in the clustering analysis is that , the genes that have the same expression mode may have similar functions ( 5 ) .
however , traditional clustering methods have a series of problems in reducing noise , mining local information , and synthesizing the heterogeneous data , etc .
the data dimension is becoming higher and higher due to the use of new biological microarray chips .
different objects of the same cluster in the data of a high dimensional space could show the similarity only in a certain subspace . when this principle is applied to gene expression data
, mutual - controlling genes could show similar expression patterns in some conditions for test samples .
in fact , in the whole input space , the gene expression pattern is different .
therefore , difficulties appear when we use traditional clustering methods to determine the object similarity by using the value approximation in high dimensional data .
the biclustering algorithm presented by cheng and church ( 6 ) is different from traditional clustering algorithms , in which the similarity is not treated as a function of pairs of genes or pairs of conditions .
instead , it is a measure of the coherence of the genes and conditions in the biclustering .
this measure can be a symmetric function of genes and conditions involved and thus the finding of biclusters is a process that groups genes and conditions simultaneously .
the most important innovation of cheng and church algorithm is that they put forward a definition called residue score
6 .
, 7 .. the algorithm divides an expression model into three parts : attribute residue , object residue , and -cluster residue ( or background residue ) .
the mathematical definitions are as follows:(1)eij=iieij|i|eij=jjeij|j|eij=ii , jjeij|i||j| where i and j are the row and column vector sets of the submatrix , respectively ; |i| and |j| are the number of rows and columns , respectively ; eij is the element of the submatrix ; eij , eij , and eij are the attribute residue , object residue , and -cluster residue , respectively .
the definition of the residue score is as follows:(2)rsij(i , j)=eijeijeij+eij let x be the set of genes and y the set of conditions .
let eij be the element of the gene - condition expression matrix representing the logarithm of the relative abundance of the mrna of the i gene under the j condition .
let i x and j y be the subsets of genes and conditions .
the pair ( i , j ) specifies a submatrix aij with the following mean squared residue score:(3)h(i , j)=ii , jjrsij2|i||j| the lowest score h(i , j ) = 0 indicates that the gene expression levels fluctuate in unison .
these trivial biclusters may not be very interesting but need to be discovered and masked so that more interesting ones can be found .
the row variance may be an accompanying score to reject trivial biclusters:(4)v(i , j)=1|j|jj(eijeij)2 the higher the value of h is , the more disordered the data is . in cheng and church algorithm , a greedy method is used to select submatrix with a low h score .
firstly , the method is to remove the row or column to achieve the largest decrease of the score .
for the current submatrix , they calculate the average residue score of each row using d(i)=1|j|jjrsij(i , j ) and the average residue score of each column using e(j)=1|i|iirsij(i , j ) , then choose the row or column with the maximal score and delete it from the current submatrix , until h(i , j ) < . also they use a parameter , so that they can delete a set of nodes each time before the score is recalculated . without updating the score after the removal of each node , the matrix may shrink too much and one may miss some large -clusters .
one may also choose an adaptive based on the score and the size during the iteration .
secondly , they add rows and columns so that the matrix with the maximal size can be obtained .
the most important innovation of cheng and church algorithm is that they put forward a definition called residue score
6 .
, 7 .. the algorithm divides an expression model into three parts : attribute residue , object residue , and -cluster residue ( or background residue ) .
the mathematical definitions are as follows:(1)eij=iieij|i|eij=jjeij|j|eij=ii , jjeij|i||j| where i and j are the row and column vector sets of the submatrix , respectively ; |i| and |j| are the number of rows and columns , respectively ; eij is the element of the submatrix ; eij , eij , and eij are the attribute residue , object residue , and -cluster residue , respectively .
the definition of the residue score is as follows:(2)rsij(i , j)=eijeijeij+eij let x be the set of genes and y the set of conditions .
let eij be the element of the gene - condition expression matrix representing the logarithm of the relative abundance of the mrna of the i gene under the j condition .
let i x and j y be the subsets of genes and conditions .
the pair ( i , j ) specifies a submatrix aij with the following mean squared residue score:(3)h(i , j)=ii , jjrsij2|i||j| the lowest score h(i , j ) = 0 indicates that the gene expression levels fluctuate in unison .
these trivial biclusters may not be very interesting but need to be discovered and masked so that more interesting ones can be found .
the row variance may be an accompanying score to reject trivial biclusters:(4)v(i , j)=1|j|jj(eijeij)2 the higher the value of h is , the more disordered the data is . in cheng and church algorithm ,
firstly , the method is to remove the row or column to achieve the largest decrease of the score . for the current submatrix
, they calculate the average residue score of each row using d(i)=1|j|jjrsij(i , j ) and the average residue score of each column using e(j)=1|i|iirsij(i , j ) , then choose the row or column with the maximal score and delete it from the current submatrix , until h(i , j ) < . also they use a parameter , so that they can delete a set of nodes each time before the score is recalculated . without updating the score after the removal of each node ,
one may also choose an adaptive based on the score and the size during the iteration .
secondly , they add rows and columns so that the matrix with the maximal size can be obtained .
because the greedy method may not always lead to correct results , we use an additional course to avoid deleting
the steps for the node addition in the original algorithm are as follows:1.compute eij ( for all i ) , eij ( for all j ) , eij , and h(i , j).2.add the columns j j with1|i|ii(eijeijeij+eij)2<h(i , j).3.recompute eij , eij , and h(i , j).4.add the rows i i with1|j|jj(eijeijeij+eij)2<h(i , j).5.for the i row that is still not in i , add its inverse if1|j|jj(eijeijeij+eij)2<h(i , j).6.if no node needs to add in the current iteration , return the final i and j. compute eij ( for all i ) , eij ( for all j ) , eij , and h(i , j ) .
recompute eij , eij , and h(i , j ) . add the rows i i with 1|j|jj(eijeijeij+eij)2<h(i , j ) .
for the i row that is still not in i , add its inverse if 1|j|jj(eijeijeij+eij)2<h(i , j ) .
if no node needs to add in the current iteration , return the final i and j. considering that the search space of cheng and church algorithm is only a subspace of the result set , we make some improvements as follows . in order to maximize the size of the result submatrix , we amend the decision condition in the original algorithm by changing the original constraint(5)1|i|ii(eijeijeij+eij)2<h(i , j)into the following form(6)1|i|ii(eijeijeij+eij)2<kh(i , j ) and when it is added into the former matrix , the value of h in the new matrix is less than the original value .
the improved algorithm extends the searching scope and increases the number of the nodes that can be added into the cluster .
our improved algorithm introduces a parameter k in equation ( 6 ) to ensure that the blind search is reduced .
cross validation shows that the improved algorithm performs better when k is taken as 3.2 . to speed up the improved algorithm
, we first express the matrix by using the idea of chromosome used in evolutionary computation , and then change the chromosomes in the matrix into two - dimensional link lists , in which we calculate the value of h and save the values of the chromosomes in the field h. to examine the efficiency of the improved algorithm , we tested it using the yeast gene expression spectrum from the gene expression data set ( 2 ) .
for the ten clusters obtained in the simulation , we calculated the average computational time of the results and found that the original algorithm cost 65 s , while the improved algorithm cost 94 s. the comparison results are shown in table 1 .
the quality of result sets of the improved algorithm is enhanced obviously , on the tolerable condition that the time cost is increased by less than 0.5 times .
the comparison of the submatrix rows and columns from the improved algorithm and the result sets of the original algorithm are also shown in table 1 .
it is obvious that from the results in the same condition , the improved algorithm can obtain better result sets and more information . in cheng and church algorithm ,
there are two important parameters and that need to be set before the algorithm running , where is a threshold of score function h and measures the extent of data consistency .
the parameter influences the quality of matrix clustering and in general it is better if the value is smaller .
but if is too small , the scale of the submatrix will be over small and easy to lose information .
hence , a balance point should be found for this parameter before running the algorithm .
the parameter is used in the deletion course of the first phase in the original algorithm , which is also an important threshold .
we determined the value ranges through experiments to provide referable information for realizing adaptive setting for the parameters .
firstly , we chose real data sets for testing . through a series of numerical experiments
, we obtained the relation between the values of parameter and submatrix size , as shown in figure 1 .
the arithmetic average of space size is used to estimate the quality of the clustering . in the experiments
, it was found that the size of the submatrix decreases monotonously with the descent of the value of . when is taken as around 120 , the trend of the descent is gentle .
even the value of goes down again , this trend does not change essentially .
therefore , we suggest that for these data sets , it is better to take the value of in the range of [ 120 , 180 ] . for the same data sets , we took the difference of the two systems clocks before and after the experiment as the time consumption of clustering , and only calculated the consuming time during the course of deletion . in this way we obtained the relation between the value of and the time consumption ( figure 2 ) .
the value of was taken in the range of [ 2.8 , 3.2 ] .
we also need to make practical clustering test at = 2.8 to avoid any misvalue .
if the clustering results are satisfied , we could keep the range , otherwise we have to increase the lower limit . for different data sets
, we can obtain a series of results . through linear regression and suitable adjustment to these calculation results
let [ a , b ] be the value range of data sets , m the number of genes , and n the number of conditions .
the simulating experiments showed that it is better to take the value of in the range of [ 3j , 4j ] .
the results of simulations showed that it is better to take the value of in the range of [ 7l , 8l ] .
the open human lymphoma b cell data set ( 8) was used to examine the proposed improved algorithm .
compared with the original algorithm , the quality of clustering results using the improved algorithm is enhanced obviously , the mining expression models are better , and the data possess stronger consistency with fluctuation on the condition that the time cost is increased a bit .
in addition , in spite that the noise level of data sets is very high of having the loss rate of 12.3% , simulation results showed that the improved algorithm could still keep a good clustering effect even if the noise interference is very strong .
because the greedy method may not always lead to correct results , we use an additional course to avoid deleting
the steps for the node addition in the original algorithm are as follows:1.compute eij ( for all i ) , eij ( for all j ) , eij , and h(i , j).2.add the columns j j with1|i|ii(eijeijeij+eij)2<h(i , j).3.recompute eij , eij , and h(i , j).4.add the rows i i with1|j|jj(eijeijeij+eij)2<h(i , j).5.for the i row that is still not in i , add its inverse if1|j|jj(eijeijeij+eij)2<h(i , j).6.if no node needs to add in the current iteration , return the final i and j. compute eij ( for all i ) , eij ( for all j ) , eij , and h(i , j ) .
recompute eij , eij , and h(i , j ) . add the rows i i with 1|j|jj(eijeijeij+eij)2<h(i , j ) .
for the i row that is still not in i , add its inverse if 1|j|jj(eijeijeij+eij)2<h(i , j ) .
if no node needs to add in the current iteration , return the final i and j. considering that the search space of cheng and church algorithm is only a subspace of the result set , we make some improvements as follows . in order to maximize the size of the result submatrix , we amend the decision condition in the original algorithm by changing the original constraint(5)1|i|ii(eijeijeij+eij)2<h(i , j)into the following form(6)1|i|ii(eijeijeij+eij)2<kh(i , j ) and when it is added into the former matrix , the value of h in the new matrix is less than the original value .
the improved algorithm extends the searching scope and increases the number of the nodes that can be added into the cluster .
our improved algorithm introduces a parameter k in equation ( 6 ) to ensure that the blind search is reduced .
cross validation shows that the improved algorithm performs better when k is taken as 3.2 . to speed up the improved algorithm
, we first express the matrix by using the idea of chromosome used in evolutionary computation , and then change the chromosomes in the matrix into two - dimensional link lists , in which we calculate the value of h and save the values of the chromosomes in the field h. to examine the efficiency of the improved algorithm , we tested it using the yeast gene expression spectrum from the gene expression data set ( 2 ) .
for the ten clusters obtained in the simulation , we calculated the average computational time of the results and found that the original algorithm cost 65 s , while the improved algorithm cost 94 s. the comparison results are shown in table 1 .
the quality of result sets of the improved algorithm is enhanced obviously , on the tolerable condition that the time cost is increased by less than 0.5 times .
the comparison of the submatrix rows and columns from the improved algorithm and the result sets of the original algorithm are also shown in table 1 .
it is obvious that from the results in the same condition , the improved algorithm can obtain better result sets and more information .
in cheng and church algorithm , there are two important parameters and that need to be set before the algorithm running , where is a threshold of score function h and measures the extent of data consistency .
the parameter influences the quality of matrix clustering and in general it is better if the value is smaller .
but if is too small , the scale of the submatrix will be over small and easy to lose information .
hence , a balance point should be found for this parameter before running the algorithm .
the parameter is used in the deletion course of the first phase in the original algorithm , which is also an important threshold .
we determined the value ranges through experiments to provide referable information for realizing adaptive setting for the parameters .
firstly , we chose real data sets for testing . through a series of numerical experiments , we obtained the relation between the values of parameter and submatrix size , as shown in figure 1 .
the arithmetic average of space size is used to estimate the quality of the clustering . in the experiments
, it was found that the size of the submatrix decreases monotonously with the descent of the value of . when is taken as around 120 , the trend of the descent is gentle .
even the value of goes down again , this trend does not change essentially .
therefore , we suggest that for these data sets , it is better to take the value of in the range of [ 120 , 180 ] . for the same data sets , we took the difference of the two systems clocks before and after the experiment as the time consumption of clustering , and only calculated the consuming time during the course of deletion . in this way we obtained the relation between the value of and the time consumption ( figure 2 ) .
the value of was taken in the range of [ 2.8 , 3.2 ] .
we also need to make practical clustering test at = 2.8 to avoid any misvalue .
if the clustering results are satisfied , we could keep the range , otherwise we have to increase the lower limit . for different data sets
, we can obtain a series of results . through linear regression and suitable adjustment to these calculation results
let [ a , b ] be the value range of data sets , m the number of genes , and n the number of conditions .
the simulating experiments showed that it is better to take the value of in the range of [ 3j , 4j ] .
the results of simulations showed that it is better to take the value of in the range of [ 7l , 8l ] .
the open human lymphoma b cell data set ( 8) was used to examine the proposed improved algorithm .
compared with the original algorithm , the quality of clustering results using the improved algorithm is enhanced obviously , the mining expression models are better , and the data possess stronger consistency with fluctuation on the condition that the time cost is increased a bit .
in addition , in spite that the noise level of data sets is very high of having the loss rate of 12.3% , simulation results showed that the improved algorithm could still keep a good clustering effect even if the noise interference is very strong .
the process of the extended space in the second stage of cheng and church algorithm is improved .
the numbers of rows and columns are increased about 20% by using the improved algorithm .
the effects of the two important parameters on the speed of the algorithm and the clustering quality are discussed . on the basis of simulated experiments , the experienced values for selecting parameter ranges | cheng and church algorithm is an important approach in biclustering algorithms . in this paper ,
the process of the extended space in the second stage of cheng and church algorithm is improved and the selections of two important parameters are discussed .
the results of the improved algorithm used in the gene expression spectrum analysis show that , compared with cheng and church algorithm , the quality of clustering results is enhanced obviously , the mining expression models are better , and the data possess a strong consistency with fluctuation on the condition while the computational time does not increase significantly . | Introduction
Cheng and Church algorithm
Results and Discussion
Improvements for Cheng and Church algorithm
Parameter selection
Application
Conclusion |
PMC4242818 | the ophthalmic segment of the internal carotid artery ( ica ) , running from the distal dural ring to the posterior communicating artery ( pcom ) , can give rise to aneurysms.1 the three subtypes are the superiorly pointing aneurysms originating at the ophthalmic artery ( i.e. , ophthalmic artery [ oa ] aneurysms ) , the medially pointing aneurysms originating from the superior hypophyseal trunk , and aneurysms originating from a perforator - free part of the ica , the so - called atypical or blister ica aneurysms.2
the incidence of oa aneurysms is somewhere between 0.5% and 11% of all intracranial aneurysms.3
4 unruptured large oa aneurysms frequently present with gradual loss of vision .
the goal of their treatment is then twofold : to exclude the aneurysm from the circulation and to preserve or restore visual function .
treatment of oa aneurysms can be surgical or endovascular , depending on the mode of presentation , aneurysm geometrics and size , as well as the patients medical condition.5 in the past , intracranial aneurysm repair was exclusively surgical,4 and in large unruptured oa aneurysms , clipping is still often the preferred method because surgical decompression of the aneurysm sac may lend the optic nerve a better prognosis.6
7 however , surgery of oa aneurysms carry inherent risks such as stroke,8 hemorrhage,9 or visual loss.10
11
12
since its introduction in 1991,13 endovascular aneurysm repair has grown in popularity and versatility.8
14
15 with the introduction of stents,16
17 endovascular treatment of oa aneurysms has made important progress .
although this development is welcomed , it also makes the choice of treatment strategy less straightforward than in the past . in this report
, we present a patient with a troublesome unruptured oa aneurysm and some lessons learned .
a 56-year - old right - handed woman presented with a 4-month history of retro - orbital headache and progressive decline in visual acuity oculus sinister ( os ) .
her ophthalmologist ( p.l . ) found only perception of light os and normal visual acuity oculus dexter ( od ) .
magnetic resonance imaging ( mri ) detected a left - sided oa aneurysm 20 12 13 mm in diameter ( fig .
1 ) .
magnetic resonance imaging ( mri ) of a 56-year - old right - handed woman who presented with a 4-month history of retro - orbital headache and progressive visual loss demonstrating a left - sided ophthalmic artery aneurysm 20 12 13 mm in diameter .
because of her long history of very poor visual acuity , we considered her left eye to be irredeemable . because the aneurysm had a wide neck and was of moderate size
the treatment plan was first to place a regular stent within the ica , running from proximal to the oa to distal to the pcom artery , with a second semipermeable stent inside the first stent in the aneurysm orifice area to enhance aneurysm thrombosis .
following 1 week of treatment with plavix and aspirin preceding the endovascular procedure , a leo ( balt extrusion , montmorency , france ) 4.5 mm 40 mm stent was placed in the ica ( s. j. b. and e. a. j. ) .
attempts to place additional stents , however , were unsuccessful . to promote aneurysm thrombosis ,
three matrix coils ( boston scientific , fremont , california , united states ) , of 24 mm 40 cm , 30 mm 16 cm , and 14 mm 30 cm , respectively , were put into the aneurysm sac ( fig .
this led to stagnation of the contrast media in the aneurysm sac , but because of the optic nerve compression , the aneurysm was filled loosely so as not to cause further optic chiasm compression .
digital subtraction angiography after a stent was placed in the interior carotid artery and the aneurysm sac coiled using three matrix coils .
after the procedure , the patient woke up with an expressive dysphasia and a right - sided hemiparesis .
mri demonstrated several punctate lesions in the left parieto - occipital region secondary to emboli and perfusion mri showed a small hypoperfusion in the left lentiform nucleus region .
her visual acuity improved to perception of hand movement ad ocolum os and acuity od remained at 1.0 . over the following months ,
the aneurysm continued to grow , measuring 22 13 14 mm ( fig .
it now caused significant compression of both optic nerves , and her ophthalmologist recorded a reduction of visual acuity to 0.8 od and an upper quadrant anopia .
a course of prednisolone 20 mg twice daily was tried for 5 days without effect .
the aneurysm continued to grow , measuring 22 13 14 mm 2 months post - coiling .
six months after her presentation , her visual acuity remained at light perception os and 0.8 od with an upper quadrant anopia . a digital subtraction angiography ( dsa ) demonstrated coil compaction and further aneurysm growth .
the patient failed an ica balloon test occlusion ( w.s.o . ) and was therefore referred to high - flow extracranial - intracranial ( ec - ic ) artery bypass grafting in berlin , germany .
preoperatively , her visual acuity od was down to 0.7 with an upper quadrant anopia , whereas os remained at light perception .
she underwent a high - flow venous graft bypass between the left external carotid artery and the left middle cerebral artery ( p.v . and t.r.m . )
4 ) . because of the combined effects of stent within the ica and the large coil mass in the aneurysm , it was difficult to put an occluding clip on the ica just distal to the oa take - off , and the ica was therefore occluded proximally in the neck .
the patient underwent a high - flow venous graft bypass between the left external carotid artery and the middle cerebral artery ( mca ) .
( a ) axial computed tomography angiography demonstrating the patent graft to mca and coil artifacts .
( b ) digital subtraction angiography anteroposterior view demonstrating filling of the extracranial - intracranial bypass .
postoperatively , she had expressive dysphasia , right - sided hemiparesis , and short - term memory loss .
postoperative t1-weighted mri demonstrated some encephalomalacia with peri - sylvian cortical laminar necrosis in the temporal and frontal lobes ( fig .
5 ) . over the next months she recovered well from these neurologic deficits , with the exception of blindness os .
postoperative fluid attenuated inversion recovery magnetic resonance imaging demonstrates some encephalomalacia with peri - sylvian cortical laminar necrosis .
contrary to what we anticipated , the aneurysm continued to expand ( 27 19 20 mm ) ; her right eye visual acuity decreased dramatically to 0.3 a month after the surgery .
a dsa demonstrated good flow through the bypass , retrograde filling of the distal ica , antegrade filling of the oa , some filling in the cavernous portion of the ica , and some filling in the bottom of the aneurysm ( fig .
digital subtraction angiography demonstrates good bypass flow , with retrograde filling of the distal internal carotid artery ( ica ) , antegrade filling of the ophthalmic artery , and some filling in the cavernous portion of the ica .
there is also some filling of the caudal part of the aneurysm ( arrow ) , the latter finding explaining the continued aneurysm growth .
because of progressive visual field defect and acuity loss od , the patient was operated again a year after her initial presentation via a 30 25 mm supraorbital craniotomy ( t.r.m . ) .
the stented ica was crushed using a debakey forceps to fit a clip across the ica proximal to the pcom .
7 ) , and the optic chiasm and both optic nerves were decompressed . the patient made an uneventful recovery , and the surgery led to a significant improvement in visual acuity od to 0.7 .
the patient was reoperated via a 30 25 mm supraorbital craniotomy , so as not to compromise the bypass .
the stented internal carotid artery ( ica ) was crushed using a debakey forceps to fit a clip across the ica proximal to the posterior communicating artery , and the coiled giant aneurysm was excised , thereby decompressing the optic chiasm and optic nerves .
surgery led to a significant improvement in visual acuity on the right side ; the left remained amaurotic .
a 56-year - old right - handed woman presented with a 4-month history of retro - orbital headache and progressive decline in visual acuity oculus sinister ( os ) .
her ophthalmologist ( p.l . ) found only perception of light os and normal visual acuity oculus dexter ( od ) .
magnetic resonance imaging ( mri ) detected a left - sided oa aneurysm 20 12 13 mm in diameter ( fig .
1 ) .
magnetic resonance imaging ( mri ) of a 56-year - old right - handed woman who presented with a 4-month history of retro - orbital headache and progressive visual loss demonstrating a left - sided ophthalmic artery aneurysm 20 12 13 mm in diameter .
because of her long history of very poor visual acuity , we considered her left eye to be irredeemable . because the aneurysm had a wide neck and was of moderate size
the treatment plan was first to place a regular stent within the ica , running from proximal to the oa to distal to the pcom artery , with a second semipermeable stent inside the first stent in the aneurysm orifice area to enhance aneurysm thrombosis .
following 1 week of treatment with plavix and aspirin preceding the endovascular procedure , a leo ( balt extrusion , montmorency , france ) 4.5 mm 40 mm stent was placed in the ica ( s. j. b. and e. a. j. ) .
attempts to place additional stents , however , were unsuccessful . to promote aneurysm thrombosis ,
three matrix coils ( boston scientific , fremont , california , united states ) , of 24 mm 40 cm , 30 mm 16 cm , and 14 mm 30 cm , respectively , were put into the aneurysm sac ( fig .
this led to stagnation of the contrast media in the aneurysm sac , but because of the optic nerve compression , the aneurysm was filled loosely so as not to cause further optic chiasm compression .
digital subtraction angiography after a stent was placed in the interior carotid artery and the aneurysm sac coiled using three matrix coils .
after the procedure , the patient woke up with an expressive dysphasia and a right - sided hemiparesis .
mri demonstrated several punctate lesions in the left parieto - occipital region secondary to emboli and perfusion mri showed a small hypoperfusion in the left lentiform nucleus region .
her visual acuity improved to perception of hand movement ad ocolum os and acuity od remained at 1.0 . over the following months ,
the aneurysm continued to grow , measuring 22 13 14 mm ( fig .
it now caused significant compression of both optic nerves , and her ophthalmologist recorded a reduction of visual acuity to 0.8 od and an upper quadrant anopia .
a course of prednisolone 20 mg twice daily was tried for 5 days without effect .
the aneurysm continued to grow , measuring 22 13 14 mm 2 months post - coiling .
six months after her presentation , her visual acuity remained at light perception os and 0.8 od with an upper quadrant anopia . a digital subtraction angiography ( dsa ) demonstrated coil compaction and further aneurysm growth .
the patient failed an ica balloon test occlusion ( w.s.o . ) and was therefore referred to high - flow extracranial - intracranial ( ec - ic ) artery bypass grafting in berlin , germany .
preoperatively , her visual acuity od was down to 0.7 with an upper quadrant anopia , whereas os remained at light perception .
she underwent a high - flow venous graft bypass between the left external carotid artery and the left middle cerebral artery ( p.v . and t.r.m . )
4 ) . because of the combined effects of stent within the ica and the large coil mass in the aneurysm , it was difficult to put an occluding clip on the ica just distal to the oa take - off , and the ica was therefore occluded proximally in the neck .
the patient underwent a high - flow venous graft bypass between the left external carotid artery and the middle cerebral artery ( mca ) .
( a ) axial computed tomography angiography demonstrating the patent graft to mca and coil artifacts .
( b ) digital subtraction angiography anteroposterior view demonstrating filling of the extracranial - intracranial bypass .
postoperatively , she had expressive dysphasia , right - sided hemiparesis , and short - term memory loss .
postoperative t1-weighted mri demonstrated some encephalomalacia with peri - sylvian cortical laminar necrosis in the temporal and frontal lobes ( fig .
5 ) . over the next months she recovered well from these neurologic deficits , with the exception of blindness os .
postoperative fluid attenuated inversion recovery magnetic resonance imaging demonstrates some encephalomalacia with peri - sylvian cortical laminar necrosis .
contrary to what we anticipated , the aneurysm continued to expand ( 27 19 20 mm ) ; her right eye visual acuity decreased dramatically to 0.3 a month after the surgery .
a dsa demonstrated good flow through the bypass , retrograde filling of the distal ica , antegrade filling of the oa , some filling in the cavernous portion of the ica , and some filling in the bottom of the aneurysm ( fig .
digital subtraction angiography demonstrates good bypass flow , with retrograde filling of the distal internal carotid artery ( ica ) , antegrade filling of the ophthalmic artery , and some filling in the cavernous portion of the ica .
there is also some filling of the caudal part of the aneurysm ( arrow ) , the latter finding explaining the continued aneurysm growth .
because of progressive visual field defect and acuity loss od , the patient was operated again a year after her initial presentation via a 30 25 mm supraorbital craniotomy ( t.r.m . ) .
the stented ica was crushed using a debakey forceps to fit a clip across the ica proximal to the pcom .
7 ) , and the optic chiasm and both optic nerves were decompressed . the patient made an uneventful recovery , and the surgery led to a significant improvement in visual acuity od to 0.7 .
the patient was reoperated via a 30 25 mm supraorbital craniotomy , so as not to compromise the bypass .
the stented internal carotid artery ( ica ) was crushed using a debakey forceps to fit a clip across the ica proximal to the posterior communicating artery , and the coiled giant aneurysm was excised , thereby decompressing the optic chiasm and optic nerves .
surgery led to a significant improvement in visual acuity on the right side ; the left remained amaurotic .
major neurologic deficits after treatment of giant intracranial aneurysms occur in 10% of patients.18
19
20
21
22 with paraclinoid giant aneurysms , visual deterioration after treatment is observed in up to 17% of patients.19
20 although surgical clipping is often the preferred treatment for aneurysms causing cranial nerve deficits,6
7 in cases of giant paraclinoid ica aneurysms , it may not be feasible in > 60% of cases.18
20
21
22 surgery on oa aneurysms is technically demanding because these aneurysms are often large and may extend into the cavernous sinus.21
22
23
24
25
26 thus they are challenging even for the most experienced of neurosurgeons . over the years , these aneurysms have therefore been treated by common carotid artery ligation,20
21
22
27
28 ica occlusion,18
19
20
22
29
30 proximal vessel ligation with ec - ic bypass,18
19
20
22 trapping,18
19
31 intra - aneurysmal balloon occlusion,32
33 coiling,34
35
36 or by stenting.37 however , because visual deterioration after coil embolization of oa aneurysms has been reported in as many as a third of the patients,14 the decision whether to clip or coil is often difficult . in the present case ,
the endovascular treatment plan was to first place a regular stent in the ica from the oa to the pcom , with a second semipermeable stent inside the first stent in the aneurysm orifice area .
retrospectively , upon failing to place the semipermeable stent , we should have discontinued the endovascular procedure because the subsequent aneurysm coiling would not make the aneurysm shrink and would make aneurysm surgery at a later stage considerably more difficult .
given a situation with a large and growing coiled aneurysm and with a failed balloon test occlusion ( bto ) , we opted for a high - flow ec - ic bypass followed by therapeutic ica closure because ica occlusion , with or without ec - ic bypass , is a good therapeutic option when treating giant ica aneurysms.29
38
39
40
41
42
43
44 however , it proved difficult to put an occluding clip on the ica just distal to the oa take - off due to the combined effects of the stent within the ica and the large coil mass within the aneurysm .
furthermore , the pcom was also not in sight due to the previously mentioned limiting structures , but also because of the high - flow vein graft in the surgical field . consequently , the ica was occluded proximally in the neck . retrospectively , after finishing the bypass and realizing that the intracranial ica closure was impossible , we should have left the ica open and closed this vessel off endovascularly by placing coils at the origin of the oa / aneurysm because it is known that proximal ica occlusion does not fully prevent subsequent hemorrhage or necessarily prevent aneurysm enlargement.10
20
21
38
41
45
46 a more distal ica closure would have eliminated blood flow in the ica below the pcom and triggered thrombosis and shrinkage of the noncoiled portion of the aneurysm .
it is difficult to tidy up the mess , as it were , if you start out wrong .
given the chance again , we would have followed our usual game plan when treating patients with large unruptured oa aneurysms : if there is reduced visual acuity and no or moderate thrombus material in the aneurysm , we opt for surgical clipping via a lateral supraorbital craniotomy with extradural clinoidectomy for proximal control of the ophthalmic ica segment,47 aneurysm dissection , suction / decompression of the aneurysm ( either direct after temporary trapping or ad modum hunt batjer with exposure of the ica in the neck48 ) , and aneurysm clipping . if there is a large thrombotic mass in the aneurysm , with or without reduced visual acuity , we generally manage our patients with semipermeable ( flow diversion ) stents or therapeutic ica closure with or without a high - flow excimer laser - assisted nonocclusive anastomosis ( elana ) bypass for flow replacement,43 depending on the bto tolerance.44
|
objective and importance when treating large unruptured ophthalmic artery ( oa ) aneurysms causing progressive blindness , surgical clipping is still the preferred method because aneurysm sac decompression may relieve optic nerve compression .
however , endovascular treatment of oa aneurysms has made important progress with the introduction of stents .
although this development is welcomed , it also makes the choice of treatment strategy less straightforward than in the past , with the potential of missteps .
clinical presentation a 56-year - old woman presented with a long history of progressive unilateral visual loss and magnetic resonance imaging showing a 20-mm left - sided oa aneurysm .
intervention because of her long history of very poor visual acuity , we considered her left eye to be irredeemable and opted for endovascular therapy .
the oa aneurysms was treated with stent and coils but continued to grow , threatening the contralateral eye .
because she failed internal carotid artery ( ica ) balloon test occlusion , we performed a high - flow extracranial - intracranial bypass with proximal ica occlusion in the neck .
however , aneurysm growth continued due to persistent circulation through reversed blood flow in distal ica down to the oa and the cavernous portion of the ica . due to progressive loss of her right eye vision , we surgically occluded the ica proximal to the posterior communicating artery and excised the coiled , now giant , oa aneurysm .
this improved her right eye vision , but her left eye was permanently blind .
conclusion this case report illustrates complications of the endovascular and surgical treatment of a large unruptured oa aneurysm . | Background
Case Report
Clinical Presentation
Discussion |
PMC2671915 | the fission yeast schizosaccharomyces pombe is a convenient model to study cell morphogenesis ( hayles and nurse , 2001 ) .
wild - type cells are simple elongated rods growing at the cell poles and dividing in the middle . yet
, previous studies have outlined an interesting interplay between shape , growth and cytoskeletal organization .
the first component is the rigid cell wall surrounding yeast cells that maintains cell shape independently of the cytoskeleton .
second , the actin cytoskeleton is essential for cell growth and cell wall remodeling ( la carbona et al , 2006 ) .
lastly , although microtubules ( mts ) are not required for growth per se , they control the location of growth sites by depositing specific marker proteins ( mata and nurse , 1997 ; brunner and nurse , 2000 ; sawin and snaith , 2004 ) .
abnormal deposition , occurring for example in mutants where mts are shorter , results in cells that are either bent or branched ( sawin and nurse , 1998 ; snaith and sawin , 2005 ) .
mts also position the nucleus ( tran et al , 2001 ; loiodice et al , 2005 ) and thus define the site of cytokinesis ( daga and chang , 2005 ; tolic - norrelykke et al , 2005 ) and the partitioning of the cell into daughter cells .
hence , by controlling cell growth and division , mts impact the evolution of shape in the cell lineage . as mts are constrained within the cell , the converse is also true with mt organization being dependent on cell shape . for the rigid s. pombe cells ,
the two processes occur on very different timescales ; with mt lifetimes being in the order of minutes , whereas cells typically double in size after 3 h. consequently , individual mts are enclosed in a boundary that is effectively constant during their lifetime .
this means that it is valid to first study how mts depend on cell shape , and to later include cell shape changes .
we use here computer simulation for the first step , calculating the dynamic spatial organization of mts within a fixed cell shape .
this approach complements other efforts where cell morphogenesis is modeled with reaction diffusion equations ( csikasz - nagy et al , 2008 ) by focusing on the mt cytoskeleton .
interphase mts in fission yeast are typically forming 26 bundles , which are usually attached to the nucleus at their middle ( tran et al , 2001 ) ( figure 1 ) .
antiparallel mts overlap at their static minus ends , whereas the plus ends are dynamic and grow from the overlap zone toward the cell poles ( tran et al , 2001 ; hoog et al , 2007 ) . such bundles transmit forces produced at the cell poles by mt polymerization to the nucleus . to position the nucleus near the middle of the cell ( tran et al , 2001 ;
loiodice et al , 2005 ) , mts should efficiently target the cell poles , and have catastrophes ( the switch to depolymerization ) that are rare enough to enable mts to reach the cell cortex but not so rare as to induce bending of the mt around the polar cell wall ( tran et al , 2001 ) , a configuration that is not observed in wild - type cells .
the most popular explanation for the timing of catastrophes involves multiple molecular activities that are assembled at the cell poles ( mata and nurse , 1997 ) .
another possibility is that forces caused by mt polymerization feed back on mt dynamics ( dogterom et al , 2005 ) .
this possibility has so far not been confirmed mainly due to the lack of experimental tools in vivo . in this study , we have circumvented this problem using stochastic computer simulations to check whether the effect of force measured in vitro can explain the observed mt plus end dynamics in s. pombe .
this involved constructing three models of mt dynamics : model f ( force ) in which forces at mt tips regulate mt growth and catastrophe rates , model l ( length ) in which mt length affects catastrophe rate and model fl which combines both effects . to simulate the mt cytoskeleton in s. pombe
, we first idealized its 3d shape as a spherocylinder ( see figure 1b and c ) . a nucleus and mt bundles
were then added and confined within this volume . in vivo , mt bundles self - organize ( janson et al , 2007 ) , to form overlap zones of 0.840.29 m ( tran et al , 2001 ) . for the purpose of this study , it was sufficient to use fixed overlap zones and four bundles , each containing four antiparallel mts , which is representative of the average situation .
the nucleus was represented by a sphere , to which the overlap zones of the bundles were attached ( supplementary figure s4 ) .
the deformations of the nuclear membrane observed in vivo ( tran et al , 2001 ; daga et al , 2006 ) were incorporated into the model by attaching mt bundles to the nucleus using hookean springs of moderate stiffness .
the points to which the bundles were attached were also able to move on the nuclear surface .
this allowed elongating bundles to align with the cell axis , as in vivo ( supplementary figure s5 ) . in summary ,
the simulation comprised bundles of flexible mts and a connected spherical nucleus that were confined within a frictionless cortex . although mt minus ends were static , plus ends grew and shrank independently of each other , thus producing polymerization forces , fiber deformation and nuclear movements .
the physical equations describing the evolution of this system were solved numerically as explained before ( nedelec and foethke , 2007 ) .
the advantage of using s. pombe as a model organism is that numerous dynamical properties of mts have been measured by light microscopy , and this enables the models to be quantitatively compared to reality .
table i lists the 10 in vivo properties of mts that were used to evaluate computational models .
these 10 traits summarize most of the currently established knowledge of the wild - type cells that are relevant for mt organization .
note that it was necessary to adjust the definitions of these traits to what is available from the literature ( see supplementary information ) .
for example , it was important to distinguish bundle catastrophes , ' that affect the longest mt in a half - bundle from other mt catastrophes .
other catastrophes are harder to observe because mts overlap in vivo , and were often not reported ( brunner and nurse , 2000 ; drummond and cross , 2000 ; tran et al , 2001 ) .
independently of the traits , characteristics of the cell geometry , mt bending elasticity and cytoplasmic viscosity were obtained from the literature or determined experimentally in the course of this study ( see supplementary information ) .
the models discussed here contain two parameters : the polymerization speed v0 and the catastrophe rate c0 .
they are evaluated by running thousands of simulations with random values of v0 and c0 , and then testing which trait was matched in each simulation .
this systematic numerical exploration of the parameter space is centered on v02.4 m / min , which is observed under standard laboratory conditions ( tran et al , 2001 ) , and extends to regions where traits start to fail .
model f corresponds to an in vitro experiment , which showed that a barrier could inhibit tubulin assembly ( dogterom and yurke , 1997 ) .
these results were described by vg = v0 exp(f / fs ) ( equation a ) , where fs1.7 pn specifies the sensitivity to force , and f is the projected force between the barrier and the mt tip ( figure 1c ) .
in addition , reduced assembly in vitro was shown to promote catastrophes according to b : c=1/(a+bvg ) ( janson et al , 2003 ; janson and dogterom , 2004 ) .
model f used equations ( a ) and ( b ) with the forces calculated for each mt .
the constants fs and a were measured experimentally with purified tubulin . the constant b was calculated as the solution of ( b ) with c = c0 and v = vo such that c0 and v0 correspond to the catastrophe and assembly rates of unconstrained mts .
the resultant value of b differs from that measured in vitro , because it represents the combined effects of mt - associated proteins ( maps ) present in the cell . a value for b corresponding to what was reported in vitro without mt regulators results in excessively long mts that curl around the cell pole .
we found that simulations with model f consistently matched t18 ( traits of the wild - type cell ) around the reference values of c0 and v0 ( supplementary figure s1 ) .
however , these simulations failed to match t9 and t10 in this region , which lead us to investigate these traits further .
experimentally , t9 was measured in cells that are made asymmetric by centrifugation , with the nucleus ending up nearer one cell pole .
it was observed that during nucleus re - centering , mts have longer contact time with the proximal than with the distal pole ( 9253 and 4636 s , respectively ; daga et al , 2006 ) .
model f showed opposite effects because , as shown by our simulation , mts experience stronger compression on the proximal side than on the distal side .
n / l ) , which indicates that the maximum force of an mt at equilibrium is inversely proportional to its length .
however , contrary to this formula , the simulation considers the rate of mts reaching the cell ends , and the motion of the nucleus and thus calculates the effective force rather than an upper limit .
the other unmatched trait t10 represents the centering precision of bundles when they are not attached to the nucleus ( carazo - salas and nurse , 2006 ) . to understand why t10 fails
, we can consider the simpler situation of an unattached bundle made of two antiparallel mts .
this bundle mechanically behaves as a single elastic beam of small viscous drag : the forces at both ends equalize very fast .
consequently , the plus tips of both mts experience similar forces no matter where the overlap zone is located : there are little centering cues .
the simulation shows that the situation with bundles of four mts is essentially similar : they are centered on average , but with a large variance of 3 m .
however , when a nucleus is present , additional averaging occurs on the positions of mt minus ends : the nucleus connects multiple independent bundles , and averages their fluctuations over time , because its mobility is much lower than that of individual bundles .
thus , t10 contains different information than t7 and t8 . in conclusion , although model f matched most measurements listed in table i , its failure to match t9 and t10 prompted us to investigate how it could be adjusted .
in the situation considered for t9 , mts are shorter on the proximal side than on the distal side , and therefore the observed asymmetry in contact times may indicate that the length of an mt influences its stability , which was not the case in model f. to test this possibility , we simulated a catastrophe rate that depends on mt length l according to c = hl/(a+bvg ) ( equation b ) .
the constant h was set to 0.2/m , so that c0 would determine the catastrophe rate at l=5 m ( the length at which mts typically undergo catastrophe in average cells ) .
this makes the resulting model fl directly comparable to model f , except that longer mts are less stable compared with short ones . with model fl , t9 was matched with , for example , contact times of 10268 s on the proximal side , and 5131 s on the distal side . the length dependence overcompensated for the effects of force in a situation where the nucleus is close to the cell pole .
in fact , model fl matched the 10 traits robustly with respect to ( v0 , c0 ) around their expected in vivo values ( figure 2a ) .
although the average mt cortex contact time in the symmetric case was already considered in t4 , comparing the shape of their distribution would provide another test of the model . to this extent
, we measured 303 in vivo contact events ( 6636 s mean and standard deviation ) .
being approximately 20% shorter , they were comparable to what had previously been reported ( 8346 s ) ( daga et al , 2006 ) .
interestingly , model fl matched the distribution ( see figure 3a ) , showing that the contact times are explained by the memory - less ( first - order ) mt catastrophe transition , because as mts continue to grow after contact , forces building up increase the instantaneous catastrophe rate .
note that this distribution is also matched by model f ( see supplementary figure s2 ) , which was anyhow discarded for other reasons .
the simulation also predicts the location of the hidden mt catastrophes ( figure 3b ) , which are frequent but difficult to observe in vivo because these events occur on the side of a longer mt in the same bundle .
such phenomenon was observed previously in xenopus egg extracts ( dogterom et al , 1996 ) , and tischer et al have confirmed experimentally that this behavior occurs in s. pombe cells , observing that catastrophe rates increase linearly from 0.1/min at l2 m up to 0.3/min at l5 m , for mts that do not contact the cell cortex ( tischer et al , in this upload ) .
the measured magnitude of the dependence of catastrophe upon mt length is identical to what we have assumed , in the center of the region in which all traits are fulfilled ( c0=0.3/min ) .
it is tempting to speculate about the molecular mechanism that could mediate the destabilization of longer mts .
the s. cerevisiae kinesin-8 kip3p produce a length - dependent destabilizing activity in vitro , because after binding at any point along the length of the mt , it moves processively to the tips , where it has a depolymerizing activity ( gupta et al , 2006 ; varga et al , 2006 ) .
it is possible that the two homologs of kip3p in s. pombe , klp5p and klp6p ( west et al , 2001 ) have a similar activity .
consistent with this view , the double deletion of klp5 and klp6 produced elongated mts that curl at cell poles ( west et al , 2001 ) , and reduces the length dependence of catastrophe ( tischer et al ) .
simulations show that length - dependent catastrophe rates offer several potential advantages to the cell .
first , nucleus repositioning is faster by a factor 2 in model fl compared with model f ( data not shown ) , because proximal mts push for a longer time than distal mts ( t9 ) .
second , bundles that are observed to detach from the nucleus ( tran et al , 2001 ; carazo - salas and nurse , 2006 ) would keep their overlap regions better centered ( their variance was 2 m instead of 3 m for model f ) , which may allow them to reattach more rapidly ( we did not simulate this hypothesis ) . finally , a model l having the length dependence present in model fl , but without any force dependence also fails to fulfill all traits ( supplementary figure s1 ) , showing that the length dependence is not sufficient to adjust mts within the cell .
hence , in model fl , the established response to force together with the length - dependent mt - destabilizing activity of maps , leads to the accurate description of the dynamics of mts in wild - type s. pombe cells .
remarkably , in both model f and fl , it was not necessary to assume that the cell poles had any localized activity associated with them that would affect mts .
this shows that , as anticipated ( dogterom et al , 2005 ) , forces in principle are sufficient to account for the location of mt catastrophes at the cell poles . in future work
, it will be necessary to explicitly distinguish maps to be able to recapitulate their mutant phenotypes . to do so
will require knowing how the dynamic equilibrium distribution of maps near mt tips is affected by force .
however , having no free parameter , our current model is already predictive in calculating how cell morphology affects a wild - type mt cytoskeleton , and this sheds light on some mutant phenotypes . in the current model , the catastrophe rate c0 represents the combined action of all maps on mts .
this in particular includes the potential effect of tea1p , tip1p or tea2p , which are located on growing mt plus ends .
these proteins are later deposited at the cell ends , but our model does not include any influence of the deposited proteins on mts in return . in the case of tea1p ,
however , such an influence has been suggested ( brunner et al , 2000 ) , because genetic deletion affects mts .
the observed defect is that mts exhibit curling at the cell poles ( see figure 3c ) . in the simulation
, we noticed that cell diameter ( but not cell length ) affected mt curling ( see figure 3d ) .
this prompted us to measure mt curling in vivo , together with the dimensions of cells .
there also , we found a clear correlation between cell diameters and the extent of mt curling ( see figure 3e ) , and no correlation between cell lengths and curling .
thus , the curling phenotype can be explained by the fact that tea1 cells were , on average , wider than wild - type cells ( figure 3f ) .
furthermore , thin tea1 cells exhibited a comparable level of curling to wild - type cells of similar diameter , which confirmed that the mt phenotype is largely the consequence of the increased diameter , and that unlike previously speculated , tea1p may not directly influence mt catastrophes .
the variability around the average behavior is smaller in the simulation than in reality , most likely because irregularities in cell shape together with measurement errors have not been modeled .
the slopes of the best linear fit were comparable in vivo and in the computational model ( figure 3d and e ) . in summary
, it seems that unbranched tea1 cells have wild - type mts in a body that is wider than wild type
this is beyond the scope of the current study however , because it requires redefining many of the traits , which are only meaningful for cells having two ends .
the simulation available on www.cytosim.org can be extended to further investigate mt organization in s. pombe .
a successful account of interphase mt dynamics , as initiated here , is a necessary step to understand the determination of cell shape in this simple organism .
this file contains
a. a list of supplementary movies and files ;
b. a description of the simulation methods ;
c. a detailed description of the traits listed in table 1 ;
d. a description of the parameters of the simulation , with details on how their values were set from the previous literature .
e. a description of the methods used to generate figure 3d and e. frequently asked questions supplementary figure s2 | the cytoskeleton is essential for the maintenance of cell morphology in eukaryotes . in fission yeast , for example , polarized growth sites are organized by actin , whereas microtubules ( mts ) acting upstream control where growth occurs .
growth is limited to the cell poles when mts undergo catastrophes there and not elsewhere on the cortex . here , we report that the modulation of mt dynamics by forces as observed in vitro can quantitatively explain the localization of mt catastrophes in schizosaccharomyces pombe
. however , we found that it is necessary to add length - dependent catastrophe rates to make the model fully consistent with other previously measured traits of mts .
we explain the measured statistical distribution of mt
cortex contact times and re - examine the curling behavior of mts in unbranched straight tea1 cells .
importantly , the model demonstrates that mts together with associated proteins such as depolymerizing kinesins are , in principle , sufficient to mark the cell poles . |
Supplementary Material |
PMC4603221 | the reasons for errors made in ultrasound diagnosis of the urinary system are usually of multifactorial nature .
generally , they result from improper technique of ultrasound ( us ) examination or its erroneous interpretation .
such errors are frequent effects of insufficient experience of the ultrasonographer , inadequate class of the scanner , insufficient knowledge of its operation and of wrong preparation of patients , their constitution and severe condition as well as the lack of cooperation during the examination .
the reasons for misinterpretations of ultrasound images of the urinary system may lie in a large polymorphism of the kidney ( defects and developmental variants ) and may result from improper access to the organ as well as from the presence of artefacts .
moreover , mistakes in ultrasound diagnosis of the urinary system are frequently related to the lack of knowledge of the management algorithms and diagnostic possibilities of other imaging modalities .
below , the authors list errors in ultrasound diagnosis of the urinary system divided into : errors resulting from improper technique of examination , incorrect preparation of patients for the examination or their constitution and errors resulting from misinterpretations of ultrasound images .
insufficient quality of ultrasound equipment precludes obtaining diagnostic images and thus , involves a risk of failing to visualize pathologies , mistaking artefacts for pathologies and making differential diagnosis impossible to conduct.inadequate selection of the transducer , i.e. wrong type of the transducer and frequency , e.g. selecting convex probes with the frequency 2.05.0 mhz in examining the urinary system in children ( fig .
1 a , b).inadequate set up selection for the examination of given organs , e.g. to examine the urinary system , one should choose the abdomen set up and to examine the kidneys , the correct choice is kidney set up .
inappropriate gain of the ultrasound beam and other parameters ( tgc , brightness of the monitor , penetration , focus ) .
each time , the image should be optimized and adjusted to the conditions of the examination and constitution of the patient.improper lighting in the ultrasound laboratory , i.e. too bright rooms or placing the ultrasound monitor opposite the windows.noise which affects concentration of the ultraso nographer.excess tiredness of the ultrasonographer which limits logical thinking and affects image interpretation.artefacts and distortions caused by electric and electromagnetic devices which affect the operation of the ultrasound scanner ( e.g. magnetic resonance , respirators etc . ) .
insufficient quality of ultrasound equipment precludes obtaining diagnostic images and thus , involves a risk of failing to visualize pathologies , mistaking artefacts for pathologies and making differential diagnosis impossible to conduct .
inadequate selection of the transducer , i.e. wrong type of the transducer and frequency , e.g. selecting convex probes with the frequency 2.05.0 mhz in examining the urinary system in children ( fig .
inadequate set up selection for the examination of given organs , e.g. to examine the urinary system , one should choose the abdomen set up and to examine the kidneys , the correct choice is kidney set up .
inappropriate gain of the ultrasound beam and other parameters ( tgc , brightness of the monitor , penetration , focus ) .
each time , the image should be optimized and adjusted to the conditions of the examination and constitution of the patient .
improper lighting in the ultrasound laboratory , i.e. too bright rooms or placing the ultrasound monitor opposite the windows . noise which affects concentration of the ultraso nographer .
artefacts and distortions caused by electric and electromagnetic devices which affect the operation of the ultrasound scanner ( e.g. magnetic resonance , respirators etc . ) .
error caused by the selection of the transducer with too low frequency in ultrasound examination of the urinary system in children . a. examination performed with the use of a convex probe with the frequency of 2.06.0 mhz visualizes solely the dilatation of the upper pelvicalyceal system .
b. examination performed with a high - frequency transducer ( 9.012.0 mhz ) visualizes the dilatation of the pelvicalyceal system also in the region of the middle and lower calyces and renal pelvis .
intestinal gas and contents cause artefacts which conceal the internal organs.the inability to take and hold breath by the patient may considerably inhibit or preclude kidney visualization , particularly in obese patients.lack of cooperation with the patient :
children lack of cooperation , crying , screaming , physical agitation;unconscious patients lack of cooperation in terms of breathing , position and taking the patient 's history .
tenderness and pain in the abdominal cavity impossibility to apply necessary pressure with the transducer.pyknic constitution
high position of the diaphragm may considerably reduce the possibility to visualize the kidneys ( an attempt should be made to visualize them by intercostal access ) ; the presence of gas in the intestine frequently produces artefacts that conceal the kidneys.patients with dyspnea impossibility to draw a deep breath and frequently , inability to remain in supine position.open wounds in the region of the abdomen , dressings , sutures or catheters optimal transducer application is impossible.empty urinary bladder impossibility to assess the bladder and other organs of the pelvis minor .
intestinal gas and contents cause artefacts which conceal the internal organs . the inability to take and hold breath by the patient may considerably inhibit or preclude kidney visualization , particularly in obese patients .
lack of cooperation with the patient :
children lack of cooperation , crying , screaming , physical agitation;unconscious patients lack of cooperation in terms of breathing , position and taking the patient 's history .
children lack of cooperation , crying , screaming , physical agitation ; unconscious patients lack of cooperation in terms of breathing , position and taking the patient 's history . tenderness and pain in the abdominal cavity impossibility to apply necessary pressure with the transducer .
high position of the diaphragm may considerably reduce the possibility to visualize the kidneys ( an attempt should be made to visualize them by intercostal access ) ; the presence of gas in the intestine frequently produces artefacts that conceal the kidneys .
patients with dyspnea impossibility to draw a deep breath and frequently , inability to remain in supine position .
open wounds in the region of the abdomen , dressings , sutures or catheters optimal transducer application is impossible . empty urinary bladder impossibility to assess the bladder and other organs of the pelvis minor .
errors concerning the number of kidneys : failure to visualize the kidney in its typical site :
agenesis / aplasia sonography does not enable the differentiation between these two pathologies;renal ectopia or dystopia;hypoplasia or aplasia the kidney is so small or so altered that its identification is impossible for various reasons ( obesity , artefacts produced by intestinal gas , kidney localization etc . ) .
the final verification is conducted on the basis of intravenous urography or computed tomography.situations occurring more rarely :
renal agenesis in neonates the possibility to mistake enlarged adrenal glands for hypoplastic kidneys;tumors of the gastrointestinal tract
pseudokidney , particularly in patients with unilateral renal agenesis;ectopic kidneys possibility to mistake pelvic kidney for a tumor - like lesion;in extreme cases , ectopic ovaries and their high , abdominal localization may mimic a hypoplastic kidney;renal hypoplasia and dysplasia with concomitant ectopia a particularly complex anomaly which prohibits the identification of the kidneys and inhibits their assessment;horseshoe kidney erroneous interpretation of the isthmus of a horseshoe kidney as a pathological mass in the extraperitoneal space ( fig .
2 a , b ) .
errors in size assessment :
errors resulting from anomalous location , structure and rotation of the kidney ; this inhibits the measurement of the longest longitudinal and transverse lengths which should be measured in these planes , e.g. horseshoe kidney;errors resulting from the selection of improper plane for the measurements renal measurements should be conducted after the longest longitudinal and transverse lengths have been obtained ; errors may be caused by taking measurements in oblique sections;errors resulting from the lack of standardization of measurement sites failure to obtain reproducible measurements ; measurements of the kidneys and dilated pelvicalyceal system ought to be performed each time in the same manner;errors resulting from the dynamics of renal conditions , influence of an ongoing treatment as well as degree of hydration and fullness of the pelvicalyceal system , e.g. erroneous interpretation of the pelvicalyceal system filled with urine with maximally filled bladder as hydronephrosis in such cases , one should assess the kidneys following miction;errors resulting from the impossibility to visualize the entire kidneys or overlapping shadows of the ribs and intestinal loops as well as faulty measurements reflecting a part of the kidney , not the entire one .
errors in the interpretation of fluid - filled spaces in the kidneys :
identification of isolated dilatation of the calyceal system / systems in the case of central cysts;mistaking central renal cysts for dilatation of the pelvicalyceal system;misinterpretation of a cyst localized at the hilum as dilated renal pelvis;erroneous differentiation between a central cyst and aneurysm of the renal artery
doppler mode is used for differential diagnosis;erroneous differentiation of the character of a cystic lesion a complex cyst versus cystic form of renal carcinoma or solid - cystic renal carcinoma;erroneous interpretation of the cyst in the upper pole as an adrenal or hepatic cyst in the case of the right kidney and as a cyst in the tail of the pancreas or spleen in the case of the left kidney;errors in interpreting a cystic lesion with abscesslike character as a cyst without obtaining clinical information ( fig .
3);erroneous interpretation of hypoechoic lesions , e.g. renal lymphoma ( fig . 4 b ) , as dilatation of the pelvicalyceal system ;
errors resulting from the morphological similarity of pathological lesions to the presentation of the kidney , which is the effect of diverse renal structures depending on the patient 's age , developmental defects , individual polymorphism and proliferative changes ( tab .
1 ) :
normal presentation of the kidneys in premature infants and neonates ( kidneys of lobar structure with hypoechoic pyramids and hyperechoic cortex ) is erroneously interpreted as lesions in the course of pyelonephritis , renal failure or renal cysts;renal scar ( elements of the connective tissue penetrating into the parenchyma ) interpreted as angiomyolipoma or postinflammatory scar;dromedary hump of the kidney mistaken for a tumor ( fig .
5 a c);hypertrophy of the pyramid and renal column mistaken for a tumor ( fig . 6
a , b);inflammatory tumors , postinfarction tumors and other lesions related to vascularity disorders may be erroneously interpreted as neoplastic tumors;renal lymphomas , leukemic infiltrations and primary proliferative lesions with echostructure and vascular pattern resembling normal renal parenchyma may be misinterpreted as a normal kidney particularly when they are small and not well - demarcated .
the essence of ultrasound imaging is the phenomenon of ultrasound wave reflection . making use of the reverse piezoelectric effect ,
the us transducers generate ultrasonic waves which are sent inside the tissues where they are reflected and return to the transducer .
depending on the presentation used by the scanner , these returning echoes are enhanced , appropriately processed and displayed .
the size of the returning echo depends on multiple factors , the knowledge of which enables accurate interpretation of ultrasound images . apart from being reflected , the ultrasonic wave also undergoes refraction , interference , scattering and absorption . in sonography
, there are other equally significant factors such as : acoustic impedance of tissues , the shape of reflecting surfaces or elastic properties of tissues .
being familiar with all these phenomena and their influence on the displayed ultrasound image is a condition for its accurate interpretation .
errors in differentiating between hyperechoic images , such as renal concretions , with calcifications , ( postinflammatory ) scars , peripapillary fibrosis and , most frequently , with lesions in the arciform arteries in young patients and children with nephrolithiasis ( fig . 8 a ,
b ) .
errors in the assessment of the site after nephrectomy :
false positive identification of neoplastic relapse caused by stool retention in the bowel loops;errors resulting from the presence of gas in the bowels , which precludes reliable assessment of the site after kidney removal .
failure to visualize the kidney in its typical site :
agenesis / aplasia sonography does not enable the differentiation between these two pathologies;renal ectopia or dystopia;hypoplasia or aplasia the kidney is so small or so altered that its identification is impossible for various reasons ( obesity , artefacts produced by intestinal gas , kidney localization etc . ) .
sonography does not enable the differentiation between these two pathologies ; renal ectopia or dystopia ; hypoplasia or aplasia the kidney is so small or so altered that its identification is impossible for various reasons ( obesity , artefacts produced by intestinal gas , kidney localization etc . ) .
situations occurring more rarely :
renal agenesis in neonates the possibility to mistake enlarged adrenal glands for hypoplastic kidneys;tumors of the gastrointestinal tract e.g. colon carcinoma manifests itself as a , so - called ,
pseudokidney , particularly in patients with unilateral renal agenesis;ectopic kidneys possibility to mistake pelvic kidney for a tumor - like lesion;in extreme cases , ectopic ovaries and their high , abdominal localization may mimic a hypoplastic kidney;renal hypoplasia and dysplasia with concomitant ectopia a particularly complex anomaly which prohibits the identification of the kidneys and inhibits their assessment;horseshoe kidney erroneous interpretation of the isthmus of a horseshoe kidney as a pathological mass in the extraperitoneal space ( fig .
the possibility to mistake enlarged adrenal glands for hypoplastic kidneys ; tumors of the gastrointestinal tract
possibility to mistake pelvic kidney for a tumor - like lesion ; in extreme cases , ectopic ovaries and their high , abdominal localization may mimic a hypoplastic kidney ; renal hypoplasia and dysplasia with concomitant ectopia a particularly complex anomaly which prohibits
erroneous interpretation of the isthmus of a horseshoe kidney as a pathological mass in the extraperitoneal space ( fig .
errors in size assessment :
errors resulting from anomalous location , structure and rotation of the kidney ; this inhibits the measurement of the longest longitudinal and transverse lengths which should be measured in these planes , e.g. horseshoe kidney;errors resulting from the selection of improper plane for the measurements renal measurements should be conducted after the longest longitudinal and transverse lengths have been obtained ; errors may be caused by taking measurements in oblique sections;errors resulting from the lack of standardization of measurement sites failure to obtain reproducible measurements ; measurements of the kidneys and dilated pelvicalyceal system ought to be performed each time in the same manner;errors resulting from the dynamics of renal conditions , influence of an ongoing treatment as well as degree of hydration and fullness of the pelvicalyceal system , e.g. erroneous interpretation of the pelvicalyceal system filled with urine with maximally filled bladder as hydronephrosis in such cases , one should assess the kidneys following miction;errors resulting from the impossibility to visualize the entire kidneys or overlapping shadows of the ribs and intestinal loops as well as faulty measurements reflecting a part of the kidney , not the entire one .
errors resulting from anomalous location , structure and rotation of the kidney ; this inhibits the measurement of the longest longitudinal and transverse lengths which should be measured in these planes , e.g. horseshoe kidney ; errors resulting from the selection of improper plane for the measurements renal measurements should be conducted after the longest longitudinal and transverse lengths have been obtained ; errors may be caused by taking measurements in oblique sections ; errors resulting from the lack of standardization of measurement sites failure to obtain reproducible measurements ; measurements of the kidneys and dilated pelvicalyceal system ought to be performed each time in the same manner ; errors resulting from the dynamics of renal conditions , influence of an ongoing treatment as well as degree of hydration and fullness of the pelvicalyceal system , e.g. erroneous interpretation of the pelvicalyceal system filled with urine with maximally filled bladder as hydronephrosis in such cases , one should assess the kidneys following miction ; errors resulting from the impossibility to visualize the entire kidneys or overlapping shadows of the ribs and intestinal loops as well as faulty measurements reflecting a part of the kidney , not the entire one .
errors in the interpretation of fluid - filled spaces in the kidneys :
identification of isolated dilatation of the calyceal system / systems in the case of central cysts;mistaking central renal cysts for dilatation of the pelvicalyceal system;misinterpretation of a cyst localized at the hilum as dilated renal pelvis;erroneous differentiation between a central cyst and aneurysm of the renal artery
doppler mode is used for differential diagnosis;erroneous differentiation of the character of a cystic lesion a complex cyst versus cystic form of renal carcinoma or solid - cystic renal carcinoma;erroneous interpretation of the cyst in the upper pole as an adrenal or hepatic cyst in the case of the right kidney and as a cyst in the tail of the pancreas or spleen in the case of the left kidney;errors in interpreting a cystic lesion with abscesslike character as a cyst without obtaining clinical information ( fig .
3);erroneous interpretation of hypoechoic lesions , e.g. renal lymphoma ( fig . 4 b ) , as dilatation of the pelvicalyceal system
identification of isolated dilatation of the calyceal system / systems in the case of central cysts ; mistaking central renal cysts for dilatation of the pelvicalyceal system ; misinterpretation of a cyst localized at the hilum as dilated renal pelvis ; erroneous differentiation between a central cyst and aneurysm of the renal artery
doppler mode is used for differential diagnosis ; erroneous differentiation of the character of a cystic lesion a complex cyst versus cystic form of renal carcinoma or solid - cystic renal carcinoma ; erroneous interpretation of the cyst in the upper pole as an adrenal or hepatic cyst in the case of the right kidney and as a cyst in the tail of the pancreas or spleen in the case of the left kidney ; errors in interpreting a cystic lesion with abscesslike character as a cyst without obtaining clinical information ( fig .
3 ) ; erroneous interpretation of hypoechoic lesions , e.g. renal lymphoma ( fig . 4 b ) , as dilatation of the pelvicalyceal system ; differential diagnosis is facilitated by doppler options .
errors resulting from the morphological similarity of pathological lesions to the presentation of the kidney , which is the effect of diverse renal structures depending on the patient 's age , developmental defects , individual polymorphism and proliferative changes ( tab .
1 ) :
normal presentation of the kidneys in premature infants and neonates ( kidneys of lobar structure with hypoechoic pyramids and hyperechoic cortex ) is erroneously interpreted as lesions in the course of pyelonephritis , renal failure or renal cysts;renal scar ( elements of the connective tissue penetrating into the parenchyma ) interpreted as angiomyolipoma or postinflammatory scar;dromedary hump of the kidney mistaken for a tumor ( fig .
5 a c);hypertrophy of the pyramid and renal column mistaken for a tumor ( fig . 6
a , b);inflammatory tumors , postinfarction tumors and other lesions related to vascularity disorders may be erroneously interpreted as neoplastic tumors;renal lymphomas , leukemic infiltrations and primary proliferative lesions with echostructure and vascular pattern resembling normal renal parenchyma may be misinterpreted as a normal kidney particularly when they are small and not well - demarcated .
normal presentation of the kidneys in premature infants and neonates ( kidneys of lobar structure with hypoechoic pyramids and hyperechoic cortex ) is erroneously interpreted as lesions in the course of pyelonephritis , renal failure or renal cysts ; renal scar ( elements of the connective tissue penetrating into the parenchyma ) interpreted as angiomyolipoma or postinflammatory scar ; dromedary hump of the kidney mistaken for a tumor ( fig .
c ) ; hypertrophy of the pyramid and renal column mistaken for a tumor ( fig . 6 a , b ) ; inflammatory tumors , postinfarction tumors and other lesions related to vascularity disorders may be erroneously interpreted as neoplastic tumors ; renal lymphomas , leukemic infiltrations and primary proliferative lesions with echostructure and vascular pattern resembling normal renal parenchyma may be misinterpreted as a normal kidney particularly when they are small and not well - demarcated .
vascular malformations ( e.g. angiomas ) may be misinterpreted as renal tumors ( fig . 7 a ,
the essence of ultrasound imaging is the phenomenon of ultrasound wave reflection . making use of the reverse piezoelectric effect , the us transducers generate ultrasonic waves which are sent inside the tissues where
depending on the presentation used by the scanner , these returning echoes are enhanced , appropriately processed and displayed .
the size of the returning echo depends on multiple factors , the knowledge of which enables accurate interpretation of ultrasound images . apart from being reflected , the ultrasonic wave also undergoes refraction , interference , scattering and absorption . in sonography , there are other equally significant factors such as : acoustic impedance of tissues , the shape of reflecting surfaces or elastic properties of tissues . being familiar with all these phenomena and
their influence on the displayed ultrasound image is a condition for its accurate interpretation .
errors in differentiating between hyperechoic images , such as renal concretions , with calcifications , ( postinflammatory ) scars , peripapillary fibrosis and , most frequently , with lesions in the arciform arteries in young patients and children with nephrolithiasis ( fig . 8 a ,
b ) .
errors in differentiating between hyperechoic images , such as renal concretions , with calcifications , ( postinflammatory ) scars , peripapillary fibrosis and , most frequently , with lesions in the arciform arteries in young patients and children with nephrolithiasis ( fig .
errors in the assessment of the site after nephrectomy :
false positive identification of neoplastic relapse caused by stool retention in the bowel loops;errors resulting from the presence of gas in the bowels , which precludes reliable assessment of the site after kidney removal .
false positive identification of neoplastic relapse caused by stool retention in the bowel loops ; errors resulting from the presence of gas in the bowels , which precludes reliable assessment of the site after kidney removal .
2d b - mode presentation focal lesion in the kidney with low echogenicity ( abscess ) mimicking a cyst .
being familiar with clinical presentation ( apart from certain clinically silent cases , such as considerable immunodeficiency ) and application of doppler options enables the differential diagnosis in these cases
a. hypoechoic regions ( lymphoma ) mimicking the dilatation of the pelvicalyceal system with concomitant dilatation of the upper pelvicalyceal system .
2d b - mode presentation
a. dromedary hump of the left kidney mimicking a tumor .
b. application of the b - flow mode reveals vascularization that is analogous to the one of the remaining renal parenchyma . c. impression of the spleen on the left kidney causing renal hump which may be misinterpreted as a renal tumor .
2d b - mode presentation
a. hyperechoic focal lesion in the kidney ( angioma ) mimicking a malignant tumor .
b. power doppler examination failed to show the signs of flow in the region of the lesion
a. hyperechoic region in the kidney ( postinflammatory scar ) mimicking a stone .
b. hyperechoic connective tissue of the renal pelvis produces acoustic shadow and mimics a stone .
2d b - mode presentation pseudotumors in the kidneys normal ureters are not usually visible on us examination , except for in very slim patients .
usually , it is possible to visualize the upper fragment of the ureter down to the level of the inferior pole of the kidney and perivesical portion of the ureter ( intramural and retrovesical parts ) provided that the urinary bladder is well filled
. the visualization of the entire ureter should raise suspicions of a pathology which inhibits urine draining from the kidney .
in normal conditions , the ureter has its peristaltic waves which in a natural way eliminate the possibility to trace the ureter on its entire length .
retroperitoneal fibrosis ( ormond 's disease ) is a disease entity which is difficult to diagnose on the basis of us examination .
one of the characteristic features observed in intravenous urography or in computed tomography ( ct ) in the urographic phase is the medial shift of the ureters caused by inflammatory process taking place in the retroperitoneal space .
the changes in the retroperitoneal space may be visible as iso- or hyperechoic focal lesions .
ct and mri are referential methods . with respect to the comments above , in us diagnosis of the ureters
the following may occur : errors resulting from the presence of artefacts generated by the intestinal contents and gas , e.g. mimicking concretions in the ureter.erroneous interpretation of the iliac vessels as dilated ureters doppler examination helps in differentiation.erroneous indication of the level of narrowing or obstruction of the ureter due to its covering by intestinal contents.errors resulting from diseases of the tissues adjacent to the ureter , e.g. extraperitoneal fibrosis .
errors resulting from the presence of artefacts generated by the intestinal contents and gas , e.g. mimicking concretions in the ureter .
erroneous interpretation of the iliac vessels as dilated ureters doppler examination helps in differentiation .
erroneous indication of the level of narrowing or obstruction of the ureter due to its covering by intestinal contents .
errors resulting from diseases of the tissues adjacent to the ureter , e.g. extraperitoneal fibrosis .
errors resulting from insufficiently or excessively filled bladder :
when the bladder is not filled sufficiently , its reliable assessment is inhibited and there is a risk of omitting proliferative changes in the wall , diverticula , vesical stones , abnormalities in the structure of the bladder neck and ureteral orifices as well as mistaking the folds of the mucous membrane of the urinary bladder for a neoplastic infiltration;the urinary bladder which is filled excessively or insufficiently does not allow for a reliable assessment of retained urine volume and measurement of the wall thickness ; assessing the thickness of the bladder wall is a measure of subvesical obstruction and to perform it , the urinary bladder must be filled with at least 250 ml of fluid .
errors resulting from the impression of the bladder by the surrounding organs :
dilated intestinal loops with the empty bladder may mimic a tumor of the bladder;the enlarged prostate gland indenting the urinary bladder may be erroneously interpreted as a tumor of the bladder;the enlarged uterine body with myomas indenting the urinary bladder may be erroneously interpreted as a tumor of the bladder;a tumor of the ovary indenting the urinary bladder may be erroneously interpreted as a tumor of the bladder;when the bladder is empty , smooth - walled ovarian cysts , embryonic cysts in the medial line of the prostate and cystic form of seminal vesicle carcinoma may be interpreted as the urinary bladder .
errors resulting from insufficiently or excessively filled bladder :
when the bladder is not filled sufficiently , its reliable assessment is inhibited and there is a risk of omitting proliferative changes in the wall , diverticula , vesical stones , abnormalities in the structure of the bladder neck and ureteral orifices as well as mistaking the folds of the mucous membrane of the urinary bladder for a neoplastic infiltration;the urinary bladder which is filled excessively or insufficiently does not allow for a reliable assessment of retained urine volume and measurement of the wall thickness ; assessing the thickness of the bladder wall is a measure of subvesical obstruction and to perform it , the urinary bladder must be filled with at least 250 ml of fluid .
when the bladder is not filled sufficiently , its reliable assessment is inhibited and there is a risk of omitting proliferative changes in the wall , diverticula , vesical stones , abnormalities in the structure of the bladder neck and ureteral orifices as well as mistaking the folds of the mucous membrane of the urinary bladder for a neoplastic infiltration ; the urinary bladder which is filled excessively or insufficiently does not allow for a reliable assessment of retained urine volume and measurement of the wall thickness ; assessing the thickness of the bladder wall is a measure of subvesical obstruction and to perform it , the urinary bladder must be filled with at least 250 ml of fluid .
errors resulting from the impression of the bladder by the surrounding organs :
dilated intestinal loops with the empty bladder may mimic a tumor of the bladder;the enlarged prostate gland indenting the urinary bladder may be erroneously interpreted as a tumor of the bladder;the enlarged uterine body with myomas indenting the urinary bladder may be erroneously interpreted as a tumor of the bladder;a tumor of the ovary indenting the urinary bladder may be erroneously interpreted as a tumor of the bladder;when the bladder is empty , smooth - walled ovarian cysts , embryonic cysts in the medial line of the prostate and cystic form of seminal vesicle carcinoma may be interpreted as the urinary bladder .
dilated intestinal loops with the empty bladder may mimic a tumor of the bladder ; the enlarged prostate gland indenting the urinary bladder may be erroneously interpreted as a tumor of the bladder ; the enlarged uterine body with myomas indenting the urinary bladder may be erroneously interpreted as a tumor of the bladder ; a tumor of the ovary indenting the urinary bladder may be erroneously interpreted as a tumor of the bladder ; when the bladder is empty , smooth - walled ovarian cysts , embryonic cysts in the medial line of the prostate and cystic form of seminal vesicle carcinoma may be interpreted as the urinary bladder .
errors concerning the number of kidneys : failure to visualize the kidney in its typical site :
agenesis / aplasia sonography does not enable the differentiation between these two pathologies;renal ectopia or dystopia;hypoplasia or aplasia the kidney is so small or so altered that its identification is impossible for various reasons ( obesity , artefacts produced by intestinal gas , kidney localization etc . ) .
the final verification is conducted on the basis of intravenous urography or computed tomography.situations occurring more rarely :
renal agenesis in neonates the possibility to mistake enlarged adrenal glands for hypoplastic kidneys;tumors of the gastrointestinal tract
pseudokidney , particularly in patients with unilateral renal agenesis;ectopic kidneys possibility to mistake pelvic kidney for a tumor - like lesion;in extreme cases , ectopic ovaries and their high , abdominal localization may mimic a hypoplastic kidney;renal hypoplasia and dysplasia with concomitant ectopia a particularly complex anomaly which prohibits the identification of the kidneys and inhibits their assessment;horseshoe kidney erroneous interpretation of the isthmus of a horseshoe kidney as a pathological mass in the extraperitoneal space ( fig .
2 a , b ) .
errors in size assessment :
errors resulting from anomalous location , structure and rotation of the kidney ; this inhibits the measurement of the longest longitudinal and transverse lengths which should be measured in these planes , e.g. horseshoe kidney;errors resulting from the selection of improper plane for the measurements renal measurements should be conducted after the longest longitudinal and transverse lengths have been obtained ; errors may be caused by taking measurements in oblique sections;errors resulting from the lack of standardization of measurement sites failure to obtain reproducible measurements ; measurements of the kidneys and dilated pelvicalyceal system ought to be performed each time in the same manner;errors resulting from the dynamics of renal conditions , influence of an ongoing treatment as well as degree of hydration and fullness of the pelvicalyceal system , e.g. erroneous interpretation of the pelvicalyceal system filled with urine with maximally filled bladder as hydronephrosis in such cases , one should assess the kidneys following miction;errors resulting from the impossibility to visualize the entire kidneys or overlapping shadows of the ribs and intestinal loops as well as faulty measurements reflecting a part of the kidney , not the entire one .
errors in the interpretation of fluid - filled spaces in the kidneys :
identification of isolated dilatation of the calyceal system / systems in the case of central cysts;mistaking central renal cysts for dilatation of the pelvicalyceal system;misinterpretation of a cyst localized at the hilum as dilated renal pelvis;erroneous differentiation between a central cyst and aneurysm of the renal artery
doppler mode is used for differential diagnosis;erroneous differentiation of the character of a cystic lesion a complex cyst versus cystic form of renal carcinoma or solid - cystic renal carcinoma;erroneous interpretation of the cyst in the upper pole as an adrenal or hepatic cyst in the case of the right kidney and as a cyst in the tail of the pancreas or spleen in the case of the left kidney;errors in interpreting a cystic lesion with abscesslike character as a cyst without obtaining clinical information ( fig .
3);erroneous interpretation of hypoechoic lesions , e.g. renal lymphoma ( fig . 4 b ) , as dilatation of the pelvicalyceal system ;
errors resulting from the morphological similarity of pathological lesions to the presentation of the kidney , which is the effect of diverse renal structures depending on the patient 's age , developmental defects , individual polymorphism and proliferative changes ( tab .
1 ) :
normal presentation of the kidneys in premature infants and neonates ( kidneys of lobar structure with hypoechoic pyramids and hyperechoic cortex ) is erroneously interpreted as lesions in the course of pyelonephritis , renal failure or renal cysts;renal scar ( elements of the connective tissue penetrating into the parenchyma ) interpreted as angiomyolipoma or postinflammatory scar;dromedary hump of the kidney mistaken for a tumor ( fig .
5 a c);hypertrophy of the pyramid and renal column mistaken for a tumor ( fig . 6
a , b);inflammatory tumors , postinfarction tumors and other lesions related to vascularity disorders may be erroneously interpreted as neoplastic tumors;renal lymphomas , leukemic infiltrations and primary proliferative lesions with echostructure and vascular pattern resembling normal renal parenchyma may be misinterpreted as a normal kidney particularly when they are small and not well - demarcated .
the essence of ultrasound imaging is the phenomenon of ultrasound wave reflection . making use of the reverse piezoelectric effect ,
the us transducers generate ultrasonic waves which are sent inside the tissues where they are reflected and return to the transducer .
depending on the presentation used by the scanner , these returning echoes are enhanced , appropriately processed and displayed .
the size of the returning echo depends on multiple factors , the knowledge of which enables accurate interpretation of ultrasound images . apart from being reflected , the ultrasonic wave also undergoes refraction , interference , scattering and absorption . in sonography
, there are other equally significant factors such as : acoustic impedance of tissues , the shape of reflecting surfaces or elastic properties of tissues .
being familiar with all these phenomena and their influence on the displayed ultrasound image is a condition for its accurate interpretation .
errors in differentiating between hyperechoic images , such as renal concretions , with calcifications , ( postinflammatory ) scars , peripapillary fibrosis and , most frequently , with lesions in the arciform arteries in young patients and children with nephrolithiasis ( fig . 8 a ,
b ) .
errors in the assessment of the site after nephrectomy :
false positive identification of neoplastic relapse caused by stool retention in the bowel loops;errors resulting from the presence of gas in the bowels , which precludes reliable assessment of the site after kidney removal .
failure to visualize the kidney in its typical site :
agenesis / aplasia sonography does not enable the differentiation between these two pathologies;renal ectopia or dystopia;hypoplasia or aplasia the kidney is so small or so altered that its identification is impossible for various reasons ( obesity , artefacts produced by intestinal gas , kidney localization etc . ) .
sonography does not enable the differentiation between these two pathologies ; renal ectopia or dystopia ; hypoplasia or aplasia the kidney is so small or so altered that its identification is impossible for various reasons ( obesity , artefacts produced by intestinal gas , kidney localization etc . ) .
situations occurring more rarely :
renal agenesis in neonates the possibility to mistake enlarged adrenal glands for hypoplastic kidneys;tumors of the gastrointestinal tract e.g. colon carcinoma manifests itself as a , so - called ,
pseudokidney , particularly in patients with unilateral renal agenesis;ectopic kidneys possibility to mistake pelvic kidney for a tumor - like lesion;in extreme cases , ectopic ovaries and their high , abdominal localization may mimic a hypoplastic kidney;renal hypoplasia and dysplasia with concomitant ectopia a particularly complex anomaly which prohibits the identification of the kidneys and inhibits their assessment;horseshoe kidney erroneous interpretation of the isthmus of a horseshoe kidney as a pathological mass in the extraperitoneal space ( fig .
the possibility to mistake enlarged adrenal glands for hypoplastic kidneys ; tumors of the gastrointestinal tract
possibility to mistake pelvic kidney for a tumor - like lesion ; in extreme cases , ectopic ovaries and their high , abdominal localization may mimic a hypoplastic kidney ; renal hypoplasia and dysplasia with concomitant ectopia a particularly complex anomaly which prohibits
erroneous interpretation of the isthmus of a horseshoe kidney as a pathological mass in the extraperitoneal space ( fig .
errors in size assessment :
errors resulting from anomalous location , structure and rotation of the kidney ; this inhibits the measurement of the longest longitudinal and transverse lengths which should be measured in these planes , e.g. horseshoe kidney;errors resulting from the selection of improper plane for the measurements renal measurements should be conducted after the longest longitudinal and transverse lengths have been obtained ; errors may be caused by taking measurements in oblique sections;errors resulting from the lack of standardization of measurement sites failure to obtain reproducible measurements ; measurements of the kidneys and dilated pelvicalyceal system ought to be performed each time in the same manner;errors resulting from the dynamics of renal conditions , influence of an ongoing treatment as well as degree of hydration and fullness of the pelvicalyceal system , e.g. erroneous interpretation of the pelvicalyceal system filled with urine with maximally filled bladder as hydronephrosis in such cases , one should assess the kidneys following miction;errors resulting from the impossibility to visualize the entire kidneys or overlapping shadows of the ribs and intestinal loops as well as faulty measurements reflecting a part of the kidney , not the entire one .
errors resulting from anomalous location , structure and rotation of the kidney ; this inhibits the measurement of the longest longitudinal and transverse lengths which should be measured in these planes , e.g. horseshoe kidney ; errors resulting from the selection of improper plane for the measurements renal measurements should be conducted after the longest longitudinal and transverse lengths have been obtained ; errors may be caused by taking measurements in oblique sections ; errors resulting from the lack of standardization of measurement sites failure to obtain reproducible measurements ; measurements of the kidneys and dilated pelvicalyceal system ought to be performed each time in the same manner ; errors resulting from the dynamics of renal conditions , influence of an ongoing treatment as well as degree of hydration and fullness of the pelvicalyceal system , e.g. erroneous interpretation of the pelvicalyceal system filled with urine with maximally filled bladder as hydronephrosis in such cases , one should assess the kidneys following miction ; errors resulting from the impossibility to visualize the entire kidneys or overlapping shadows of the ribs and intestinal loops as well as faulty measurements reflecting a part of the kidney , not the entire one .
errors in the interpretation of fluid - filled spaces in the kidneys :
identification of isolated dilatation of the calyceal system / systems in the case of central cysts;mistaking central renal cysts for dilatation of the pelvicalyceal system;misinterpretation of a cyst localized at the hilum as dilated renal pelvis;erroneous differentiation between a central cyst and aneurysm of the renal artery
doppler mode is used for differential diagnosis;erroneous differentiation of the character of a cystic lesion a complex cyst versus cystic form of renal carcinoma or solid - cystic renal carcinoma;erroneous interpretation of the cyst in the upper pole as an adrenal or hepatic cyst in the case of the right kidney and as a cyst in the tail of the pancreas or spleen in the case of the left kidney;errors in interpreting a cystic lesion with abscesslike character as a cyst without obtaining clinical information ( fig .
3);erroneous interpretation of hypoechoic lesions , e.g. renal lymphoma ( fig . 4 b ) , as dilatation of the pelvicalyceal system
identification of isolated dilatation of the calyceal system / systems in the case of central cysts ; mistaking central renal cysts for dilatation of the pelvicalyceal system ; misinterpretation of a cyst localized at the hilum as dilated renal pelvis ; erroneous differentiation between a central cyst and aneurysm of the renal artery
doppler mode is used for differential diagnosis ; erroneous differentiation of the character of a cystic lesion a complex cyst versus cystic form of renal carcinoma or solid - cystic renal carcinoma ; erroneous interpretation of the cyst in the upper pole as an adrenal or hepatic cyst in the case of the right kidney and as a cyst in the tail of the pancreas or spleen in the case of the left kidney ; errors in interpreting a cystic lesion with abscesslike character as a cyst without obtaining clinical information ( fig .
3 ) ; erroneous interpretation of hypoechoic lesions , e.g. renal lymphoma ( fig . 4 b ) , as dilatation of the pelvicalyceal system ; differential diagnosis is facilitated by doppler options .
errors resulting from the morphological similarity of pathological lesions to the presentation of the kidney , which is the effect of diverse renal structures depending on the patient 's age , developmental defects , individual polymorphism and proliferative changes ( tab .
1 ) :
normal presentation of the kidneys in premature infants and neonates ( kidneys of lobar structure with hypoechoic pyramids and hyperechoic cortex ) is erroneously interpreted as lesions in the course of pyelonephritis , renal failure or renal cysts;renal scar ( elements of the connective tissue penetrating into the parenchyma ) interpreted as angiomyolipoma or postinflammatory scar;dromedary hump of the kidney mistaken for a tumor ( fig .
5 a c);hypertrophy of the pyramid and renal column mistaken for a tumor ( fig . 6
a , b);inflammatory tumors , postinfarction tumors and other lesions related to vascularity disorders may be erroneously interpreted as neoplastic tumors;renal lymphomas , leukemic infiltrations and primary proliferative lesions with echostructure and vascular pattern resembling normal renal parenchyma may be misinterpreted as a normal kidney particularly when they are small and not well - demarcated .
normal presentation of the kidneys in premature infants and neonates ( kidneys of lobar structure with hypoechoic pyramids and hyperechoic cortex ) is erroneously interpreted as lesions in the course of pyelonephritis , renal failure or renal cysts ; renal scar ( elements of the connective tissue penetrating into the parenchyma ) interpreted as angiomyolipoma or postinflammatory scar ; dromedary hump of the kidney mistaken for a tumor ( fig .
c ) ; hypertrophy of the pyramid and renal column mistaken for a tumor ( fig . 6 a , b ) ; inflammatory tumors , postinfarction tumors and other lesions related to vascularity disorders may be erroneously interpreted as neoplastic tumors ; renal lymphomas , leukemic infiltrations and primary proliferative lesions with echostructure and vascular pattern resembling normal renal parenchyma may be misinterpreted as a normal kidney particularly when they are small and not well - demarcated .
vascular malformations ( e.g. angiomas ) may be misinterpreted as renal tumors ( fig . 7 a ,
the essence of ultrasound imaging is the phenomenon of ultrasound wave reflection . making use of the reverse piezoelectric effect , the us transducers generate ultrasonic waves which are sent inside the tissues where
depending on the presentation used by the scanner , these returning echoes are enhanced , appropriately processed and displayed .
the size of the returning echo depends on multiple factors , the knowledge of which enables accurate interpretation of ultrasound images . apart from being reflected , the ultrasonic wave also undergoes refraction , interference , scattering and absorption . in sonography , there are other equally significant factors such as : acoustic impedance of tissues , the shape of reflecting surfaces or elastic properties of tissues . being familiar with all these phenomena and
their influence on the displayed ultrasound image is a condition for its accurate interpretation .
errors in differentiating between hyperechoic images , such as renal concretions , with calcifications , ( postinflammatory ) scars , peripapillary fibrosis and , most frequently , with lesions in the arciform arteries in young patients and children with nephrolithiasis ( fig . 8 a ,
b ) .
errors in differentiating between hyperechoic images , such as renal concretions , with calcifications , ( postinflammatory ) scars , peripapillary fibrosis and , most frequently , with lesions in the arciform arteries in young patients and children with nephrolithiasis ( fig .
errors in the assessment of the site after nephrectomy :
false positive identification of neoplastic relapse caused by stool retention in the bowel loops;errors resulting from the presence of gas in the bowels , which precludes reliable assessment of the site after kidney removal .
false positive identification of neoplastic relapse caused by stool retention in the bowel loops ; errors resulting from the presence of gas in the bowels , which precludes reliable assessment of the site after kidney removal .
2d b - mode presentation focal lesion in the kidney with low echogenicity ( abscess ) mimicking a cyst .
being familiar with clinical presentation ( apart from certain clinically silent cases , such as considerable immunodeficiency ) and application of doppler options enables the differential diagnosis in these cases
a. hypoechoic regions ( lymphoma ) mimicking the dilatation of the pelvicalyceal system with concomitant dilatation of the upper pelvicalyceal system .
2d b - mode presentation
a. dromedary hump of the left kidney mimicking a tumor .
b. application of the b - flow mode reveals vascularization that is analogous to the one of the remaining renal parenchyma . c. impression of the spleen on the left kidney causing renal hump which may be misinterpreted as a renal tumor .
2d b - mode presentation
a. hyperechoic focal lesion in the kidney ( angioma ) mimicking a malignant tumor .
b. power doppler examination failed to show the signs of flow in the region of the lesion
a. hyperechoic region in the kidney ( postinflammatory scar ) mimicking a stone .
b. hyperechoic connective tissue of the renal pelvis produces acoustic shadow and mimics a stone .
normal ureters are not usually visible on us examination , except for in very slim patients .
usually , it is possible to visualize the upper fragment of the ureter down to the level of the inferior pole of the kidney and perivesical portion of the ureter ( intramural and retrovesical parts ) provided that the urinary bladder is well filled . the visualization of the entire ureter should raise suspicions of a pathology which inhibits urine draining from the kidney .
in normal conditions , the ureter has its peristaltic waves which in a natural way eliminate the possibility to trace the ureter on its entire length .
retroperitoneal fibrosis ( ormond 's disease ) is a disease entity which is difficult to diagnose on the basis of us examination .
one of the characteristic features observed in intravenous urography or in computed tomography ( ct ) in the urographic phase is the medial shift of the ureters caused by inflammatory process taking place in the retroperitoneal space .
the changes in the retroperitoneal space may be visible as iso- or hyperechoic focal lesions .
ct and mri are referential methods . with respect to the comments above , in us diagnosis of the ureters
the following may occur : errors resulting from the presence of artefacts generated by the intestinal contents and gas , e.g. mimicking concretions in the ureter.erroneous interpretation of the iliac vessels as dilated ureters doppler examination helps in differentiation.erroneous indication of the level of narrowing or obstruction of the ureter due to its covering by intestinal contents.errors resulting from diseases of the tissues adjacent to the ureter , e.g. extraperitoneal fibrosis .
errors resulting from the presence of artefacts generated by the intestinal contents and gas , e.g. mimicking concretions in the ureter .
erroneous interpretation of the iliac vessels as dilated ureters doppler examination helps in differentiation .
erroneous indication of the level of narrowing or obstruction of the ureter due to its covering by intestinal contents .
errors resulting from diseases of the tissues adjacent to the ureter , e.g. extraperitoneal fibrosis .
errors resulting from insufficiently or excessively filled bladder :
when the bladder is not filled sufficiently , its reliable assessment is inhibited and there is a risk of omitting proliferative changes in the wall , diverticula , vesical stones , abnormalities in the structure of the bladder neck and ureteral orifices as well as mistaking the folds of the mucous membrane of the urinary bladder for a neoplastic infiltration;the urinary bladder which is filled excessively or insufficiently does not allow for a reliable assessment of retained urine volume and measurement of the wall thickness ; assessing the thickness of the bladder wall is a measure of subvesical obstruction and to perform it , the urinary bladder must be filled with at least 250 ml of fluid .
errors resulting from the impression of the bladder by the surrounding organs :
dilated intestinal loops with the empty bladder may mimic a tumor of the bladder;the enlarged prostate gland indenting the urinary bladder may be erroneously interpreted as a tumor of the bladder;the enlarged uterine body with myomas indenting the urinary bladder may be erroneously interpreted as a tumor of the bladder;a tumor of the ovary indenting the urinary bladder may be erroneously interpreted as a tumor of the bladder;when the bladder is empty , smooth - walled ovarian cysts , embryonic cysts in the medial line of the prostate and cystic form of seminal vesicle carcinoma may be interpreted as the urinary bladder .
errors resulting from insufficiently or excessively filled bladder :
when the bladder is not filled sufficiently , its reliable assessment is inhibited and there is a risk of omitting proliferative changes in the wall , diverticula , vesical stones , abnormalities in the structure of the bladder neck and ureteral orifices as well as mistaking the folds of the mucous membrane of the urinary bladder for a neoplastic infiltration;the urinary bladder which is filled excessively or insufficiently does not allow for a reliable assessment of retained urine volume and measurement of the wall thickness ; assessing the thickness of the bladder wall is a measure of subvesical obstruction and to perform it , the urinary bladder must be filled with at least 250 ml of fluid .
when the bladder is not filled sufficiently , its reliable assessment is inhibited and there is a risk of omitting proliferative changes in the wall , diverticula , vesical stones , abnormalities in the structure of the bladder neck and ureteral orifices as well as mistaking the folds of the mucous membrane of the urinary bladder for a neoplastic infiltration ; the urinary bladder which is filled excessively or insufficiently does not allow for a reliable assessment of retained urine volume and measurement of the wall thickness ; assessing the thickness of the bladder wall is a measure of subvesical obstruction and to perform it , the urinary bladder must be filled with at least 250 ml of fluid .
errors resulting from the impression of the bladder by the surrounding organs :
dilated intestinal loops with the empty bladder may mimic a tumor of the bladder;the enlarged prostate gland indenting the urinary bladder may be erroneously interpreted as a tumor of the bladder;the enlarged uterine body with myomas indenting the urinary bladder may be erroneously interpreted as a tumor of the bladder;a tumor of the ovary indenting the urinary bladder may be erroneously interpreted as a tumor of the bladder;when the bladder is empty , smooth - walled ovarian cysts , embryonic cysts in the medial line of the prostate and cystic form of seminal vesicle carcinoma may be interpreted as the urinary bladder .
dilated intestinal loops with the empty bladder may mimic a tumor of the bladder ; the enlarged prostate gland indenting the urinary bladder may be erroneously interpreted as a tumor of the bladder ; the enlarged uterine body with myomas indenting the urinary bladder may be erroneously interpreted as a tumor of the bladder ; a tumor of the ovary indenting the urinary bladder may be erroneously interpreted as a tumor of the bladder ; when the bladder is empty , smooth - walled ovarian cysts , embryonic cysts in the medial line of the prostate and cystic form of seminal vesicle carcinoma may be interpreted as the urinary bladder .
authors do not report any financial or personal links with other persons or organizations , which might affect negatively the content of this publication and/or claim authorship rights to this publication . | the article presents the most frequent errors made in the ultrasound diagnosis of the urinary system .
they usually result from improper technique of ultrasound examination or its erroneous interpretation .
such errors are frequent effects of insufficient experience of the ultrasonographer , inadequate class of the scanner , insufficient knowledge of its operation as well as of wrong preparation of patients , their constitution , severe condition and the lack of cooperation during the examination .
the reasons for misinterpretations of ultrasound images of the urinary system may lie in a large polymorphism of the kidney ( defects and developmental variants ) and may result from improper access to the organ as well as from the presence of artefacts .
errors may also result from the lack of knowledge concerning clinical and laboratory data .
moreover , mistakes in ultrasound diagnosis of the urinary system are frequently related to the lack of knowledge of the management algorithms and diagnostic possibilities of other imaging modalities .
the paper lists errors in ultrasound diagnosis of the urinary system divided into : errors resulting from improper technique of examination , artefacts caused by incorrect preparation of patients for the examination or their constitution and errors resulting from misinterpretation of ultrasound images of the kidneys ( such as their number , size , fluid spaces , pathological lesions and others ) , ureters and urinary bladder .
each physician performing kidney or bladder ultrasound examination should possess the knowledge of the most frequent errors and their causes which might help to avoid them . | Introduction
Errors resulting from improper technique of examination
Errors resulting from inadequate preparation of patients for examination or from their constitution
Errors resulting from misinterpretation of ultrasound images
Errors in kidney assessment
Errors in ureter assessment
Errors in the assessment of the urinary bladder
Conflict of interest |
PMC4324440 | pancreatic cancer ( pc )
is one of the most fatal cancers , with a
5 year survival of less than 5% .
an estimated
46 420 new cases of pc are expected in the u.s . in 2014 as
well as 39 590 deaths from this disease .
a major hurdle toward improving clinical outcome of pc
is the lack of diagnostic biomarkers at early stages of the disease .
given the high mortality associated with pc ,
novel and cost - effective biomarkers to improve treatment and survival
outcomes of pc patients are urgently needed . to date , the only
treatment that provides significant survival
benefit is surgical resection , but only 2025% of patients
are diagnosed at early disease stages when resection is appropriate .
the clinical symptoms of pc are usually vague
and nonspecific until progression to advanced stages has occurred .
attempts to reduce pc deaths have therefore relied
greatly on early cancer detection and treatment , generally through
imaging examination , such as magnetic resonance imaging ( mri ) , computed
tomography , endoscopic retrograde cholangiopancreatography ( ercp ) ,
or endoscopic ultrasound ( eu )
. however , the specificity and sensitivity
of these modalities are not adequate for tumors of less than 2 cm
in diameter .
the traditional tumor marker ca19 - 9 ,
the sensitivity of which can reach 80% for pc , is unsuitable for early
detection of pc due to low sensitivity for patients at resectable
stages and especially because of its weak
specificity .
numerous efforts have been
made in the search for pc biomarkers during recent decades , and , as
a result , tumor - specific growth factor ( tsgf ) , ca242 , mic-1 , platelet factor 4 , peanut
agglutinin ( pna)-binding glycoprotein , cell adhesion molecule 17.1 , and serum
immune signatures found by affinity proteomics have been identified as candidate biomarkers .
unfortunately ,
these biomarkers display low sensitivity for resectable disease , and
their accuracy for detecting resectable stage cancer has not been
evaluated .
metabonomics , a new member of omics technologies
that quantitatively
measures altered metabolites resulting from pathophysiological changes ,
is rapidly becoming a discovery tool for new diagnostic and prognostic
biomarkers of human diseases .
we have previously shown the
use of metabonomics for diagnosis and evaluation of pathologic conditions
of various cancers .
recently , metabonomics studies of pc have identified biomarkers
in plasma or tissue that differentiate pc from controls .
however , these studies have
had relatively small sample sizes and a small number of early stage
or resectable cancers . in this study , we used a combination
of liquid chromatography time - of - flight
mass spectrometry ( lc tofms ) and gas chromatography time - of - flight
mass spectrometry ( gc tofms ) to profile plasma metabolites
of pc patients and controls from the u.s . and china .
the aim of this
study was to identify plasma metabolites as potential markers for
early detection of pc and to test the diagnostic performance of these
markers .
blood samples
used in this study
were from 100 pc patients and 100 age- and gender - matched population
controls in connecticut ( ct ) , usa , and from 100 pc patients and 100
similarly matched population controls from shanghai ( sh ) , china ( table 1 ) .
the pc patients were newly diagnosed with pancreatic
ductal adenocarcinoma and were not recurrent or on any medication
prior to sample collection .
patient characteristics , staging of the
disease , and other parameters are shown in table 1 .
these studies were approved by the state of connecticut department
of public health as well as by the institutional review boards of
30 connecticut hospitals ( ct ) , the institutional human subjects review
board of the shanghai cancer institute ( sh ) , and the human investigation
committee of yale university ( ct ) and shanghai cancer institute ( sh ) .
blood specimens
were collected
from all participants and returned on ice to our laboratories within
2 h of collection for blood processing .
tofms
were used for the metabonomic profiling of all samples in the study .
the profiling procedure ( sample preparation , metabolite separation
and detection , metabonomic data preprocessing , metabolite annotation ,
and statistical analysis for biomarker identification ) was performed
following our previously published protocols with minor modifications .
quality control ( qc ) samples ,
which were prepared by mixing equal amounts of plasma from all subject
samples , were used to control intra- and interbatch variability .
details of plasma
sample preparation and lc / gc ms analysis are provided in the supporting information .
the metabonomic data obtained
were normalized using internal standard p - chlorophenylalanine
and calibrated using qc samples .
tofms
and lc tofms data sets were combined and exported to simca - p+
12.0 software ( umetrics , ume , sweden ) for multivariate statistical
analysis .
orthogonal partial least squares - discriminant
analysis ( opls - da ) were performed to discriminate between pc patients
and controls . on the basis of a threshold of variable importance in
the projection ( vip , value > 1 ) from the 7-fold cross - validated
opls - da
model ,
a panel of metabolites responsible for the difference in the
metabolic profiles of patients and controls was obtained .
in addition
to the multivariate statistical method , student s t - test was also applied to measure the significance of each metabolite .
the resultant p values for all metabolites were subsequently
adjusted to account for multiple testing by a false discovery rate
( fdr ) method .
metabolites with both multivariate
and univariate statistical significance ( vip > 1 and p < 0.05 ) were considered to be potential markers capable of differentiating
pc from controls .
the corresponding fold change was calculated to
show how these selected differential metabolites varied in the cancer
samples relative to the controls . altered metabolic pathways in pc
were analyzed by means of the quantitative enrichment analysis ( qea )
algorithm represented in the metabolite set enrichment analysis ( msea )
method .
receiver operating characteristic ( roc ) curve analysis
and binary logistic regression were conducted using spss software
( ibm spss statistics 19 , usa ) following our previously published data
analysis protocols .
briefly , a logistic
regression model constructed using the binary outcome of pc and control
as dependent variables was used to determine the best combination
of plasma markers for pc prediction .
the forward stepwise regression ,
the procedure to select the strongest variables ( metabolites ) until
there are no more significant predictors in the data set , was used
for potential biomarker selection .
the wald test was used to assess
significance in logistic regression , and this test assigns a p value to each metabolite to assess significance .
roc curves
for the logistic regression model were plotted with the fitted probabilities
from the established model as possible cut - points for the computation
of sensitivity and specificity .
demographic ,
lifestyle , and clinical information of the study subjects is listed
in table 1 .
patients and controls were well - matched
for age and gender within each study site .
, 202 metabolites were identified
( supporting information table s1 ) from
the detected spectral features of samples ; of these , 109 metabolites
( 53.7% , 70 metabolites from gc ms and 39 from lc
ms )
were validated with reference standards , whereas the others were annotated
by comparing with available databases including the nist library and
the human metabolome database ( hmdb ) . a one - predictive component and
two - orthogonal component opls - da model ( r2x = 0.170 , r2y(cum ) = 0.757 ,
q2(cum ) = 0.565 )
was constructed with satisfactory discriminating
ability using the metabonomics data of the 202 identified plasma metabolites
in ct samples ( figure 1a ) .
similarly , a one - predictive
component and four - orthogonal component opls - da model ( r2x = 0.258 ,
r2y(cum ) = 0.880 , q2(cum ) = 0.679 ) was constructed with satisfactory
discriminating ability using the metabonomics data of the 202 identified
plasma metabolites in sh samples ( figure 1b ) .
pc patients from both ct and sh sample sets could be separated from
their control counterparts .
metabolic profiles depicted by opls - da scores
plots of lctofms
and gctofms spectral data ( 202 metabolites ) from ( a ) ct plasma
samples , ( b ) sh plasma samples , and ( c ) 3d opls - da scores plot of
plasma metabolic profiles of pc patients and controls from ct and
sh . using the vip values ( vip >
1 ) derived from the opls - da model and
the p values ( p < 0.05 ) , 65 differentially
expressed metabolites in the ct set and 62 in the sh set were obtained ,
among which 31 metabolites were the same and were significantly altered
in the same direction ( table 2 ) .
pc patients
can be discriminated from control subjects with the 31 differential
metabolites identified both in ct and sh samples , as evidenced by
a 3d opls - da scores plot of plasma metabolic profiles of pc patients
and controls from ct and sh shown in figure 1c .
variable
importance in the projection
( vip ) was obtained from opls - da with a threshold of 1.0 . fold change ( fc )
was obtained by
comparing those metabolites in the pc group to the control group ;
fc with a value > 1 indicates a relatively higher concentration
present
in the pc group , whereas a value < 1 indicates a relatively lower
concentration compared to the control group .
the 31 significantly altered plasma metabolites in
both ct and
sh patients ( adjusted p < 0.05 , supporting information figure s1a ) include amino acids , carbohydrates ,
lipids , nucleosides , organic acids , aromatic heteropolycyclic compounds ,
aliphatic acyclic compounds , and aliphatic heteromonocyclic compounds
( table 2 and supporting
information figure s1a ) .
thirty six metabolic pathways were
found to be dysregulated in pc based on the analysis of the qea algorithm
of the msea method ( bonferroni - corrected p < 0.05 , supporting information figure s1b ) . to evaluate the potential utility of plasma metabolites
for the discrimination between pc patients and control subjects
, we
developed a logistic regression model based on the 31 validated biomarkers
from the ct set . through a forward stepwise analysis , we identified
glutamate , choline , 1,5-anhydro - d - glucitol , betaine , and
methylguanidine as being the best predictors of disease status in
the regression model ( table 3 ) . using these
metabolites , we established a regression model as follows : p values were calculated
using the wald test .
next ,
we generated roc curves to assess the potential usefulness
of plasma metabolite signatures as noninvasive biomarkers for the
diagnosis of pc .
our roc analyses revealed that plasma metabolite
biomarkers were robust in discriminating patients with pc from controls
in ct , with an area under the curve ( auc ) value of 0.943 ( 95%
ci =
0.9080.977 ) ( figure 2a ) . using a cutoff
value of 0.3598 ,
the sensitivity , specificity , and positive and negative
predictive values are given in table 4 .
( a ) roc curve
analysis for the predictive power of combined plasma
biomarkers for distinguishing pc from controls in the ct set .
the
final logistic model included five plasma biomarkers : glutamate , choline ,
1,5-anhydro - d - glucitol , betaine , and methylguanidine .
( b )
roc curve analysis for the predictive power of combined plasma biomarkers
for distinguishing pc from control in the sh set . at the cutoff value
determined in the ct set
, plasma metabolite biomarkers yielded an
auc value of 0.835 ( 95% ci , 0.7770.893 ) with 77.4% sensitivity
and 75.8% in discriminating pc from controls .
( c ) plots of the diagnostic
values of the constructed diagnostic model and tumor marker levels
in 100 pc patients and 100 controls in the ct set and 100 pc patients
and 100 controls in the sh set , according to disease stage .
third , the parameters obtained from the ct set were
used to predict
the probability of pc diagnosis in the sh set .
similarly , the roc
curve was constructed with the predicted probability for the sh set
and is shown in figure 2b .
at the same cutoff value of 0.3598 , its sensitivity and
specificity values were still substantial ( table 4 ) .
the predictive values obtained from this classification
method in the ct and sh samples are also shown in scatter plots in
figure 2c . to investigate whether the
pc - associated plasma metabolite signatures
can differentiate between patients and controls independently of possible
confounding risk factors ,
the pc - associated plasma metabolite
signatures remained significant ( p < 0.001 ) after
adjustment for bmi and use of tobacco and alcohol ( supporting information table s2 ) .
genetic alterations enable cancer
cells to reprogram metabolism to meet increased energy demands for
cell proliferation and to survive in hypoxic and nutrient - deprived
tumor microenvironments . in this
regard ,
a better understanding of metabolic dysregulation in pc is important
and necessary .
metabonomics allows for global assessment of
the cellular metabolic state within the context of the immediate environment ,
taking into account genetic regulation , altered kinetic activity of
enzymes , and changes in inflammatory and stress levels . in this study , we used combined lc
the combination of different analytical platforms takes advantage
of complementary analytical outcomes and , therefore , provides an unrivaled
number of identified metabolites for explaining biological variations
associated with pathophysiological conditions and obtains cross - validated results as well .
we also included two
groups of subjects with different ethnic backgrounds , from usa and
china , to identify and validate the metabolite biomarkers .
although
there are ethnic differences in metabonomic profiles between ct and
sh subjects ( figure 1c ) , pc patients can still
be readily discriminated from the controls .
the opls - da models
derived from the current metabonomic analysis
were able to differentiate between pc and controls in both the ct
and sh cohorts , highlighting the diagnostic potential of this noninvasive
analytical approach .
our results also demonstrated the potential role
of metabolite biomarkers in the early detection of pc , which is supported
by the markedly high auc values of 0.943 and 0.835 from comparisons
between pc patients and controls ( sensitivity = 97.7 and 77.4% ; specificity
= 83.1 and 75.8% ) in the ct and sh cohorts , respectively .
even more
important for potential early diagnosis of pc , our metabonomics - based
diagnostic model was sensitive at detecting early stage pc in the
models that were adjusted for bmi and history of smoking and drinking .
the pathogenesis of pancreatic disease
can cause significant decreases
in plasma levels of amino acids , fatty acids , aliphatic acyclic compounds ,
and aromatic heteropolycyclic compounds ( valine , glutamine , proline ,
tryptophan , monoisobutyl phthalic acid , propionylcarnitine , urea ,
and uric acid ) and increases in glutamic acid and glycocholic acid ,
which have been demonstrated by other research groups that have found
alterations in plasma / serum metabolites similar to ours .
it is well - known that uptake
and catabolism of amino acids and fatty acids are enhanced to support
rapid cell proliferation in cancer tissues and that these changes may be explained as a result of the enhanced
usage in tumors ( supporting information figures
s2 and s3 ) .
metabolic pathway analysis also suggests that glycine / serine / threonine / methionine
metabolism ( supporting information figure s2 ) , glutamate pathway ( supporting information
figure s3 ) , tyrosine metabolism ( supporting
information figure s4 ) , tca cycle ( supporting
information figure s5 ) , choline metabolism ( supporting information figures s2 and s6 ) , and bile acid metabolism
( supporting information figure s7 ) are
markedly altered .
pc may result from a mutation in either the
exocrine or endocrine
function of the pancreas .
therefore ,
there is a possibility that the decreases in their plasma metabolite
levels also reflect malnutrition .
consistent with results found by
kobayashi and colleagues , we observed
that plasma levels of 1,5-anhydro - d - glucitol were significantly
reduced in pc patients compared to controls ( table 2 ) .
1,5-anhydro - d - glucitol is reported to be a biomarker
of short - term glycemic control , and decreased
plasma levels of 1,5-anhydro - d - glucitol suggest the presence
of hyperglycemia and glycosuria .
these results indicate some impairment
of glucose tolerance in these patients because of pancreatic insufficiency .
glutamate has been implicated in tumorigenesis through activation
of alpha - amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa )
receptors ( ampar ) , and glutamate concentration plays a key role in
pc cell invasion and migration .
our observation
of a significantly elevated plasma level of glutamate is in accordance
with serum analyses of pc by bathe et al .
choline - containing
metabolites have already been chosen as biomarkers in various carcinoma
studies and have been reported to be
decreased in pc .
choline deficiency can
also produce severe acute pancreatitis in animal models . in our results , reduced betaine enhanced the
discrimination of pc from controls .
betaine donates methyl groups
for remethylation of homocysteine to methionine and dimethylglycine ,
which support proper liver and pancreatic function , cellular replication ,
and detoxification reactions . because choline is a precursor of betaine
,
the depletion of both betaine and choline in pc may be interrelated
( supporting information figures s2 and s6 ) .
it has been reported that urinary levels of methylguanidine are
significantly increased in chronic pancreatitis patients , yet this metabolite has not been studied in
pc to date .
glycocholic acid was not selected in the panel of
markers ( glutamate ,
choline , 1,5-anhydro - d - glucitol , betaine , and methylguanidine )
for the prediction of pc using the forward stepwise regression method .
because its level is remarkably increased in pc patients ( 3.65-fold
increase in the ct set and 7.35-fold increase in the sh set ) , we added
glycocholic acid manually into the logistic regression model , and
as a result , there is no statistical significance for glycocholic
acid in the model ( p = 0.229 ) ( supporting information table s3 ) .
although our current
assay may stimulate the development of tools
for early diagnosis of pc , there are a number of limitations to consider .
first , the identified markers used to build the logistic regression
model were selected from both the ct and sh data sets .
therefore ,
the marker panel needs to be further validated with a new independent
sample set .
we did not have access to subjects with chronic pancreatitis
and thus our marker panel needs to be examined in such patients to
see how much they differ from pc patients .
third , recent metabolomics
studies of pc have provided metabolite biomarkers that can differentiate pc patients from controls . however , whether
the identified metabolite biomarkers are specific to pc , rather than
involving other malignancies or inflammatory disease in general , is
not clear .
future metabonomics studies need to evaluate the specificity
of metabolite biomarkers for pc versus other malignancies .
fourth ,
we observed the impact of only two ethnic backgrounds on the performance
of diagnostic markers .
a new sample set with more diverse ethnic backgrounds
would be useful to increase generalizability .
in addition , the sensitivity
and specificity of this metabolite panel needs to be compared with
the performance of existing markers , such as ca19 - 9 , in future studies .
in summary , we identified a panel of five plasma metabolite markers
of pc and developed a diagnostic model using logistic regression analysis
of the biomarker panel .
additional studies are still necessary for further evaluation and
validation of the biomarkers identified in the current study
. however ,
this novel approach holds potential to improve patient prognosis by
early detection of pc , when it may still be at a resectable stage . | patients
with pancreatic cancer ( pc ) are usually diagnosed at late
stages , when the disease is nearly incurable .
sensitive and specific
markers are critical for supporting diagnostic and therapeutic strategies .
the aim of this study was to use a metabonomics approach to identify
potential plasma biomarkers that can be further developed for early
detection of pc . in this study , plasma metabolites of newly diagnosed
pc patients ( n = 100 ) and age- and gender - matched
controls ( n = 100 ) from connecticut ( ct ) , usa , and
the same number of cases and controls from shanghai ( sh ) , china , were
profiled using combined gas and liquid chromatography mass spectrometry .
the metabolites consistently expressed in both ct and sh samples were
used to identify potential markers , and the diagnostic performance
of the candidate markers was tested in two sample sets .
a diagnostic
model was constructed using a panel of five metabolites including
glutamate , choline , 1,5-anhydro - d - glucitol , betaine , and
methylguanidine , which robustly distinguished pc patients in ct from
controls with high sensitivity ( 97.7% ) and specificity ( 83.1% ) ( area
under the receiver operating characteristic curve [ auc ] = 0.943 , 95%
confidence interval [ ci ] = 0.9080.977 ) .
this panel of metabolites
was then tested with the sh data set , yielding satisfactory accuracy
( auc = 0.835 ; 95% ci = 0.7770.893 ) , with a sensitivity of
77.4% and specificity of 75.8% .
this model achieved a sensitivity
of 84.8% in the pc patients at stages 0 , 1 , and 2 in ct and 77.4%
in the pc patients at stages 1 and 2 in sh .
plasma metabolic signatures
show promise as biomarkers for early detection of pc . | Introduction
Materials and Methods
Results
Discussion
Conclusions |
PMC5270605 | in teleosts , the sex differentiation pattern may be classified as synchronous hermaphroditism
( protandrous and protogynous ) or gonochorism ( undifferentiated and differrentiated ) ( yamamoto , 1969 ) .
both genetic and environmental factors can
significantly impact the sex determination of fish ( devlin &
nagahama , 2002 ) .
the physicochemical factors relevant to the sex determination of fish
include the water temperature , salinity , and ph ( baroiller et al . ,
1999 ) , while the related chemical factors include polychorinated biphenyls ( psbs ) ,
nonylphenol , bisphenol , and dioxin by endocrine disruptor ( gray &
metcalf , 1997 ) .
to improve the economics of aquaculture , these parameters may be used to
render fish populations artificially mono - sexual ( e.g. , by adjusting the water temperature , controlling
the ph , or applying hormone treatments ) . the korean rose bitterling , rhodeus uyekii , is an indigenous species in korea . falling
within the cypriniformes , cyprinidae , and rhodeus ,
these fish live on the muddy floor
at the edges of reservoirs , plant - filled and/or slow - moving streams , and ponds .
they are distributed in
the watershed of the nakdong river and other streams in the southern part of korea ( jeon , 1982 ) .
the korean rose bitterling breeds between the end of
april and mid - june , and shows special features in breeding in the fresh water bivalves of the family
unionidae by extending the ovipositor in the female ( uchida ,
1939 ; nakamura , 1969 ) .
seen from the side , the body of the
korean rose bitterling is relatively compressed from nose to tail and relatively tall from the caudal to
dorsal aspect , with the male generally being taller than the female . in color , the body is light brown
on the back , darker on the front back of the dorsal fin and the caudal peduncle , and reddish white
silver on the flank .
the male is black on the outside of its rear fin and exhibits a small white spot
inside .
given its excellent visual effect , the
male is far preferred over the female as an aquarium fish , besides the taxonomic group with urgent
preservation due to easy exposure to the recent biological and environmental pollution ( kang et al .
, researchers have studied : the early life history of laboratory - reared
fish ; egg and larval development ; the seasonal sex ratio in the field ; the reproductive cycle of the
spring - spawning bitterling ; osteology ; elongation of the ovipositor ; the temperaturedependent index of
the mitotic interval ( 0 ) for chromosome manipulation ; morphometric traits ; cytogenetics of
induced crosses and reciprocal hybrids between korean rose bitterling and r. notatus ;
and the use of lidocaine hydrochloride and clove oil as an anesthetic ( for both korean rose bitterling
and oily bitterling , acheilognathus koreensis ) ( kim
& han , 1990 ; park & kim , 1990 ; an , 1995 ; kim , 1997 ; chae , 2001 ; kim et al . , 2011 ,
2012 ) .
studies have also examined trends in the early
development of the gonad and the potential for sex change , but the process of sex differentiation in the
korean rose bitterling is not yet fully understood .
here , we investigated the sex differentiation and gonadal development of the korean rose bitterling , and
their dependence on the initial growth and integral water temperature at the constant breeding water
temperature for the hatching fry through the artificial insemination as a part of production of the
monosexual population and the seed production of the korean rose bitterling .
brood stock of the korean rose bitterling , rhodeus uyekii , were obtained from the
inland aquaculture research center , national institute of fisheries science ( nifs ;
changwon , korea ) and housed in a mariculture facility at the fishery genetics and breeding sciences
laboratory of the korea maritime and ocean university in busan , korea .
the fish were maintained in a
glass tank ( w 120 cm l 30 cm h 50 cm ) at a water temperature of 201c until the
experiments were initiated . during the 2013 spawning season ( late april to midjune ) , densely colored males and
ovipositor - extended females were selected and used as brood stock .
eggs were harvested by abdominal
compression of ovipositor - extended females , semen was harvested by abdominal compression of males
and inseminated within 2 minutes .
plastic tanks ( w 30 cm l 20 cm h 15 cm ; fresh plus , mirae
chemical , korea ) were loaded with 200 eggs each and maintained at a water depth of 10 cm and a l : d
ratio of 13:11 until hatching .
the water was replaced twice daily with aerated bottled water
( natural mineral water , pulmuone , korea ) , and the water temperature was maintained at 220.5
oc .
hatched larvae were divided into groups of 10 and distributed to transparent plastic containers ( w 10
cm l 5 cm h 3 cm ; coolrara , easyfilm , korea ) .
the larvae began to feed once the yolk was completely
absorbed , at 21 days post - hatching ( dph ) .
the hatchlings ( n = 20 ) were sampled daily from hatching to 5 dph , at 2-day
intervals from 7 to 21 dph , at 4-day intervals from 25 to 41 dph , at 6-day intervals from 47 to 83
dph , and at 30-day intervals from 110 to 170 dph .
the fish were sampled at different stages and
anesthetized with 300 ppm lidocaine - hcl / nahco3 at 25c according to method of
kang et al .
body weight ( bw ) was measured to the
nearest 0.01 g using an electric balance ( ax 200 , shimadzu corp . ,
japan ) , the total length ( tl ) was
measured to the nearest 0.01 cm using a vernier caliper ( cd-20 cp , mitutoyo , japan ) , and growth was
estimated using an exponential growth curve and the calculation of a condition factor . a mimetic diagram was produced according to the growth for indicate morphological changes .
each
sample of whole fish was fixed in bouin s solution for 24 h , washed thoroughly with water , exposed
to decalcification solution for 24 h , washed again , and dehydrated through a graded alcohol series
( 70% , 80% , 90% , and 100% ethanol 1 h each ) . the samples were then cleared with xylene , impregnated
with soft paraffin , impregnated with hard paraffin , embedded , trimmed , and cut ( 6 m ) .
eosin , mounted with canadian balsam , and
examined / photographed under optical microscopy ( axiocam mr , carl zeiss , germany ) .
the korean rose bitterling , rhodeus uyekii , hatched at two days post - fertilization with
a post - fertilization integral water temperature ( iwt ) of 43c .
fig . 1 , fig . 2 , and 3 present the changes in total length ( tl ) , body weight ( bw ) , condition factor , and
morphology from
just - hatched ( iwt post - hatching 0c ) to 170 days post - hatching ( dph ; iwt
post - hatching 3,740c ) . at hatching ,
the average tl and bw were 6.10.09 ( sd ) mm and 4.90.07
mg , respectively , while at 83 dph , these values were 18.90.28 mm and 48.20.72 mg , respectively ( fig . 1 and fig .
3 ) . tl increased
continuously from hatching to 83 dph , and could be represented by the growth equation :
tl=4.914e ( r = 0.961 ; t , time ) ( fig .
1 , upper panel ) .
bw showed a slight reduction between hatching and the completion of yolk
absorption ( 21 dph , see below ) , but increased rapidly once the hatchling began to feed ; it could be
represented by the growth equation : bw=2.200e ( r=0.725 ) ( fig .
the condition factor decreased from just - hatching to 21 dph ,
but remained steady thereafter ( fig .
yolk absorption was complete at 21 dph ( 9.20.14 mm tl ,
4.80.07 mg bw ; 462c iwt post hat ching ) ( fig .
abbreviations : tl , total length ; bw , body weight ; and t , time .
condition factor =
( a / b)1,000 where a is bw and
b is tl .
bars = 3 mm . a : at 17 dph , 374c iwt ; inset shows a high - power view of a primordial germ cell ( pgc ; inset bar
= 10 m ) .
abbreviations : ab , air bladder ; cc , condensed chromatin ; ec ,
endoovarian canal ; g , gut ; gr , genital ridge ; m , mesentery ; mf , me - iotic figure ; mp , meiotic
prophase ; mpgc , mitotic primordial germ cell ; pgc , primordial germ cell ; sc , somatic cell ; sd ,
sperm duct ; sg , spermatogonia ; and y , yolk .
bars = 20 m . at 17 dph ( 7.90.12 mm tl , 3.70.06 mg bw , 374c iwt ) ( fig .
2-g ) when the first feeding
commenced following complete absorption of the yolk , gonads became apparent on both sides , running
beneath the air bladder mesentery to the genital ridge in the abdominal cavity ( fig .
the primordial germ cells ( pgcs ) were oval , approximately 7.2 m in
diameter , and enclosed by loose connective tissue .
their cytoplasm was weakly stained by hematoxylin ,
and they contained round nuclei of 2 m in diameter ( fig .
3a
insert ) . by 19 dph ( 8.20.12 mm tl , 3.60.05 mg bw , 418c iwt ) ( fig .
2-h ) the number of pgcs was increased and numerous mitotic pgcs and condensed chromatin
were apparent ( fig .
3b ) . at 21 dph ( fig . 2-i : 9.20.14 mm tl , 4.80.07 mg bw ,
462c iwt ) , some of the pgcs contained condensed chromatin ; oocytes were observed in meiotic
prophase ; and the pgcs and oocytes were enclosed by somatic cells ( fig .
2-k : 11.20.17 mm tl , 10.5 0.16 mg
bw , 726c iwt ) , numerous oocytes were observed in meiotic prophase , meiotic figures were
present , and the oocytes were in the chromatin - nucleolus stage , with blood vessels and endo - ovarian
canals beginning to fill the ovary ( fig .
13.60.20 mg bw , 1,166c iwt ) ,
the oocytes were in the perinucleolus stage and had increased to 50 m in diameter ( fig .
perinucleolus - stage oocytes increased in number and size to 83 dph . from
110 to 170 dph ,
abbrevia - tions : ec , endoovarian canal ; pno , oocyte in
the perinucleolus stage ; sc , somatic cell ; sg , spermatogonia ; smc , spermatocyte .
bars : in a and b = 30 m ; in c = 500 m ; and in d = 20 m .
the korean rose bitterling appeared to begin its sex differentiation when yolk absorption was completed
and the first feeding began ( 21 dph ) . at 25 dph ( fig .
2-j :
10.10.15 mm tl , 5.40.08 mg bw , 550c iwt ) , spermatogonia could be distinguished from pgcs
( fig .
3d ) . at 33 dph ( fig .
2-k : 11.20.17 mm tl , 10.50.16 mg bw , 726c iwt ) , the spermatogonia had increased
in number ( fig .
no significant development of the testes was
seen up to 83 dph ( 18.9 mm tl , 48.2 mg bw , 1,826c iwt ) .
spermatocytes and sperm cells were
first observed at 140 dph ( 31.2 mm tl , 265.8 mg bw , 3,080c iwt ) and continued to mature
through 170 dph ( fig .
these observations indicate that the
korean rose bitterling belongs to the differentiated type of gonochoristic teleosts ( fig .
the present study shows that although the tl of the korean rose bitterling , rhodeus
uyekii , increased continuously post - hatching , the bw decreased somewhat until yolk
absorption was complete and feeding began ( at 21 dph ) .
this likely reflects the dependence of the
metabolism on energy from the yolk rather than the intake of exogenous food .
the sex differentiation of teleostei varies and can show irregular features , and many gonochoric fishes ,
including some hermaphroditic fishes , easily change sex ( devlin &
nagahama , 2002 ) . in aquaculture , this has been used to produce monosexual populations with
improved economics .
thus , we need to fundamentally understand the sex differentiation and sex change of
fishes ( park et al . , 2004 ) . here , the sex differentiation of the
korean rose bitterling was analyzed with respect to tl and bw at various dph .
result of gonadogenesis
and sex differentiation in european eel , anguilla anguilla and bagrid catfish ,
pseudobagrus fulvidraco .
these results relatively correct better than pre - vious
studies based on simply dph ( colombo & grandi , 1996 ; park et al . , 2004 ) .
moreover , as include the integral water
temperature for check factor in this study , it will be more correctly standard than previous
studies .
those of the tilapia ,
tilapia zillii , appear at hatching ; those of the rainbow trout , salmo
gairdneri , are first seen at 36 dph under the mesonephric duct ; those of the ussurian
bullhead , leiocassis ussuriensis , and black bullhead , p. koreanus ,
appear at 1 dph ; those of the ko - rean bullhead appear at 3 dph ; and those of the chinese minnow ,
moroco oxycephalus , appear at 8 dph under the air bladder mesentery ( yoshikawa & oguri , 1978 ; takashima et al .
, 1980 ; park et al . , 1998 , 2001 , 2004 , 2008 ) .
the present study shows that the pgcs of the korean rose
bitterling first appear at 17 dph under the air bladder mesentery , and formation genital ridge thus
confirmed early gonad .
undifferentiated gonads of fish during gonadal differentiation in the ovary or testis differentiation
into different duct can be determined by forming patterns .
ovarian differentiation in the korean rose
bitterling began at 21 dph ( 9.20.14 mm tl , 4.80.07 mg bw , 462c iwt ) , as indicated by the
appearance of meiotic prophase oocytes .
the timing of sex differentiation differs by species , beginning
at 40 dph ( 326c iwt ) in the cherry salmon , oncorhynchus masou , but at
( 270c iwt ) in the coho salmon , o. kisutch ( pifferrer & donaldson , 1989 ; park et al . ,
however , there seems to be some consistency in that the sex differentiation of various
fish species begins at the time the yolk sac is completely absorbed and the first feeding occurs , such
as in the rainbow trout , the cherry salmon ( 40 dph ) ( park et al .
,
1997 ; van den hurk & solf , 1981 ) , and the korean
rose bitterling ( 21 dph ; this work ) .
the sex differentiation of teleosts can be grouped at the level of species or family , with the process
occurring at 10 ~40 dph in cichlids , 10~30 dph in cyprinodontids , and 3~40 dph in anabantids ( yamazaki , 1976 ; shelton &
jensen , 1979 ; pandian & sheela , 1995 ) . the sex
differentiation of the ussurian bullhead occurs at 15~20 dph and that of the korean bullhead occurs in a
similar range of 12 - 20 dph , while those of the mandarin fish , siniperca scherzeri , and
cherry salmon begin somewhat later , at 40 dph ( park et al . , 1997 ,
2001 , 2004 ) .
the
present study shows that the sex differentiation of the korean rose bitterling occurs at 21~25 dph , with
ovaries first seen at 21 dph and testes observed at 25 dph . in some fishes , such as the ocean perch
,
diterma temmincki , testicular differentiation is seen first ; however , most fish
show initial differentiation of the ovary ( takashima et al . ,
1980 ;
lee & lee , 1996 ; park et al . , 2004 ) , as observed here for the korean rose bitterling .
the entovarian sac forms at the center of the ovarian
cavity , while the parovarian sac forms on the edge of ovary ( lee &
lee , 1996 ) . the sweet fish , plecoglossus altivelis , and the threespine
stickleback , gasterosteus aculeatus , reportedly form parovarian sacs ( bang et al . , 2000 ) , while the white spotted char , salvelinus
leucomaenis , the rainbow trout , and the ussurian bullhead reportedly form entovarian sacs .
the present study shows that the korean rose bitterling forms its sacs in the middle of the ovary
pattern , indicating that this species forms entovarian sacs ( takashima
et al . , 1980 ; park et al . , 2001
the observation that the korean rose bitterling began ovarian differentiation at 21 dph and testicular
differentiation at 25 dph indicates that this species exhibits gonochorism . in this , the korean rose
bitterling resembles the rainbow trout , the chum salmon , o. keta , the medaka ,
oryzias latipes , the ussurian bullhead , the chinese minnow , and the ocean perch
( robertson , 1953 ; tuzuki et
al .
, 1966 ; yoshikawa & oguri , 1978 ; takashima et al . , 1980 ;
lee &
lee , 1996 ; park et al . , 1998 , 2001 ) .
the korean rose bitterling is endemic species to korea and the importance in the resources enforcement
has been emerged ( jeon , 1982 ) .
therefore , it is important that we
understand the timing of sex differentiation timing and the sex ratio in this species .
future studies
should focus on tracking the differentiation and development of the gonad in the korean rose bitterling
to see how the sex ratio and timing of sex differentiation can be altered by artificial factors ( e.g. ,
hormones and environmental factors ) .
the results of the present study could be utilized as fundamental
data for the resources proliferation and the ecological changes in this species . | abstractthis report describes the sex differentiation of the korean rose bitterling , rhodeus
uyekii , from hatching to 170 days post - hatch ( dph ) in relation to total length ( tl ) ,
body weight ( bw ) , and integral water temperature ( iwt ) .
the growth curve of tl from just hatching to
83 dph was 5.144e0.045 t ( r = 0.961 ; t , time ) , and that of bw was
2.398e0.086 t ( r = 0.725 ) .
primordial germ cells ( pgcs ) were
observed at 17 dph ( 7.9 mm tl , 3.74 mg bw , 374c iwt ) , and thereafter began to protrude into the
peritoneal cavity . at 21 dph ( 9.20.14 mm tl , 4.80.07 mg bw , 462c iwt ) ,
some pgcs contained
condensed chromatin and oocyte were observed in meiotic prophase .
in contrast to the ovaries , which
grew gradually after sexual differentiation , testes began multiplying at 25 dph ( 10.1 mm tl , 5.42 mg
bw , 550c iwt ) , when testicular differentiation was first identified , and multiplied continuously
thereafter . at 33 dph ( 11.2 mm tl , 10.5 mg bw , 726c iwt ) ,
the developing testes contained
spermatogonia that exhibited mitotic activity .
no spermatocyte or sperm cell was observed until 83
dph ( 18.9 tl , 48.2 mg bw , 1,826c iwt ) . at 170 dph ( 32.5 mm tl , 270.1 mg bw , 3,740c iwt ) , which was
the end point of this study , the mature ovaries showed germinal vesicle breakdown , while the mature
testes contained observable spermatocytes and sperm cells .
these results allow us to identify the
sex differentiation type of the korean rose bitterling as differentiated gonochoristic . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION |
PMC3916837 | as mentioned before , the increase in phase measurement sensitivity in the nci is not due to noise reduction but due to signal enhancement . in order to demonstrate this ,
we experimentally compare it with a ci , which works at the snl . as the snl is related to the phase - sensing photon number .
the schematic diagrams of an su(1,1 ) nci and a conventional mz interferometer ( ci ) are shown in fig .
beam splitters are replaced by parametric amplifiers ( pa1 , pa2 ) , which amplify the incoming
idler input field is labelled as and is in vacuum . in the present realization of nci ,
the parametric amplifier is based on non - degenerate four - wave mixing in hot rb-85 atomic vapour cells29 ( see methods for the details of the experimental arrangement ) .
output relation for a parametric amplifier is given by : where g is the amplitude gain and g = g1 .
with an overall phase shift in the idler arm , we obtain the output fields of the interferometer : with ( )=ge+g , ( )=gg(1+e ) as the phase - sensitive gains . for a coherent state | injected in the signal input and vacuum in the idler input
, we obtain the output intensity in the idler port or one of the outputs of the nci as where g(+1 ) is the intensity of the phase - sensing field ( idler field in fig .
1a ) with || as the input intensity . for > > 1 , g . a conventional mz interferometer shown in fig .
1b , on the other hand , has a fringe given by with 50:50 beam splitters and a coherent state | input .
since the sensitivity of an interferometer is related to the photon number inside the interferometer , for a fair comparison between nci and ci , we set = , which can be achieved by adjusting , independently .
therefore , we see from equations ( 3 ) and ( 4 ) that , under the condition of the same phase - sensing intensity , the output of the nci is 2 g times that of the ci .
however , as is well known , for any amplifier , the amplification of signal is also accompanied by the amplification of the noise .
so , the signal - to - noise ratio at best does not change in the process31 .
fortunately , it is not the case for the nci discussed here . from equation ( 2 ) , at dark fringe when =(2n+1) , we have . however , since is in vacuum , the output of the nci is at vacuum noise level , just like the ci at dark fringe .
thus , the signal in the nci is increased while the noise level is not and we should expect an enhancement of 2 g in signal - to - noise ratio of the nci over the ci .
what happens here for the nci is that although each amplifier increases signal and noise together , the first parametric amplifier ( pa1 ) produces two fields ( two arms of the nci ) that are quantum mechanically entangled323334 so that their noises are also correlated , and a destructive interference occurs at the second parametric amplifier ( pa2 ) when the interferometer is operated at dark fringe so that most of the amplified noise from pa1 are cancelled and only the vacuum noise is left35 .
therefore , the nci increases the signal while it keeps the noise unchanged , leading to an enhancement in signal - to - noise ratio for phase measurement as compared with the ci .
figure 1c shows the interference fringes observed for both nci and ci with the same phase - sensing intensity = .
the fringe size of the nci is bigger than that of the ci because of the amplification at pa2 , which leads to an enhancement in the phase signal .
when operated at dark fringe with a small phase change : =(2n+1)+ ( <<1 , n = integer ) , we obtain from equations ( 3 ) and ( 4 ) the change in the output intensities of the two interferometers due to as : so , the signal is enhanced by bsinci/ici=2 g for the same and = . the fringe enhancement in fig
the noise performance of the nci is measured by homodyne detection ( hd ) method at the idler output port , as shown in fig .
2c shows the noise level at the output of the nci when the phase is scanned .
the minimum noise occurs at dark fringe when there is a destructive quantum interference at pa2 .
this is in contrast to the trace in yellow when one arm of the nci is blocked and there is no interference effect .
the trace in yellow corresponds to two uncorrelated amplifiers in series , as depicted in fig .
respectively show the noise levels for individual amplifiers when the other one is not active .
notice that at some phases , the output noise level ( the trace in dark blue ) of the nci is even lower than the amplified noise levels of individual amplifiers ( the traces in green and dark brown ) as a result of noise cancelation .
2c respectively show the measured noise levels at dark fringe setting for the nci and the ci under the same condition when the fringes in fig .
the observed noise level for the nci is 3.30.3 db higher than that of the ci .
this is not what is predicted in previous paragraphs , that is , the noise level of the nci is the same as that of the ci at the vacuum noise level .
the higher measured output noise of the nci is due to internal losses of the interferometer ( more on this later ) .
30 that the signal due to a small phase shift in hd of an interferometer has the same form as that in direct intensity detection ) , we still obtain an enhancement of 4.10.3 db in signal - to - noise ratio for the nci over the ci , that is , snrnci / snrci=4.1 db=2.5 . from equation
( 5 ) , we know that the signal is proportional to , whereas the noise measured in the experiment is the noise power .
so , we have the phase measurement sensitivity as in rms value . as the ci has a phase measurement sensitivity at snl3 , our nci beats the shot noise level by a factor of = 1.6 in rms value .
the simple theory in the previous part does not include losses in the system and predicts the continuous increase of the snr with the gain of the amplifiers .
losses , however , will introduce extra vacuum noise , which is also amplified by pa1 and pa2 but is not correlated and can not be cancelled , thus leading to higher noise level for the nci than the ci but at a manageable level , as observed in fig .
2c . the extra amplified vacuum noise will increase with the gain of the amplifiers in the same way as the phase signal .
3 where we plot in blue diamond shape the ratio of the output noise level of the interferometer at dark fringe ( the light blue trace in fig .
2c ) as a function of the average noise gain of individual amplifiers ( the traces in green and dark brown in fig .
since the vacuum noise level is also the output noise level of the ci at dark fringe , this is also the noise level ratio of the nci to the ci . shown also in fig .
3 in black square shape are the ratio of the signal level of the nci to that of the ci , or the signal enhancement factor bs extracted from the fringes similar to fig . 1c .
therefore , the ratio of the black square to the blue diamond gives the enhancement of the signal - to - noise ratio of the nci over the ci , which is shown as red triangle in fig .
3 . as seen in the figure , both the signal enhancement factor ( black ) and the noise level ( blue ) at the output increase with the amplifier gain in about the same rate at high gain .
so , the signal - to - noise improvement factor ( red ) levels out at a gain of ~4 db , in qualitative agreement with ref . 30
next , we characterize the effect of losses on the performance of the nci by introducing some extra losses .
first , we place an attenuator with loss l in front of the homodyne detector and vary the attenuation .
the signal enhancement factor bs and noise n above vacuum noise level after the attenuator will change with the loss l by where bs0 and n0 are the respective values before the attenuator .
so , the signal - to - noise ratio after the attenuator becomes the solid lines in fig .
4a are fitted to equations ( 6 ) and ( 7 ) with bs0=7.5 db and n0=3.2 db .
so , the signal - to - noise improvement factor does not change much at small loss for large n0 .
this is well known for amplification process : the amplified noise is above the vacuum level so that the noise will decrease under loss just like the signal , making snr less sensitive to external losses .
this is quite different from the squeezed state scheme56 in which the improvement will suffer under loss . for large loss , however , snr will drop with the increase of the loss because the noise level is mostly vacuum noise already and wo n't decrease further . for the effect of internal loss , we place a variable attenuator in the paths of both arms of the nci .
we need to readjust the gain of pa2 to re - balance the interferometer so that it produces fringe visibility close to 100% . in the meantime , gain balancing also leads to optimized output noise levels shown fig .
4b , the output noise level ( blue diamond ) increases as the loss increases more uncorrelated vacuum noise enters the interferometer and gets amplified .
therefore , the improvement factor in signal - to - noise ratio decreases more quickly with internal loss ( fig .
the effect of internal loss on the nci is complicated so we can not fit the data to a theoretical prediction .
as mentioned before , the increase in phase measurement sensitivity in the nci is not due to noise reduction but due to signal enhancement . in order to demonstrate this ,
we experimentally compare it with a ci , which works at the snl . as the snl is related to the phase - sensing photon number .
the schematic diagrams of an su(1,1 ) nci and a conventional mz interferometer ( ci ) are shown in fig .
beam splitters are replaced by parametric amplifiers ( pa1 , pa2 ) , which amplify the incoming
idler input field is labelled as and is in vacuum . in the present realization of nci ,
the parametric amplifier is based on non - degenerate four - wave mixing in hot rb-85 atomic vapour cells29 ( see methods for the details of the experimental arrangement ) .
output relation for a parametric amplifier is given by : where g is the amplitude gain and g = g1 .
with an overall phase shift in the idler arm , we obtain the output fields of the interferometer : with ( )=ge+g , ( )=gg(1+e ) as the phase - sensitive gains . for a coherent state | injected in the signal input and vacuum in the idler input
, we obtain the output intensity in the idler port or one of the outputs of the nci as where g(+1 ) is the intensity of the phase - sensing field ( idler field in fig .
1a ) with || as the input intensity . for > > 1 , g . a conventional mz interferometer shown in fig .
1b , on the other hand , has a fringe given by with 50:50 beam splitters and a coherent state | input .
since the sensitivity of an interferometer is related to the photon number inside the interferometer , for a fair comparison between nci and ci , we set = , which can be achieved by adjusting , independently .
therefore , we see from equations ( 3 ) and ( 4 ) that , under the condition of the same phase - sensing intensity , the output of the nci is 2 g times that of the ci .
however , as is well known , for any amplifier , the amplification of signal is also accompanied by the amplification of the noise .
so , the signal - to - noise ratio at best does not change in the process31 .
fortunately , it is not the case for the nci discussed here . from equation ( 2 ) , at dark fringe when =(2n+1) , we have . however , since is in vacuum , the output of the nci is at vacuum noise level , just like the ci at dark fringe .
thus , the signal in the nci is increased while the noise level is not and we should expect an enhancement of 2 g in signal - to - noise ratio of the nci over the ci .
what happens here for the nci is that although each amplifier increases signal and noise together , the first parametric amplifier ( pa1 ) produces two fields ( two arms of the nci ) that are quantum mechanically entangled323334 so that their noises are also correlated , and a destructive interference occurs at the second parametric amplifier ( pa2 ) when the interferometer is operated at dark fringe so that most of the amplified noise from pa1 are cancelled and only the vacuum noise is left35 .
therefore , the nci increases the signal while it keeps the noise unchanged , leading to an enhancement in signal - to - noise ratio for phase measurement as compared with the ci .
figure 1c shows the interference fringes observed for both nci and ci with the same phase - sensing intensity = .
the fringe size of the nci is bigger than that of the ci because of the amplification at pa2 , which leads to an enhancement in the phase signal .
this is the key difference between an nci and a ci . when operated at dark fringe with a small phase change : =(2n+1)+ ( <<1 , n = integer ) , we obtain from equations ( 3 ) and ( 4 ) the change in the output intensities of the two interferometers due to as : so , the signal is enhanced by bsinci/ici=2 g for the same and = . the fringe enhancement in fig .
the noise performance of the nci is measured by homodyne detection ( hd ) method at the idler output port , as shown in fig .
2c shows the noise level at the output of the nci when the phase is scanned .
the minimum noise occurs at dark fringe when there is a destructive quantum interference at pa2 .
this is in contrast to the trace in yellow when one arm of the nci is blocked and there is no interference effect .
the trace in yellow corresponds to two uncorrelated amplifiers in series , as depicted in fig .
respectively show the noise levels for individual amplifiers when the other one is not active .
notice that at some phases , the output noise level ( the trace in dark blue ) of the nci is even lower than the amplified noise levels of individual amplifiers ( the traces in green and dark brown ) as a result of noise cancelation .
2c respectively show the measured noise levels at dark fringe setting for the nci and the ci under the same condition when the fringes in fig .
the observed noise level for the nci is 3.30.3 db higher than that of the ci .
this is not what is predicted in previous paragraphs , that is , the noise level of the nci is the same as that of the ci at the vacuum noise level .
the higher measured output noise of the nci is due to internal losses of the interferometer ( more on this later ) .
30 that the signal due to a small phase shift in hd of an interferometer has the same form as that in direct intensity detection ) , we still obtain an enhancement of 4.10.3 db in signal - to - noise ratio for the nci over the ci , that is , snrnci / snrci=4.1 db=2.5 . from equation
( 5 ) , we know that the signal is proportional to , whereas the noise measured in the experiment is the noise power .
so , we have the phase measurement sensitivity as in rms value . as the ci has a phase measurement sensitivity at snl3 , our nci beats the shot noise level by a factor of = 1.6 in rms value .
the simple theory in the previous part does not include losses in the system and predicts the continuous increase of the snr with the gain of the amplifiers .
losses , however , will introduce extra vacuum noise , which is also amplified by pa1 and pa2 but is not correlated and can not be cancelled , thus leading to higher noise level for the nci than the ci but at a manageable level , as observed in fig .
2c . the extra amplified vacuum noise will increase with the gain of the amplifiers in the same way as the phase signal .
3 where we plot in blue diamond shape the ratio of the output noise level of the interferometer at dark fringe ( the light blue trace in fig .
2c ) as a function of the average noise gain of individual amplifiers ( the traces in green and dark brown in fig .
since the vacuum noise level is also the output noise level of the ci at dark fringe , this is also the noise level ratio of the nci to the ci . shown also in fig .
3 in black square shape are the ratio of the signal level of the nci to that of the ci , or the signal enhancement factor bs extracted from the fringes similar to fig . 1c .
therefore , the ratio of the black square to the blue diamond gives the enhancement of the signal - to - noise ratio of the nci over the ci , which is shown as red triangle in fig .
3 . as seen in the figure , both the signal enhancement factor ( black ) and the noise level ( blue ) at the output increase with the amplifier gain in about the same rate at high gain .
so , the signal - to - noise improvement factor ( red ) levels out at a gain of ~4 db , in qualitative agreement with ref . 30 .
next , we characterize the effect of losses on the performance of the nci by introducing some extra losses .
first , we place an attenuator with loss l in front of the homodyne detector and vary the attenuation .
the signal enhancement factor bs and noise n above vacuum noise level after the attenuator will change with the loss l by where bs0 and n0 are the respective values before the attenuator .
so , the signal - to - noise ratio after the attenuator becomes the solid lines in fig .
4a are fitted to equations ( 6 ) and ( 7 ) with bs0=7.5 db and n0=3.2 db .
so , the signal - to - noise improvement factor does not change much at small loss for large n0 .
this is well known for amplification process : the amplified noise is above the vacuum level so that the noise will decrease under loss just like the signal , making snr less sensitive to external losses .
this is quite different from the squeezed state scheme56 in which the improvement will suffer under loss . for large loss , however , snr will drop with the increase of the loss because the noise level is mostly vacuum noise already and wo n't decrease further . for the effect of internal loss , we place a variable attenuator in the paths of both arms of the nci . we need to readjust the gain of pa2 to re - balance the interferometer so that it produces fringe visibility close to 100% . in the meantime , gain balancing also leads to optimized output noise levels shown fig .
4b , the output noise level ( blue diamond ) increases as the loss increases more uncorrelated vacuum noise enters the interferometer and gets amplified .
therefore , the improvement factor in signal - to - noise ratio decreases more quickly with internal loss ( fig .
the effect of internal loss on the nci is complicated so we can not fit the data to a theoretical prediction
we have demonstrated an su(1,1 ) nci with 4 db improvement in signal - to - noise ratio in phase measurement over a ci operated under the same condition .
the improvement is mostly limited by the loss inside the interferometer and is less sensitive to external loss than the squeezed state method .
the improvement in signal - to - noise ratio comes from the increase in signal rather than the reduction of noise .
furthermore , if we inject squeezed states into the unused idler input port of pa1 ( see fig .
1a ) , reduction of the noise of the interferometer is possible , leading to further improvement of signal - to - noise ratio . therefore , the current scheme and the squeezed state scheme are complementary to each other . notice that our nci involves amplification of the input field whereas a conventional mz interferometer ( ci ) does not .
however , according to refs 21 , 25 it is the photon number of the phase probing field that counts for the limit of sensitivity .
therefore , when we compare the performance of our nci with the ci , we make sure that the light fields probing the phase change have the same intensity in both nci and ci .
when we reduce the intensity of the injected coherent state , this interferometer will approach to the originally proposed su(1,1 ) interferometer by yurke et al.19 , which can have a phase measurement sensitivity reaching the heisenberg limit .
3 , the internal loss of the interferometer is the main limiting factor in achieving higher sensitivity at higher gain .
30 and demkowicz - dobrzanski et al.36 then , it is natural to ask what kind of phase measurement sensitivity limit we can reach in our new interferometer . to answer that
unfortunately , our measurement technique , that is , the hd method , does not allow us to perform an analysis with fisher information method , which requires photon counting technique .
phase estimate with fisher information method for this new type of interferometer will be the topic in future researches .
what we did in current work is to compare the performance of our new interferometer with a conventional mz interferometer , which has the well - known snl ( 1/-dependence for phase measurement uncertainty ) , and show a 4-db improvement in signal - to - noise ratio .
the use of a nonlinear process to mix the signal and idler fields means that the two fields may have different frequencies . indeed , in our experiment , the frequency difference between the signal and the idler fields is ~6 ghz .
this opens up a new way to construct hybrid interferometers involving different waves and thus broadens the scope of precision measurement involving interferometry .
5 . the two parametric amplifiers ( pa1 , pa2 ) employed for the nci as shown in fig .
1a are based on non - degenerate four - wave mixing process in two hot rb-85 atomic vapour cells temperature - stabilized at 117 c ( ref .
29 ) ; see inset of fig . 5 for energy diagram of relevant components ) .
they are respectively pumped by a vertically polarized beam ( p1 , p3 ) at a maximum power of 400 mw with a waist of 500 m .
the pump beams are from a ti : sapphire laser frequency - stabilized to a stable reference cavity .
the pump beams are detuned about 0.8 ghz above the transition line of rb-85 f=2f at 795 nm .
the pump power can be adjusted with a combination of polarization beam splitters ( pbs ) and half - wave plates . a horizontally polarized seed beam ( also known as
signal ) with waist of 200 m is injected with an angle of 0.4 relative to the pump .
signal beam is 3.04 ghz red shifted from the pump beam by an acousto - optic modulator in double - pass configuration .
the pump and signal beams are combined with a glan - laser pbs before being sent into the atomic cell . at the output of the atomic cell , the seed signal beam
is amplified with a gain that depends on the power and frequency detuning of the pump beam .
the amplification of the signal beam is accompanied by a conjugate beam ( called idler ) with a frequency of 3.04 ghz blue shift from the pump at the other side of the pump beam .
the second cell is identical to the first one except that both the signal and the idler fields have inputs that are from the first cell .
a 4-f imaging system ( l1 , l2 ) is used to mode match the fields of the two cells .
a ci of mz type is formed in the path of the idler field with a pair of flipping pbs ( pbs1 , pbs2 ) and a beam - redirecting mirror . in this way
we block the signal arm and the pump to cell 2 ( p3 ) when we operate the ci .
a pzt - mounted mirror ( pzt ) is placed in both interferometers as the phase modulator so that we can compare the performance of both interferometers under the same phase change .
since the pbs splits the idler beam into two equal parts in the ci and reduces the phase - sensing intensity to half , we need to increase the injected signal at cell 1 to bring the phase - sensing intensity of the ci to the same level as that of the nci ( ips=60 w with iseed=16 w ) . a regular silicon photo - detector ( d ) is placed in the idler output port of the nci , which is also the output port for the ci .
this detector records the fringes of the nci and the ci in a digital scope , as shown in fig .
the noise performance of the nci is measured by hd ( with a 98% homodyne efficiency ) at the output of the idler side of the nci under different circumstances .
the local oscillator of 726 w for hd is obtained from cell 1 with another seed ( 196 w ) and pump ( p2=300 mw)34 .
the local oscillator goes through the same imaging system to match its mode with those in the interferometer .
5 . the two parametric amplifiers ( pa1 , pa2 ) employed for the nci as shown in fig .
1a are based on non - degenerate four - wave mixing process in two hot rb-85 atomic vapour cells temperature - stabilized at 117 c ( ref .
29 ) ; see inset of fig . 5 for energy diagram of relevant components ) .
they are respectively pumped by a vertically polarized beam ( p1 , p3 ) at a maximum power of 400 mw with a waist of 500 m .
the pump beams are from a ti : sapphire laser frequency - stabilized to a stable reference cavity .
the pump beams are detuned about 0.8 ghz above the transition line of rb-85 f=2f at 795 nm .
the pump power can be adjusted with a combination of polarization beam splitters ( pbs ) and half - wave plates . a horizontally polarized seed beam ( also known as
signal ) with waist of 200 m is injected with an angle of 0.4 relative to the pump .
signal beam is 3.04 ghz red shifted from the pump beam by an acousto - optic modulator in double - pass configuration .
the pump and signal beams are combined with a glan - laser pbs before being sent into the atomic cell . at the output of the atomic cell , the seed signal beam
is amplified with a gain that depends on the power and frequency detuning of the pump beam .
the amplification of the signal beam is accompanied by a conjugate beam ( called idler ) with a frequency of 3.04 ghz blue shift from the pump at the other side of the pump beam .
the second cell is identical to the first one except that both the signal and the idler fields have inputs that are from the first cell .
a 4-f imaging system ( l1 , l2 ) is used to mode match the fields of the two cells .
a ci of mz type is formed in the path of the idler field with a pair of flipping pbs ( pbs1 , pbs2 ) and a beam - redirecting mirror . in this way
we block the signal arm and the pump to cell 2 ( p3 ) when we operate the ci .
a pzt - mounted mirror ( pzt ) is placed in both interferometers as the phase modulator so that we can compare the performance of both interferometers under the same phase change .
since the pbs splits the idler beam into two equal parts in the ci and reduces the phase - sensing intensity to half , we need to increase the injected signal at cell 1 to bring the phase - sensing intensity of the ci to the same level as that of the nci ( ips=60 w with iseed=16 w ) . a regular silicon photo - detector ( d ) is placed in the idler output port of the nci , which is also the output port for the ci .
this detector records the fringes of the nci and the ci in a digital scope , as shown in fig .
the noise performance of the nci is measured by hd ( with a 98% homodyne efficiency ) at the output of the idler side of the nci under different circumstances .
the local oscillator of 726 w for hd is obtained from cell 1 with another seed ( 196 w ) and pump ( p2=300 mw)34 .
the local oscillator goes through the same imaging system to match its mode with those in the interferometer . | conventional interferometers usually utilize beam splitters for wave splitting and recombination .
these interferometers are widely used for precision measurement .
their sensitivity for phase measurement is limited by the shot noise , which can be suppressed with squeezed states of light . here
we study a new type of interferometer in which the beam splitting and recombination elements are parametric amplifiers .
we observe an improvement of 4.10.3 db in signal - to - noise ratio compared with a conventional interferometer under the same operating condition , which is a 1.6-fold enhancement in rms phase measurement sensitivity beyond the shot noise limit .
the improvement is due to signal enhancement .
combined with the squeezed state technique for shot noise suppression , this interferometer promises further improvement in sensitivity .
furthermore , because nonlinear processes are involved in this interferometer , we can couple a variety of different waves and form new types of hybrid interferometers , opening a door for many applications in metrology . | Results
Comparison between different types of interferometers
Experimental results
Discussions
Methods
Detailed experimental arrangement
Author contributions
Additional information |
PMC4328094 | approximately 20 to 30% of the patients with epilepsy , ultimately develop refractory seizures that do not respond adequately to pharmacological treatment ( 1 ) .
these patients have only a small chance of obtaining seizure control with one of the established anticonvulsant drugs .
one of the groups of drugs that have come into focus recently is the group of diuretics ( 2 ) .
although the first reports of anticonvulsant effects of the loop diuretic furosemide date back nearly thirty years ( 3 , 4 ) , it was discovered only a decade ago that furosemide in high concentrations shows anticonvulsant properties in vitro ( 5 ) . in the following , anticonvulsant effects of diuretics , particularly of furosemide ,
were shown using a variety of in vitro models of epilepsy ( 5 - 8 ) . at the same time
, there was epidemiological evidence that diuretic use could protect from the risk of new - onset seizures in a community - based sample of elderly patients ( 9 ) .
most investigators have focused on furosemide , most likely because of its high diuretic potency and the availability of solutions for intravenous or intraperitoneal injections .
this is in contrast to the fact that in the population - based study as well as in the maximum electroshock model , the protective effect of thiazide diuretics was stronger than of furosemide ( 9 ) .
in addition , it has been shown that hct as well as other benzothiazide derivates potentiate the effect of low - dose phenytoin on the seizure threshold in the maximum electroshock model .
the same study also showed that there is a tendency to elevate the seizure threshold by hct alone ( 10 ) .
the mechanisms of anticonvulsant effect of hct remain to be elucidated . whereas , there is increasing evidence that furosemide has direct effect on neurons and glia via the electroneutral na - k-2cl- co - transporters ( nkcc ) that modulate intracellular chloride concentration and cell volume ( 11 , 12 ) .
thiazides do not have influence on the nkcc or the electroneutral k - cl - cotransporters ( kcc ) , either .
carbonic anhydrase ( ca ) regulates the diffusion of hco3- and protons across the cell membrane ( 2 , 15 ) and its inhibition increases the hyperpolarizing effect of long - lasting gaba - a currents ( 16 ) .
thus , the most favoured hypothesis is that the epidemiological effect of seizure protection by hct intake may at least partially be an effect of ca inhibition ( 2 ) .
no in vitro experiments investigating the anticonvulsant properties and mechanisms of thiazide diuretics in epilepsy models have been published so far .
the aim of the present study was to evaluate whether hydrochlorothiazide ( hct ) , probably the most common used thiazide diuretic , plays anticonvulsant role in brain tissues in vitro .
furthermore , we investigated if hct could pass blood - brain - barrier ( bbb ) to clear if its effect on cns is a neuronal modulatory action or a systemic effect .
the experiments were performed on rat hippocampal slices . they have been performed according to national guidelines for animal research .
after introduction of deep pentobarbital anesthesia , the animal was decapitated and the brain was removed .
then the slices were preincubated in artificial cerebrospinal fluid ( acsf ) at 28c for 1
the preincubation acsf contained ( in mmol / l ) : nacl 124 , kcl4 , cacl2 1.0 , nah2po4 1.24 , mgso4 1.3 , nahco3 26 , and glucose 10 .
after preincubation , cacl2 was elevated to 2.0 mmol / l and the slices were transferred into an interphase - type recording chamber . in the experimental chamber , the temperature was increased to 32c . during the experiments , the ph , the bath temperature and the flow rate ( 1.5 to 2 ml / min ) remained constant . for the preparation of the zero - magnesium solution , we omitted mgso4 .
hydrochlorothiazide ( hct ) solution was prepared by adding dimethylsulfoxide ( 0.5 ml per mmol / l of the crystalline hct substance ) to achieve water solubility .
extracellular field potential recordings were recorded with glass microelectrodes ( resistance 2 - 5 m ) in the stratum radiatum of the ca1 and ca3 hippocampal subregions connected with dc amplifiers .
extracellular recordings were obtained using a custom made differential amplifier ( with band - pass filters at 0.5 - 10 khz , sampling rate 0.3 - 100 hz ) . the reference electrode and
field potentials were recorded by an ink - writer ( capable to record between 10 v ) as well as by a digital oscilloscope .
traces were digitized by nicolet 460 digitizing oscilloscope ( nicolet instruments ) and analyzed with accompanying software .
a continuous single pulse of electrical stimulation was applied through a bipolar platinum electrode attached to the schaffer collaterals of hippocampal slices .
evoked field excitatory post - synaptic potentials ( fepsp ) were recorded in hippocampal ca1 region ( stratum radiatum ) .
the fepsp was elicited by adjusting the intensity of stimulation to ~50% of the maximum response ( 0.1 - 0.5 ma ) .
the amplitude of fepsp 1 m sec after the onset was measured for data analysis . in ltp experiments ,
tetanic stimulation ( 100 hz , for 1 sec , at baseline stimulation intensity ) was operationally defined as the mean change in fepsp amplitude for five stimuli given 30 min after tetanic stimulation compared with the mean fepsp to five test pulses given immediately before the stimulation .
tetanic stimulation was applied 60 min after application of hct ( 2 mmol / l ) .
the protocol for the zero - magnesium model experiments consisted of four periods : 1 ) superfusion with acsf for 30 min to test for spontaneously occurring epileptiform field potentials ( efp ; control period ) .
2 ) superfusion with zero - magnesium acsf until there was a stable efp frequency and amplitude for at least 20 min ( baseline period ) .
3 ) addition of hct ( 2 mmol / l , n=5 ; 0.2 mmol / l , n=5 ; 0.02 mmol / l , n=5 ) to the magnesium - depleted acsf for 60 min ( hct period ) .
4 ) superfusion with zero - magnesium acsf for 60 min ( wash - out period ) .
the efp amplitude was measured peak - to - peak and efp duration was determined as the width at half - maximal amplitude .
owing to the small group sizes that make normal distribution and variance homogeneity unlikely , we chose median and 25/75 percentiles for cohort description .
the data were processed and analyzed using commercially available software ( spss version 12.1 , chicago , ill .
interval - and ordinal - scaled data were compared using the wilcoxon test , and nominal - scaled data with the chi - square test or fisher 's exact test ( 2x2 tables ) .
four animals were injected intraperitoneally ( ip ) with hct ( 2 mmol / l , 5 ml ) , two animals only received saline .
two hr later , the animals were sacrificed with high doses of isoflurane followed by decapitation .
the brain tissue was stored at -20 c for 12 to 48 hr until hplc analysis .
hplc was performed using a method developed by zendelovska et al ( 17 ) for determination of hct in human plasma .
the system included a lichrospher 100 rp8 column ( 125x5 , 5 ) with the mobile phase consisting of 0.025 m phosphate buffer ( adjusted to a ph of 5 with triethylamine ) and acetonitile at a relation of 85/15 , a pump , an uv - detector with variable wavelength and interface and dedicated software ( lachrom series , merck - hitachi , darmstadt ) running on a personal computer with intel pentium iii processor .
the clear supernatant was transferred into an eppendorf cap , and 40 l were injected into the hplc system .
the flow rate was 1ml / min , and the detection wave length was 230 nm .
the experiments were performed on rat hippocampal slices . they have been performed according to national guidelines for animal research .
after introduction of deep pentobarbital anesthesia , the animal was decapitated and the brain was removed .
then the slices were preincubated in artificial cerebrospinal fluid ( acsf ) at 28c for 1
the preincubation acsf contained ( in mmol / l ) : nacl 124 , kcl4 , cacl2 1.0 , nah2po4 1.24 , mgso4 1.3 , nahco3 26 , and glucose 10 .
after preincubation , cacl2 was elevated to 2.0 mmol / l and the slices were transferred into an interphase - type recording chamber . in the experimental chamber , the temperature was increased to 32c . during the experiments , the ph , the bath temperature and the flow rate ( 1.5 to 2 ml / min ) remained constant . for the preparation of the zero - magnesium solution , we omitted mgso4 .
hydrochlorothiazide ( hct ) solution was prepared by adding dimethylsulfoxide ( 0.5 ml per mmol / l of the crystalline hct substance ) to achieve water solubility .
extracellular field potential recordings were recorded with glass microelectrodes ( resistance 2 - 5 m ) in the stratum radiatum of the ca1 and ca3 hippocampal subregions connected with dc amplifiers .
extracellular recordings were obtained using a custom made differential amplifier ( with band - pass filters at 0.5 - 10 khz , sampling rate 0.3 - 100 hz ) . the reference electrode and
field potentials were recorded by an ink - writer ( capable to record between 10 v ) as well as by a digital oscilloscope .
traces were digitized by nicolet 460 digitizing oscilloscope ( nicolet instruments ) and analyzed with accompanying software .
a continuous single pulse of electrical stimulation was applied through a bipolar platinum electrode attached to the schaffer collaterals of hippocampal slices .
evoked field excitatory post - synaptic potentials ( fepsp ) were recorded in hippocampal ca1 region ( stratum radiatum ) .
the fepsp was elicited by adjusting the intensity of stimulation to ~50% of the maximum response ( 0.1 - 0.5 ma ) .
the amplitude of fepsp 1 m sec after the onset was measured for data analysis . in ltp experiments ,
tetanic stimulation ( 100 hz , for 1 sec , at baseline stimulation intensity ) was operationally defined as the mean change in fepsp amplitude for five stimuli given 30 min after tetanic stimulation compared with the mean fepsp to five test pulses given immediately before the stimulation .
tetanic stimulation was applied 60 min after application of hct ( 2 mmol / l ) .
the protocol for the zero - magnesium model experiments consisted of four periods : 1 ) superfusion with acsf for 30 min to test for spontaneously occurring epileptiform field potentials ( efp ; control period ) .
2 ) superfusion with zero - magnesium acsf until there was a stable efp frequency and amplitude for at least 20 min ( baseline period ) .
3 ) addition of hct ( 2 mmol / l , n=5 ; 0.2 mmol / l , n=5 ; 0.02 mmol / l , n=5 ) to the magnesium - depleted acsf for 60 min ( hct period ) .
4 ) superfusion with zero - magnesium acsf for 60 min ( wash - out period ) .
the efp amplitude was measured peak - to - peak and efp duration was determined as the width at half - maximal amplitude .
owing to the small group sizes that make normal distribution and variance homogeneity unlikely , we chose median and 25/75 percentiles for cohort description .
the data were processed and analyzed using commercially available software ( spss version 12.1 , chicago , ill .
interval - and ordinal - scaled data were compared using the wilcoxon test , and nominal - scaled data with the chi - square test or fisher 's exact test ( 2x2 tables ) .
four animals were injected intraperitoneally ( ip ) with hct ( 2 mmol / l , 5 ml ) , two animals only received saline .
two hr later , the animals were sacrificed with high doses of isoflurane followed by decapitation .
the brain tissue was stored at -20 c for 12 to 48 hr until hplc analysis .
hplc was performed using a method developed by zendelovska et al ( 17 ) for determination of hct in human plasma .
the system included a lichrospher 100 rp8 column ( 125x5 , 5 ) with the mobile phase consisting of 0.025 m phosphate buffer ( adjusted to a ph of 5 with triethylamine ) and acetonitile at a relation of 85/15 , a pump , an uv - detector with variable wavelength and interface and dedicated software ( lachrom series , merck - hitachi , darmstadt ) running on a personal computer with intel pentium iii processor .
the clear supernatant was transferred into an eppendorf cap , and 40 l were injected into the hplc system .
the flow rate was 1ml / min , and the detection wave length was 230 nm .
application of mg - free acsf resulted in efp after a median latency of 41 min ( 2 mmol / l hct ) , 47 min ( 0.2 mmol / l hct ) and 43 min ( 0.02 mmol / l hct ) , respectively . after reaching a stable level over 15 min ,
median baseline repetition rate was 13/min ( 2 mmol / l hct ) , 11/min ( 0.2 mmol / l hct ) and 10/min ( 0.02 mmol / l hct ) respectively .
addition of hct to the bath resulted marked increase of repetition rate , amplitude and duration of the epileptiform field potentials ( figure 1 ) .
after 1 hr , the increase of the repetition rate was highest with 2 mmol / l hct ( median 26/min , 25 - 75 quartiles 21.5/min-38/min ) , slightly lower with 0.2 mmol / l hct ( median 21/min , 25 - 75 quartiles 18.5 - 24.5/min ) and with 0.02 mmol hct ( median 15/min , 25 - 75 quartiles 14,5 - 20.5/min ) .
the inter - group differences were statistically significant , as were the differences between baseline and 1 hr exposition within each group ( p<0.05 ) .
the amplitude increased from a median of 0.18 mv to a median of 0.55 mv in the 2 mmol group , from a median of 0.24 mv to a median of 0.37 mv in the 0.2 mmol / l group , and from a median of 0.21 mv to a median of 0.23 mv in the 0.02 mmol / l group .
only the within - group differences of the 2 mmol / l and the 0.2 mmol / l groups were significant ( p<0.05 ) .
discharge duration also increased from 120 msec to 340 msec ( median ) in the 2 mm group , from 190 msec to 230 msec ( median ) in the 0.2 mmol / l group , and from 270 msec to 320 msec ( median ) in the 0.02 mmol / l group .
after 60 min of wash - out , repetition rate , discharge amplitude and discharge duration gradually returned to baseline levels ( figure 2 ) .
a conditioning tetanic stimulation was delivered to the schaffer collaterals pathway followed by pulses with stimulation parameters identical to control values .
the evoked fepsp was stable for at least 30 min ( with mean amplitude of 0.3 0.03 mv ) before application of tetanic stimulation ( less than 10% variation ; figure 3 ) .
administration of tetanic stimulation produced a rapid , stable , and lasting enhancement of the amplitude of the fepsp in all tested preparations ( n=6 , 1471.6% control ; figure 3 b ) .
the potentiation rose within 2 to 3 min and stabilized within 5 - 10 min after the train of stimulations .
mmol / l ; n = 8) 60 min before tetanic stimulation did not change ltp induction in all tested slices ( 146 1.4% control , figure 3b ) .
enhancement of ltp after application of hct did not significantly differ from control slices ( p<0.05 ) . in hplc ,
when brain homogenate of hct - treated rats was injected , hplc showed no significant peak at the hct retention time ( figure 4 ) .
there was also no peak when the brain of rats injected with saline ( control ) was analyzed .
application of mg - free acsf resulted in efp after a median latency of 41 min ( 2 mmol / l hct ) , 47 min ( 0.2 mmol / l hct ) and 43 min ( 0.02 mmol / l hct ) , respectively . after reaching a stable level over 15 min ,
median baseline repetition rate was 13/min ( 2 mmol / l hct ) , 11/min ( 0.2 mmol / l hct ) and 10/min ( 0.02 mmol / l hct ) respectively .
addition of hct to the bath resulted marked increase of repetition rate , amplitude and duration of the epileptiform field potentials ( figure 1 ) .
after 1 hr , the increase of the repetition rate was highest with 2 mmol / l hct ( median 26/min , 25 - 75 quartiles 21.5/min-38/min ) , slightly lower with 0.2 mmol / l hct ( median 21/min , 25 - 75 quartiles 18.5 - 24.5/min ) and with 0.02 mmol hct ( median 15/min , 25 - 75 quartiles 14,5 - 20.5/min ) .
the inter - group differences were statistically significant , as were the differences between baseline and 1 hr exposition within each group ( p<0.05 ) .
the amplitude increased from a median of 0.18 mv to a median of 0.55 mv in the 2 mmol group , from a median of 0.24 mv to a median of 0.37 mv in the 0.2 mmol / l group , and from a median of 0.21 mv to a median of 0.23 mv in the 0.02 mmol / l group .
only the within - group differences of the 2 mmol / l and the 0.2 mmol / l groups were significant ( p<0.05 ) .
discharge duration also increased from 120 msec to 340 msec ( median ) in the 2 mm group , from 190 msec to 230 msec ( median ) in the 0.2 mmol / l group , and from 270 msec to 320 msec ( median ) in the 0.02 mmol / l group .
after 60 min of wash - out , repetition rate , discharge amplitude and discharge duration gradually returned to baseline levels ( figure 2 ) .
a conditioning tetanic stimulation was delivered to the schaffer collaterals pathway followed by pulses with stimulation parameters identical to control values .
the evoked fepsp was stable for at least 30 min ( with mean amplitude of 0.3 0.03 mv ) before application of tetanic stimulation ( less than 10% variation ; figure 3 ) .
administration of tetanic stimulation produced a rapid , stable , and lasting enhancement of the amplitude of the fepsp in all tested preparations ( n=6 , 1471.6% control ; figure 3 b ) .
the potentiation rose within 2 to 3 min and stabilized within 5 - 10 min after the train of stimulations .
application of hct ( 2 mmol / l ; n = 8) 60 min before tetanic stimulation did not change ltp induction in all tested slices ( 146 1.4% control , figure 3b ) .
enhancement of ltp after application of hct did not significantly differ from control slices ( p<0.05 ) .
in hplc , the peak of hct appeared at a retention time of approximately 3.5 min .
when brain homogenate of hct - treated rats was injected , hplc showed no significant peak at the hct retention time ( figure 4 ) .
there was also no peak when the brain of rats injected with saline ( control ) was analyzed .
in contrast to our expectations , hct increased repetition rate , amplitude and duration of the epileptiform discharges elicited by omission of mg from the bath solution in a dose - dependent manner .
hct also increased the amplitude of electrically evoked field potentials , but did not have any influence on the ltp induced by tetanic stimulations applied to schaffer collaterals .
when intraperitoneally injected , hplc analysis did not show evidence of hct in the brain even at high doses .
our recordings used the zero - mg model of epileptiform activity ( 18 - 20 ) .
this model initiates robust epileptiform activity in both normal and lesioned cortex by augmenting the nmda - component of epsp , without pharmacological blockade of synaptic receptors ( i.e. , gabaa receptors in the bicuculline model ) , and represents a model for pharmacoresistant status epilepticus .
this model represents a well - controlled analysis of drug effects independent of systemic influences ( 18 - 20 ) .
this model quite closely imitates in vivo conditions because recurrent and lateral inhibition remains intact .
in addition , low - magnesium activity induces post - synaptic calcium influx , resulting in activation of the same calcium dependent second messenger mechanisms that underlie activity - dependent neuronal plasticity ( 20 ) .
it has been found that diazoxide , another benzothiazide diuretic , is a potent and reversible gating modifier of ampa - type glutamate receptor channels ( 22 ) that inhibits the rapid desensitization of the response to a constant agonist concentration .
the most potent agent of the group of thiazide desensitizers is cyclothiazide ( ctz ) ( 23 , 24 ) that recently has been proposed as an in vitro epilepsy model ( 25 ) .
although there is chemical similarity between different benzothiazide diuretics , the effect on quisqualat - induced ampa - desensitation has been shown to be quite variable . in concentrations of 500umol
, hct also potentiated the steady - state component of quisqualat - activated currents ( 23 ) , but to a much smaller amount than cyclothiazide .
it remains undetermined whether hct also - like ctz - increases pre - synaptic glutamate release ( 26 ) or directly inhibits gaba - a receptors ( 27 ) .
ltp is a phenomenon in which a constant pre - synaptic high stimulation of excitatory amino acids neuronal pathways results in prolonged enhanced post - synaptic responses .
it is well established that nmda receptors are a molecular detector of the coincidence of both the pre - synaptic release of glutamate and a post - synaptic depolarization at the origin of ltp induction ( 28 ) .
, it has been shown that the hct ( 500 m ) potently inhibits non - nmda receptors desensitization in cultured hippocampal neurons ( 23 ) .
cyclothiazide ( 300 m ) , a thiazide diuretic and an allosteric inhibitor of non - nmda receptor desensitization , induced potentiated low mg- induced seizure - like events recorded from the ca3 pyramidal layer of juvenile rat hippocampal slices ( 29 ) . in line with our results , cyclothiazide administered intracerebral ventricle induced epileptiform activities from an initial of multiple evoked population spikes , progressed to spontaneous spikes and finally to highly synchronized burst activities in hippocampal ca1 neurons ( 30 , 31 ) .
in contrast to in vivo studies , systemic administration of thiazides , including hydrochlorothiazide ( 100 mg ) , enhanced the anticonvulsant action of carbamazepine ( 32 ) and gabapentin ( 33 ) in the maximal electroshock seizure model in mice . in our experiments , we could not find evidence that hct does permeate bbb in the rat .
this is in agreement with studies of dogs where dosages as high as 500 mg / kg administered twice daily did not result in measurable quantities ( <3 mg
unfortunately , there is no data available in the literature regarding the bbb permeability for hct in humans .
in addition , there is no evidence for central nervous effects of hct even in the setting of overdose intoxication ( 35 ) .
one could argue that hct may influence ion transport at the bbb without actually permeating it . however , hct at a relatively high concentration of 1.5
mmol / l does not have any effect on the blood to brain sodium transport in vitro ( 36 ) .
the anticonvulsant effect of hct in vivo most likely is mediated via mechanisms based on effects outside the brain .
dashputra and colleagues found that there is no correlation between the effect on the carbonic anhydrase and the potentiation of the anticonvulsant effect of phenytoin in the maximal electroshock model in rats ( 10 ) .
systemic acidosis seems equally unlikely to be related to the protective effect of hct and other benzothiazides because acetazolamide raises the seizure threshold before there has been time for substantial bicarbonate loss ( 37 ) .
it is very unlikely that the protective effect of hct in humans is due to enhancing the action of standard anticonvulsants because the population under study did not suffer from epilepsy ( 9 , 38 ) .
first , we did not investigate the effect of hct in other in vitro models of epilepsy .
mmol / l to 2 mmol / l . we intended to look across a large window of possible concentrations .
however , hct concentration in plasma ( or brain ) of humans when exposed to hct in clinical practice may be much lower ( 39 ) .
thus , we may have missed relevant effects of concentrations below our lowest concentration , or between concentrations .
however , the effects we have observed seem rather robust , which makes it unlikely that with larger groups we would have seen different results .
the protective effect of hct and the other diuretics is rather mild in the epidemiological studies .
moreover , in the animal studies presented by hesdorffer et al ( 9 ) , hct ( as well as furosemide ) was applied in doses that are 100 to 200 times higher than the doses used in clinical practice of hypertension treatment ( hct 165 mg / kg in rats vs. hct 0.3 - 1 mg / kg in humans ) .
therefore , the use of diuretics as alternative convulsant agents can not be recommended yet .
although hct seems to have anticonvulsive properties on in vivo experimental models , exposure of hippocampal slices to hct resulted in an enhancement of the epileptiform burst discharges .
in addition , hct at the same concentration did not cross the bbb in rats .
thus , the anticonvulsive effect of hct most likely is not through direct neuronal effects . | objective(s):protective effects of diuretics , particularly of hydrochlorothiazide ( hct ) , for the development of epilepsy have been described in vivo .
however , its mechanism of action is unknown .
materials and methods : extracellular field potentials were recorded from the ca1- and ca3-subfields of the hippocampus of rats .
epileptiform discharges were induced by omission of mg2 + from the artificial cerebrospinal fluid ( acsf ) .
hct was added to the acsf at a concentration of 2 mmol / l , 0.2 mmol / l or 0.02 mmol / l .
frequency , amplitude and duration of the epileptiform discharges were evaluated .
long - term potentiation ( ltp ) was induced with and without the presence of hct ( n=6 ; 2 mmol / l ) .
in addition , rats were injected with hct ( n=4 ) or saline ( n=2 ) , and the brain tissue was analyzed using hplc .
results : application of 0.02 , 0.2 , and 2 mmol / l hct accelerated the frequency of discharges by 50% , 91% , and 100% , respectively .
the amplitude of burst discharges also increased by 9% , 54% , and 300% , and the duration of epileptiform discharges increased by 10% , 30% and 120% .
all parameters returned close to the basal levels after 60min washout of the substance .
hct increased the electrical evoked potentials but did not affect the ltp in hippocampal tissues .
there was no evidence of hct in the rat brain after intraperitoneal injection .
conclusion : exposure of hippocampal slices to hct enhanced epileptiform activity in a dose - dependent manner .
in addition , hct does not seem to cross the blood brain barrier in rats .
thus , the anticonvulsive effect of hct most likely is not through direct neuronal effect .
| Introduction
Materials and Methods
Preparation of the slices and solutions
Electrophysiological recordings
Evoked field potentials and long-term potentiation (LTP)
Experimental protocol
Results
The effect of HCT on 0-Mg2+-induced EFP
The effect of HCT on LTP
Measurement of HCT concentration in the brain after IP injection
Discussion
Conclusion |
PMC5080600 | the nervous system relies on conserved endocytic and synaptic vesicle ( sv ) recycling mechanisms to sustain high activity .
defective endocytosis and sv recycling have long been implicated in neurodegeneration and neurodegenerative diseases , yet the molecular mechanisms remain unclear ( esposito et al . , 2012 , heutink and verhage , 2012 , saheki and de camilli , 2012 , schreij et al . , 2016 ) .
endophilin - a ( henceforth endophilin ) endocytic adaptors are key players in membrane dynamics at the neuronal synapse ( ringstad et al . , 1999 ,
, 2002 , milosevic et al . , 2011 , saheki and de camilli , 2012 ) .
endophilin-1 and 3 are brain - specific , whereas endophilin-2 is ubiquitous ( ringstad et al . , 1997 , ringstad et al . , 1999 ) .
all three contain a highly conserved bar domain and have a central role in both clathrin - mediated endocytosis ( farsad et al . , 2001 ,
, 2001 , verstreken et al . , 2002 , milosevic et al . ,
2011 ) and clathrin - independent endocytosis ( boucrot et al . , 2015 ) . in clathrin - mediated endocytosis , endophilin recruits synaptojanin-1 to the necks of coated vesicles prior to scission by dynamin ( milosevic et al . , 2011 ) .
furthermore , availability of endophilin is rate limiting for vesicle uncoating and sv recycling , and neurons without endophilin show reduced neurotransmission ( milosevic et al . , 2011 ) .
total absence of endophilin is perinatally lethal , whereas mice lacking endophilin-1 and 2 ( 1,2 double knockout [ dko ] ) show ataxia , neurodegeneration , and early lethality ( milosevic et al . , 2011 ) .
it can not be easily explained by defective neurotransmission , because even mice with paralysis and abolished neurotransmission do not develop neurodegeneration ( heutink and verhage , 2012 ) .
thus , it is unlikely that neurodegeneration in endophilin 1,2 dko mice is solely the result of defective sv recycling .
mice without endophilin also show increased protein ubiquitination ( cao et al . , 2014 ) , and neurodegeneration
is frequently associated with impaired proteolytic systems , most notably the ubiquitin - proteasome system ( ups ) and autophagy ( nakamura and lipton , 2009 , nixon , 2013 , menzies et al . , 2015 ) .
endophilin has been linked to the ups through direct interaction with the parkinson s disease ( pd)-related e3-ubiquitin ligase parkin ( trempe et al . , 2009 ) and as a key component of the cbl - cin85-endophilin complex known to downregulate receptors ( soubeyran et al .
additional links between endophilin and neurodegeneration have recently emerged . the most commonly disrupted gene in familial pd ( cookson , 2010 ) , lrrk2 , encodes a protein that phosphorylates endophilin ( matta et al . , 2012 ) .
lrrk2 appears in lewy bodies and plays a role in apoptosis and autophagy ( iaccarino et al .
in addition , a number of case studies described synaptojanin-1 and auxilin mutations in early - onset parkinsonism ( edvardson et al . , 2012 , quadri et al . , 2013 , krebs et al . , 2013 ) .
auxilin is recruited to the clathrin - coated vesicles directly after the action of synaptojanin-1 , which is brought to the neck of the clathrin - coated pit by endophilin ( milosevic et al . , 2011 ) .
interestingly , endophilin is upregulated in the brains of pd patients ( shi et al . , 2009 ) .
in addition , endophilin-1 and 3 have been shown to interact with ataxin-2 , mutations in which lead to spinocerebellar ataxia type 2 ( ralser et al . , 2005 , nonis et al . , 2008 ) . despite direct parkin - endophilin interaction and a striking upregulation of parkin in mice lacking all three endophilin genes ( endophilin triple knockout [ tko ] ; cao et al . , 2014 ) , elevated parkin
we subjected endophilin mutants to rna sequencing and found upregulation of another e3-ubiquitin ligase , f - box - only protein 32 ( fbxo32 , also called atrogin-1/mafbx ) .
fbxo32 has previously been studied in muscles , where its induction occurs prior to the onset of rapid atrophy ( sandri et al . , 2004 ) .
while studying how the fbxo32-endophilin - a interaction balances protein degradation and neuronal health in the brain , we discovered a direct role of endophilin - a in autophagosome formation .
mice lacking all three endophilin genes die perinatally , whereas endophilin 1,2 dko animals show ataxia , neurodegenerative changes , and lethality around 3 weeks ( movie s1 ; milosevic et al . , 2011 ) . whereas single knockout ( ko ) and heterozygous ( 1ht-2ht ; 1ht-2ht-3ht ) mice appear phenotypically normal , endophilin 1ko-2ht , 1ko-2wt-3ko , and particularly 1ko-2ht-3ko
mice exhibit shorter lifespans ( 19 , 21 , and 18 months , respectively ; 1ht-2ht mice lived over 27 months ; figure s1a ) . observing 1ko-2ht and
1ko-2ht-3ko mice revealed progressive impairments in motor coordination and increasingly obvious ataxia as these animals age . to systematically measure the progression of ataxia , we used a composite phenotype scoring system that incorporates hindlimb clasping , ledge walking , kyphosis , and gait ( guyenet et al . , 2010 ) .
when applied to four endophilin lines ( 1ko-2wt , 1ko-2ht , 1ko-2wt-3ko , and 1ko-2ht-3ko ) and wild - type ( wt ) controls , it revealed phenotypic differences between endophilin mutants and within the same animals over the course of aging ( figure 1a ) ; i.e. , the onset and progression of ataxia correlate with age and severity of endophilin deficiency .
next , we systematically evaluated locomotion by accelerating rotarod ( arr ) performance test and gait analysis . whereas the performance of 1ko-2wt mice on the arr was comparable to wt , fall latency in 1ko-2ht-3ko mice was already significantly lower at a very young age ( figure 1b ) .
intermediate phenotypes were observed in 1ko-2ht and 1ko-2wt-3ko mice , where fall latency compared to wt was decreased significantly at 3 and 15 months , respectively .
gait analysis revealed that ability to run on a motorized treadmill and walking pattern also depended on the number of missing alleles and age ( figures 1c , 1d , and s1b ; see movie s2 ) .
1ko-2ht-3ko gait was impaired at 3 months , whereas the littermate 1ko-2wt-3ko mice showed impaired gait at 15 months ( figure 1d ) .
overall , onset and progression of movement impairments correspond to age and the extent of endophilin deficiency . to evaluate whether neurodegenerative changes could cause these motor impairments , we inspected neuronal cell death and gliosis in our model . whereas overall neuronal cell morphology was unaltered in these mice ( figure s1d ) , tunel assay revealed slightly increased cell death in endophilin 1ko-2wt and more prominently in 1,2 dko motor cortex and hippocampus at postnatal day ( p)18 ( figure 1e ) . increased cell death was also found in 18-month 1ko-2ht-3ko motor cortex and hippocampus ( figure s1c ) .
glial fibrillary acidic protein immunostaining revealed prominent upregulation of gliosis in 1,2 dko motor cortex ( figure 1f ) . because endophilin tko mice die a few hours after birth , we probed for early markers of apoptosis in endophilin tko brains and found an increase in caspase 3 activity ( figure 1 g ) . in summary , newborn endophilin tkos , juvenile 1,2 dko , and aged 1ko-2ht-3ko mice display signs of mild to moderate neurodegeneration .
we were interested in the molecular mechanisms underlying neurodegeneration in our endophilin - deficient models , so we employed rna sequencing ( experimental strategy in figure s2a ) .
we first sequenced hippocampal rna from tko mice that show altered sv recycling and controls ( n = 89 mice / condition ) .
the data are of high technical quality ( 27 m aligned reads ) and samples clustered well according to genotype ( table s1 ; figures s2b and s2c ) .
182 differentially expressed genes ( degs ) between endophilin tko and littermate controls and 964 degs between tko and wt were detected ( adjusted p value < 0.01 ; log2 fold change > 0.4 ) ( tables s2 and s3 , respectively ) .
given the pronounced movement defects of endophilin mutants , we first looked for movement - disorder - related genes or pathways .
interestingly , tko versus wt degs showed significant enrichment of genes involved in ataxia and pd ( figures 2a and 2b ; table s4 ) . because endophilin 1,2 dko mice present first signs of neurodegeneration by the end of the 2 postnatal week , hippocampi from dko , littermate 1ko-2wt , and wt mice were extracted during the 3 week and subjected to rna sequencing and transcriptome analysis .
the data are again of high technical quality ( 28 m reads ; table s1 ; samples clustered well according to genotype ; figures s2d and s2e ) .
1,479 degs between 1,2 dko and littermate controls , and 1,749 degs between dko and wt were detected ( adjusted p value < 0.01 ; log2 fold change > 0.4 ; tables s5 and s6 , respectively ) . both 1,2 dko versus littermate and dko versus wt lists were significantly enriched for genes involved in ataxia and pd ( table s4 ; figures 2c and 2d ) .
ataxia - related genes include atm interactor ( atmin ) , e2f1 , and rad51 ; pd - related genes include park2 ( encoding parkin ) , lrrk2 , the tyrosine hydroxylase gene ( th ) , and parl . these findings further support endophilin s association with neurodegeneration , ataxia , and pd ( arranz et al . , 2015 , edvardson et al . , 2012 ,
, 2013 , matta et al . , 2012 , milosevic et al . , 2011 , quadri et al . , 2013 , ralser et al .
, 2005 , shi et al . , 2009 , trempe et al . , 2009
we next analyzed tko versus both littermate and wt controls ( genes significantly changed in the same direction in tko against both littermates and wts ; table s7 ; figure s2f ) by ingenuity pathway analysis ( ipa ) software to identify altered pathways and networks .
the resulting most - represented pathways were related to compromised synaptic transmission , protein homeostasis , phagosome maturation , mitochondrial function , dopamine metabolism , and apoptosis ( figure 2e ) . altered protein homeostasis has been strongly linked to neurodegeneration ( e.g. , deger et al .
, 2015 ) and might be relevant to the ataxic and neurodegenerative phenotypes observed in endophilin - deficient mice .
the differential expression of several genes was validated by quantitative real - time pcr in endophilin tko hippocampi versus wt , and a strong correlation was observed ( r = 0.83 ; figure 2f ) .
we also examined regulators of the transcriptional profile in endophilin tko hippocampi and found that , among those statistically enriched , fbxo32 presents the most - robust change in gene expression ( 1.9-fold increase ; table s8 ) .
this was interesting because fbxo32 is an e3-ubiquitin ligase linked to autophagy in skeletal and cardiac muscle ( sandri et al .
its expression is known to be regulated by the transcription factors foxo1 and foxo3a ( sandri et al . , 2004 ,
webb and brunet , 2014 ) : whereas foxo1 was unchanged , foxo3a was significantly upregulated in tko hippocampi ( table s8 ) .
given the observed changes in the foxo3a - fbxo32 network and that fbxo32 is known to promote cell death and is required for muscle atrophy ( sandri et al .
, 2004 , wu et al . , 2011 , xie et al . , 2009 )
, we decided to follow up on this network at the rna and protein level .
as in the rna - sequencing data , foxo3a and fbxo32 were upregulated in tko hippocampi when measured by qpcr ( figure 3a ) whereas foxo1 was unchanged ( figures s2 g and s2h ) .
a striking increase in fbxo32 protein levels in tko whole - brain extracts relative to littermate controls and to wt mice was detected ( figure 3b ; figure s2i displays elevated fbxo32 in the hippocampus ) .
levels of foxo3a[ser253 ] phosphorylation ( which represses foxo3a binding to dna ) were also decreased ( figure 3b ) , suggesting an increase in the foxo3a transcriptional activity , and the distribution of foxo3a[ser253]-p is altered in tko hippocampus in comparison to controls ( figure 3c ) . to determine whether foxo3a activation is directly responsible for the upregulation of fbxo32
, foxo3a was silenced in tko primary hippocampal neurons by small interfering rna ( sirna ) knockdown , using two sirna constructs .
near - complete ablation of foxo3a affects neuronal survival , so we used a milder condition ( 45% reduction ) to probe for fbxo32 levels ( figure 3d ) .
the elevated fbxo32 levels in tko neurons were lowered upon foxo3a knockdown , implying that foxo3a is a sufficient regulator of fbxo32 in this system ( figure 3d ) .
fbxo32 was also strongly upregulated in 1,2 dko brain samples , both at p0 ( whole brain ) and 3 weeks ( cortex ; figure 3e ) , as well as in the cortex of 1ko-2ht-3ko mice at 18 months ( figure 3f ) .
thus , the foxo3a - fbxo32 network originally described in muscle atrophy is operative in the brain of endophilin mutant mice . given the reported role of fbxo32 in the regulation of protein homeostasis via the ups , we investigated whether the ubiquitin system is also affected in endophilin mutants .
the pd - related e3-ubiquitin ligase parkin monoubiquitinates endophilin , and tko brains show increased levels of parkin and ubiquitinated proteins ( trempe et al . , 2009 , cao et al . , 2014
however , 1,2 dko mice in which the parkin gene park2 was also knocked out displayed a similar neurological phenotype to 1,2 dko mice ( cao et al . , 2013 ) , suggesting that other ubiquitin ligases might be involved .
given that fbxo32 is also an e3-ubiquitin ligase , we hypothesized that its robust upregulation in endophilin - deficient brain might contribute to the loss of protein homeostasis in endophilin knockouts .
first , we tested whether fbxo32 can ( mono)ubiquitinate endophilin-1 and 2 , but this was not the case ( figures s3a and s3b ) .
we observed that 1,2 dko brains also display increased levels of ubiquitinated proteins ( figure 4a ) , similar to tko brains
( cao et al . , 2013 ) . to distinguish whether this was due to proteasome - related or localization-/signaling - related ubiquitination , we measured k48-(proteasome - related ) and k63-(signaling - related)-linkage - specific polyubiquitination ( herhaus and dikic , 2015 ) . whereas k48-linked ubiquitination was prominently increased in 1,2 dko cortical extracts ( figure 4b )
, there was no significant change in the levels of k63-linked ubiquitination ( figure 4c ) , suggesting that the accumulation of ubiquitinated proteins is associated with defective processing by the proteasome .
given the apparent inability of the proteasome to cope with the level of ubiquitinated proteins in endophilin knockouts , we probed the proteasomal system using a ubiquitinated substrate that is targeted to the proteasome and degraded ( ubiquitin - g76v - gfp ; dantuma et al . , 2000 ) , as well as by measuring the enzyme activity of the proteasome itself .
we observed an accumulation of gfp - fluorescence originating from the substrate in tko primary hippocampal neurons as well as fibroblasts ( mouse embryonic fibroblasts [ mefs ] ) in comparison to respective controls ( figures 4d and 4e ) , denoting proteasome saturation in the endophilin ko cells .
interestingly , we noticed that the increase in ubiquitinated proteins elicited by the expression of ubiquitin - g76v - gfp results in significantly more apoptosis in the tko mefs than in controls ( figure 4 g ) .
altogether , these results suggest a causal link between proteasome saturation in endophilin mutants , higher levels of ubiquitinated proteins , and increased cell death susceptibility . to determine whether the proteasome saturation was due to decreased activity of the proteasome , we measured the proteasome enzyme activity .
while there was a trend toward a decrease in proteasomal activity , we observed no significant difference in the proteasome activity per se in the brain extracts ( figure 4f ) .
thus , the proteasome system in tko brains seems functional but unable to keep up with the high demand . in light of the upregulation of fbxo32 in the absence of endophilin
first , we tested whether fbxo32 interacts with endophilin family members by immunoprecipitation and pull - downs .
an interaction between fbxo32 and all three endophilins in vivo was revealed by specific enrichment of fbxo32 in anti - gfp immunoprecipitates from hela expressing fbxo32-mcherry and endophilin 1 , 2 , or 3-egfp ( figures 5a5c ) .
similar results were obtained by glutathione s - transferase ( gst ) pull - downs from mouse brain lysate using the gst - fbxo32 as bait ( figure s3e ) .
we demonstrated that fbxo32 is able to pull - down purified endophilin-1 protein ( figure 5d ) , suggesting a direct interaction .
furthermore , the interaction is mediated through endophilin s sh3 domain , as revealed by another set of pull - downs from mouse brain lysate using the gst - sh3 domains of endophilin-1 and 3 as baits ( an enrichment of fbxo32 with stronger binding to endophilin-1 and 3 sh3s was observed ; figure s3f ) .
we examined the intracellular localization of fbxo32 and its overlap with endophilins in cultured clonal cells ( hela and mefs were used to control for cell line variability ) and primary hippocampal neurons .
it is noteworthy that expressing fbxo32 ( either fbxo32-egfp or fbxo32-mcherry ) induced cell death : 50% of fbxo32-transfected
hela and 40% of fbxo32-transfected mefs survived 30 hr post - transfection ; less than 15% of fbxo32-transfected primary neurons were detected on day in vitro ( div ) 14 ( detailed below ) .
fbxo32 distribution showed substantial cell - to - cell variability : although mainly cytosolic , fbxo32 was enriched at dynamic organelle - like structures of various sizes and often accumulated in the nucleus ( movie s3 ) .
native fbxo32 distribution was similar to that of expressed fbxo32 : immunostaining against fbxo32 revealed nuclear as well as cytosolic protein distribution in hela cells ( figure s4a ) .
surprisingly , both in hela cells and mefs , fbxo32-labeled tubular structures were observed ( examples in figures 5f , 5i , and s4c ; see also movie s5 ) . given that fbxo32 interacts with endophilins , and the propensity of endophilins to tubulate membranes , we examined the colocalization of fbxo32 with endophilin 1 , 2 , and 3 .
the distribution of endophilins is mainly cytosolic : sparse puncta were attributed to endophilin s role in endocytosis ( milosevic et al .
, 2011 , perera et al . , 2006 , boucrot et al . , 2015 ,
when endophilin-1 and fbxo32 were coexpressed at similar levels , fbxo32 protein distribution changed : fbxo32 became predominantly cytoplasmic and excluded from the nucleus ( figures s4b and s6e ; also see figures 7b and 7d ) .
interestingly , we also observed that cell death due to fbxo32 overexpression was abolished upon endophilin coexpression , which we detail further below . fbxo32 and endophilin-1
were also found to colocalize sparsely at dynamic organelle - like structures ( figures 5l and s4b ) .
these were found throughout the cytoplasm and not only in the vicinity of the plasma membrane ( as would be expected for endophilin-1 when being recruited to endocytic structures ) . when endophilin-2 and fbxo32 were coexpressed , they colocalized transiently at both organelle - like structures and tubuli ( figures 5e , 5l , and s4c ; movie s5 ) .
we further observed that endophilin-2 colocalized with fbxo32 on dynamic tubulated structures , although endophilin-2 was not needed for the recruitment of fbxo32 to tubuli ( figure 5f at 24 s ; movie s5 ) .
similarly to endophilins , fbxo32 also directly binds and tubulates membranes in vitro ( b. kroppen , m. meinecke , and i.m .
lastly , fbxo32 and endophilin-3 colocalized partially on organelle - like structures ( figures 5l and s4d ) .
we examined the overlap between fbxo32 and endophilin-1 or endophilin-2 in primary hippocampal cultures . fbxo32-egfp and endophilin-1-mrfp or endophilin-2-mrfp signal overlapped partially both in the cell bodies and in neuronal processes ( figures 5 g and 5h ; again fbxo32 colocalized to a greater extent with endophilin-2 than endophilin-1 ) . in sum , the data show that fbxo32 interacts directly with endophilin - a proteins ( possibly through their sh3 domain ) , both proteins are involved in membrane tubulation , and both transiently colocalized on dynamic organelle - like structures of various sizes .
in addition to being an e3-ubiquitin ligase , fbxo32 is known to regulate autophagy in muscle , raising the question of whether the fbxo32-labeled organelle - like structures were autophagosomes .
we coexpressed fbxo32-egfp together with lc3-mrfp and observed a prominent colocalization of the two probes in starved cells ( figures 5i and 5 m ; movie s4 ) .
a similar result was observed when fbxo32 was immunostained ( figure s4a ) , irrespective of type of cell ( hela cells were also used ) or fluorescent labeling ( figure s4e ) .
next , when endophilin-2 was coexpressed with the autophagosomal marker lc3 , sparse but significant colocalization was observed in starved hela cells , suggesting that endophilin-2 , like fbxo32 albeit to a lesser extent , transiently associates with autophagosomes ( figures 5j and 5 m ) .
similar observations were made for endophilin-1 and endophilin-3 , which were also occasionally seen on lc3-labeled autophagosomes ( figures 5 m , s4f , and s4 g ; please note that the values represent colocalization above coincidental colocalization , which is prominent in this system given that endophilins are cytosolic proteins ) .
for example , a kymograph from endophilin-3- and lc3-expressing hela cell shows that endophilin-3 colocalizes with an lc3-labeled autophagosome transiently ( figure 5n ; arrows indicate colocalization ) .
importantly , fbxo32 , lc3 , and endophilin-2 could be simultaneously detected at the same dynamic organelle ( movie s6 ) .
notably , endophilin-2 showed much stronger overlap with atg5 ( an early autophagosomal marker ) than with lc3 ( figure 5k ; 43% colocalization ; see movie s7 ) , implying a role for endophilin - a in autophagosome formation .
given that both fbxo32 and endophilins - a were occasionally found on lc3-positive autophagosomes , we examined whether autophagy was altered in brains of tko and 1,2 dko mice .
notably , both lc3b - ii ( lower band is the active form ) , a classical marker for autophagosomes , and atg5 , a marker for early autophagosomes , were prominently decreased in tko brains ( figure 6a ) and in aged endophilin 1ko-2ht-3ko mice ( figures 6c and 6d ) .
interestingly , the atg5 protein level in endophilin 1ko-2ht-3ko mice decreases progressively between 3 and 18 months of age ( figure 6d ) . a reduction in lc3b - ii and atg5
was also observed in 1,2 dko cortex ( figure 6b ) , suggesting that endophilin-3 can not effectively compensate for the loss of endophilin-1 and 2 in autophagy .
we prepared primary neuronal cultures from endophilin tko and wt hippocampi and labeled autophagosomes either by mildly expressing lc3-egfp ( figures 6e and s5c ) or by immunostaining for lc3 ( figure s5e ) .
we detected an overall decrease in the number of lc3-labeled autophagosomes when autophagy was simultaneously induced by starvation and mtor inhibitor torin-1 ( figures 6e , 6f , s5c , and s5d ) .
interestingly , the effect was more obvious at the synapses , as apparent from colocalization of lc3 and synaptobrevin-2 , a presynaptic marker ( figure 6e ; insets in figure s5c ) .
altogether , the biochemical and imaging data suggest that autophagy depends on endophilin in vivo and in vitro and imply a role for endophilin in the formation and/or maturation of autophagosomes .
notably , endophilin - b has a known role in autophagosome formation , and its cdk5-mediated phosphorylation is required for induced autophagy in models of pd ( wong et al . , 2011 ) .
endophilin - a proteins are closely related to endophilin - b : they share an identical domain structure with a high homology , and both protein subfamilies are enriched at the synapses and able to tubulate membranes ( farsad et al . , 2001 ,
thus , we tested the levels of endophilin - b , cdk5 , and several autophagy initiators .
no change in the protein level of endophilin - b in endophilin - a tko brains was detected ( figure 6 g ) , whereas the levels of cdk5 were decreased ( figure 6a ) .
however , in 1,2 dko brains , the cdk5 level was unchanged ( figure 6b ) .
protein levels of uvrag , beclin , atg3 , and hdac6 were not altered , either in endophilin tko brains or endophilin 1,2 dko cortex ( figures s5a and s5b ) .
these results imply that endophilin - b can not compensate for autophagosome formation in the absence of endophilin - a and that autophagy is not altered due to repression of signaling pathways , because the key autophagy initiation regulators are not affected by absence of endophilin - a . the key autophagy factors atg9a and atg16l have been shown to undergo clathrin - mediated endocytosis ( ravikumar et al .
, 2010a , puri et al . , 2014 ) . in the light of endophilins
well - established role in endocytosis , it is possible that the observed phenotype is an indirect effect of defective endocytosis in endophilin mutants .
however , we found that both atg9a and atg16l were not significantly changed in our mutants ( figures 6h and s5f ) , indicating that availability of these two factors does not influence the findings we describe here . to further characterize the role of fbxo32 in autophagy , we employed the drosophila melanogaster neuromuscular junction ( nmj ) to see whether fbxo32 is relevant for autophagy in neuronal cells in vivo . when fbxo32 is silenced in drosophila in vivo , there was a very obvious decrease in the number of autophagosomes detected at the nmj ( figures s6a s6c ; quantification in figure s6d ) .
thus , fbxo32 seems necessary for autophagosome formation at the fruit fly nmj . given that , in the endophilin mutant brains there is more fbxo32 and yet still less autophagosome formation
, the interaction between endophilin and fbxo32 may play a key role in this process . to determine whether upregulation of fbxo32 is an important step in the brain pathology of endophilin mutants
, we explored the consequences of fbxo32 upregulation in clonal ( hela and mef ) and primary ( neurons ) cell lines .
it has been reported that fbxo32 elevation induces apoptosis in cardiomyocytes and cancer cells ( wu et al . , 2011 , xie et al . , 2009 ) .
we observed that expression of fbxo32 causes substantial cell death in hela and mefs , as determined by the number of remaining egfp - positive cells and by the activity of caspases 3 and 7 ( figures 7a7c , 7e , and s6e ) .
the detrimental effects of fbxo32 were independent of its fluorescent tag and cell - transfection method ( electroporation / lipofectamine / fugene ) .
strikingly , when endophilin-1 was coexpressed with fbxo32 in mefs , fbxo32-induced cell death was rescued , as demonstrated by the number of egfp - positive cells ( normalized [ norm . ] to total dapi count ) 30 hr after transfection and healthy cell appearance ( figure 7b ; also in hela cells : figure s6e ) , the quantification of cell survival ( figure 7c ) , and the activity of caspases 3 and 7 ( figure 7e ) .
importantly , the levels of fbxo32 expression were elevated when fbxo32 and endophilin-1 were coexpressed , implying that the rescue of fbxo32-induced apoptosis is not due to a reduction in fbxo32 levels ( figures 7a and 7b ) .
however , coexpression with endophilin-1 seemed to relocate fbxo32-egfp out of the nucleus ( figures 7d and s6e ) in agreement with the data presented in figure 5 .
thus , fbxo32-induced apoptosis is associated with aberrant intracellular fbxo32 localization , and both the localization and the apoptosis susceptibility can be rescued by simultaneous upregulation of endophilin-1 .
these data show that fbxo32 and endophilin-1 interact not only at the protein but also at the genetic level . in neurons ,
when either wt primary hippocampal or cortical neurons were transfected with fbxo32-egfp , less than 15% of all transfected neurons were detected at div14 ( figures 7f7h ) .
the surviving fbxo32-expressing neurons displayed a lower number of processes and appeared unhealthy ( figure s6f ) .
tko hippocampal and cortical neurons in culture were comparable to wt and littermate endophilin 1ko-2wt-3ko cells and did not show increased cell death under standard culturing conditions .
however , when tko cortical neurons were transfected with fbxo32 , less than 2% of transfected neurons were detected at div14 ( in comparison to egfp - expressing tko neurons ) , with the surviving cells presenting a reduction in the number of processes and vacuoles in cell bodies .
lastly , coexpression of endophilin-1 or endophilin-2 was able to rescue fbxo32-induced apoptosis in cortical neurons : when endophilin-1-mrfp or endophilin-2-mrfp were coexpressed with fbxo32-egfp , either in wt and endophilin tko cortical neurons , the majority of neurons thrived ( over 80% and 67% of all fbxo32-transfected neurons , respectively ; figures 7f7h ) .
imbalance in fbxo32/endophilin - a levels is likely a key mechanistic step in the brain pathology of endophilin mutants , and fbxo32-endophilin interplay seems important for maintaining neuronal health and overall protein homeostasis in the brain .
to date , the known roles of endophilin - a have been limited to endocytosis and sv recycling . here , we show that endophilin also affects the ups and autophagy in the brain .
the robust induction of the foxo3a / fbxo32 network ( known to mediate muscle atrophy ) in the brain of mice with endophilin deficiency promotes apoptosis and likely mediates brain pathology .
similarly to muscle , the foxo / fbxo32 network seems relevant for regulating protein degradation in the brain and for maintaining neuronal health .
if fbxo32 is expressed in cell lines and primary neurons , it causes apoptosis that can be rescued by simultaneous coexpression of endophilin .
we also report that partial endophilin absence in mice results in neurodegeneration and in perturbations of a significant number of ataxia- and pd - related genes .
these observations corroborate a number of recent findings that indirectly link endophilin to neurodegeneration and pd ( arranz et al . , 2015 , edvardson et al . , 2012 , krebs et al . , 2013 , matta et al . , 2012 , milosevic et al . , 2011 ,
endophilin models may also be of relevance for neurodegeneration research because they may provide insights into signaling pathways affected prior to overt manifestation of neurodegeneration .
fbxo32 was thus far studied mainly in skeletal muscle , where this e3-ubiquitin ligase was found to mediate rapid atrophy as a result of foxo3a transcriptional activation .
interestingly , foxo3a activation is associated with cellular stress responses and neurodegeneration ( webb and brunet , 2014 ) .
foxo3a stimulates autophagy in neurons ( xu et al . , 2011 ) , and
fbxo32 is necessary for autophagy ; activation of the foxo - fbxo32 axis should thus lead to increased autophagy .
nevertheless , this is not the case when endophilin is lacking : the number of autophagosomes is sharply decreased .
also , endophilin colocalizes transiently with mature and even more impressively with early autophagosomes , suggesting that endophilin - a has a previously unreported role in the formation and/or maturation of autophagosomes .
this is supported by the recent identification of endophilin - a as one of the regulators of autophagosome formation ( strohecker et al . , 2015 ) .
interestingly , mice without endophilin also display the same pattern of perinatal lethality observed in mice lacking proteins necessary for autophagosome formation ( kuma et al . , 2004 ) .
given endophilin s involvement in endocytic processes , the decrease in the number of autophagosomes might also result from compromised endocytosis .
specifically , it has been shown that blockage of endocytosis upstream of clathrin coat assembly results in decreased autophagosome formation ( ravikumar et al .
. however , endophilin deficiency results in the accumulation of clathrin - coated vesicles , and the levels of clathrin are not altered ( milosevic et al . , 2011 ) .
thus , clathrin is available to interact with atg16l1 , and rna and protein levels of atg16l1 are not altered in this model .
in addition , the rna and protein levels of atg9 , a key autophagic protein known to traffic via the plasma membrane and endosomal compartments ( mari et al . , 2010 , puri et al . , 2014 ) are not altered in endophilin mutant brains . altogether
, whereas we can not entirely exclude endophilin s role in endocytosis also affecting autophagy , we consider other mechanism(s ) besides endocytic defects to be underlying the decrease in autophagosomes in endophilin mutants . decreased autophagosome availability and , consequently
, lower autophagic flux are known to affect protein homeostasis ( korolchuk et al . , 2009 ) .
we observed an accumulation of k48-linked ubiquitinated proteins in the cytoplasm , suggesting saturation of the ups ( due to overloading rather than deficiencies in the proteasome ) .
there is possibly a double hit on this pathway : decreased autophagy and increased availability of ubiquitin ligases ( fbxo32 and parkin ) .
both fbxo32 and parkin are involved in selective autophagy of ubiquitinated proteins ( zaglia et al . ,
2014 ) , and both function under the regulation of foxos ( webb and brunet , 2014 ) .
upregulation of fbxo32 and parkin may thus be a compensatory response to defective autophagy , an attempt to shunt more of the burden to the ups , or possibly both .
interestingly , the lower abundance of autophagosomes in the absence of endophilin - a can not be ( fully ) compensated for by a related protein with the same domain organization , endophilin - b , an established mediator of autophagosome formation ( takahashi et al . , 2011 ) .
endophilin - a primarily operates in the endocytic processes at the plasma membrane , whereas endophilin - b has prominent roles in apoptosis , mitochondrial membrane dynamics , and autophagosome formation ( kjaerulff et al . , 2011 ) .
furthermore , cdk5-mediated phosphorylation of endophilin - b is required for inducing autophagy in models of pd ( wong et al . , 2011 ) .
more research is needed to understand endophilin - a and endophilin - b links to autophagy , neurodegeneration , and pd .
we propose a model in which endophilin s absence , likely due to its membrane - curvature - inducing / stabilizing properties , leads to reduced availability of membranes for autophagosome formation and decreased autophagic activity .
interestingly , fbxo32 and endophilin interact at both the gene and protein level and both are required for the autophagy process .
like endophilin , fbxo32 can tubulate membranes and is recruited to membrane tubules in vivo . thus
, endophilin and fbxo32 membrane - tubulating activity may contribute to the formation / maturation of autophagosomes .
this model is in concordance with the reported role of endophilin - a as necessary and sufficient for macroautophagy at presynaptic terminals of fruit flies ( soukup et al .
in summary , our study addresses the origin of neurodegeneration in a key mammalian endocytic model and reveals that the foxo / fbxo32/endophilin network may play a role in maintaining neuronal health by balancing autophagy and the ups in the brain .
the results are consistent with the following plausible scenario : reduced endophilin availability results in impaired capacity to form autophagosomes , resulting in decreased autophagy , accumulation of ubiquitinated proteins in the cytoplasm , proteasome saturation , and further accumulation of unwanted proteins over time that eventually affect neuronal function and health , leading to neurodegeneration .
accordingly , we show that imbalanced autophagy also develops in aged endophilin heterozygote mice that display age - dependent progressive ataxia .
our data reveal a role in autophagy for a key endocytic adaptor previously thought to be strictly dedicated to endocytosis and invite further research addressing the physiological and pathological implications of these findings .
supplemental experimental procedures detail gene - targeting and cloning strategies and list the antibodies and plasmids used .
animal experiments were conducted according to the european guidelines for animal welfare ( 2010/63/eu ) with approval by the lower saxony landesamt fr verbraucherschutz und lebensmittelsicherheit ( laves ) , registration number 14/1701 . for rotarod experiments ,
620 males / group were tested on a setup by tse systems ( 3375 - 5 ) . a training phase consisted of four trainings over 2 days ( intertrial interval [ iti ] was 25 hr ; 180 s at 5 rpm ) .
the testing phase consisted of four tests over 2 days ( iti 25 hr ) .
mice were placed on the rod , which then accelerated from 5 to 40 rpm in 180 s. fall latency was measured .
a simple composite phenotype scoring system for evaluating mouse models of ataxia was applied as described ( guyenet et al . ,
eight to twelve mice were used per condition , and scoring was performed by two researchers independently .
ventral plane videography for gait analysis was performed using instrumentation ( digigait ) and software from mouse specifics , according to manufacturer s instructions . for each group ,
tunel assay was performed on cryosections of the brains of perfused mice to test for apoptotic and necrotic cells .
mice were anesthetized with chloral hydrate ( 40 mg / ml ) before cardiac perfusion with 4% paraformaldehyde ( pfa ) .
after slow dehydration with sucrose , brains were embedded in oct ( tissue tek ) and frozen in liquid n2 .
sagittal brain sections were cut with a cryostat ( 10 m thick ) . sections were post - fixed and permeabilized with 0.1%
in situ cell death detection kit ( fluorescein ) was used according to manufacturer s instructions .
slides were counterstained with dapi , embedded with mowiol 4 - 88 , and imaged with a lsm 710 confocal setup ( zeiss ) .
immunofluorescence of frozen brain sections and cultured neurons ( div 1420 ) was carried out as described ( ringstad et al . , 2001 , milosevic et al . , 2011
live mefs , hela cells , and neurons were imaged either using a zeiss axioobserver z1 total internal reflection fluorescence ( tirf ) microscope with an evolve charge - coupled device ( ccd ) camera ( photometrics ) and 100 plan apo zeiss objective or perkin elmer ultraview spinning - disk confocal setup that consists of nikon ti - e eclipse inverted microscope equipped with perfect focus , 60 cfi plan apo vc nikon objective , and 14-bit electron - multiplied ccd camera ( hamamatsu c9100 ) .
autophagy induction and colocalization analysis was performed as detailed in the supplemental experimental procedures and below .
whole - brain or cortex samples for western blotting were prepared by homogenization of brain tissue ( whole brain for p0 animals ; one cortex for p14p21 animals and aged animals ) as detailed in supplemental experimental procedures . for trap - gfp immunoprecipitation
, hela cells were transfected with endophilin ( 1 , 2 , or 3)-egfp and mcherry / fbxo32-mcherry using lipofectamine 3000 ( invitrogen )
. we used chromotek trap - gfp agarose beads ( chromotek ) and followed manufacturer s protocol . alternatively , hela cells were transfected with fbxo32-egfp and coupled to chromotek trap - gfp agarose beads .
purified endophilin-1 was added , and all fractions ( input , non - bound , and bound ) were collected .
primary hippocampal and cortical cultures were prepared as previously described ( ferguson et al . , 2007 ) .
primary neurons expressed constructs , following amaxa ( lonza)-based transfection , under the control of the chicken--actin promoter to allow for long - term expression .
a minimum of 15 images from three to five experiments were analyzed for each genotype / condition .
given that the majority of respective fluorescent signals were cytosolic , colocalization was evaluated semi - automatically using object - based overlap analysis and imagej jacop plugin ( bolte and cordelires , 2006 ) . in short , the image was first segmented into objects and background ( i.e. , bright fluorescent objects are segmented from the image ) , and then their spatial relationship ( overlap ) was measured .
the same analysis was performed with the mirror image , and random colocalization was subtracted in each case .
, circles were superimposed on bright fluorescent spots in the fbxo32 ( or lc3 or atg5 ) channel and transferred to identical image locations in the endophilin channel .
if the fluorescence intensity maximum in the endophilin channel was located in the same circle and the morphology of the signal resembled that of the fbxo32 signal , the circle was rated as positive ( colocalized ) ; if not , it was rated as negative ( not colocalized ) . to correct for accidental colocalization , circles were also transferred to a mirror image of the endophilin channel ( detailed in supplemental experimental procedures ) .
we used wt and endophilin tko mefs , immortalized with e6/e7 viral proteins , grown on supplemented dmem at 5% co2 at 37c .
cells were pelleted and resuspended with n - dodecylmaltoside 1.5% in pbs with complete protease inhibitor cocktail ( roche ) and phosstop phosphatase inhibitor cocktail ( roche ) and lysed at 4c in a rotator for 30 min . the lysate
was then centrifuged to pellet debris and placed in 96-well glass - bottomed plates for use in the assay . the fluorimetric proteasome assay ( proteasome 20s activity assay kit ) was performed according to manufacturer s instructions using a synergy h1 plate reader ( biotek ) at 37c .
caspase 3/7 apoptosis assay was performed with the apotox - glo triplex assay ( promega ) kit according to a modified version of the manufacturer s protocol for 96-well plates , detailed in supplemental experimental procedures .
figures 2e [ with n.r . ] , 3a , 3b , 3e , and 6a6c ; c.m.r . : figures 1 [ with i.m . ] , 3c , 3f , 4a4c , 6d , 6 g , and 6h ) ; supervision , p.v . | summaryendophilin - a , a well - characterized endocytic adaptor essential for synaptic vesicle recycling , has recently been linked to neurodegeneration .
we report here that endophilin - a deficiency results in impaired movement , age - dependent ataxia , and neurodegeneration in mice .
transcriptional analysis of endophilin - a mutant mice , complemented by proteomics , highlighted ataxia- and protein - homeostasis - related genes and revealed upregulation of the e3-ubiquitin ligase fbxo32/atrogin-1 and its transcription factor foxo3a .
fbxo32 overexpression triggers apoptosis in cultured cells and neurons but , remarkably , coexpression of endophilin - a rescues it . fbxo32
interacts with all three endophilin - a proteins . similarly
to endophilin - a , fbxo32 tubulates membranes and localizes on clathrin - coated structures . additionally , fbxo32 and endophilin - a are necessary for autophagosome formation , and both colocalize transiently with autophagosomes .
our results point to a role for endophilin - a proteins in autophagy and protein degradation , processes that are impaired in their absence , potentially contributing to neurodegeneration and ataxia . | Introduction
Results
Discussion
Experimental Procedures
Author Contributions |
PMC4489318 | however , there continues to be a substantial gap between the avalanche of genomic data , which are abundant but not reliable , and our limited biological knowledge , which can only be discovered through careful , small - scale techniques .
this disparity has been exacerbated with the development and popularity of next - generation technologies , such as rna - seq , which enable genome - wide measurements at unprecedented resolution and cost ( 1 ) .
a paucity of biological knowledge ( i.e. experimentally validated gene function ) limits the coverage and accuracy of computational methods that require prior knowledge to learn novel biology , even when large - scale genomic data are available ( 2 ) .
thus , these methods are limited to performing well on processes and pathways that are already well characterized in an organism .
imp ( integrated multi - species prediction ) was originally developed to address the growing need to interpret and analyze results from genome - wide studies and generate hypotheses for experimental follow - up in the context of integrated functional gene networks , even when prior knowledge is limited in an organism or for a specific biological context ( 3 ) .
imp is an exploratory tool that provides a high - quality , interactive interface for functional prediction and interrogation .
for example , biologists can overlay their genes from a high - throughput experiment onto imp 's functional gene networks , expanding or contracting the network and identifying enriched , unifying functional themes .
alternatively , researchers can generate specific functional hypotheses by querying imp 's collection of gene - pathway predictions , identifying candidate genes for a biological context of interest . in all of these analyses ,
imp systematically applies a previously developed network - based method that identifies functionally similar homologs to transfer annotations ( i.e. gene - pathway membership ) between organisms .
this more specific homology detection method extends beyond simple annotation transfer by sequence similarity and enables accurate gene pathway predictions , even for processes that have few or no experimental annotations in an organism ( 2 ) .
there are several successful web servers that allow researchers to analyze their gene sets in the context of gene networks ( 46 ) , however , they are either constrained by the availability of prior knowledge in an organism and biological process of interest or limited to sequence - based transfers of input data ( 7,8 ) .
imp is unique in its systematic incorporation of a functional genomics - based homology transfer of prior knowledge ( 9 ) in all of its analyses , improving the accuracy and coverage of functional interrogation ( 2 ) .
imp has been continuously maintained and developed since the original publication and here we describe major updates to the server .
we have extensively updated the gene networks and functional predictions across all seven organisms , adding publicly available gene expression experiments from the subsequent years , and updating the already included data sources . additionally , we extend imp 's functional coverage to include human diseases , allowing biologists to analyze disease contexts and predictions in human and across model organisms .
human disease gene knowledge is transferred to other organisms and predictions are made using each organism 's gene network . by exploring disease gene predictions across the model organisms , biologists can find candidate genes to serve as targets for follow - up in human and in potential animal models for their disease of interest .
additionally , we have created a flexible tool that furthers the original goal of the web server : to enable biologists to analyze their experimental results in the context of massive - scale integrated data compendia .
we developed a prediction platform that allows biologists to bring their larger experimental result ( for example , a list of hundreds of genes identified as over - expressed in a microarray experiment ) and run a sophisticated machine - learning method for classification .
this tool can be used to answer many pertinent questions , for example , identifying additional candidate disease genes from a microarray experiment , or additional players in a biological process of interest .
such an analysis might otherwise be infeasible due to biologists limited computational resources or expertise .
the software is maintained and executed on imp 's servers and only requires a list of genes from the user .
genome - wide results are available by email , if provided , or directly on the web server .
imp is a flexible tool that can be used to answer diverse biological questions posed in the form of a biological context ( a process or a disease ) , a single gene , or a set of genes of interest .
these questions can be broad and exploratory , for example , determining the shared pathways among a set of genes that are co - expressed in an mrna experiment .
alternatively , researchers can generate specific experimentally testable hypotheses , such as identifying functional partners of a gene of interest or possible pathways that the gene participates in . as an exploratory tool ,
imp provides interactive visualizations of gene - gene functional relationships , gene - process predictions and cross - organism network alignments .
imp is both a collection of gene - pathway predictions that users can query for specific targeted results and a suite of user - driven tools that can be customized for broad discovery .
all of imp 's diverse analyses leverage an organism 's functional gene network , which integrates thousands of genome - wide experiments from an array of public data sources ( 1013 ) and describes whether genes participate in similar biological processes .
these networks are constructed using previously described methods ( 2,6,14 ) and have been extensively updated in the subsequent years since imp was originally released .
we use a new expert - curated set of gene ontology ( go ) terms ( 15 ) to define the gold standard for learning gene gene functional relationships and have updated the standard to include the latest experimental annotations .
imp networks now integrate 3540 data sets , a 49% increase in the number of data sets from imp 's original release ( 3 ) , and include over 70 000 experimental conditions .
in addition to adding gene expression experiments from the last three years , imp networks have been updated with the most recent releases of popular functional genomic databases .
for example , biogrid ( 10 ) has been updated to include 196 909 additional protein protein interactions , an increase of 78% from the original networks .
biologists can query imp with a gene set or biological context of interest to retrieve putative gene - pathway assignments .
we have extended imp 's biological contexts to include human diseases , in addition to go biological processes .
imp applies the same machine - learning method for predicting genes to biological processes ( 2,3 ) as it does to diseases , which uses a functional network as input to a support vector machine ( svm ) to classify genes ( figure 1 ) .
we showed previously that this method is accurate and competitive among state - of - the - art methods in predicting genes to biological processes ( 2,3 ) .
disease gene predictions are inferred directly in human from disease genes curated by online mendelian inheritance in man ( omim ) ( 16 ) and using the human functional network and in the six model organisms .
the disease predictions inferred in the other organisms leverage biological knowledge from human by transferring omim knowledge using our previously described method to identify the appropriate homologs for gene annotation transfer ( 2,9 ) .
these human - transferred gene - disease annotations are then used as training data for prediction with the organisms functional network , and the subsequent gene predictions are specific to that organism . by applying a model organism 's functional network to predict disease genes ,
imp can help biologists address an important challenge in the study of human disease : identifying the best model system for a given disease and the appropriate orthologs for a disease of interest .
( a ) imp constructs a functional network for each of seven organisms by integrating heterogeneous genomic data .
( b ) disease - gene annotations from human are transferred to the functionally similar homologs in other organisms .
( c ) additional disease genes are predicted using the human - transferred disease genes in the organism - specific functional networks .
using imp , users search by disease ontology ( do ) ( 17 ) term or by gene to retrieve gene - disease predictions .
omim disease genes are mapped to do , using the mapping provided by do , to leverage the unified naming and hierarchical structure of the ontology .
figure 2 shows queries for hypertrophic cardiomyopathy ( hcm ) in both human ( figure 2a ) and mouse ( figure 2b ) .
many of the top genes in the human query are known to be involved in the disease ( highlighted rows ) , and the others are potential disease candidates .
for example , the second novel gene prediction is trim63 , which encodes an e3 ubiquitin ligase and plays a role in the atrophy of skeletal and cardiac muscle ( 18,19 ) .
the gene has recently been suggested ( independent of imp ) as a candidate for hcm with several mutations observed in patients with the disease ( 20 ) .
hypertrophic cardiomyopathy in human returns a list of genes predicted to be involved in the disease , sorted by probability .
imp applies known hypertrophic cardiomyopathy genes in human ( from omim ) to predict additional genes from the human functional network .
( b ) the same disease query can be performed in mouse , returning predicted mouse genes .
these predictions were learned using human disease genes transferred to mouse with the mouse functional network .
these gene predictions , which leverage human disease knowledge transferred to mouse , are potentially informative as a mouse model for the disease .
in fact , the most confidently predicted gene , csrp3 , was a target in the first model for dilated cardiomyopathy with hypertrophy in a genetically manipulatable organism .
csrp3-deficient mice reproduce the same morphologic and clinical features of the disease as in human ( 21 ) .
the csrp3 mouse model serves as a valuable resource for understanding the pathophysiology of heart failure and for identifying potential therapies for the disease ( 22,23 ) .
thus , in these example use cases , imp independently , and in a data - driven predictive fashion , identifies a candidate human gene for hcn and a mouse gene that is already a model for understanding human hcm .
many biological questions can not be posed as a predefined gene set , such as a go biological process or omim disease , or expressed as a small gene set ( i.e. < 50 genes ) , requiring more advanced and flexible data - mining techniques .
for example , a researcher with results from a genetic screen may be interested in identifying additional candidate genes .
alternatively , a biologist may want to combine her private experimental result with public gene pathway annotations to make customized predictions .
most biologists lack the computational resources or expertise to implement and support the necessary machine learning software and data compendia for such an analysis .
with imp 2.0 , we provide a flexible platform for researchers to run state - of - the - art machine learning methods and pose customized , sophisticated biological questions .
users provide a gold standard , in the form of a set of relevant genes , or use imp provided gene sets , which include go biological process and do terms .
imp uses the same previously described and validated method for predicting go function ( 2,3 ) , which applies a svm with features constructed from the organism of interest 's functional gene network for classification .
the svm classifies all genes in the genome based on its pattern of functional relationships with the provided genes of interest , up - weighting the parts of the network that are informative for membership in the gene set .
this method has been previously shown to be accurate in predicting genes to biological processes and phenotypes , with corresponding estimates of prediction performance ( 2,24 ) .
a user starts an analysis by specifying an organism and her genes of interest , either manually , from a user - saved gene set , or pre - defined by imp .
pre - defined gene sets can be from go or do , and can include annotations transferred from other organisms by selecting the corresponding checkbox .
figure 3a shows the input for an analysis of five user - provided breast cancer genes .
these genes are treated as positive examples for classification , with random negative gene examples selected by imp for classification .
the researcher runs the analysis on imp 's servers using the human functional gene network ( figure 3b ) .
each gene in the genome is assigned a probability based on its five - fold cross - validated svm result , and results are sent by email , if provided , or viewed directly on the server though a result - specific url ( figure 3c ) .
performance is evaluated as the area under the receiver - operator curve ( auc ) and provided with the genome - wide prediction results . as we continue to update imp 's collection of functional networks in the future
, the prediction performance of this tool is expected to improve even further , and we encourage biologists to rerun their analyses . with these features , imp enables biologists to both pose complex biological questions and easily run sophisticated machine - learning tools to help answer them .
genes can be pasted , selected from a saved gene set , or chosen from a pre - defined set .
( b ) imp applies an svm with the provided gene set as positive examples and predicts additional genome - wide genes likely to be functionally related .
( c ) the output is a list of genome - wide genes , ranked by their probability of functional relationship with the provided gene set .
this result can be emailed to the user or accessed directly on the web server .
imp is a flexible , user - friendly web server that serves as an intuitive and accessible resource for molecular biologists who want to leverage heterogeneous biological big data collections to explore predictions of gene function and disease association in human and model organisms .
the described updates add substantial value to imp as a unique resource and suite of analysis tools for biological researchers . in the future
, we plan to continue to add additional organisms ( arabidopsis thaliana ) and additional data sources for our functional gene networks .
we continue to develop additional tools that leverage our cross - organism collection of networks and predictions with the goal of making complex tools and analyses accessible to biological researchers .
national science foundation ( nsf ) career [ dbi-0546275 ] ; national institutes of health [ r01 gm071966 , r01 hg005998 , t32 hg003284 ] ; national institute of general medical sciences ( nigms ) center of excellence [ p50 gm071508 ] . | imp ( integrative multi - species prediction ) , originally released in 2012 , is an interactive web server that enables molecular biologists to interpret experimental results and to generate hypotheses in the context of a large cross - organism compendium of functional predictions and networks .
the system provides biologists with a framework to analyze their candidate gene sets in the context of functional networks , expanding or refining their sets using functional relationships predicted from integrated high - throughput data .
imp 2.0 integrates updated prior knowledge and data collections from the last three years in the seven supported organisms ( homo sapiens , mus musculus , rattus norvegicus , drosophila melanogaster , danio rerio , caenorhabditis elegans , and saccharomyces cerevisiae ) and extends function prediction coverage to include human disease .
imp identifies homologs with conserved functional roles for disease knowledge transfer , allowing biologists to analyze disease contexts and predictions across all organisms .
additionally , imp 2.0 implements a new flexible platform for experts to generate custom hypotheses about biological processes or diseases , making sophisticated data - driven methods easily accessible to researchers .
imp does not require any registration or installation and is freely available for use at http://imp.princeton.edu . | INTRODUCTION
IMP SYSTEM DATA UPDATES
DISEASE PREDICTIONS
PLATFORM FOR CUSTOM PREDICTIONS
SUMMARY
FUNDING |
PMC4325607 | mosquitoes were sampled in a rural area , on the santa rosa farm ( 24.78951s 047.78068w ,
south american datum 69 ) , located at the serra azul neighbourhood , prefeito ivo zanella
state highway , sp-222 , km 87 , iguape ( fig .
1 ) .
fig . 1 : location of collection area in the municipality of iguape , state of so
paulo , brazil , 2012
.
mosquito collections were carried out monthly over the course of three summer months and
three autumn months , totaling six months of field collections ( from january - june 2012 ) .
a latin square collection design was adopted to avoid potential biases caused by
ecological and climatic peculiarities associated with the traps locations ( kline 2002 ) that might influence their performance -
either positively or negatively of the traps .
three locations were randomly selected on
the santa rosa farm , except for the distance among the traps that was somewhat fixed .
accordingly , each trap was separated from each other by approximately 200 m. based on
the published literature records , in order to avoid interference among the traps they
should be kept separate by distances that normally vary from 30 - 50 m ( cilek & hallmon 2005 , hiwat et al .
to ensure that there was no interference among
the traps , we adopted a distance of approximately 200 m. all traps were installed and
removed at the same time and period in day one and rotated in the consecutive two days .
the cdc - lt and cdc - a were installed at approximately 1.5 m above ground level in a way
that the light source , the lurex and co2 release point were at the same
distance of the mmi inlet relative to the ground level .
the traps were switched among
the three locations over consecutive days and were left running for 12 h per day ( from
06:00 pm-06.00 am ) , totaling 36 h per month for each trap .
the cdc - lt trap was chosen because it is widely used in entomological surveys .
another
choice was the cdc trap without light source , but with attractive . according to forattini et al .
( 1990 ) , the use of the co2 and lactic acid
as attractive in traps increases mosquitoes capture .
the mmi was chosen to compare its
effectiveness with other traps used in most entomological studies .
thus , in the present
study , we evaluated a cdc - lt , a cdc - a and an mmi with co2 plus
lurex3 .
the co2 used in the cdc was obtained from a compressed
gas cylinder with a controlled flow rate of 450 ml co2 per minute .
the
release of the co2 in the trap was controlled by a low - pressure valve
( swagelok , usa ) .
the cartridge of the lurex3contained 4.88 g
of lactic acid incorporated to 13.8 g of a gel matrix , thus releasing 230 mg / day of
lactic acid ( hoel et al .
the mmi simulates
the human presence by releasing co2 , heat , humidity and lactic acid provided
by the cartridge of the lurex3 , identical to that used in the cdc - a .
the data obtained with the cdc - lt were employed as the baseline for comparative
analyses .
this procedure was adopted because the cdc - lt has been widely employed for
surveying mosquito fauna ( hutchings et al .
mosquitoes were individually identified employing the morphological keys
proposed by lane ( 1953 ) , galindo et al .
( 1954 ) , correa and
ramalho ( 1956 ) , consoli and
loureno - de - oliveira ( 1998 ) and forattini
( 2002 ) .
the efficiency of traps was measured using two parameters : the diversity of species
sampled by each trap and the performance of each trap , i.e. , how each trap undertook the
mosquito sampling . for this ,
the time spent working on the basis of abundance and
species selectivity at the level of subfamily and tribe was analysed .
biodiversity ( kindt & coe 2005 ) and venneuler ( chen & boutros 2011 ) of the program r v.2.15.2 .
the homogeneity of variances was assessed by levene s test and the normality of
abundance data was examined by the shapiro - wilk and kolmogorov - smirnov statistical
tests .
the diversity of species per trap was estimated by the diversity index obtained
in the profile of the rnyi series ( melo 2008 )
that generalises total richness ( = 0 ) , diversity ( shannon - weiner = 1 ; simpson - yule
= 2 ) and dominance ( berger - parker = inf ) .
the kruskal - wallis ( kw ) test ( p > 0.05 )
was used to assess differences among the rnyi diversity index estimated for each trap .
the bonferroni test was employed to perform multiple comparisons among the traps testing
each pair of means .
bootstrap values were used to estimate the total species richness
expected for each trap .
jaccard ( cj ) and sorensen ( cn ) indexes were used to address the
similarity of the species captured by the traps .
the selectivity of traps estimated as
part of trap performance was evaluated by the pearson chi - squared test ( )
using the spss v.17.0 program .
correspondence analysis ( spss program v.15.0 ) which seeks
to synthesise the mass of data with ease of implementation and interpretation was
employed as a complement to the test and can graphically display the
association observed , because of similar categories are placed more closely to each
other ( hoffman & franke 1986 , carvalho et al .
this study showed the
relationship among the selectivity of each trap relative to species grouped on the basis
of the subfamilies and tribes they belong to and the trap employed .
the venn diagram was constructed to graphically illustrate the species shared by each
pair of traps , by all the traps and those captured exclusively by each individual
trap .
of
these , 860 individuals ( 4.25% ) were damaged and were not identified .
consequently ,
18,156 specimens were identified and grouped into 64 species and 11 taxonomic units
( table i ) .
the cdc - lt captured 18.82%
( 3,417 ) of all samples , with an average of 625 mosquitoes per month or 17 insects per
hour .
the cdc - a captured 26.10% ( 4,739 ) of the sample , with an average of 838
individuals per month , i.e. , 23 mosquitoes / hour .
the mmi trap captured 55.08% ( 10,000 )
of the insects representing an average of 1,706 specimens per month or 47
culicidae / hour . the statistical test to check the homoscedasticity of the variances of the observed data
between the three traps showed a p value = 0.001 with a statistical significance of p
> 0.05 . regarding the tests for assessing the normality of the data distribution , it
was seen that for each of the trap the values presented by the shapiro - wilk and
kolmogorov - smirnov tests were p = 0.000 for both tests , with a 5% level of significance ,
indicating that the mosquito abundance observed in the traps did not have a normal
distribution .
thus the necessary conditions for the employment of the parametric tests
were met ; accordingly non - parametric statistics were adopted for the analysis of the
data .
table ispecies and taxonomic units abundance per sampling trap in rural areas in
the tropical atlantic rainforest , southeastern brazil traps n ( % ) species / taxonomic unitabctotal ( n)anophelinae
anopheles ( anopheles ) costai
3 ( 1.08)64 ( 23.02)211 ( 75.9)278
anopheles ( anopheles ) intermedius
1 ( 7.14)1 ( 7.14)12 ( 85.71)14
anopheles ( kerteszia ) bellator
0 ( 0)9 ( 13.85)56 ( 86.15)65
anopheles ( kerteszia ) cruzii
0 ( 0)54 ( 21.86)193 ( 78.14)247
anopheles ( nyssorhynchus ) evansae
1 ( 100)0 ( 0)0 ( 0)1
anopheles ( nyssorhynchus ) galvaoi
0 ( 0)0 ( 0)2 ( 100)2
anopheles ( nyssorhynchus ) oswaldoi
0 ( 0)0 ( 0)1 ( 100)1
anopheles ( nyssorhynchus ) triannulatus
0 ( 0)0 ( 0)4 ( 100)4 total5 ( 0.82)128 ( 20.91)479 ( 78.27)612aedini
ochlerotatus ( protomacleaya ) argyrothorax
0 ( 0)0 ( 0)1 ( 100)1
ochlerotatus ( chrysoconops ) fulvus
0 ( 0)2 ( 100)0 ( 0)2
ochlerotatus ( ochlerotatus ) scapularis
4 ( 2.26)56 ( 31.64)117 ( 66.1)177
ochlerotatus ( protoculex ) oligopistus
0 ( 0)1 ( 100)0 ( 0)1
ochlerotatus ( protoculex ) serratus
11 ( 16.67)42 ( 63.63)13 ( 19.7)66
ochlerotatus ( protoculex ) serratus / nubilus
23 ( 11.79)112 ( 57.44)60 ( 30.77)195
psorophora ( grabhamia ) cingulata
1 ( 4.35)10 ( 43.48)12 ( 52.17)23
psorophora ( janthinosoma ) ferox
2 ( 15.38)3 ( 23.08)8 ( 61.54)13
psorophora ( janthinosoma ) lutzii
0 ( 0)7 ( 50)7 ( 50)14
sallumia hortator
1 ( 100)0 ( 0)0 ( 0)1 total42 ( 8.52)233 ( 47.26)218 ( 44.22)493culicini
culex ( aedes ) amazonensis
10 ( 30.30)15 ( 45.45)8 ( 24.24)33
culex ( culex ) chidesteri
3 ( 100)0 ( 0)0 ( 0)3
culex ( culex ) eduardoi
1 ( 100)0 ( 0)0 ( 0)1
culex ( culex ) nigripalpus
228 ( 13.02)381 ( 21.76)1,142 ( 65.22)1,751
culex ( culex ) quinquefasciatus
2 ( 8.33)1 ( 4.17)21 ( 87.5)24
culex ( culex ) sp.30 ( 81.08)3 ( 8.11)4 ( 10.81)37
culex ( culex ) sp . coronator group6 ( 100)0 ( 0)0 ( 0)6
culex ( microculex ) imitator
1 ( 100)0 ( 0)0 ( 0)1
culex ( microculex ) neglectus
1 ( 100)0 ( 0)0 ( 0)1
culex ( microculex ) pleuristriatus
0 ( 0)0 ( 0)1 ( 100)1
culex ( melanoconion ) akritos
71 ( 55.9)5 ( 3.94)51 ( 40.16)127
culex ( melanoconion ) eastor
5 ( 100)0 ( 0)0 ( 0)5
culex ( melanoconion ) evansae
1 ( 100)0 ( 0)0 ( 0)1
culex ( melanoconion ) faurani
0 ( 0)1 ( 100)0 ( 0)1
culex ( melanoconion ) misionensis
1 ( 100)0 ( 0)0 ( 0)1
culex ( melanoconion ) oedipus
1 ( 100)0 ( 0)0 ( 0)1
culex ( melanoconion ) pedroi
39 ( 59.1)27 ( 40.9)0 ( 0)66
culex ( melanoconion ) putumayensis
2 ( 100)0 ( 0)0 ( 0)2
culex ( melanoconion ) rabelloi
6 ( 100)0 ( 0)0 ( 0)6
culex ( melanoconion ) ribeirensis
691 ( 43.51)628 ( 39.55)269 ( 16.94)1,588
culex ( melanoconion ) sacchettae
1,360 ( 32.04)1,862 ( 43.86)1,023 ( 24.1)4,245
culex ( melanoconion ) sp . atratus group1 ( 100)0 ( 0)0 ( 0)1
culex ( melanoconion ) sp . melanoconion
section171 ( 72.15)46 ( 19.41)20 ( 8.44)237
culex ( melanoconion ) spissipes
4 ( 57.14)3 ( 42.86)0 ( 0)7
culex ( melanoconion ) vaxus
2 ( 100)0 ( 0)0 ( 0)2
culex ( melanoconion ) zeteki
1 ( 50)0 ( 0)1 ( 50)2
lutzia ( lutzia ) bigoti
1 ( 100)0 ( 0)0 ( 0)1 total2,639 ( 32.38)2,972 ( 36.46)2,540 ( 31.16)8,151mansoniini
coquillettidia ( rhynchotaenia ) albicosta
4 ( 22.22)3 ( 16.67)11 ( 61.11)18
coquillettidia ( rhynchotaenia ) chrysonotum
227 ( 3.46)1,144 ( 17.45)5,186 ( 79.09)6,557
coquillettidia ( rhynchotaenia ) hermanoi
17 ( 13.39)34 ( 26.77)76 ( 59.84)127
coquillettidia ( rhynchotaenia ) juxtamansonia
0 ( 0)2 ( 50)2 ( 50)4
coquillettidia ( rhynchotaenia ) venezuelensis
352 ( 20.29)151 ( 8.7)1,232 ( 71.01)1,735
mansonia ( mansonia ) titillans
1 ( 20)1 ( 20)3 ( 60)5 total601 ( 7.11)1,335 ( 15.81)6,510 ( 77.08)8,446sabethinii
limatus durhami
0 ( 0)6 ( 28.57)15 ( 71.73)21
limatus flavisetosus
1 ( 11.11)0 ( 0)8 ( 88.89)9
runchomyia ( runchomyia ) reversa
1 ( 5.26)6 ( 31.58)12 ( 63.16)19
wyeomyia ( phoniomyia ) davisi
0 ( 0)0 ( 0)4 ( 100)4
wyeomyia ( phoniomyia ) fuscipes
0 ( 0)0 ( 0)1 ( 100)1
wyeomyia ( phoniomyia ) galvaoi
0 ( 0)0 ( 0)18 ( 100)18
wyeomyia ( phoniomyia ) longirostris
0 ( 0)1 ( 20)4 ( 80)5
wyeomyia ( phoniomyia ) mulhensi
0 ( 0)0 ( 0)1 ( 100)1
wyeomyia ( phoniomyia ) pallidoventer
0 ( 0)0 ( 0)1 ( 100)1
wyeomyia ( phoniomyia ) palmata
0 ( 0)1 ( 50)1 ( 50)2
wyeomyia ( phoniomyia ) pilicauda / incauda
0 ( 0)0 ( 0)5 ( 100)5
wyeomyia ( phoniomyia ) near lassali
0 ( 0)0 ( 0)1 ( 100)1
wyeomyia ( phoniomyia ) near
longilostris
0 ( 0)0 ( 0)3 ( 100)3
wyeomyia ( phoniomyia ) quasilongirostris
0 ( 0)2 ( 16.67)10 ( 83.33)12
wyeomyia ( phoniomyia ) theobaldi
0 ( 0)0 ( 0)88
wyeomyia confusa
2 ( 1.1)55 ( 30.22)125 ( 68.68)182
wyeomyia felicia / pampeithes
0 ( 0)0 ( 0)26 ( 100)26
wyeomyia mystes / finlayi
0 ( 0)0 ( 0)3 ( 100)3
wyeomyia airosai / howardi / luteoventralis
0 ( 0)0 ( 0)6 ( 100)6 total4 ( 1.22)71 ( 21.71)252 ( 77.06)327uranotaeniini
uranotaenia ( uranotaenia ) geometrica
10 ( 100)0 ( 0)0 ( 0)10
uranotaenia ( uranotaenia ) incognita
1 ( 50)0 ( 0)1 ( 50)2
uranotaenia ( uranotaenia ) mathesoni
7 ( 100)0 ( 0)0 ( 0)7
uranotaenia ( uranotaenia ) natalie
5 ( 100)0 ( 0)0 ( 0)5
uranotaenia ( uranotaenia ) pulcherrima
103 ( 100)0 ( 0)0 ( 0)103 total126 ( 99.21)0 ( 0)1 ( 0.79)127total specimens3,417 ( 18.82)4,739 ( 26.1)10,000 ( 55.08)18,156total species / taxonomic unit47355075a : cdc light trap ; b : cdc with co2 and lactic acid ; c : mosquito
magnet independence .
a : cdc light trap ; b : cdc with co2 and lactic acid ; c : mosquito
magnet independence .
the results of the test showed the presence of a positive association
between the cdc - lt and species of the culicini and uranotaeniini tribes .
the
species / taxonomic units belonging to the culicini tribe represented 77.23% of the total
abundance sampled by the trap ( = 4594.040 ; p < 0.000 ) . as compared with
the other traps , the cdc - lt was highly selective for species of the ura- notaeniini
tribe , capturing 126 individuals , representing 99.21% of the total species of the genus
uranotaenia collected by the traps .
a , there was a positive association between the trap and
representatives of the culicini and aedini tribes .
thus 62.72% of the mosquitoes
captured in the trap belonged to the culicini ( 2,972 insects ) , whereas 4.91% were of the
aedini ( 233 individuals ) .
the mmi showed a positive association with species of the
mansoniini and sabethini tribes and the anophelinae subfamily .
mosquitoes of the
mansoniini represented 65.51% ( 6,510 individuals ) of the specimens obtained in the mmi ,
252 mosquitoes were of the sabethini and 479 belonged to the anophelinae subfamily .
the
mmi was capable of capturing a greater number of anopheles than the
cdc - lt and the cdc - a ( table ii ) , about 78% of
anopheles collected . the results of the correspondence analysis are
given in fig .
2 , which shows the associations of
the traps with the subfamilies and tribes .
table iispecies and taxonomic units abundance grouped into tribes and subfamily per
sampling trap in rural areas in the tropical atlantic rainforest , southeastern
brazil a n ( % ) b n ( % ) c n ( % ) culicinae aedini42 ( 1.23)233 ( 4.91)218 ( 2.18 ) culicini2,639 ( 77.23)2,972 ( 62.72)2,540 ( 25.4 ) mansoniini601 ( 17.58)1,335 ( 28.17)6,510 ( 65.1 ) sabethini4 ( 0.12)71 ( 1.5)252 ( 2.52 ) uranotaeniini126 ( 3.69)0 ( 0)1 ( 0.01)anophelinae5 ( 0.15)128 ( 2.7)479 ( 4.79)total3,417 ( 100)4,739 ( 100)10,000 ( 100)a : cdc light trap ; b : cdc with co2 and lactic acid ; c : mosquito
magnet independence ; = 4594.040 ; gl = 10 ; p =
0,000 .
a : cdc light trap ; b : cdc with co2 and lactic acid ; c : mosquito
magnet independence ; = 4594.040 ; gl = 10 ; p =
0,000 .
2 : correspondence analysis graphically represents the associations of the
traps ( cdc - a : cdc with co2 and lactic acid ; cdc - lt : cdc light trap ; mm :
mosquito magnet ) and the tribes and subfamily .
to address the diversity of culicids per trap , only individuals identified to the
species level
the results of the kw test indicated that the observed
differences between the rnyi diversity index ( fig .
3 ) were statistically significant only as regards richness ( kw =
19.338 ; p = 6.321 e-05 ) .
the results of the bonferroni test indicated that the mmi
presented a significant difference as compared to the cdc - lt ( p = 6e-05 ) and the cdc - a
( p = 0.0012 ) . however , the cdc - a showed no significant difference ( p = 0.6059 ) compared
with the cdc - lt .
the other rnyi diversity index were not statistically significant ,
shannon - weiner ( kw = 0.9298 ; p = 0.6282 ) , simpson - yule ( kw =
2.3813 ; p = 0.304 ) and berger - parker dominance ( kw = 3.4419 ; p = 0.1789 ) .
the expected values obtained by the bootstrap analyses were closer to the observed
richness in the mmi data because the species captured represented 88.7% of the expected
species ( sobserved = 44 ; bootstrap = 49.6 ) .
the cdc - lt was able to sample
83.5% ( sobserved = 44 , bootstrap = 52.7 ) and the cdc - a 76.7%
( sobserved = 33 , bootstrap = 43 ) of the expected species .
the mmi and
cdc - lt traps presented the same number of species , with some species common to both
traps and other species that were captured by one specific trap .
the cj ( 0.57 ) and the
cn ( 0.72 ) values showed similarities between species captured by the cdc - a and mmi traps
( supplementary data 1 ) . the venn diagram ( fig .
4 )
illustrated species distribution in each trap , as well as those that were shared by two
or three traps .
a : cdc light trap ; b : cdc with co2 and lactic acid ; c : mosquito magnet
independence .
4 : venn diagram illustrating the similarity of the mosquito species captured
and shared between traps in the municipality of iguape , state of so paulo ,
brazil .
a : cdc light trap ; b : cdc with co2 and lactic acid ; c : mosquito magnet
independence .
results of the analyses conducted for the present study clearly demonstrated that the
mmi is more efficient at capturing mosquitoes than the cdc - lt or the cdc - a . in this
context , the mmi captured 2.0 and ~2.7 times more culicids than the cdc - a and the
cdc - lt , respectively .
( 2010 ) when comparing the performance of different models of mm with the cdc
trap .
recently , de s and sallum ( 2013 ) obtained
similar results by employing the set of traps in different locations and with lower
sampling effort .
the traps used in this study captured species of importance to public health . among them
it is worth mentioning the coquillettidia venezuelensis that is
competent to transmit the oropuche virus and the eastern equine encephalitis virus
( eeev ) ( forattini 1965 , rosa et al .
anopheles cruzii and
anopheles bellator are local vectors of plasmodium
sp . ( deane et al
. 1984 , forattini et al . 1996 ) that can cause disease in humans .
ochlerotatus scapularis , captured in great abundance by mmi , is
involved in the transmission of several viruses , as for instance , the melon virus ,
ilhus virus , rocio virus and venezuelan equine encephalitis virus ( spence et al . 1962 , arnell 1976 , forattini et al .
culex nigripalpus is a vector of the saint louis virus , west nile
virus and eeev ( forattini et al .
the cdc - a captured a larger
number of ochlerotatus serratus and oc .
serratus / nubilus with vector competence for the yellow
fever virus ( yfv ) , ilhus , oropuche , aura , tocara and san luis viruses ( forattini 1965 , vasconcelos et al .
culex ribeirensis with vector competence for the eeev
( santos - neto & lozovei 2008 ) was captured
with greater abundance by the cdc - lt . regarding selectivity ,
the cdc - lt showed a statistically significant positive
association with species of the culicini and uranotaeniini tribes .
as the uranotaeniini
species feed on the blood of amphibians ( cupp et al .
2004 ) , the attractant factor was the light source of the cdc - lt that was
absent from the cdc - a .
the traps were linked to kairomones that attract insects that
carry the blood obtained from mammals , including humans ( dugassa et al .
2013 ) , thus the absence of mosquitoes of the genus
uranotaenia in the mmi was also observed by de s and sallum ( 2013 ) .
the cdc - a showed a positive association
with species of the culicini and aedini tribes .
it is noteworthy that the cdc - a
estimated the greatest abundance of oc .
the presence of these mosquitoes in the cdc - a indicates
the possibility of employing it for monitoring mosquito vector species of medical
importance .
for instance , for surveillance of species that is potentially involved in
the introduction , establishment and dispersion of the yfv in new areas . according to
cardoso et al .
( 2010a ) , a new subtype of yfv had been isolated from specimens of
oc .
. results of a study conducted by laporta and sallum
( 2011 ) in a preserved area of the atlantic forest in southeastern sp , using
cdcs traps associated with co2 and 1-octeno-3-ol , showed that oc .
serratus was the second most abundant species sampled in the preserved
forest area . considering that oc .
serratus is present in both preserved
forest areas as well as in rural areas surrounded by secondary forests we may be
permitted to hypothesise that the species acts as a bridge vector for dispersing the yfv
from forest to rural cycles .
the mmi was positively associated with species of the mansoniini tribe and with
coquillettidia chrysonotum as the most abundant species .
chrysonotum has not yet been incriminated in the transmission of pathogens
to humans ; however , its aggressive blood feeding behavior ( forattini 2002 ) is a cause of discomfort and annoyance to humans and
domestic animals .
as has been seen , the mmi trap performed well in sampling species of
mosquitoes that have the potential to become pests in urban and rural environments .
a
second model of trap , the mm professional ( mmpro ) , has been employed for the control of
culicoides spp in the peridomestic environment in coastal areas of
florida , united states of america ( cilek & hallmon
2005 ) .
the mmpro is capable of sampling several other groups of bloodsucking
insects as well as mosquitoes .
furthermore , the mmpro has been utilised for assessing
biodiversity parameters and species distribution in urban , rural and natural
environments in belgium ( versteirt et al
2013 ) . in relation to the subfamily anophelinae ,
the mmi trap sampled the largest number of
representatives of the genus anopheles ( 479 individuals ) .
.
currently , almost 99.5% of human malaria cases reported in brazil are the amazon region .
some cases do occur also outside the area of active transmission , in extra - amazonian
areas , where malaria is manifested as isolated cases or in small outbreaks occurring
mainly under the conditions associated with the atlantic forest biome ( oliveira - ferreira et al .
bellator are local vectors of
species of the genus plasmodium sp . that cause malaria in humans .
the
transmission cycle of the pathogen may involve monkeys of the genus
cebus and allouata ( oliveira - ferreira et al . 2010 , yamasaki et
al .
2011 ) . in the study area , the mmi collected a higher number of an .
thus , in the light of the results of this study within the context of
entomological surveillance , the mmi was more effective for monitoring mosquito species
of public health importance .
other models of mm trap have been tested for surveillance of those
anopheles species that are vectors of plasmodium .
( 2012 ) examined the
effectiveness of mm liberty plus ( mmlp ) regarding the method of capture by human
attractant in an area where malaria is endemic , in the state of bolivar , venezuela .
they
argued that the mmlp can be employed for monitoring anopheles
populations . in a study conducted in the state of mato grosso , brazil ,
2011 ) compared the mm defender with
human attraction for the sampling of anopheles .
the results showed that
mm defender was efficient because it captured the highest number of species and
estimated the greater abundance of anopheles triannulatus ,
anopheles benarrochi , anopheles oswaldoi ,
anophles peryassui and anopheles rangeli .
interestingly , this trap did not have the same positive effect in collecting
anopheles darlingi .
( 2011a ) , in a study in suriname ,
found that the mm trap has potential for use as an alternative for collecting
anopheles aquasalis .
the various mm models have been developed to capture synanthropic mosquitoes in open
domiciliary environments ( cilek & hallmon
2005 ) .
however , multiple studies have been conducted to address the
effectiveness of the trap in environments with distinct environmental , climatic and
ecological conditions , including areas of tropical forest .
despite the presence of
variations , the results suggest that the trap has potential for use in monitoring
activities of mosquito fauna in different regions of the world , since it is able to
capture diverse species compositions . in this study ,
when the composition of species sampled by the mmi was compared with that
observed in the cdc - a , greater similarity could be observed between them .
this was
proven by the similarity index , a result also observed by de s and sallum ( 2013 ) . the similarity is probably related to
attractants ( co2 or lurex3 ) used in the traps , which permitted
the collection of species that can carry blood from mammals and other vertebrates .
traps
which have specific odours attractive to anthropophilic species , particularly combined
with co2 , may provide improved results in the sampling of these species and
have the potential to be effective as methods for capture , for example , in entomological
monitoring and ecological studies of malaria vectors ( jawara et al . 2009 , 2011 , hiwat et al .
it is noteworthy that the flow rate of co2 might influence the
mosquitoes collected .
on the other hand , the species composition
was different ( supplementary data 2 ) .
the use of cdc - lts for capturing diurnal mosquitoes is not appropriate
because it depends on the attractiveness of a point source of light . to improve the
performance of the collections made with the cdcs , chemicals attractants ( laporta & sallum 2011 ) or
associating
ultraviolet ( uv ) light with the cdc increases the effectiveness of the trap , both for
the species sampled and for obtaining males ( hutchings
et al .
however , other groups of insects may be also captured by a
cdc - uv trap , what is not adequate in studies conducted in preserved or even rural
environments .
insects outside the focus group may be unnecessarily removed from the
environment , sometimes in great number .
it is noteworthy that neither the mmi nor the
cdc - a captured specimens of any other group of insects besides culicidae - due to the
use of specific attractants .
it is necessary to adopt different , new methods of capture if one is to achieve better
sampling in order to assist study of the epidemiological surveillance of vectors .
the
mmi , despite requiring greater care in its installation , use and transport , presented
good performance both in its ability to sequester a large number of mosquitoes from the
environment , as in the capture of culicidae of importance to public health . | traps are widely employed for sampling and monitoring mosquito populations for
surveillance , ecological and fauna studies . considering the importance of assessing
other technologies for sampling mosquitoes
, we addressed the effectiveness of
mosquito magnet independence ( mmi ) in comparison with those of the cdc
trap with co2 and lurex3 ( cdc - a ) and the cdc light trap ( cdc - lt ) .
field
collections were performed in a rural area within the atlantic forest biome ,
southeastern state of so paulo , brazil . the mmi sampled 53.84% of the total number
of mosquitoes , the cdc - a ( 26.43% ) and cdc - lt ( 19.73% ) .
results of the pearson
chi - squared test ( 2 ) showed a positive association between cdc - lt and species of
culicini and uranotaeniini tribes . additionally , our results suggested a positive
association between cdc - a and representatives of the culicini and aedini tribes ,
whereas the mmi was positively associated with the mansoniini and sabethini as well
as with anophelinae species .
the mmi sampled a greater proportion ( 78.27% ) of
individuals of anopheles than either the cdc - lt ( 0.82% ) or the cdc - a
traps ( 20.91% ) .
results of the present study showed that mmi performed better than
cdc - lt or cdc - a in sampling mosquitoes in large numbers , medically important species
and assessing diversity parameters in rural southeastern atlantic forest . | MATERIALS AND METHODS
RESULTS
DISCUSSION |
PMC4961814 | meprins are zinc metalloproteases that are most abundantly expressed in the brush - border membranes ( bbms ) of proximal kidney tubules .
several studies have demonstrated a role for meprins in the pathology of ischemia / reperfusion ( ir ) induced renal injury .
pretreatment of mice with the meprin inhibitor , actinonin , and disruption of the meprin gene both protect mice against ir - induced renal injury [ 13 ] , suggesting that presence of meprins enhances acute kidney injury .
however , the mechanisms by which meprins modulate kidney injury in ir are not fully understood .
meprins consist of two subunits , and , resulting in two protein isoforms .
meprin a is a homooligomer of - subunits or a heterodimer of - subunits , while meprin b is a homooligomer of - subunits .
while meprin a and meprin b are highly similar and have several shared substrates , there are unique substrates specific for each isoform . identifying meprin substrates in the kidney
has provided insights on how meprins modulate kidney injury in ir . based on the currently known substrates
, meprins could enhance kidney injury via proteolytic processing / degrading of cytoskeletal proteins ( e.g. , villin and actin ) and tight junction proteins ( e.g. , occludin and e - cadherin ) [ 5 , 6 ] . in ir , meprins have been shown to be redistributed from the bbm to the cytosol and basolateral membranes .
this redistribution brings meprins in close proximity to proteins in other cell compartments that could be proteolytically processed and thus impact the cellular response to ir .
meprin targets in the cytosol and basement membrane include cell signaling molecules ( e.g. , the catalytic subunit of protein kinase a , pka c ) [ 7 , 8 ] and the extracellular matrix ( ecm ) proteins ( e.g. , laminin , fibronectin , nidogen-1 , and collagen ) [ 912 ] .
the injury associated with ir is due , in large part , to reduced oxygen delivery to renal tissue .
the normal response to hypoxia is mediated by hypoxia response genes , which include hypoxia inducible factors ( hifs ) , for example , hif-1 and hif-2. hif-1 and hif-2 have cell - type specific effects on gene expression . in the kidney
, hif-1 is the predominant form in epithelial cells , while hif-2 is predominant in interstitial fibroblast and endothelial cells .
hif-1 target genes encode proteins that enable cells to survive oxygen deprivation by providing oxygen - independent means of atp production such as glucose transporters and glycolytic enzymes or by inhibiting hypoxia - induced apoptosis for example , through survival factors like insulin - like growth factor 2 ( igf2 ) .
studies with a yeast two - hybrid system showed that os-9 interacts with the carboxyl - terminal tail of meprin .
this interaction is significant because os-9 has also been shown to interact with hif-1 and prolyl hydroxylase , proteins which mediate the cell 's response to changes in oxygen concentration .
although os-9 is localized in the endoplasmic reticulum , it is present in both nuclear and cytoplasmic protein extracts , suggesting that it could traffic hif proteins between the nucleus and the er during the hypoxic response .
the current study was conducted to determine if os-9 is a meprin substrate and whether there is a correlation between meprin expression and proteolytic processing of os-9 in vivo and in vitro .
the findings will provide new insights on how meprins modulate kidney injury under hypoxic conditions such as ir - induced acute kidney injury .
twelve - week - old wild - type and meprin double knockout ( ko ) mice on a c57bl/6 background were used for these studies .
the mice were housed at the north carolina a&t state university laboratory animal resource unit ( laru ) , greensboro , north carolina .
all the mice were kept under a 12 : 12 light : dark cycle and provided rodent chow and fresh water ad libitum .
the animal procedures used were approved by the north carolina a&t state university institutional animal care and use committee ( iacuc ) .
ischemia reperfusion was induced in wt and meprin ko mice ( n = 6 for each genotype ) by bilateral clamping of the renal pedicle for 26 minutes followed by reperfusion for 6 h or 24 h as previously described [ 1 , 4 ] . control mice ( n = 4 for each genotype ) were sham - operated without clamping the renal pedicle .
the mice were sacrificed at 6 h or 24 h after ir by using isoflurane asphyxiation followed by cervical dislocation .
sections of the kidney were fixed in carnoy 's fixative ( 60% ethanol , 30% formalin , and 10% acetic acid ) overnight and then transferred to 70% ethanol .
the kidney tissues were subsequently paraffin embedded at the penn state hershey histology core laboratories .
the remaining kidney tissues were wrapped in aluminum foil , snap - frozen in liquid nitrogen , and stored at 80c until used for protein extraction . to confirm kidney injury , the levels of blood urea nitrogen ( bun ) were determined using bun slides ( cat .
# 1532332 , ortho - clinical diagnostics , rochester ny ) read on a vitro dt60 ii analyzer ( ortho - clinical diagnostics ) .
the kidney tissues were thawed on ice and homogenized in ice - cold edta - free lysis buffer with protease inhibitors .
proteins were fractionated by differential centrifugation as previously described [ 4 , 7 , 17 ] to obtain a bbm- and cytosolic - enriched protein fractions .
some kidney tissues were extracted in ripa buffer without any fractionation ( herein referred to as total protein ) . extracted kidney proteins were aliquoted and stored at 80c until used for western blot analysis .
we first determined the kidney protein fraction that had the most abundant os-9 levels by loading 60 g of total ( nonfractionated kidney proteins ) , cytosolic- , and bbm - enriched kidney proteins onto 10% sds - page gels .
the proteins were transferred to nitrocellulose membranes and western blot analysis was used to evaluate levels of os-9 .
our data showed that os-9 proteins were most abundant in the cytosolic - enriched protein fraction ( figure 1(a ) ) .
for this reason , subsequent analysis utilized cytosolic - enriched protein fractions . to determine whether meprins are capable of proteolytically processing os-9 present in kidney tissue , activated purified forms of recombinant homomeric meprin a ( - ) and meprin b ( - ) were incubated with 60 g of cytosolic - enriched kidney proteins from meprin double ko mice ( which lack endogenous meprins ) in tris buffer ( 20 mm tris and 150 mm nacl , ph 7.5 ) at 37c for 4 h. the recombinant homomeric meprin a and meprin b used were purified from stably transfected human embryonic kidney ( hek293 ) cells and activated using limited trypsin proteolysis as previously described [ 4 , 18 ] .
briefly , meprins were incubated with trypsin ( 1 : 20 ratio ) in 20 mm tris , 150 mm nacl , ph 7.5 , at 37c for 30 min ( meprin a ) or 1 h ( meprin b ) .
the trypsin and sti were removed by running the reaction mixtures through a sephadex g25 column .
meprin activity was measured by using the substrate azocasein ( sigma - aldrich , mi ) .
product fluorescence was measured with a hitachi f2000 fluorescence spectrophotometer using an excitation wavelength of 320 nm and an emission wavelength of 417 nm . to confirm that the cleavage was meprin b - specific , tris buffer and latent forms of meprin a and meprin b that were not trypsin - activated were used as negative controls .
additional negative controls used meprin b which was preincubated with 30 m of the meprin inhibitor , actinonin for 1 h. the products obtained from coincubating meprins with protein lysates were electrophoretically separated and western blot analysis with anti - os-9 specific antibodies ( cat .
# ta301503 , origene , rockville md ) was used to evaluate for degradation / fragmentation of os-9 .
our preliminary data showed that meprins cleave os-9 present in kidney proteins . to confirm that degradation of os-9 was meprin - specific , we incubated purified human os-9 ( cat .
# tp316820 , origene , rockville , md ) with activated forms of meprin a and meprin b and used western blot analysis to evaluate protein fragmentation / degradation . to this end , 4
nm activated homomeric meprin a ( - ) and 4 nm meprin b ( - ) were incubated with 92 nm purified os-9 in a total reaction volume of 5065 l . equal volumes of reactants ( 7 l ) were taken out at 0 , 0.25 , 0.5 , 1 , 2 , 3 , and 4 h. additional incubations were done for shorter time points totaling 1 h , with samples being taken out at 5 minute intervals .
proteolysis was stopped by addition of sds sample buffer followed by boiling for 5 minutes .
the proteins were electrophoretically separated on 10% sds gels , and western blot analysis with anti - os-9 specific antibodies ( origene , rockville , md ) was used to evaluate changes in os-9 levels and fragmentation over time . because os-9 has been shown to interact with the hypoxia response factor , hif-1 , we chose to work with an in vivo hypoxia model ( ischemia / reperfusion ) which causes acute kidney injury . to evaluate meprin degradation of os-9 in vivo , cytosolic - enriched kidney protein fractions from wt and meprin ko mice subjected to ir
the levels of os-9 in individual protein samples from sham - operated control kidneys and kidneys subjected to 6 h ir were quantified by optic densitometry using bio - rad 's gs800 calibrated densitometer . to determine whether meprin b interacts with os-9 in vitro
, mdck cells were transfected with a meprin cdna construct using the lipofectamine method as previously described [ 1921 ] .
the cdna construct sequence was analyzed at the penn state hershey genomic core laboratories and the sequence confirmed using blast alignment before transfecting the cells .
stably transfected cell lines were established using hygromycin b supplemented dulbecco 's modified essential medium ( dmem ) .
western blot analysis and immunofluorescence staining were used to confirm expression of meprin b proteins by the transfected mdck cells .
meprin transfected and mock transfected control mdck cells were seeded at 1 10 in 100 mm dishes and cultured in dmem supplemented with 10% fbs and antibiotics / antimycotics at 37c and 5% co2 until they were 8090% confluent .
judith bond , penn state college of medicine , hershey , pa ) were also used .
the hek293 cells were similarly cultured in mem media supplemented with 10% fbs and antibiotics / antimycotics . to induce hypoxia , the cells were serum - starved by overnight incubation in serum - free media supplemented with 0.1% bsa .
the cells were then exposed to 125 m cobalt chloride ( cocl2 ) , a hypoxia mimic for 03 h. cells for immunofluorescence staining were cultured in 8-well slide chambers and similarly exposed to 125 m cocl2 .
cell protein lysates were fractionated into cytosolic- , nuclear- , and membrane - enriched fractions using previously described protocols [ 5 , 20 ] .
all cell lysis buffers used were supplemented with protease inhibitors with edta ( roche diagnostics , indianapolis , in ) .
bio - rad 's protein reagent ( cat . # 500 - 0006 ) was used to determine the protein concentrations .
western blot analysis was used to confirm meprin b protein expression by the transfected mdck cells , to evaluate the levels of hif-1 and os-9 in kidney proteins and mdck protein lysates , and for evaluating fragmentation and/or degradation of os-9 .
the protocols used were as previously described [ 4 , 7 ] . to confirm that cocl2 treatment induced hypoxia , nuclear - enriched proteins were probed for hif-1 by incubation in anti - hif-1 antibody ( mouse monoclonal , cat .
# ab16066 , abcam , cambridge , uk ) , diluted 1 : 1000 in tbs - t .
to probe for os-9 , cytosolic- and nuclear - enriched protein fractions were incubated in rabbit polyclonal anti - os-9 antibodies ( cat .
# ta301503 , origene , rockville , md ) , diluted 1 : 1000 in tbs - t .
the intensities of the protein bands on the x - ray film were quantified by optic densitometry using bio - rad 's gs800 calibrated densitometer and quantity one software .
the levels of tubulin were used as a loading control for determining the relative ods for os-9 and hif-1. immunofluorescence staining was used to evaluate protein expression for hif-1 and os-9 in cultured cells exposed to cocl2 .
the mdck cells were cultured in slide chambers until 8090% confluent and exposed to cocl2 as described above .
the cells were fixed by heating at 70c for 10 minutes in freshly made 1% paraformaldehyde .
the cells were then permeabilized in 0.2% triton - x 100 in pbs for 10 min at room temperature ( rt ) .
nonspecific binding sites were blocked in 5% normal goat serum in pbs with 0.1% triton - x-100 for 1 h at room temperature .
this was followed by incubation in primary antibodies at rt for 1 h or overnight at 4c in a humidified chamber .
the antibodies used were rabbit polyclonal anti - meprin b ( hmc77 , a gift from dr .
judith bond , pennsylvania state university , hershey , pa ) diluted 1 : 400 , mouse monoclonal anti - hif1 ( abcam , cambridge , uk ) diluted 1 : 200 , and rabbit polyclonal anti - os-9 ( origene , rockville , md ) diluted 1 : 200 .
boston , ma ) diluted 1 : 1000 or anti - mouse alexa fluor555 ( cat .
the slide sections were imaged using a confocal microscope ( carl zeiss microscopy , llc , thornwood , ny ) with axiovision software .
two - way anova with pairwise comparisons was employed to evaluate protein levels following quantitation of the protein bands by optic densitometry .
western blot analysis of mouse kidney proteins detected os-9 ( ~88 kda ) predominantly in the cytosolic - enriched kidney protein fractions ( figure 1(a ) ) .
incubation of cytosolic - enriched kidney proteins from meprin ko mice with activated meprin b resulted in degradation of os-9 present in the kidney proteins .
this degradation was not observed when proteins were incubated with activated meprin a ( figure 1(b ) ) . to confirm that the degradation of os-9 was meprin - specific , activated forms of recombinant homomeric mouse meprin a ( - ) and rat meprin b ( - ) were incubated with purified human os-9 for 04 h at 37c
. a time - dependent degradation of the purified os-9 was observed in reactions with activated meprin b , but not in proteins incubated with activated meprin a or tris buffer without meprins ( figures 2 and 3 ) .
degradation of os-9 was not observed in control reactions incubated with latent meprin b or reactions in which activated meprin b was preincubated with the meprin inhibitor , actinonin ( figure 3(b ) ) .
however , we did not observe accumulation of os-9 intermediate fragments when purified os-9 was incubated with meprin b. to determine whether meprins proteolytically process os-9 present in kidney tissue in vivo , western blot analysis was used to evaluate fragmentation of os-9 in cytosolic - enriched kidney proteins from wt and meprin ko mice subjected to ir - induced acute kidney injury .
kidney injury was first confirmed by assays for blood urea nitrogen ( bun ) ( figure 4(b ) ) .
the bun levels were significantly higher in mice subjected to ir injury when compared to sham - operated controls at 6 and 24 h after ir ( p 0.05 and p 0.0001 , resp . ) .
bun levels at 24 for wt were also significantly higher than for the meprin ko at the same time point ( p 0.05 ) .
the pattern for markers of kidney injury was consistent with data from previous studies where the plasma creatinine levels were significantly higher in wt mice when compared to meprin knockout counterparts at 6 and 24 h after ir .
the western blot data showed fragmentation of the os-9 present in samples from wt mouse kidneys ( which express normal levels of meprin a and meprin b ) at 6 h after ir .
the ir - associated fragmentation produced a unique ~60 kda os-9 fragment in proteins from wt kidneys at 6 h after ir .
this fragmentation was not observed in proteins from meprin ko kidneys ( which are deficient in meprin a and meprin b ) subjected to ir or wt control kidneys not subjected to ir ( figure 4(a ) ) .
the data suggest that the presence of both meprins and hypoxia / atp - depletion associated with ir plays a role in the cleavage of os-9 to produce the 60 kda fragment . a second fragment ( ~37 kda )
however , its presence did not correlate to ir - induced kidney injury . to evaluate in vitro interactions between meprin b and os-9 , we used meprin transfected madin - darby canine kidney ( mdck ) cells and human embryonic kidney ( hek293 ) cells .
we first used western blot analysis to determine localization of os-9 in mdck cells exposed to cocl2 .
our data showed that os-9 is predominantly present in the nuclear - enriched protein fraction ( figure 5(a ) ) .
western blot analysis also confirmed expression of meprin b in the meprin transfected mdck ( figure 5(b ) ) and hek293 cells ( data not shown ) .
exposure of the cells to the hypoxia mimic , cocl2 , resulted in a time - depended stabilization of nuclear hif-1 leading to increased levels of hif-1 in both mdck ( figure 6 ) and hek293 ( figure 7 ) cells .
we then determined changes in the nuclear levels of os-9 in mdck and hek293 following exposure to cocl2 . in mock transfected cells
this increase was observed at 0.5 h after cocl2 exposure and was sustained through the 3 h time course . in meprin
transfected cells on the other hand , the levels of os-9 significantly decreased ( 5075% decrease ; p 0.001 ) following exposure to cocl2 ( figures 6 and 7 ) .
immunofluorescence staining and confocal microscopy also showed that both the nuclear and cytosolic levels of os-9 increased following exposure to cocl2 in nontransfected mdck cells ( figure 8(a ) ) .
however , this increase was not observed in meprin transfected cells , where in contrast the os-9 levels decreased in a time - dependent manner following exposure to cocl2 ( figure 8(b ) ) .
an equally important observation was the correlation between meprin b degradation of os-9 and the relative levels for nuclear hif-1 ( figures 6 and 7 ) .
in mock transfected mdck cells , we observed a more modest increase in hif-1 ( ~2-fold at 2 h ) .
however for meprin transfected mdck cells , the fold - change in hif-1 was significantly higher ( ~3 fold at 2 h ) ( p 0.0001 ) .
mdck cells also had higher basal levels of hif-1 when compared to hek293 cells , for which hif-1 levels were below detectable levels .
the relative quantities for hif-1 were significantly higher in meprin transfected hek293 cells when compared to the mock transfected control cells .
acute kidney injury caused by ischemia / reperfusion ( ir ) occurs in nearly 5% of hospitalized patients .
the reperfusion phase of ir is associated with oxidative stress , cellular dysfunction , and altered signal transduction . in ir - induced renal injury , disruption in the cytoskeleton leads to loss of the brush - border , a breakdown of cell junctions , and the incorrect relocalization of sodium - potassium atpases from the basal surface to the apical surface .
this reperfusion event has been shown to be the causative agent of oxidative injury to tubular cells .
however , specific proteases that are activated in ir and their substrates are not fully understood .
meprins are metalloprotease that are abundantly expressed in the brush - border membranes ( bbms ) of proximal kidney tubules .
changes in the level of expression and localization of meprins have been associated with the pathology of ir - induced kidney injury in mice and rats [ 1 , 10 , 24 , 25 ] . while meprins are normally localized in the bbm of proximal tubules , redistribution of meprins to the cytosol and basolateral compartments
this redistribution brings meprins in close proximity with protein targets in these compartments , which include mediators of the hypoxia response . the cells response to hypoxia
is primarily mediated by hypoxia inducible factors ( hifs ) , which consist of two subunits , an oxygen - regulated subunit and a constitutively expressed subunit . under normoxia conditions , hif - prolyl - hydroxylase promotes degradation of hif. however , hypoxia inhibits hif - prolyl - hydroxylases , resulting in rapid accumulation of hif , which then binds to hif to form a heterodimer .
the hif dimers translocate into the nucleus where they activate genes that promote adaptation to hypoxia by counteracting oxidative stress and improving cell survival . in the kidney ,
hif-1 is expressed in tubular cells while hif-2 is predominantly in peritubular cells , renal interstitial fibroblast cells , and endothelia cells .
while hifs are central in mediating the response to hypoxia , multiple mechanisms are involved in the pathophysiology of hypoxia - induced renal injury .
osteosarcoma amplified 9 ( os-9 ) , an endoplasmic reticulum ( er ) lectin , is a protein coding gene located on 12q13 chromosome of humans .
os-9 is part of the endoplasmic reticulum - associated degradation ( erad ) machinery and erad substrates and aids in the transfer of misfolded proteins .
using rnai , os-9 was shown to be required for efficient ubiquitination of glycosylated erad substrates .
there is increasing evidence that os-9 is a critical component of the oxygen - signaling that upregulates hif-1 .
os-9 negatively regulates hif-1 by binding to the hif-1 and prolyl - hydroxylases ( phds ) promoting degradation of one of its subunits .
the presence of os-9 promotes interaction of hif-1 with prolyl - hydroxylase domain proteins phd2 or phd3 , leading to hydroxylation and von hippel - lindau ( vhl ) binding . by promoting degradation of hif-1
, os-9 could potentially modulate the hypoxia response and is a key player in the pathophysiology of renal ir .
studies using a yeast two - hybrid system showed that meprin interacts with os-9 .
however , it was not known whether meprins are capable of cleaving os-9 and what the significance of such cleavage would be .
the present study evaluated the interaction between meprin metalloproteases and os-9 in vivo and in vitro .
our data shows that meprin metalloproteases are capable of proteolytically processing os-9 in vitro and in vivo .
coincubation of meprins with kidney proteins from meprin ko mice ( which lack endogenous meprins ) and purified os-9 confirmed that the os-9 cleavage was meprin b - specific and was inhibited by the meprin inhibitor , actinonin , at molar concentrations previously demonstrated to inhibit meprin activity . to the best of our knowledge
previous studies have demonstrated differences in substrate and cleavage site preferences for the meprin protein isoforms , with meprin a selecting a variety of small and hydrophilic amino acids ( e.g. , proline residues ) while meprin b prefers acidic residues ( e.g. , asp / glu - n peptidase ) .
the present data further shows that hypoxia could induce meprin b degradation of os-9 in vitro and in vivo . a distinct 60 kda os-9 fragment
was observed in the cytosolic kidney proteins from wild - type mice subjected to ir - induced renal injury , but not in samples from meprin ko mice counterparts ( which lack endogenous meprins ) .
interestingly , we did not observe accumulation of the 60 kda os-9 fragment when meprin b was incubated with purified os-9 .
this could suggest that while meprin b cleaves os-9 , other factors present in the kidney tissue could play a role in stabilizing the cleavage products .
degradation of os-9 was similarly observed in meprin transfected mdck and hek293 cells exposed to the hypoxia mimic , cocl2 , suggesting that hypoxia enhances meprin degradation of os-9 .
studies that identify the meprin cleavage sites on os-9 are thus needed to determine the functional implications of the meprin - os-9 cleavage . because os-9 is believed to shuttle the hypoxia inducible factor ( hif ) between the nucleus and er
using immunofluorescence staining and confocal microscopy , we further showed that , in nontransected kidney cell lines , there was a time - dependent redistribution of os-9 from the nucleus to the cytosol over a 3 h period following exposure to cocl2 .
however , in meprin transfected cells , the hypoxia - induced cytosolic accumulation of os-9 did not occur .
taken together , the data suggest that meprin b is partly responsible for the in vivo os-9 fragmentation and that meprin b degradation of os-9 blocks the cytosolic accumulation of os-9 in the mdck and hek293 cells .
the current data thus demonstrates a link between the previously reported interaction between os-9 and meprin to the pathology of acute kidney injury induced by ir .
indeed , the fold - change in nuclear hif-1 levels was significantly higher in meprin expressing cells under hypoxic conditions than in the nontransfected cells .
previous work from our lab has shown that meprins are capable of proteolytically processing / degrading cytoskeletal kidney proteins , for example , actin and villin , the catalytic subunit of protein kinase a ( pka c ) , and protein kinase c ( pkc ) , all in an isoform - specific manner . furthermore , ir - induced acute kidney injury resulted in meprin - associated changes in pka c profiles in mouse kidney tubules , leading us to conclude that the pka signaling pathway is involved in the hypoxia response .
other known kidney meprin substrates include extracellular matrix ( ecm ) proteins [ 912 ] , tight junction proteins , for example , e - cadherin and occludin [ 5 , 31 ] , and proinflammatory cytokines , for example , il-1 , il-6 , and il-18 [ 21 , 32 , 33 ] .
of all the meprin substrates identified to date , os-9 provides the most direct link to the hypoxia response and provides new insights on how meprins modulate tubular injury in ir .
the hif-1/os-9 interaction also links the hypoxia response to mitochondrial energy expenditure and reactive oxygen species ( ros ) formation .
this is supported by previous studies which showed upregulation of os-9 in response to er stress .
the present study and previous work from our group have provided evidence that meprins modulate the pathophysiology of ir - induced kidney injury via multiple mechanisms .
further investigations are needed in elucidating these mechanisms because the knowledge is important in development of therapeutic targets for acute kidney injury . | meprin metalloproteases play a role in the pathology of ischemia / reperfusion- ( ir- ) induced renal injury . the endoplasmic reticulum - associated protein , osteosarcoma-9 ( os-9 ) ,
has been shown to interact with the carboxyl - terminal tail of meprin . more importantly , os-9 interacts with the hypoxia inducible factor-1 ( hif-1 ) and the prolyl - hydroxylase , proteins which mediate the cell 's response to hypoxia . to determine
if os-9 is a meprin substrate , kidney proteins from meprin knockout mice ( ko ) ( which lack endogenous meprins ) and purified human os-9 were incubated with activated forms of meprin a and meprin b , and western blot analysis was used to evaluate proteolytic processing of os-9 .
fragmentation of os-9 was observed in reactions with meprin b , but not meprin a. to determine whether meprin b cleaves os-9 in vivo , wild - type ( wt ) and meprin ko mice were subjected to ir - induced renal injury .
fragmentation of os-9 was observed in kidney proteins from wt mice subjected to ir , but not in meprin ko counterparts .
transfection of kidney cells ( mdck and hek293 ) with meprin cdna prevented accumulation of os-9 following exposure to the hypoxia mimic , cocl2 .
these data suggest that meprin interaction with os-9 plays a role in the hypoxia response associated with ir - induced renal injury . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion |
PMC4508475 |
overview . ovarian cancer ( oc ) is a common condition in women scenario and it represents the principal cause of death from gynaecologic cancer in united states .
it is estimated that 21.290 cases will have been diagnosed in 2015 and that 14.180 women will have died due to this malignancy .
about 90% of oc are epithelial carcinomas and 70% of those have a serous histology .
death rate from oc declined from 1970s to 1990s but it has since then remained stable . in light of these discouraging data , the development of novel therapies for oc has become a priority .
recent better molecular characterization and immune system identification are the starting point of future research in immunotherapy .
undoubtedly , the next decade will see immunotherapy coming to the clinic use alongside standard regimes and it is possible that it could replace cytotoxic chemotherapy in combination strategies .
therefore , in addition to possessing expertise with immunotherapy , oncologists will be expected to conduct trials of novel agents in combination with standard treatment .
thus , it is paramount to focus the attention on maximising the knowledge of the more important component of the immune system .
loss in this challenge could run the risk that oncologists will take a passive role in the development of new strategies .
this paper reviews the rationale for immunotherapy and the main approaches under investigation in oc , with a special focus on the role of checkpoint inhibitors
. we will briefly describe the human immune system in an attempt to provide a means of understanding how it relates specifically to the clinical practice .
the role of immunotherapy in cancer treatment has been identified decades ago , because of beneficial effect of severe induced infection on tumour regression . at that time
, coley showed that inoculation with streptococcal organisms resulted in the shrinkage of inoperable bone and soft - tissue sarcomas .
however , severe criticisms due to the inconsistency of the method and results emerged in the scientific community , across the years .
one explanation was that other physicians , who tested his treatment , did not report the same excellent effect .
these results , as well as the concurrent development of radiotherapy and chemotherapy , determined immunotherapy to slowly disappear from treatment cancer scenario . since coley 's death
, immunology has represented an active research field and , nowadays , immunotherapy is considered again a valid treatment option in different types of cancer .
the human immune system can be divided in two components : the innate and the adaptive immunity .
the innate immune system consists of natural killer ( nk ) cells , dendritic cells , and macrophages and neutrophils , whereas b cells and t cells , including cytotoxic ( cd8 + t or ctl ) cells , helper ( cd4 + t ) cells , and nk t cells , are specific of the adaptive immunity .
the innate immunity provides a first line response against pathogens in a nonspecific manner ; it has no immunologic memory and it is not able to recognize antigen .
thus , in terms of tumor immunology , its contribution is marginal and limited to secreted cytokines that recruit immune cells . on the other hand ,
the adaptive immune system plays a central role in the antitumor immune response , due to its capability in processing nonself cells .
firstly , the presentation of an antigen to a t cell receptor ( tcr ) by a major histocompatibility complex ( mhc ) molecule on antigen presenting cells ( apcs ) . secondly , the interaction of the cd28 receptor on t cells to b7 costimulatory molecules ( b7 - 1 and b7 - 2 ) on apcs .
the distinction between self and nonself is provided by cancer - specific antigens that are expressed by tumor cells .
tumor antigens are traditionally divided in two classes : tumor specific antigens ( tsas ) and tumor associated antigens ( taas ) .
for instance , oncospermatogonal antigens are expressed by tumor cells as well as normal spermatocytes ; carcinoembryonic antigen ( cea ) is expressed on fetal tissues and in several cancer types .
theoretically , the immune system recognizes nascent transformed cells , in order to prevent progression to clinical tumor
it is an extrinsic tumor suppressor mechanism that consists of 3 sequential phases : elimination , equilibrium , and escape .
transformed cells are recognized and eradicated by the innate and adaptive immune system . cd8 + t cells , cd4 + t cells , natural killer ( nk ) cells , and nk t cells secrete interferon- ( inf- ) to inhibit tumor cell proliferation and angiogenesis , whereas macrophages and dendritic cells are processed to phagocytise and remove tumor cells killed .
the cells that are not eliminated in this phase may then enter the equilibrium phase , in which their development is prevented by adaptive immunologic mechanism .
cd8 + t cells and dendritic cells secrete inf- and interleukin- ( il- ) 12 , respectively , and preserve tumor cells in a steady state .
this is a functional state in which latent tumor cells are specifically controlled by the adaptive immunity .
this dynamic balance can persist for long period , sometimes exceeding 20 years . in response to immune system , tumor cells can change their characteristics in immune resistant cells and therefore escape from immune system suppression . in this final phase ,
tumor cells emerge and become clinically apparent , because they are no longer blocked by immunity .
the generation of immune resistant tumor cells can occur in several ways : through loss of tumor antigens expression ; through downregulation of mhc ; through the overactivation of the prooncogenic transcription factor stat3 ; through the overexpression of antiapoptotic effector bcl-2 ; through the expression of inhibitory cell surface molecules , such as programmed cell death 1 ligand 1 ( pd - l1 ) , cytotoxic t - lymphocyte associated protein-4 ( ctla-4 ) , and fas ligand ( fasl ) , which directly kill cytotoxic cd8 + t cells .
otherwise tumor cell escape can be a consequence of an immunosuppressive state established in the tumor microenvironment .
this condition may result from the secretion of immunosuppressive cytokines , like il-4 , il-1 , vascular endothelial growth factor ( vegf ) , and prostaglandin - e2 ( pge2 ) , which recruit regulatory cells . particularly , the secretion of il-4 recruits macrophages that inhibit cd4 + t cells , by expressing transforming growth factor- ( tgf- ) , il-10 , and platelet - derived growth factor ( pdgf ) , whereas the secretion of il-1 , vegf , and pge2 determines the accumulation of myeloid - derived suppressor cells that blocks t cell function . over the last decade
we need to integrate the potential understanding of the immunoediting process from the 3es and the tumor characteristics to conduct the optimal treatment .
it is difficult to define a clear role of immunotherapy ; nonetheless it is reasonable to hypothesise that any immune molecule capable of activating this process might have a useful role in eradication of nascent tumor cells . at this time it is paramount that oncologists are familiar with the immunoediting process so that they can have a role in the rational development of innovative clinical trials .
the stabilization of equilibrium state , as well as the inhibition of tumor escape mechanisms , should be clinical endpoints .
current immunotherapies for cancer treatment include therapeutic vaccines , cytokines , immune modulators , immune checkpoint inhibitors , and adoptive t cell transfer .
therapeutic vaccines are designed to treat established cancers and may be used in the induction of the tumor - directed immune response of the patients through the introduction of tumor antigens .
the other approaches such as immune checkpoint inhibitors and adoptive t cell transfer are designed to augment anticancer immunity against cancer .
nowadays , one of the most promising strategies seems to be the takeover of immune cell - intrinsic checkpoints that are induced on t cells activation .
the blockade of one of these checkpoints , such as ctla-4 or the programmed death 1 ( pd-1 ) receptor , has recently been found to be active to achieve an immune - modulation approach in the treatment of solid tumors [ 15 , 16 ] .
the immune checkpoint blockade targeted agents might represent breakthrough drugs in the treatment of solid tumors and have generated greater expectations in the field of cancer immunotherapy , even in oc [ 17 , 18 ] .
t cells activity is regulated by a great number of different molecules , as well as immune - modulatory signals , both costimulatory and coinhibitory . to avoid inappropriate t cell activation , resulting in autoimmunity , negative regulators of t cell immunity , including ctla-4 and pd-1 , are needed .
in fact both ctla-4 and pd-1 are key immune checkpoint proteins and represent a further promising immunotherapeutic target .
ctla-4 is a member of the cd28:b7 immunoglobulin superfamily , typically low - expressed on the surface of naive effector t cells and regulatory t cells ( tregs ) .
when naive t cells are stimulated through the tcr , ctla-4 is upregulated and competes with cd28 for b7 and , finally , determines the suppression of t cell activity .
it was found that the antitumor effect of ctla-4 blockade might be obtained also by depletion of treg , as revealed in a model of mouse melanoma , in which both the augmentation of t effector cell function and inhibition of treg activity through the blockade of ctla-4 manage to obtain a strong antitumor response .
pd-1 is expressed on chronically stimulated t cells , as well as tregs , activated b cells , and nk cells .
differently from ctla-4 , which regulates t - lymphocytes at the level of initial activation , pd-1 regulates immunity at multiple phases of the immune response , including its effect on effector t - lymphocyte activity in the peripheral tissues .
experimental models showed that pd-1 deficient mice present enhanced immunity with phenotypes characterized by autoimmune cardiomyopathy and a lupus - like syndrome [ 24 , 25 ] .
the activity of pd-1 is related to its interaction with its ligands , pd - l1 ( b7-h1 ) and pd - l2 ( b7-dc ) . both ligands , especially pd-1 , are expressed on many hematologic and nonhematologic human tumors .
generally , in human cancer , when pd-1 binds with cells bearing one of its ligands , t cell activity is attenuated ( phenomena known as peripheral tolerance ) , which prevents these t cells from rejecting the tumor at the tissue level , and tumors can thereby employ the pd-1 inhibitory pathway to silence the immune system .
based upon the findings of preclinical studies , suggesting the involvement of these molecules in immune control , various agents blocking ctla-4 , pd-1 , or pd - l1 or other immune molecules are currently investigated in ovarian cancer ( oc ) treatment .
the ctla-4 is currently being investigated as a single or combinatorial therapy in clinical trials involving several cancer types .
ipilimumab and tremelimumab are fully human igg1 or igg2 antibodies , respectively , that antagonize the ctla-4 immune checkpoint . the majority of clinical data derived from studies in patients with melanoma .
in these studies ctla-4 blockade has yielded objective responses to such an extent that ipilimumab was fda approved to treat metastatic or unresectable melanoma in 2011 [ 29 , 30 ] .
experience in oc is actually based on small population studies but results seem to be interesting .
firstly showed [ 31 , 32 ] antitumor effects in patients with stage iv oc patients .
initially , they reported that a single infusion of ipilimumab ( 3 mg / kg ) in two - stage iv oc patients previously vaccinated with granulocyte - macrophage colony - stimulating factor modified irradiated autologous tumor cells ( gvax ) , was well tolerated , and triggered a decrease or stabilization of ca-125 levels of several months ' duration . in order to clarify the toxicity and antitumor efficacy , they treated additional 9-stage iv oc subjects by using the same antibody dose and schedule ( with the exception of one patient ) . in one patient ,
an objective radiographic response was noted and multiple infusions of anti - ctla-4 antibody every 3 to 5 months have maintained disease control over 4 years ; furthermore , 3 out of 9 patients had stable disease of 6 ( ongoing at the moment of paper 's publication ) , 4 , and 2 months ' duration , as measured by ca-125 levels and radiographic criteria , in the absence of serious toxicities .
tumor regression correlated with the cd8+/treg ratio , suggesting that other forms of therapy that target treg depletion might have a synergistic effect when combined with the tumor vaccine and ctla-4 antibody molecules .
these findings prompted a phase ii clinical trial to evaluate ipilimumab as monotherapy in platinum - resistant oc patients ( nct01611558 ) .
ipilimumab can cause significant immune - related adverse events ( aes ) , and the more common observed side effects include diarrhea , colitis , and dermatitis .
less common severe immune - related adverse events include hypophysitis , thyroiditis , and hepatitis .
tremelimumab ( previously known as ticilimumab ) is a fully human igg2 monoclonal antibody to ctla-4 .
in contrast to ipilimumab , a large phase iii trial in melanoma did not demonstrate improved pfs or os compared with cytotoxic chemotherapy although durable responses were observed in some patients .
much speculation have been done about the potential reasons for this clinical result , because both phase iii clinical trials [ 14 , 29 ] testing ipilimumab succeeded in showing improved os .
it has been proposed that human igg1 ( the ipilimumab subclass ) binds with a higher affinity to fcrs than human igg2 ( the subclass of tremelimumab ) does , therefore suggesting that tremelimumab might determine a ctla-4 antibody mediated treg - cell depletion to a lesser extent [ 36 , 37 ] .
the combination of tremelimumab and a pd-1 inhibitor ( see below ) is currently ongoing , in a phase i study including ovarian and cervical cancer patients .
the therapeutic benefit obtained with ctla-4 inhibition led to the effort in identifying other potential immune checkpoint inhibitors that should have been more specific and equally efficacious and have less immune toxicity .
pd-1 and pd - l1 inhibitors were identified as potentially accomplishing those requirements . differently from ctla-4 , which regulates t - lymphocytes at the level of initial activation
, pd-1 regulates immunity at multiple steps , including exerting its effect on effector t - lymphocyte activity in the peripheral tissues .
several monoclonal antibodies have been developed that block the pd-1 system , either by interactions with the pd-1 receptor or with its specific ligands .
nivolumab ( also known as bms-936558 or mdx1106 ) is a fully human igg4 monoclonal antibody that targets pd-1 .
a phase i / ii clinical trial tested the safety and efficacy of nivolumab at doses of 0.1 to 10.0 mg / kg of body weight intravenously every 2 weeks for up to 12 cycles until disease progression or a complete response occurred .
patients with advanced melanoma , non - small - cell lung cancer , prostate cancer , renal cancer , and colorectal cancer were enrolled . among the 296 patients , those with metastatic melanoma achieved the higher rates of objective responses ( 27.6% ) , with a median os of 16.8 months ; conversely responses were not observed in colon and prostate cancer patients .
responses were seen in both pd - l1 positive and negative patients , even if with lower extent .
common treatment - related adverse events included fatigue , diarrhea , pruritus , rash , nausea , and decreased appetite .
grade 3 or 4 treatment - related adverse events were seen in 14% of patients .
treatment - related serious adverse events were noted in 11% of patients and included pneumonitis ( 3% , and grade 3 or 4 in 1% ) , colitis , hepatitis , thyroiditis , and hypophysitis .
recently , at the 2014 asco meeting , the first clinical trial of nivolumab treatment against platinum - resistant oc has been presented .
a total of 18 evaluable patients were treated with nivolumab : 10 patients were administered 1 mg / kg and 8 patients were administered 3 mg / kg , each every 2 weeks for 1 year . starting at week 8 , patients were assessed every 8 weeks and patients with disease progression were taken off study .
there were two serious treatment - related aes : one patient in the 1 mg / kg group experienced grade 3 fever , disorientation , and gait disorder and one patient in the 3 mg / kg category experienced grade 3 fever and deep - vein thrombosis .
other grade 3/4 treatment - related aes included hypothyroidism ( two patients , both in the 1 mg / kg group ) ; heart arrhythmia ( one patient , in the 3 mg / kg group ) ; and lymphocytopenia ( one patient , in the 1 mg / kg group ) .
the 3 mg / kg dose may be more favourable ( 25% ) than 1 mg / kg ( 10% ) .
two patients in the 3 mg / kg group experienced complete response ( cr ; response rate 25% ) . among those receiving 1 mg / kg nivolumab , one experienced a partial response ( 10% response rate ) and two patients experienced stable disease ( sd ) .
further researches are investigating biomarkers predicting response . a further molecule investigated in oc is pembrolizumab ( mk-3475 , formerly known as lambrolizumab ) , a humanized igg4 monoclonal antibody against pd-1 .
it was found to be active in treating both melanoma and nsclc [ 4143 ] , similar to nivolumab .
actually , no randomized trial has compared the two agents , which are surely different in binding affinities , nivolumab being a fully human igg4 , and pembrolizumab is humanized
recently , an interim analysis with pembrolizumab showed preliminary signal for clinical efficacy in recurrent oc .
in addition to antibodies targeting pd-1 , several different anti - pd1-l1 monoclonal antibodies , such as bms-936559 , mpdl3280a , medi4736 , and msb0010718c , have been developed which might enhance immune function .
it was found that the ligand / receptor interaction inhibits the t - lymphocyte response by inhibiting the kinases involved in t - lymphocyte activation via phosphatase activity and other signaling pathways .
bms-936559 is a high - affinity , fully human igg4 monoclonal antibody that binds pd - l1 and that blocks pd - l1 from binding its two known receptors pd-1 and cd8 .
it was safe in a phase i trial that included 17 oc patients in escalating doses of 0.310 mg / kg iv every 14 days in 6-week cycles for up to 16 cycles or until the patient had a complete response or confirmed disease progression and observed durable tumor regression and prolonged stabilization of disease .
common side effects included fatigue , infusion reactions , diarrhea , arthralgia , pruritis , rash , nausea , and headache . in the trial , only oc patients at the 10 mg / kg
dose achieved objective responses : 1 ( 6% ) with a partial response and 3 ( 18% ) with stable disease lasting more than 24 weeks .
msb0010718c is a fully human igg1 monoclonal antibody targeting pd - l1 . unlike other pd - l1 targeting agents
, it is a native fc receptor , allowing for antibody dependent cell mediated cytotoxicity . in a phase
i trial 27 patients with refractory malignancies were treated with msb0010718c at 1 , 3 , 10 , and 20 mg / kg twice weekly .
eleven patients in the study had received prior treatment with an immunotherapy . at the 3 and 10 mg / kg doses ,
the drug was found to inhibit 93.8% and 93.2% of the pd - l1 receptor on peripheral leukocytes .
additionally , a linear pk profile was found , with a maximum concentration of the drug achieved at 1.52 hours following infusion . at the 20 mg / kg dose , a dose - limiting immune - related adverse event was noted . in this trial
also oc patients were included and , interestingly , a larger subsequent meta - analysis of the company developing the drug ( merck ) , including 23 patients ' oc cohort , showed 48% of patients reaching stable disease and 17% getting a partial response within 30 weeks of treatment start , though 13 had been taken off the drug .
noteworthily , the responses came despite 77% of patients having already failed at least three lines of therapy .
more recently , efficacy data from the 23 patients followed up for more than 2 months ( range 28 months ) were presented .
four patients ( 17.4% ) achieved an unconfirmed partial response , 11 ( 47.8% ) patients had stable disease , and 2 patients had > 30% tumor shrinkage after progression .
toxicity was manageable and only 2 patients ( 8.7% ) experienced grade 3 drug - related aes .
other molecules , such as mpdl3280a and medi4736 , are currently investigated in phase i trials including oc patients .
tumor tissue can be considered a darwinian microenvironment that selects the better strategy to elude the immune system .
expression of specific ligands , such as pd - l1 and ctla-4 , in the stroma or in the tumor cells associated , is paramount to improve growth and resistance to immune attack .
it depends on both tumor type and histology , and therefore it also represents the major limitation of immunotherapy .
maybe the identification and characterization of similar patients population , as well as tumor histology , could provide data to facilitate the development of novel treatment strategies .
immune check point inhibitors may have a synergic mechanism in multimodality treatment and thus a positive effect on overall survival , with a tolerable toxicity profile .
in the last years , immunotherapy has achieved an important role in the fight against cancer and also , in oc immunological phenomena , has been demonstrated to play a central role .
immune checkpoint inhibitors have shown clinical activity in several cancers , especially melanoma , and they represent a major step forward in the fight against cancer .
these novel therapies will likely play a role also in oc given the potential for rapid , durable responses and their favourable toxicity profiles .
their function in the treatment of patients with oc remains to be defined but initial results seem to be promising .
next challenges should be the clinical development of combinatorial approaches and further defining patients who benefit from immune checkpoint monotherapy and patients who require potentially more active albeit more toxic combination regimens .
finally , the definition of potential biomarkers that can determine which immune checkpoint pathway or pathways dominate in a particular tumor will be crucial to guide the choice of inhibitor .
the possibility of using immunotherapy in oc is still restricted to clinical trials but it is reasonable to expect that over the next years important advances in oc immunotherapy will be made , running further phase ii and iii trials development . | ovarian cancer is the most important cause of gynecological cancer - related mortality , with the majority of women presenting with advanced disease .
although surgery and chemotherapy can improve survival rates , it is necessary to integrate alternative strategies to improve the outcomes .
advances in understanding the role of immune system in the pathogenesis of cancer have led to the rapid evolvement of immunotherapy , which might establish a sustained immune system response against recurring cancer cells .
recently , it has emerged that powerful immunologic effector cells may be blocked by inhibitory regulatory pathways controlled by specific molecules often called immune checkpoints , which turn off the immune system .
similarly , cancer cells are able to use these checkpoints to avoid immune control and rejection .
inhibition of these inhibitory pathways represents a potent strategy in the fight against cancer and is currently under investigation with encouraging results in some cancers , such as melanoma . in ovarian cancer researches
are still in an early phase , but with promising results . in this review
we will explore the rationale of immunotherapy in ovarian cancer with a special focus on these emerging molecules . | 1. Introduction
2. Immune System and Cancer Disease
3. Cancer Immunotherapy: The Role of Immune Checkpoint
4. Clinical Trials with Immune Checkpoint Blockade Targeted Agents in OC
5. Conclusions |
PMC4739139 | the animals were 24 one- to two - year - old thoroughbreds ( twelve males , twelve females ) clinically free of any
orthopaedic disorders and history . prior to a breaking and training period that begins in september and
ends in
april every year , twelve horses ( six males , six females ) were selected as subjects in the first study from
december 2013 to april 2014 .
the other twelve horses were used in the second study from december 2014 to april
2015 .
the running ( that is at a canter and a gallop ) distances for 1 month before the dates of the
ultrasonographic examination are presented in table 1table 1.changes of running distance for 1 month before the dates of the ultrasonographic examination in
december , february , and april2013 ( km / month)2014 ( km / month)december9.6 6.39.8 5.2february47.7 1.834.0 9.6april54.0 18.145.6 7.6running was performed at a canter and a gallop .. running was performed at a canter and a gallop .
the horses sdfts were ultrasonographically scanned on the same dates in december , february , and april .
before the examination ,
the horses had been kept in pasture for at least 3 hr following their daily exercise .
the horses were retained in a treatment stall , and then sedated with medetomidine ( 2 mg / head intravenous
injection ) .
after completely clipping the palmar aspect from the proximal to the distal metacarpus , a linear
array transducer ( 50 mm of effective aperture length ) with a broadband frequency between 5 and 12 mhz was
directly placed onto the skin with abundant gel . using a mobile ultrasound system ( iu22 , philips medical
systems corporation , tokyo , japan ) , three longitudinal ( three equal lengths of the labelled 1 to 3 in order
from proximal to distal metacarpus ) and
six transversal ( separated by equal distance along the labelled 1a ,
1b , 2a , 2b , 3a and 3b in order from proximal to distal , fig .
after completely clipping the palmar aspect of the metacarpus , a linear
array transducer was directly placed on the skin with abundant gel .
the rounded sdft was clearly
outlined in the 1a , 1b and 2a images ; however , the lateral and medial margins could not be visualized in
the flattened sdft of the 2b , 3a and 3b images . )
images from the proximal end ( bottom of the accessory carpal bone ) to the distal end ( top of the
proximal sesamoid bone ) of the sdft were scanned .
the maximum depth of the image was 3.5 cm , and the depth of
focus was adjusted to the sdft .
both gs and cd images were recorded for 15 and 30 sec at frame rates of 45 hz
and 6 to 11 hz , respectively .
after completely clipping the palmar aspect of the metacarpus , a linear
array transducer was directly placed on the skin with abundant gel .
the rounded sdft was clearly
outlined in the 1a , 1b and 2a images ; however , the lateral and medial margins could not be visualized in
the flattened sdft of the 2b , 3a and 3b images .
blood flows in the tendon bundles
were visualized in the longitudinal and transversal cd images , in which the region of interest was set and the
gain setting was similar for all tendons , just below the noise level ( 7785% of maximal gain ) .
the positive signals of cd flow are grade 1 ( indicated by white
arrows ) , small color activities that were fixed and rhythmically blinking . )
was recorded and graded , as previously reported : grade 1 , tiny to small color activities that were
rhythmically blinking at a site , but for which flow rates were not measurable by pulsed doppler ; grade 2 ,
unequivocal color activities that were pulsatile dots and for which flow rates were measured by pulsed
doppler ; and grade 3 , unequivocal linear color activities appearing as blood streams in a regular direction
due to periodic changes in color .
the longitudinal cd scan was
inferior to the transversal one in detecting and recording the small color activities of grade 1 and 2 ,
therefore we used only the transversal images for the cd data analysis . a transversal cd image of the sdft
. the positive signals of cd flow are grade 1 ( indicated by white
arrows ) , small color activities that were fixed and rhythmically blinking .
the horses sdfts were ultrasonographically scanned on the same dates in december , february , and april .
before the examination ,
the horses had been kept in pasture for at least 3 hr following their daily exercise .
the horses were retained in a treatment stall , and then sedated with medetomidine ( 2 mg / head intravenous
injection ) .
after completely clipping the palmar aspect from the proximal to the distal metacarpus , a linear
array transducer ( 50 mm of effective aperture length ) with a broadband frequency between 5 and 12 mhz was
directly placed onto the skin with abundant gel . using a mobile ultrasound system ( iu22 , philips medical
systems corporation , tokyo , japan ) , three longitudinal ( three equal lengths of the labelled 1 to 3 in order
from proximal to distal metacarpus ) and six transversal ( separated by equal distance along the labelled 1a ,
1b , 2a , 2b , 3a and 3b in order from proximal to distal , fig .
after completely clipping the palmar aspect of the metacarpus , a linear
array transducer was directly placed on the skin with abundant gel .
the rounded sdft was clearly
outlined in the 1a , 1b and 2a images ; however , the lateral and medial margins could not be visualized in
the flattened sdft of the 2b , 3a and 3b images . )
images from the proximal end ( bottom of the accessory carpal bone ) to the distal end ( top of the
proximal sesamoid bone ) of the sdft were scanned .
the maximum depth of the image was 3.5 cm , and the depth of
focus was adjusted to the sdft .
both gs and cd images were recorded for 15 and 30 sec at frame rates of 45 hz
and 6 to 11 hz , respectively .
after completely clipping the palmar aspect of the metacarpus , a linear
array transducer was directly placed on the skin with abundant gel .
the rounded sdft was clearly
outlined in the 1a , 1b and 2a images ; however , the lateral and medial margins could not be visualized in
the flattened sdft of the 2b , 3a and 3b images .
blood flows in the tendon bundles
were visualized in the longitudinal and transversal cd images , in which the region of interest was set and the
gain setting was similar for all tendons , just below the noise level ( 7785% of maximal gain ) .
the positive signals of cd flow are grade 1 ( indicated by white
arrows ) , small color activities that were fixed and rhythmically blinking . ) was recorded and graded , as previously reported : grade 1 , tiny to small color activities that were
rhythmically blinking at a site , but for which flow rates were not measurable by pulsed doppler ; grade 2 ,
unequivocal color activities that were pulsatile dots and for which flow rates were measured by pulsed
doppler ; and grade 3 , unequivocal linear color activities appearing as blood streams in a regular direction
due to periodic changes in color .
the longitudinal cd scan was
inferior to the transversal one in detecting and recording the small color activities of grade 1 and 2 ,
therefore we used only the transversal images for the cd data analysis . a transversal cd image of the sdft
. the positive signals of cd flow are grade 1 ( indicated by white
arrows ) , small color activities that were fixed and rhythmically blinking .
the running distance for 1 month before the date of the ultrasonographic examination was increased in the order
of december , february , and april in both of the two training periods . as the transversal gs images were obtained
using a linear array transducer that was directly placed onto the skin with abundant gels and not attached to a
coupler ,
the rounded sdft was clearly outlined in 1a , 1b , and 2a images , however the lateral and medial margins
could not be visualized in the flattened sdft of 2b , 3a and 3b ( fig .
1 ) .
there were no longitudinal or transversal gs images indicating injury in the sdfts in either of the
two training periods .
of 864 ( 6 images 2 forelimbs 24 horses 3 examinations ) transversal cd images , 56 images ( 6.4% ) showed the
cd flows in sdft bundles ( table 2table 2.among 864 transversal cd images , 56 images ( 6.4% ) showed positive cd flows in sdft bundles ) , which were categorized as rhythmically blinking ( grade 1 , 32 images , as presented in fig .
the cd flows
were unevenly distributed at 1a , 1b , 2a and 2b , and were more frequently detected in april than in either
december or february ( table 3table 3.the distribution of transversal cd images showing positive cd flows in sdft bundles1a1b2a2b3a3btotaldecember 4210007february104000014april1461230035total28121330056 ) .
there were 7 , 8 and 15 horses showing cd flows in december , february and april ,
respectively .
a normal tendon is hypovascular , and angiogenesis or increased vascularity are associated with an injured
tendon for much of the healing process . within the first 24 hr after injury ( inflammatory phase ) , inflammatory
cells are recruited to the injured site , and vasoactive and chemotactic factors are associated with an increase
in vascular permeability and the initiation of angiogenesis [ 17 , 22 ] . within a few days ,
tenocytes gradually migrate to the injury site ,
proliferate , and synthesize type iii collagen ( proliferative phase ) [ 19 ,
22 ] . after approximately six weeks , the repair tissue changes from
cellular to fibrous ( remodelling phase ) .
the fibrous tissue gradually changes to scar - like tendon tissue over
the course of one year ( maturation stage ) [ 10 , 22 ] , and the vascularity finally declines in the latter half of this stage [ 1 , 22 ] .
therefore , we hypothesised that
the blood flows would also increase in equine sdft from a few days to six weeks after the injury , and that they
might be indicative of tendon injury and the subsequent healing process
. however , in this study of one- to
two - year - old horses , no macroscopic injury was found in the 56 transversal gs images in which positive signals
of blood flow were seen within sdft bundles .
some of the positive flow signals disappeared and/or then
reappeared during the study period .
therefore , we speculate that the positive signals of blood flow observed in
this study might be due to transient increases of blood flows in the inherent vessels in response to hypoxia
and/or hyperthermia [ 11 ,
20 , 25 ] of tendon tissue which
were related to the increased exercise ( the increased mechanical behaviour of the tendon ) , rather than increased
vascularization associated with macroscopic injury of tendon bundles . based on the observance of increased
doppler flow in achilles tendons that was associated with weekly badminton hours and badminton years , it was
suggested that doppler flow may be a response to mechanical load on the tendon .
since doppler activity increased in human achilles tendons after running exercise performed by
both non - symptomatic subjects and symptomatic subjects in a rehabilitation program , it was proposed that all
tendons of both healthy and diseased subjects , have a certain amount of vascularization , which may be manifested
on demand up to a certain level . considering our result in reference
to these human studies ,
it is possible that the blood flows observed in the sdft of the one- to two - year - old
thoroughbreds were response to the increases in the intensity and distance of the running exercise during the
training period .
the main sources of blood supply are : the intrinsic systems at the myotendinous and osteotendinous junctions ,
and the extrinsic system through the paratenon or the synovial sheath .
the ratio of the blood supply from the
intrinsic systems to that from the extrinsic system varies in the different parts of the tendon .
for example ,
the central third , which could be a zone of hypovascularity , receives 35% of its blood supply from the extrinsic
system in the rabitt achilles tendon [ 14 , 18 ] .
an equine study reported that the blood supply of the normal sdft is primarily intratendinous
rather than paratendinous as previously thought . in this study ,
the
cd flows mainly increased in 1a ( 28 images ) , 1b ( 12 images ) , and 2a ( 13 images ) , which are proximal parts of the
sdft .
this result could be suggestive of increased blood supply from the intrinsic systems at the myotendinous
junction . at the myotendinous junction ,
perimysial vessels from the muscle continue between the fascicles of the
tendon , and extend to the proximal third of the human achilles
tendon [ 6 , 22 ] .
although we have no
data regarding the blood vessel distribution from the superficial digital flexor myotendinous junction to the
proximal third of metacarpal sdft in horses , this result might be explained as the perimysial blood supply to
the proximal third physiologically increasing in response to the increases in the running distance of the
clinically healthy horses . in conclusion , the increase of intratendinous blood flows in the proximal parts of the sdft could be responses
to the increase in exercise intensity as the training advanced . because no longitudinal or transversal gs image
indicating macroscopic injury of the sdft were found in either of the two training periods , increase of cd flows
in the proximal parts of the sdft are not necessarily predictive of tendon injury in the near future in the
training period of young thoroughbreds . | abstractaim of this study was to evaluate the relationships of exercise and tendon injury with doppler flows
appearing in the superficial digital flexor tendon ( sdft ) of young thoroughbreds during training periods .
the
forelimb sdfts of 24 one- to two - year - old thoroughbreds clinically free of any orthopaedic disorders were
evaluated using grey - scale ( gs ) and color doppler ( cd ) images during two training periods between december
2013 to april 2015 .
twelve horses per year were examined in december , february , and april in training periods
that began in september and ended in april .
the sdft was evaluated in 3 longitudinal images of equal lengths
( labelled 1 , 2 , 3 in order from proximal to distal ) , and 6 transversal images separated by equal lengths
( labelled 1a , 1b , 2a , 2b , 3a and 3b in order from proximal to distal ) of the metacarpus using both gs and cd .
the running ( canter and gallop ) distance for 1 month before the date of the ultrasonographic examinations was
increased in december , february , and april in both of the two training periods .
cd flows defined as
rhythmically blinking or pulsatory colored signals were found in 56 of 864 ( 6.4% ) transversal cd images , in
28 , 12 , 13 , and 3 images of 1a , 1b , 2a and 2b , respectively , and in 7 , 14 , and 35 images captured in december ,
february , and april , respectively .
there were no longitudinal or transversal gs images indicating injury in
the sdfts in either of the two training periods .
the increase of cd flows in the proximal regions of the sdft
are possibly related to the increase of the running distance during the training periods of the one- to
two - year - old thoroughbreds . because no injury was diagnosed in the sdfts by gs images during the training
periods , the increase of cd flows in the proximal parts of sdft is not necessarily predictive of tendon injury
in the near future during the training period of young thoroughbreds . | Materials and Methods
Ultrasonographic examination
Results
Discussion |
PMC4923579 | color stability of composite resin is an important property influencing its clinical longevity , which continues as a challenge inherent to material .
color changes can occur due to various etiologic factors ; extrinsic discoloration can occur due to staining in the superficial layer of resin composite , water absorption , surface roughness , smoking , and diet [ 2 , 3 ] .
intrinsic discoloration could occur as a result of physicomechanical reaction within the material ( e.g. , the filler and the resin matrix properties ) . in oral conditions ,
composite resins are exposed to different dietary beverages such as coffee which might result in absorption and adsorption of colorants in coffee into the resin surface and consequently undesirable color change .
previous investigations reported the influence of coffee on the color stability of composite resins [ 610 ] .
generally , it is recommended that the resins should be placed in 2 mm increments to obtain sufficient light transmittance and complete curing of composite resins .
placing the resin in 2 mm increments has some merits including the reduction of the polymerization shrinkage and the voids incorporation between the layers .
however , the application of composite resins in an incremental technique and light curing each increment individually is a time - consuming procedure .
there is also an increasing possibility of air bubble inclusion or moisture contamination between individual increments of resin composite restorations . to overcome such fallibility ,
, these materials can be sufficiently light cured up to 4 mm in a single increment , without influencing the polymerization shrinkage , degree of conversion , or cavity adaptation .
in addition , it is claimed that these materials have lower polymerization shrinkage compared to conventional composite resins .
thus , some postoperative problems , such as gap formation and the incidence of recurrent carries , may be diminished .
such merits are probably due to the increased translucency of the bulk - fill composites , which permits greater light transmission .
additionally , the formulation of these materials allows for modulation of the polymerization reaction by applying the stress - relieving monomers , the use of more reactive photoinitiators , and the incorporation of different types of fillers , such as prepolymer particles and fiberglass rod segments . while the manufacturers recommend bulk - filling of these materials up to 4 mm , many clinicians suspect that the depth of cure and mechanical properties might not be suitable for clinical use
there are few reports on of the effect of increment thickness and the storage media on the discoloration of these bulk - fill resin composites . since color changes are a concern that affects the population and one of the main reasons for replacing resin - based restorations , this study investigated the effects of the increment thickness and the storage media on the discoloration of one of these bulk - fill resin composites .
two a3 shade light cured composite resins , conventional and bulk - fill tetric evoceram , were selected for this study .
the specimens of conventional ( 6 mm diameter and 2 mm thickness , n = 20 ) and bulk - fill composite resins at two different thicknesses ( 6 mm diameter and 2 mm and 4 mm thickness , n = 20 ) were prepared using a polyethylene mold . after applying the composite resin , a mylar strip was placed and pressed with a glass slide to obtain a flat surface .
the glass slide was removed and the specimens were cured for 40 s using a halogen light curing unit ( 1086.67 mw / cm , demetron l.c ; kerr corporation , orange , ca , usa ) . after removing the specimens from the molds , baseline color was measured using a digital image analysis method as described later .
specimens were stored at 37c in distilled water for 28 d. half of the specimens remained in distilled water and the other half were immersed in coffee solution 20 min / d and remained in distilled water in the interval between cycles . to prepare the coffee solution , 25 g of coffee ( taster 's choice , nestl usa , inc . ,
glendale , ca , usa ) was poured in 250 ml of boiling water , and after 10 min of stirring , the solution was filtered through a filter paper .
the color of the specimens was assessed in the commission international de i'eclairege lab ( cielab ) color space using a digital image analysis method .
the cielab system is a chromatic value color space that measures chroma and value in three coordinates : l , the lightness measured from 0 ( black ) to 100 ( white ) , a , color in the red ( a < 0 ) and green ( a > 0 ) axis , and b , color in the blue ( b < 0 ) and yellow ( b > 0 ) axis .
a setup was designed in a dark room while two 60 w light sources were positioned from the sides ( 45-degree angle to the samples ) . at each interval ,
the specimens were rinsed with distilled water for 5 seconds , blotted dried with tissue paper , and placed on a dark background .
the digital images connected with the computer running adobe photoshop cs5 ( adobe , san jose , ca , usa ) as color assessment software .
the results were statistically analyzed using repeated measures two - way anova with factors including materials ( conventional , 2 mm and 4 mm bulk - fill resin composite ) and immersion media ( coffee and distilled water ) for each time interval .
for multiple comparisons , tukey 's hsd post hoc test was used to compare different materials ( p < 0.05 ) ( spss v12.0 ; spss inc . ,
the mean e values of specimens at different thicknesses and media are shown in table 2 .
the results of this study showed the significant color changes among all groups ( p < 0.001 ) .
tukey 's hsd test revealed that bulk - fill composite resins showed the significantly greater e values compared to those of conventional resins in all time intervals ( p < 0.001 ) .
regarding color changes of bulk - fill composite resin at increasing depths , 4 mm thick specimens showed a significant increase in mean e values at 14 d ( p < 0.001 ) and 28 d ( p < 0.001 ) .
however , the difference was not significant at 1 d and 7 d. immersion in coffee and distilled water provided significant color changes .
immersion in coffee solution resulted in greater and significant discoloration over time , when compared to that of water storage ( p < 0.001 ) .
color stability of dental restorations is one of the most important characteristics of composite resin materials in terms of longevity .
although there have been several studies on the effect of different beverages on the color stability of resin composites , there is little information regarding the color stability of a new bulk - fill resin composite , which has been introduced for applying in thicker layers . in the present study the effect of coffee staining on the color stability of bulk - fill ( with different thicknesses ) and conventional composite resins was evaluated over a period of 28 days .
coffee ( 20 minutes daily ) and distilled water were chosen as the storage media in this study since they mimic the liquids that are constituently in contact with resin composite restorations in the oral environment .
the e value presents relative color differences of dental materials or tooth surfaces before and after an intervention . according to literature , values of e
. values 1 < e < 3.3 are considered appreciable by skilled operators but clinically acceptable , whereas values of e > 3.3 are considered appreciable by nonskilled persons and are , hence , not clinically acceptable . therefore , color changes above a value of e = 3.3 were considered clinically unacceptable . in this study
coffee staining produced higher color changes in the specimens than those of water storage , which was in line with the previous investigations ; erta et al . and villalta et al .
immersed composite resin into the different beverages and reported that coffee showed greater color changes compared to water storage [ 6 , 7 ] .
the present result seems reasonable because when specimens were submitted to coffee staining , discoloration occurred due to the adsorption and absorption of pigments into the organic phase of resin - based materials .
additionally , coffee contains significant amounts of staining agents such as gallic acid , which facilitate staining .
the findings of our study present that water storage can also lead to slight discoloration , slightly perceptible , which is in line with the other investigations .
the staining susceptibility of specimens after being immersed in distilled water might be due to their degree of water absorption and the hydrophilic / hydrophobic nature of the resin matrix .
excessive water absorption can decrease the life of a composite resin by plasticizing and expanding the resin component , causing microcrack formation . as a result ,
microcracks or interfacial gaps at the interface between the filler and matrix allow stain penetration and discoloration .
in addition , discoloration might be due to the differences in the refractive index of filler and matrix which might increase after water absorption .
others factors that have been shown to have a significant impact on the color stability of material are the composition of composite resins and the characteristics of filler particle . in this
study the color susceptibility of bulk - fill composite resin was significantly higher than that of conventional composite resin after immersion in storage media ; it is claimed that bulk - fill tetric evoceram resins consist of a variety of fillers , prepolymer shrinkage stress reliever , different photoinitiator system , and light sensitivity system .
the mechanism of the interaction between the bulk - fill composite resin and the storage media is unknown and requires further investigation . altering the resin thickness is one of the other variables that can affect the final appearance of composite restoration . in the present study we tested the different thicknesses ( 2 and 34 mm ) on the color stability of bulk - fill composite resin .
we found that bulk - fill materials showed significantly greater discoloration at increasing increment thickness at 14 d and 28 d. this can be explained by the polymerization process of resin - based composites .
depth of cure can be influenced by different factors such as the monomer composition , filler content , and photoinitiator system .
light exposure can lead to causing activation of the photoinitiator which is attenuated by composite absorption and scattering .
thus , depth of cure relies on the material 's capacity to transfer light into its depths , as well as on the polymerization kinetics .
a previous study showed that , in the case of applying composite resin incrementally , no significant difference was observed in values of depth of cure at different depths .
but a bulk - fill technique might result in a greater number of particle / resin matrix interfaces and increased light scattering due to the differences in their refractive indices .
therefore , lesser amount of photons would reach deeper layers of composite resin and consequently a lower depth of cure value would be obtained at the deepest depths . in accordance with these results , flury et al . reported lower depths of cure of the bulk - fill specimens with 4 mm thickness compared to the values asserted by the manufacturers . in part ,
differences in the depth of cure , and even the overall degree of conversion , might affect the uncured monomer released and influence the composite discoloration . as a consequence
, mechanical properties are deteriorated leading to greater monomer elution which can result in more water absorption and , hence , discoloration .
our findings demonstrated that bulk - fill composite resin had greater color susceptibility after immersion in coffee than conventional composites .
considering the increment thickness it can be noted that the discoloration is increased with greater increment thickness .
we demonstrated that greater staining susceptibility of thicker specimens might be due to their lower depth of cure when placing bulk - fill materials . | we aimed to evaluate the color stability of bulk - fill and conventional composite resin with respect to thickness and storage media .
twenty specimens of a conventional composite resin ( 6 mm diameter and 2 mm thick ) and 40 specimens of the bulk - fill tetric evoceram composite resin at two different thicknesses ( 6 mm diameter and 2 mm thick or 4 mm thick , n = 20 ) were prepared .
the specimens were stored in distilled water during the study period ( 28 d ) .
half of the specimens were remained in distilled water and the other half were immersed in coffee solution 20 min / d and kept in distilled water between the cycles .
color changes ( e ) were measured using the cie lab color space and a digital imaging system at 1 , 7 , 14 , and 28 days of storage .
data were analyzed using two - way anova and tukey 's hsd post hoc test ( p < 0.05 ) .
composite resins showed significant increase in color changes by time ( bulk - fill > conventional ; p < 0.001 ) .
coffee exhibited significantly more staining susceptibility than that of distilled water ( p < 0.001 ) .
there was greater color changes with increasing the increment thickness , which was significant at 14 ( p < 0.001 ) and 28 d ( p < 0.01 ) .
color change of bulk - fill composite resin was greater than that of the conventional one after coffee staining and is also a function of increment thicknesses . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions |
PMC3495277 | the human papillomavirus ( hpv ) belongs to the papillomaviridae family and persistent infection of high - risk hpv is the direct cause of cervical carcinoma , which is the second most common malignancy among women worldwide .
hpv detection and genotyping is the most effective and accurate approach in screening of the early cervical lesions and cervical cancer . with hpv genotyping becoming more prevalent , over 120 types of hpv
have been identified , of which at least 40 types are indicated to infect the genital epithelium .
its genotypes are generally classified into high - risk ( hr- ) and low - risk ( lr- ) groups based on their carcinogenic potential .
hr - hpvs include hpv 16 , 18 , 31 , 33 , 35 , 39 , 45 , 51 , 52 , 56 , 58 , 59 , 68 , 73 , 82 , and so on , and the lr - hpvs include 6 , 11 , 40 , 42 , 43 , 44 , 54 , 61 , 70 , 72 , 81 , cp6108 , and so on . it should be noted that it is of particular interest to know the difference in the clinical properties of cervical neoplasia according to hpv types , which will help us in estimating the gravity of the disease and evaluating the prognosis after therapy according to different hpv types . although there are geographical differences in the distribution of hpv types among populations , globally , it has been shown that hpv 16 , 18 , 45 , 33 , and 31 are the most prevalent hpv types associated with cervical cancer .
it is of indicated that a vaccine that included the 7 most common hpv types can prevent 87.4% of cervical cancer worldwide .
therefore , it is of considerable clinical value to establish a reliable and convenient method to detect and genotype hpv .
food and drug administration ) commercially available method for the detection of hpv dna , the well - established hybrid capture ii system ( hc - ii ) has been proven to be a sensitive and reliable assay , which can detect 13 types of carcinogenic - hpv types or 5 types of low - risk hpv in a single test [ 6 , 7 ] . however , its main undeniable limitation is that hc - ii can not distinguish between different hpv genotypes definitely [ 5 , 8 ] .
recently , a new hpv - genotyping method combining two advanced techniques , the flow - through hybridization and gene chip ( hybrimax ) has been used to detect and genotype hpv , which can distinguish 21 different types of hpv dna in a single test and diagnose multiple infections [ 911 ] . in this study , we have evaluated the efficacy of hybrimax on cervical hpv genotyping through comparison of the results with hybrid capture ii ( hc - ii ) and in situ hybridization ( ish ) .
we showed that the most common hpv types tested by hybrimax in different grades of cervical disease could be determined , suggesting that hybrimax is an efficient method for hpv genotyping and is more suitable for clinical use .
591 out of 7520 women who accepted liquid - based cytology examination in china - japan friendship hospital from august 2004 to may 2005 were randomly selected for detection of the 21 hpv genotypes by hybrimax , and their mean age was 35.4 7.7 ( ranging from 20 to 64 ) . among them , 138 women ( mean age was 35.8 7.8 ) , who diagnosed within normal limits with cervical cytology for at least two years without any cervical disease or operation , were described as
a total of 453 women were diagnosed with abnormal cytology , and the mean age was 34.1 6.9 .
cytological diagnosis ( according to the 2001 bethesda system ) of those 453 patients were as follows : 161 cases with atypical squamous cells ( asc ) , 187 cases with low - grade squamous intraepithelial lesion ( lsil ) , 105 cases with high - grade squamous intraepithelial lesion ( hsil ) , or squamous cell carcinoma ( scc ) .
patients were classified into 6 groups according to their histopathology diagnosis from specimens of olcposcopic biopsy , loop electrosurgical excision procedure ( leep ) , or cold - knife conization .
these 6 groups include 152 cases of chronic cervicitis , 101 cases of cervical intraepithelial neoplasia ( cin , which indicates that dysplasia is seen on a biopsy of the cervix ) i ( mild dysplasia ) , 77 cases of cin ii ( moderate to marked dysplasia ) , 76 cases of cin iii ( severe dysplasia to carcinoma in situ ) , 27 cases of scc , and 20 cases of condyloma acuminata . with liquid - based cytology
, samples were taken with the cervical brush at gynecological examination for hpv dna testing .
it is prohibitive to apply vaginal douching three days prior to the collection of samples or to have sexual intercourse within one day .
hpv genotyping by hybrimax was performed using an hpv genoarray test kit ( hybribio ltd . ,
this assay can determine 21 hpv types , including 14 high - risk hpv types ( 16 , 18 , 31 , 33 , 35 , 39 , 45 , 51 , 52 , 56 , 58 , 59 , 66 , and 68 ) , five low - risk hpv types ( 6 , 11 , 42 , 43 , and 44 ) , and two unknown - risk types ( 53 and cp8304 ) , by the flow - through hybridization technique using hpv dna amplified by pcr .
in brief , 0.5 ml specimen was centrifuged at 13,000 g for 15 min with kubota 6930 . then
, the supernatant was removed and the pellet was resuspended in 200 l pbs buffer .
high - quality dna was yielded from lysis of cells by isolation of dna , precipitation , and purification .
the instrument used for pcr amplification was pe 9600 thermal cycler ( applied biosystems , usa ) .
we prepared the pcr master mix by mixing 19.25 l pcr - mix solution and 0.75 l dna taq polymerase for each reaction , adding 5 l dna template in each tube , and then running the amplification program .
the amplification program was denaturing at 95c for 9 min , 40 cycles at 95c for 20 seconds , 55c for 30 seconds , and 72c for 30 seconds , and finally extension at 72c for 5 min .
the flow - through hybridization was made on a prewarmed instrument at 45c , and the hybrimem hpv-21 dna microarray membrane was placed , which is marked with 21 hpv - genotype probes including hpv 6 , 11 , 42 , 43 , 44 , 16 , 18 , 31 , 33 , 35 , 39 , 45 , 51 , 52 , 53 , 56 , 58 , 59 , 66 , 68 , and cp8304 .
the number of samples tested in a batch could be adjusted from 1 to 15 as required .
the pcr products were denatured at 95c for 5 min just before hybridization and then was chilled on ice for at least 2 min .
we mixed the pcr products with hybridization solutions and then added the mixture into sample wells to proceed with flow - through hybridization for about 510 min .
the membrane was washed with hybridization solution , and the empty space was blocked without reaction . adding nbt / bcip solution to display the results , a positive result was indicated by a clearly visible indigo dot .
the hpv - genotype result was determined according to the position of the hpv - genotype probes on the microarray chip .
413 samples were detected by the commercially available hc - ii assay ( digene co. , gaitherburg , md , usa ) .
the probes used were designed to detect 13 types of high - risk hpv , including 16 , 18 , 31 , 33 , 35 , 39 , 45 , 51 , 52 , 56 , 58 , 59 , and 68 .
this enzyme - linked immunosorbent assay is based on a sandwich hybridization followed by a nonradioactive alkaline phosphatase reaction with chemiluminescence in the microplate .
samples were classified as positive for hpv dna if their chemiluminescence was more than 1.0 pg / ml of control .
it was performed with rembrandt universal dish and ap detection kit ( panpath , the netherlands ) . after being dewaxed and hydrated , paraffin sections ( 5 - 6 m )
specimens were denatured at 95c for 5 min by applying the probe solution to them and hybridized at 37c for 2 hours .
then , they were incubated with pan wash at 37c for 15 min except the positive control .
we dropped the conjugate by heating for 30 min and the nbt / bcip substrate by heating for 10 min at 37c in the dark and finally counterstaining and mounting .
the statistical analysis was performed using the spss software ( version 10.0 ) for chi - square test and kappa index was calculated to evaluate the accordance of the results .
the positive rate of hybrimax for 13 types of hr - hpv ( detected by hc - ii ) was 73.7% ( 303/413 ) and the positive rate of hc - ii was 70.7% ( 292/413 ) ( table 1 )
. in general , hpv - detection rates by hybrimax agreed well with those of hc - ii with a total accordance rate of 92.5% .
it is shown that the accordance rates were excellent in general ( kappa index ( ki ) = 0.814 ) in the group with normal cytological diagnosis ( ki = 0.750 ) , chronic cervicitis ( ki = 0.781 ) , condyloma acuminata ( ki = 0.80 ) , group cini ( ki = 0.755 ) , cin ii ( ki = 0.723 ) , and good at group cin iii ( ki = 0.547 ) ( 0.75 > ki > 0.4 , good ; ki 0.75 , excellent ) .
in addition , we found that there were 21 cases with hc - ii - negative and hybrimax - positive diagnoses , including 8 cases of hpv18 ( or multiple infections that include hpv18 ) , 5 cases of hpv 68 ( or that include hpv 68 ) , 4 cases of hpv 31 ( or that include hpv 31 ) , 2 cases of hpv 33 ( or that include hpv 33 ) , and 2 cases of hpv 51 ( or that include hpv 51 ) .
another two cases with genotyping results of hpv 66 , 2 cases with hpv 6 , one case with hpv 6/11 , and one case with hpv 44 by hybrimax were hc - ii positive , the results of which may be out of the hc - ii testing limits .
it is shown that the accordance rate of the two methods was 89.1% , and their kappa index was 0.776 .
the total hpv - positive rate of patients with abnormal cytological diagnosis was 89.6% ( 406/453 ) , with 80.9% in group a , 90.1% in group b , 92.2% in group c , 97.4% in group d , and 100% in groups e and f. in the group of patients with normal cytological diagnosis , the positive rate was 30.4% .
the 10 most common genotypes and their infection rates with abnormal cytological diagnosis in turn ( descending ) were hpv 16 ( 28.9% ) , 52/58 ( 19.0% ) , 18 ( 16.8% ) , 33 ( 9.9% ) , 31 ( 9.7% ) , 81 ( 8.4% ) , 53 ( 8.6% ) , 68 ( 8.4% ) , 66 ( 5.1% ) , and 43 ( 0% ) .
the 10 most common genotypes in normal groups were hpv 16 ( 8.0% ) , 68 ( 7.2% ) , 18 ( 6.5% ) , 52/58 ( 3.6% ) , 11 ( 2.9% ) , 53 ( 2.2% ) , 31/39 ( 1.4% ) , and 33 ( 0.7% ) , while hpv 35 , 45 , 59 , 66 , 42 , 43 , and 44 were not detected .
the 10 most common genotypes in different groups ( in descending order ) were as follows : hpv 16 , 18/58 , 52 , 31 , 53 , 68 , 81 , 33 , and 39 in the group of chronic cervicitis ; hpv 58 , 16/52 , 18 , 33 , 68 , 53 , 56/81 , and 31 in the group of cin ; hpv 16 , 52 , 58 , 18 , 33/81 , 31/51 , 53 , and 68 in cin ; hpv 16 , 58 , 52 , 18 , 31 , 33 , 81 , 53/68 , and 66 in cin iii ; hpv 16 , 18 , 52 , 58 , 33 , 66 , 68 , and 31/51/53 in group of scc .
we come to the conclusion that hpv 16 , 18 , 52 , 58 , 33 , and 31 were the 6 most common hpv types that can infect the patients with cervical lesions .
the most common hpv types causing condyloma acuminata were hpv 11 ( with a total positive rate of 55.0% ) and hpv 6 ( with a total positive rate of 30.0% ) .
the positive rates of the 6 most common genotypes in different groups were shown in figure 1 .
hpv 16 was the most frequent type in almost all the groups ( except for cin i , less than hpv 58 ) .
hpv 18 was the second most frequent type in scc , but the fourth most frequent type in all groups of cin .
similarly , the positive rates of hpv 18 increase with the development of disease . hpv 52 and hpv 58 were the third and fourth most common types in the group of scc , but the second and third most frequent types in all groups of cin .
hpv 33 was the fifth most frequent type in the group of scc , and the positive rates increase with the progress of disease in general .
previous studies have documented that hpv plays a central role in the etiology of cervical cancer [ 12 , 13 ] . it is indicated that women positive for hpv dna have a risk of developing cervical cancer 1550 times higher than those without hpv dna .
therefore , it is a preferred approach to combine the liquid - based cytology diagnosis with hpv dna testing in cervical - cancer screening .
traditional hpv - genotyping methods , such as southern - blot hybridization , direct sequencing , and restriction fragment length polymorphisms ( rflp ) based on polymerase chain reaction ( pcr ) are unsuitable for clinical use due to various reasons , such as low sensitivity , difficultly of handling , and time consumption .
ish is an easy to handle , reliable method for hpv detection and typing , working on pap smears and paraffin - embedded sections [ 17 , 18 ] ; however , its sensitivity and genotype detection are limited .
hc - ii , as the most wildly accepted hpv - dna - testing method for clinical use , is considered to be reliable , sensitive and easy to handle [ 6 , 7 ] .
however it has a limitation in discriminating hpv genotype and multiple infections , for hpv infection can only be attributed to a low - risk or high - risk group [ 5 , 8 ] .
flow - through hybridization is the most efficient method for molecular hybridization [ 911 ] .
a newly developed biotechnology named hybrimax , combining two advanced techniques , the flow - through hybridization and gene chip , is developeded to be used in clinical practice for the detection and genotyping of 21 different types of hpvs at one test .
the hpv genotypes detected by hybrimax include not only the 5 low - risk hpvs and 13 high - risk hpvs that hc - ii can detect , but also hpv 66 , 53 , and cp8304 that hc - ii can not detect .
in addition , hybrimax provides much more information than that afforded by hc - ii . in this study
, our results indicated that hybrimax was highly comparable to hc - ii in the detection of 13 types of hr - hpv , and has good accordance with ish in the detection of hpv 16/18 .
therefore , it is suggested that hybrimax can serve as an ideal method for hpv genotyping . among the 21 cases which hc - ii diagnosed negative while hybrimax revealed positive
, there were 8 cases of hpv 18 and 5 cases of hpv 68 , which probably suggested that hc - ii was less sensitive to those hpv types .
there were some samples detected hpv 6 , 11 , 44 , and 66 positive by hybrimax , present also positive by hc - ii , which should be negative ( not included in the range of genotype which can detected by hc - ii ) .
it was indicated that there was cross - reaction between the probes of the hc - ii hpv types .
it had been reported that the probes of hc - ii can have a cross - reaction with less than 22 types of hpv dna other than 13 types of hc - ii .
a potential disadvantage of hybrimax comes from the procedure of pcr , which generally was confronted with the problem of contamination .
in addition , this study revealed that , in china , the 6 most common genotypes in cervical lesions were hpv 16 , 18 , 52 , 58 , 33 , and 31 included in cervical cancer .
the recent international prevalence surveys by the international agency of research on cancer ( iarc ) reported that the most common hpv types of invasive cancer were 16 ( 57.4% ) , 18 ( 16.6% ) , 45 ( 6.8% ) , 31 ( 4.3% ) , 33 ( 3.7% ) , 52 ( 2.5% ) , 58 ( 2.3% ) , 35 ( 2.2% ) , 59 ( 1.5% ) , and 56 ( 1.3% ) , but the study did not include the chinese population . a meta - analysis made by clifford in 2003 revealed that in cases from asia , hpv 58 ( 5.8% ) and 52 ( 4.4% ) were more common than hpv 45 , 31 , and 33 , which supports our results .
a large - scale survey on the hpv types of 809 cervical cancer cases in china showed that hpv 16 and hpv 18 were the first and second most common hpv types , and hpv 58 and 52 were the third and fourth most common genotypes , followed by 31 and 33 , which also supported our results .
this study also found that the positive rates of hr - hpvs in groups of abnormal cytological diagnoses were prominently higher than that of normal groups .
therefore , it is suggested that hpv detection is especially important for women with abnormal cytological findings . | persistent infection of high - risk human papillomavirus ( hpv ) has been recognized as the direct cause of cervical carcinoma . therefore , detection and genotyping of hpv are important to cervical - cancer screening . in this study , we have evaluated the efficacy of flow - through hybridization and gene chip ( hybrimax ) on hpv genotyping through comparison of the results with hybrid capture ii ( hc - ii ) and in situ hybridization ( ish ) .
591 women were classified into 6 groups according to their histological diagnoses .
the overall accordance rate on 13 types of hpv genotypes between hybrimax and hc - ii were 92.5% and 100% in the cancer group .
the overall accordance was excellent with the kappa index ( ki ) of 0.814 .
the value of ki in each group was 0.750 ( normal cytological diagnosis ) , 0.781 ( chronic cervicitis ) , 0.80 ( condyloma acuminatum ) , 0.755 ( cervical intraepithelial neoplasia ( cin )
i ) , 0.723 ( cin ii ) , and 0.547 ( cin iii ) ( 0.75
> ki > 0.4 , good ; ki 0.75 , excellent ) . the 10 most common hpv subtype detected by hybrimax
were 16 , 52/58 , 18 , 33 , 31 , 81 , 53 , 68 , and 66 in patients , and 16 , 68 , 18 , 52 , 58 , 11 , 53 , 31/39 , and 33 in normal controls . in conclusion
, hybrimax is an efficient method for hpv genotyping and more suitable for clinical use . | 1. Introduction
2. Material and Methods
3. Results
4. Discussion |
PMC4497473 | lightning provides the most powerful natural accelerator available on earth for producing high - energy particles .
intense millisecond - scale bursts of gamma - rays produced by upward moving electrons accelerated to energies of tens of mev or more have been detected with satellite instruments .
these terrestrial gamma flashes ( tgfs ) have been shown to be associated mainly with positive polarity intracloud lightning , with the particle acceleration occurring at altitudes of 1015 km .
we show here that negative polarity cloud - to - ground lightning accelerates particles downward and produces gamma - rays with energies of at least 2 mev .
we present a sample of 24 tgfs detected at ground level associated with nearby ( < 5 km ) lightning observed over approximately 2.6 years mainly during spring and summer thunderstorms in louisiana .
tgfs were first observed by the burst and transient source experiment ( batse ) aboard the compton gamma - ray observatory [ fishman et al . , 1994 ; gjesteland et al . ,
2012 ] and have now been observed by five additional satellite detectors the reuven ramaty high energy solar spectroscopic imager ( rhessi ) [ smith et al . , 2005 ; grefenstette et al . ,
2009 ] , the gamma - ray imaging detector [ marisaldi et al . , 2010 ] and
minicalorimeter [ marisaldi et al . , 2011 ; tavani et al . , 2011 ] on astro - rivelatore gamma a immagini leggero ( agile ) , and the gamma - ray burst monitor ( gbm ) [ cohen et al . , 2010 ;
fishman et al . , 2011 ; briggs et al . , 2013 ] and large area telescope [ grove et al . ,
events are typically detected close to the subsatellite point [ grefenstette et al . , 2009 ] and are correlated both with regions of high thunderstorm activity [ cohen et al .
2011 ] and with individual positive polarity intracloud ( + ic ) and possibly positive cloud - to - ground ( + cg ) lightning discharges to within 12 ms [ inan et al . , 2006 ;
( positive polarity is needed to produce the upward beam of electrons and secondary photons necessary for detection of tgfs from space [ dwyer , 2003 ; cohen et al . ,
lightning flashes are known to emit a large fraction of their electromagnetic energy into low - frequency ( 0.330 khz ) atmospheric radio signals ( sferics ) , which can be located accurately by arrival time measurements in a worldwide radio receiver network [ rodger et al . ,
tgfs are well correlated both with sferics [ inan et al . , 2006 ; connaughton et al . ,
2013 ] and the lightning imaging sensor - optical transient detector and world wide lightning location network ( wwlln ) high - resolution lightning data [ hazelton et al . , 2009 ; smith et al . , 2010 ; fuschino et al . ,
gbm has also demonstrated that in some cases , as the original gammas propagate upward through the atmosphere , they produce secondary e via pair production [ cohen et al . , 2010 ; briggs et al . ,
these secondaries are then able to spiral around magnetic field lines to the spacecraft far from the lightning location , producing terrestrial electron beams characterized by 511 kev signals and both long - duration pulses and delayed pulses resulting from particles moving past the spacecraft and then reflecting from magnetic mirror points and returning to be detected by gbm . given the altitude of the satellites around 500 km , the observations point to beaming of the photons upward with a 30 half - angle cone coupled with attenuation of wide - angle photons passing through greater atmospheric path lengths [ grefenstette et al .
2005 ] , dwyer and smith performed detailed monte carlo simulations showing that the spectra were consistent with bremsstrahlung from electrons accelerated by the relativistic runaway electron avalanche ( rrea ) mechanism [ gurevich et al . , 1992 ; dwyer , 2003 ] at altitudes near thunderstorm tops . over the 0.110 mev range , the spectrum observed by agile [ marisaldi et al . , 2011 ]
has been well fit by a cutoff power law of the form f(e ) e
e with eo compatible with the 7 mev electron energies predicted by rrea , but the observation of individual gamma - rays with energies in excess of 40 mev has posed a challenge for the emission models [ tavani et al . , 2011 ; celestin et al . ,
rrea models of tgfs in the atmosphere [ dwyer , 2003 , 2008 ; gjesteland et al . ,
2011 ] start with mev seed electrons accelerated when lightning - associated electric fields overcome local energy losses .
these accelerated electrons produce photons , secondary electrons , positrons , and x - rays by bremsstrahlung , pair creation , and compton scattering .
the avalanche may be further seeded by the relativistic feedback mechanism , in which backward propagating positrons and x - rays lengthen the tgf durations up to several milliseconds as seen in satellite observations [ dwyer , 2008 ; dwyer et al . ,
ground - based lightning observations and comparisons of model calculations with the measured spectra indicate that the tgfs are produced at altitudes 1025 km [ dwyer and smith , 2005 ; grefenstette et al .
, 2008 ; shao et al . , 2010 ; gjesteland et al . , 2010 ; cummer et al .
a more detailed review of tgf models and observations is presented by dwyer et al .
tgf observations from satellite platforms are limited to events apparently beamed upward and large enough to be detected even in the presence of attenuation and compton scattering by the atmosphere .
although these events observed from space are extremely intense ( gamma - ray rates in excess of 300 khz measured with batse ) , the bulk of the events are presumably smaller events which can only be observed much closer to the lightning i.e . , at aircraft or balloon altitude or at ground level [ smith et al . , 2011 ; briggs et al . , 2013 ; gjesteland et al . , 2012 ;
[ dwyer , 2012 ] has suggested a possible downward directed positron and gamma - ray signature from tgfs .
observations at ground level are necessary to observe the downward component , to better understand the tgf intensity distribution and emission pattern , to understand whether the observed 30 beaming is intrinsic to the emission process or is the result of atmospheric attenuation , and to measure the spectrum versus altitude relationship . as a practical consideration
, it has been suggested that lightning - induced gamma - rays might produce a significant radiation exposure for airplane passengers flying close to a lightning stroke [ dwyer et al . ,
the majority of ground - level observation projects currently focus on correlating satellite - observed tgfs with lightning and measuring possible associated magnetic signatures [ cummer et al . , 2011 ; lu et al . ,
the international center for lightning research and testing ( iclrt ) project , however , has reported two gamma - ray bursts , one in association with triggered lightning of negative polarity [ dwyer et al .
, 2004 ] and another in association with nearby negative polarity cloud - to - ground ( cg ) lightning [ dwyer et al . ,
tgfs associated with negative polarity lightning strikes , as with these iclrt events , produce downward beams of photons which can be detected from the ground .
iclrt operates in a triggered mode , requiring either a triggered lightning current above 6 ka or the simultaneous trigger of two optical sensors .
the array of particle detectors at aragats space environment center has detected thunderstorm - associated ground enhancements above 7 mev with timescales of microseconds and tens of minutes [ chilingarian et al .
these have been detected approximately once per year and seem to be correlated with ic lightning .
in addition , a mountaintop detector has observed 3 ms bursts of x - rays associated with cg lightning [ moore et al . , 2001 ] .
longer duration ( 40 s to minutes or longer ) x - ray and gamma - ray events have been reported previously from the ground [ tsuchiya et al . , 2011 , 2013 ] , but the only other case in which a tgf - like event with millisecond emission of mev gammas has been observed from within the atmosphere is the observation by the airborne detector for energetic lightning emissions aboard an aircraft at an altitude of 14 km [ smith et al . ,
we present observations from july 2010 through february 2013 of 24 tgf - like events in which 50 kev2 mev gamma - rays are observed at ground level in shorter than 5 ms bursts associated with nearby negative polarity lightning .
the tgf and energetic thunderstorm rooftop array ( tetra ) consists of an array of twelve 19 cm 19 cm 5 mm nai(tl ) scintillators designed to detect the gamma - ray emissions from nearby lightning flashes over the range 50 kev2 mev .
the scintillators are mounted in four detector boxes , each containing three nai detectors viewed by individual photomultiplier tubes ( pmts ) .
the boxes are spaced at the corners of a 700 1300 m area on four high rooftops at the baton rouge campus of louisiana state university ( lsu ) at latitude 30.41 and longitude 91.18. unlike iclrt , tetra operates in a self - triggered mode , allowing for events to be recorded without requiring the direct detection of lightning .
each tetra detector box contains three nai scintillator plates oriented at 30 from the zenith direction and separated by 120 in azimuth .
each nai(tl ) crystal is hermetically sealed between a 6.4 mm thick glass optical window on one flat face and a 0.75 mm thick aluminum entrance window on the other face .
an ultraviolet transmitting lucite light guide is coupled to the glass window , and the light is viewed by an electron tubes 9390 kb 130 mm photomultiplier tube with a standard bialkali photocathode .
the scintillator - pmt assemblies are housed in 2.5 cm thick plastic foam insulation to prevent rapid temperature changes .
electronics boards in each detector box supply high voltage , amplify and shape the pmt outputs , provide an internal trigger for the data acquisition software , digitize the data , assign timestamps , and record adc values for each event .
once triggered , each pmt anode output is integrated and assigned a 12 bit analog - to - digital converter ( adc ) value .
a 32 channel adc board , a lassan iq gps board , and a mesa field - programmable gate array ( fpga ) board are incorporated onto a pc104 stack controlled by a microcomputer systems vdx-6357 800 mhz 486 cpu board running a qnx operating system .
the fpga is programmed to handle trigger logic , clock functionality , and event time stamping .
we refer to the mesa board together with its fpga as the trigger logic module .
each is capable of detecting events at a sustained rate of 30 khz and a burst rate of up to 70 khz .
the data are then transferred over a wireless link to a central station for analysis .
the initial version of the data acquisition software , used from october 2010 to january 2013 , utilized a network time protocol to keep timestamps accurate to within approximately 2 ms and to monitor the absolute timing uncertainty .
the current version of the software , implemented in january 2013 , uses a gps - disciplined clock to produce timestamps accurate to within 200 ns .
the adc - to - energy conversion is calibrated with radioactive sources ( na , cs , and co ) .
individual detector energy resolution ranges from 9 to 13.5% full width at half maximum ( fwhm ) at 662 kev and from 5.5 to 10.8% at 1.3 mev .
the total interaction probability in the nai scintillators is 95% at 100 kev , 82% at 500 kev , and 10% at 1 mev ( with photoelectric interaction probabilities 93% , 26% , and 0.63% , respectively ) .
in addition to the three nai scintillators , one detector box contains a 2.5 cm diameter by 2.5 cm thick cerium - doped lanthanum bromide ( labr3:ce ) scintillator that provides high - energy - resolution measurements ( 3.5% fwhm at 662 kev ) of intense events . beginning in october 2012 ,
data are accumulated for a day at a time for each of the four detector boxes individually .
the daily analysis software selects events with signals corresponding to at least 50 kev deposited energy within 1 s .
tetra triggers are selected with counts/2 ms at least 20 standard deviations above the mean for the day .
once days with excessive electronic noise or other instrumental problems are removed , there are 835.09 days of live time and 1303 tetra triggers .
in figure 1 , the heavy black line shows a time history of the count rates for the three nai photomultiplier tubes of > 50 kev events in a single detector box for 1 day .
the total count rate , plotted in counts per minute , is reasonably constant for the first 17 h and then increases by a factor of approximately 2 beginning at about 1800 cst .
the small peak in the count rate seen at about 1200 cst is due to noise in the system seen only in a single pmt on a 60 s timescale .
the thin black histogram near the bottom shows the local radar reflectivity in decibels acquired from www.wunderground.com , indicating rain , thunderstorms , hail , or strong winds .
the gamma - ray spectrum , measured during a rain event with the high - resolution labr3:ce detector mounted together with the nai detectors in one of the detector boxes , shows a clear indication of 295 , 352 , 609 , 1120 , and 1764 kev bi and pb lines characteristic of radon decay ( figure 2 ) .
summed nai counting rate per minute in box 3 on 18 august 2011 ( heavy black line , left - hand scale ) .
thin black histogram near the bottom ( right - hand scale ) shows radar reflectivity .
the row of filled circles near the top marks intervals in which the count rate in 60 s bins exceeds the day 's average by 3 ; the open square marks the tetra trigger , i.e. , the interval when the rate in 2 ms bins exceeds the day 's average by 20 . labr3:ce rain spectrum .
labr3:ce background - subtracted spectrum during a 6 h precipitation event showing radon lines at 295 kev , 352 kev , 609 kev , 1120 kev , and 1764 kev .
the filled rectangle near the bottom of figure 1 at approximately 1800 cst marks the times of lightning strikes detected by the u.s .
precision lightning network ( uspln ) unidata program within 8 km of the lsu campus .
these are mainly cloud - to - ground events with positions accurate to approximately 0.4 0.8 km . in the upper section of the diagram ,
the line of filled circles marks 60 s intervals in which the nai detector count rate is 3 standard deviations higher than the average rate for the day ; these are correlated with the peak of the extended rise at the time of the rainstorms .
tetra triggers are defined as intervals during which the rate in a 2 ms window exceeds the day 's average by 20 . the tetra trigger observed is indicated near the top of the plot as an open square .
( for a typical average counting rate of 8900 min in a detector box above 50 kev , a 20 excess corresponds to 10 counts in the three pmts in a detector box within a 2 ms window . ) .
figure 3 shows an expanded view of the data on the same day , illustrating the correlation of the triggers in individual boxes with lightning and cloud density overhead .
figure 3b shows the rate per second of lightning strikes within 8 km of the detectors , and figure 3c shows the distance of all lightning strikes recorded by the uspln network within 160 km .
( a ) triggers detected on 18 august 2011 ( nai signals above 50 kev in a single detector box with count rate per 2 ms in excess of 20 above the 18 august 2011 daily mean counting rate ) .
box 1 triggers are indicated by plus signs , box 3 by triangles , and box 4 by squares .
( b ) rate per second of uspln lightning strikes within 8 km ( c ) distance to each recorded lightning strike within 160 km ( d ) overhead cloud density . from july 2010 through february 2013
, tetra has recorded a total of 24 events with triggers occurring within several minutes of thunderstorm activity producing at least one lightning flash within 8 km of the detectors .
such events are classified as event candidates ( ecs ) and are listed in table 1 . in this table each event trigger time is listed , along with the number of lightning flashes detected within 2.5 min and 8 km and the cloud density above tetra .
also listed for each ec is the time difference to the lightning stroke closest in time to the event trigger , the distance to that lightning stroke , the current , the number of gamma - rays detected in the ec , and the t90 duration of the event ( i.e. , the time over which a burst emits from 5% to 95% of its total measured counts in a single detector box ) .
the number of sigma above the mean is listed in the second to last column for each event .
( for the first three events in the table , observed simultaneously in multiple detector boxes , the smallest number of sigma above the mean is listed .
these coincident events , labeled coincident event candidates cecs are discussed in more detail below . ) .
properties of the 24 event candidatesa cecs are listed in the top section ; ecs for which the absolute timing uncertainty is known are listed in the middle section ; and ecs for which the absolute timing uncertainty is unknown are listed in the bottom section of the table .
the date and time of each ec trigger are listed , along with the properties of the storm associated with each event .
the properties of the associated lightning , event duration , number of gamma - rays detected , total energy , and event significance are also listed for each event .
the probability of each cec occurring is listed in the last column for the cecs .
tetra 's events , with an average of 20 2 photons detected , are significantly smaller than the typical events observed in space . for tetra 's events ,
the t90 duration was calculated by considering all events detected within a 3 ms window around the trigger time , discarding the first and last 5% of timestamps for each event , and recording the time difference between the first and last events remaining .
the uncertainty in the t90 determination is approximately 200 s based on monte carlo simulations of the data . in each of the 24 events ,
7 to 45 -rays were detected within a time window of less than 5 ms , with the total energy deposited per event ranging from 2 to 32 mev .
the distances to the nearest lightning flashes were 0.64.7 km . for 14 events , absolute timing was available with 2 ms accuracy .
for each of these 14 events , lightning was observed within 7 s of the trigger time .
nine of these events were associated with cg lightning detected within 6 ms of the trigger .
july 2012 during a period when accurate trigger - lightning time differences were not recorded due to network timing difficulties .
eight of the ecs during that period were correlated with two intense thunderstorms that passed directly over tetra on 6 june 2012 .
the accidental rate of triggers coincident within 7 s of a lightning flash that is less than 8 km distant ( i.e. , events masquerading as ecs ) is calculated based on the rate of tetra triggers ( due mainly to cosmic ray showers ) , the live time , and the duration of storm activity . the storm activity time is taken to be the sum of all time windows where there was lightning within 8 km and 7 s , and there was no electronic noise or other instrumental problems . for a total storm time of 12.65 h , we calculate the expected number of ecs due to accidental triggers to be 0.82 .
the efficiency of the uspln in our area has not been tested ; however , if we assume a similar sensitivity to that measured by jacques et al . for cloud - to - ground lightning with peak current in excess of 20 ka of approximately 25% to account for undetected lightning flashes
figure 4 compares data acquired within 7 s of lightning to the remaining data with accurate timing information .
the distribution of events versus within 7 s of a uspln lightning strike within 8 km is shown in black .
the significance distribution of the remaining data has been normalized to the total storm activity time of the lightning distribution for comparison , shown in grey .
the excess of events above 20 sigma in the lightning distribution ( black ) as compared to the normalized distribution ( grey ) indicates the association of the gamma - ray events with nearby lightning .
( note that since three events involve seven separate coincident triggers in individual detector boxes , there are 18 individual triggers shown in figure 4 compared to the 14 ecs with accurate timing information in table 1 . )
a kolmogorov - smirnov test of the two distributions results in a d parameter of 0.25 , corresponding to high confidence that the two distributions are distinct .
distribution of events with significance . distribution of events within 7 s of nearby ( < 8 km ) lightning is shown in black .
distribution of all data , normalized to 0.52 days of live time , is shown in grey , showing excess of lightning - associated ecs at > 20 .
the dark solid line in figure 5 shows the deposited energy spectrum of the 24 event candidates , with events observed up to 2.7 mev deposited energy .
it should be emphasized that with tetra 's thin detectors , only a portion of the incident gamma - ray energy is actually detected . between 200 kev and 1.2 mev , the ec spectrum is fit with a power law e , with = 1.20 0.13 and /degree of freedom = 0.4 ( dashed dark line ) . on the same figure ,
the grey line shows the spectrum of non - ec triggers ( i.e. , triggers not associated with lightning within 5 mi and 7 s ) ; this spectrum is softer , with a best fit power law index = 1.79 0.04 and /degree of freedom = 1.0 ( dashed grey line ) .
the associations of the events reported here with negative polarity lightning strikes and the low likelihood that these are background events , along with the durations observed , are indicative of downward directed tgfs produced by the rrea mechanism .
spectrum of non - ec tetra triggers ( triggers not associated with lightning nearby in time and distance ) is shown in grey .
power law fits between 200 kev and 1200 kev of the form e are shown with dotted lines , where = 1.20 0.13 and 1.79 0.04 for ec and non - ec events , respectively . in three of the 24 ecs , triggers were recorded in two or more boxes separated by 1000 m within less than 2 ms .
all three of these coincident event candidates ( cecs ) occurred in july and august of 2011 , when storms in southern louisiana tend to be associated with disturbances in the gulf of mexico rather than frontal lines .
the plot shows a 50 ms window centered on the event trigger time , defined as the center of the first 2 ms bin containing a trigger .
the counts for each box ( i.e. , the number of phototubes detecting a signal with amplitude in excess of 50 kev within the 1 s pmt anode output integration time ) are plotted versus time relative to the event trigger time .
for the two events on 31 july 2011 ( figures 6a and 6b ) , the lightning strikes closest in time occurred within approximately 6 and 4 ms of the event trigger .
for those cases , the time of the lightning strike is shown as a cross with a timing uncertainty of 2 ms near the top of the plot . in the first 31 july 2011 event ( figure 6a ) , one pmt in box 3 fired , followed by two pmts in box 4 2.3 ms later .
the distance between the two boxes was 1500 m , corresponding to a gamma - ray travel time difference of up to 5 s .
in fact , we believe the differences between the event times in the separate boxes in figure 6 are a direct measure of the absolute timing differences between the boxes . nai time histories over 50 ms window centered on the trigger time for each cec .
lightning strikes within 8 km in the 50 ms window have a 2 ms timing uncertainty and are shown with crosses .
the box 3 time history is centered at 0 ms and box 4 at 2 ms .
a uspln lightning strike within 8 km is indicated by the cross at 6 ms .
the box 3 time history is centered at 0 ms and box 4 at 2 ms .
a uspln lightning strike within 8 km is indicated by the cross at 4 ms .
( c ) the event on 18 august 2011 at 17:57:38.984 cst with the box 3 time history centered at 0 ms , box 1 at 2 ms , and box 4 at 4 ms .
no uspln lightning was detected within 8 km within this 50 ms window .
the expected number of cecs due to random triggers is small : given an initial ec with counting rate in one box in excess of 20 above the daily average , the likelihood that a second or third trigger occurred at random in another box within the timing uncertainty of 2 ms on the same day is estimated as ( 4 ms n/86,400 s ) , where n is the total number of random 20 triggers detected per day through february 2013 and b is the number of boxes triggered in the event .
( for simplicity , we neglect here the increase in trigger rate during a thunderstorm shown in figure 1 . )
multiplying by the number of ecs then gives the expected number of spurious cecs involving two boxes occurring by chance as 1.7 10 , as listed in table 1 .
a composite energy spectrum summed over the three cecs is shown in figure 7 .
a total of 80 gamma - ray pulse heights above 50 kev and within the t90 interval of each coincidence trigger are shown .
the average photon energy detected is approximately 0.5 mev , an energy at which the fraction that passes through a nominal 1.6 km of atmosphere at ground level ( stp ) without interaction is 10 .
this average energy is low compared to the typical energies observed by the orbiting detectors [ dwyer et al .
, 2012a ] and is presumably biased to low energies by the 0.5 cm thickness of the tetra nai scintillators .
eighty photons were detected within the t90 interval of each individual detector box 's trigger time .
figure 8 shows the distance from the detectors and the measured current for each lightning flash within 8 km of tetra from 1 july 2010 to 28 february 2013 . there were a total of 5360 flashes within 8 km . for each of the 10 ecs and cecs with lightning within 8 km and 100 ms of the trigger time ,
although all the tetra events correspond to lightning less than 5 km away , the two lightning flashes within 100 ms of a cec are both more than 2 km away .
if all discharges produce tgfs [ stgaard et al . , 2012 ] , then the rate of detection and the cec distances point to either a range of intensities extending below the sensitivity limit of tetra , strongly nonisotropic emission , or the possibility that the gamma - ray emission is only indirectly associated with the lightning [ connaughton et al . , 2013 ] .
this can also occur if some gamma - ray events are produced by intracloud ( ic ) strikes , since the uspln data record primarily cloud - to - ground strikes .
all lightning activities within 8 km of tetra from 1 july 2010 to 28 february 2013 .
the current and distance for all uspln lightning flashes within 8 km of tetra are indicated by grey crosses .
lightning strikes that are within 8 km and 100 ms of an ec or cec are considered coincident strikes and are plotted with black crosses .
the vertical line at 0 ka indicates ic lightning . out of the 10 ecs shown in figure 7 ,
nine were found to be within 6 ms of a negative polarity cg lightning strike within 5 km with current above 20 ka ( table 1 ) .
for the two cecs that occurred on 31 july 2011 , lightning strikes are recorded at 6 ms and 4 ms before the tetra triggers . in both cases , these were nearby , cloud - to - ground events at 2.3 km distance with current 43.6 ka and 2.9 km distance with current 29.1 ka . for four ecs with accurate timing information ,
the lightning strikes closest in time to the tetra triggers were in excess of 100 ms before or after the nai signal and so are not considered coincident with a uspln observed strike .
again , this can occur if some gamma - ray events are produced by intracloud ( ic ) strikes or if the gamma - rays are not all directly associated with the lightning .
the gamma - ray events described here have durations ranging from 24 s to 4.2 ms .
the similarity of these event durations observed by tetra to those reported by batse , rhessi , agile , fermi , and iclrt suggests that the tetra events are also generated by the rrea mechanism .
dwyer et al . [ 2012b ] compared the spectrum of x - rays from lightning to gamma - rays from tgfs , showing a marked difference above 2 mev , but the restricted energy range of tetra and the low statistics make it impossible to draw strong conclusions from the observed tetra spectra .
fermi gbm data suggest that wwlln detects shorter duration tgfs more efficiently than the longer ones because of the frequency constraints of the network ( between 6 and 18 khz ) . for the sferic signals found within a 400 s window around the tgf gammas ,
the stronger sferics appear due to the tgf itself while the weaker sferics are due to associated + ic lightning [ connaughton et al . ,
the brightest tgfs seen by batse , rhessi , and gbm produce 10 runaway electrons with a source altitude 13 km [ briggs et al . ,
in contrast , the two tgfs previously reported from the ground by iclrt are associated with cg lightning [ dwyer et al . , 2004 , 2012b ] .
the 2009 iclrt event produced 10 runaway electrons and was observed at a distance of 2 km . if the tetra events are characterized by typical energy 500 kev and distance 1.6 km , then atmospheric absorption attenuates the flux by a factor of 4 10 at sea level .
assuming isotropic emission at a distance of 1.6 km , a typical total of 20 photons observed in an event by tetra then requires in excess of 10 photons at the source . either the ground level tetra events are beamed or they are distinctly different from the iclrt events .
here we have presented data for a series of gamma - ray events observed with a self - triggered ground array , suitable for observing weak events from nearby distances without a bias caused by a lightning trigger , and find that events with durations < 5 ms and detected individual photon energies up to at least 2 mev appear to be produced in conjunction with nearby cg lightning . in two cecs ,
these are most closely associated with cg events 2.3 and 2.9 km away . in the other cec event ,
the nearest detected lightning strike in time is more than 6 s after the gamma - ray event .
either this gamma - ray event is not correlated with nearby lightning , the associated cg lightning strike was missed by the lightning network , or the event was due to ic lightning that was not detected by the lightning network . | [ 1 ] terrestrial gamma - ray flashes ( tgfs)very short , intense bursts of electrons , positrons , and energetic photons originating from terrestrial thunderstorms have been detected with satellite instruments .
tgf and energetic thunderstorm rooftop array ( tetra ) , an array of nai(tl ) scintillators at louisiana state university , has now been used to detect similar bursts of 50 kev to over 2 mev gamma - rays at ground level .
after 2.6 years of observation , 24 events with durations 0.024.2 ms have been detected associated with nearby lightning , three of them coincident events observed by detectors separated by 1000 m. nine of the events occurred within 6 ms and 5 km of negative polarity cloud - to - ground lightning strokes with measured currents in excess of 20 ka .
the events reported here constitute the first catalog of tgfs observed at ground level in close proximity to the acceleration site . | 1. Introduction
2. Detector Description
3. Results
4. Conclusions |
PMC4091449 | sequence assembly is the first challenge encountered in a typical computational genomics pipeline and it involves the merging and the ordering of shorter sequence fragments ( reads ) with the aim to get as close as possible to the original larger sequence ( genome ) .
advent of next - generation sequencing platforms has allowed the sequencing a huge number of organisms and species at reasonable costs .
however , many issues regarding the computational assembly of large - scale sequencing data have remained unsolved 1 and , actually , the number of draft genomes in databases greatly overtakes the number of completely sequenced ( sometimes referred also as finished or closed ) ones ( www.genomesonline.org ) .
the output of a de novo assembly is typically a draft genome , consisting of a set of contigs ( i.e. contiguous sequence fragments ) that may be ordered and oriented into scaffold sequences , with gaps between them , representing regions of uncertainty or missing sequence 2 .
alternatively , draft genomes can be represented in the form of ( de bruijn ) graphs , an approach that is currently exploited by a number of assembly algorithms ( reviewed in 3 ) .
the difficulty in obtaining a closed genome may be due to several causes , including the presence of repetitive fragments along the genome and/or the absence of enough reads to produce a reliable assembly .
it is also known that the most rapidly evolving regions are often absent or incorrectly rendered in finished genomes 4 .
furthermore , from a mathematical viewpoint , the de novo genome assembly problem can be proven to be difficult , falling within a class of problems ( np - hard ) for which no efficient computational solution is yet known 5 . in this context , it must be added that some traditional assemblers like newbler ( 454 sequencing , roche diagnostics , indianapolis , in , usa ) usually perform a trimming of the contig edges depending on the quality of the supporting reads .
this conservative procedure , however , may result in a loss of information , discarding many correct bases characterized by a sub - optimal quality . to overcome these limitations ,
we have developed a tool called enly that allows increasing the length of the contigs deriving from de novo assemblies and the closure of part of the gaps commonly present in the draft genome .
enly is sequencing platform - independent since it be used with any kind of sequence type , as long as files are provided in fasta format . accordingly , even hybrid datasets ( obtained with different sequencing technologies ) can be used as input for enly pipeline .
finally , by taking into account scaffold information , enly drastically reduces the probability of generating chimeric assemblies .
the overall procedure requires ( at least ) two multi - fasta files as input , one containing all the contigs deriving from the first assembly and the other all the raw reads resulting from the sequencing run . by applying the strategy described below to each contig in the input file ,
enly tries to increase their length and ( possibly ) to merge them into scaffolds .
more in detail , the pipeline is based on the iteration of multiple cycles ( figure 1 ) and during each of them : 1 ) a fragment of length l1 ( specified by the user ) is detached from the 5'-end of each contig and is used as an input for a blast search 6 against a database embedding all the original reads resulting from the sequencing run ; 2 ) the blast output is parsed to identify reads that can be used to extend the contig ( i.e. those partially aligned at the end of the contig and protruding from its extremity ) . by default identity threshold among the reads and
overlap length between each read and the contig extremity is , instead , specified by the user ( with the -s parameter ) .
this means that , for example setting -s to 70% , only reads aligned for at least 70% of their length and sharing more than 97% of their sequence with the contig extremity will be used to extend contigs .
intuitively , setting this overlapping threshold to higher values will increase accuracy , although reducing the number of closed gaps ( see additional file 1 : supplementary material ) .
the use of higher thresholds ( i.e. higher stringency ) is , therefore , recommended when a reference genome / scaffold is not available for checking possible chimeric joints ( see point 8) .
when a reference scaffold is present , contigs merging will still be reliable even when low overlap thresholds ( e.g. 30% ) are selected , although it must be pointed out that the extended contigs not merged into any ( sub ) scaffold would not be checked for the presence of wrongly incorporated sequence .
3 ) the selected reads and the original contig are assembled together using either phrap 7 or minimo 8 assemblers , possibly resulting in an enlarged
4 ) the very same procedure is repeated for the 3'-end of the contig and for all the other contigs of the input file ; 5 ) the extended contigs are used as inputs for a second step , in which a fragment of length l2 ( with l2 < l1 ) is detached from each contig end and used as an input for a further blast against the reads database , assembling the matching reads with the enlarged contig .
this procedure , performed in order to compensate for the presence of reads with a heterogeneous length distribution ( resulting from a typical 454 run 9 ) , is repeated for fragments of decreasing length , until the minimum length ( specified by the user through -m option ) has been reached ; 6 ) if a reference genome ( in fasta format ) has been provided by the user , extended contigs are mapped onto it , taking advantage of an ad hoc modified version of the contiguator tool 10 , allowing launching this tool in an iterative fashion and storing results from each run in a separate folder .
7 ) contigs are mapped by blast one against each other for identifying possible overlaps and gaps closure . no threshold for contigs overlap is set in this stage since scaffold information is used ( see next point ) to discard possible chimeric joints .
8) gap closures are validated against a reference scaffold , provided by the user ( -y option ) or , alternatively , generated by the contiguator tool .
only contigs overlaps that are consistent with scaffold information are merged ( using the megamerger tool from the emboss suite ) . in case neither a scaffold nor a reference genome
have not been provided by the user , possible overlaps for each of the enly cycles are stored in a specific output files ( see enly 's manual for details ) that the user can manually inspect for eventual gap closures .
9 ) these contigs , together with those that have not been enlarged / merged are then used as input for the following cycle of the pipeline .
the first cycle of the enly pipeline is completed when all the contigs have been processed , mapped and saved to a new multi - fasta file .
the procedure is then repeated ( re - starting from point 1 ) for a user - specified number of cycles or , alternatively , until no more bases have been added to the contigs during the last cycle .
output files from the mapping procedure ( contiguator ) are saved in separate folders ( one per cycle ) , ready for being loaded by the artemis comparison tool 11 , in order to visually inspect contigs alignment against the reference genome .
reads used for extending contigs may derive from any of the currently available sequencing technologies ( illumina , 454 , iontorrent , etc . ) , as long as they are provided in the form of fasta files .
moreover , reads obtained by different sequencing technologies may be pooled in a single file to be used as input for enly pipeline . intuitively , since enly uses the sequence of the reads partially protruding from each contig , the probability of extending its length increases with the average length of the reads dataset .
it should be noticed that , besides 454 pyrosequencing , other sequencing platforms are now starting to generate reads of length comparable to the ones of the datasets used in this work ( e.g. illumina miseq , iontorrent ) , thus paving to the use of this alternative datasets for gap - filling through enly 's approach . to evaluate the reliability of the pipeline
, we tested enly on three different 454 reads datasets retrieved from either the ncbi short read archive database ( sra , http://www.ncbi.nlm.nih.gov/sra ) , namely escherichia coli ko11 ( srs084754 ) and staphylococcus aureus 649 ( srs114535 ) , or from a previous sequencing run on streptococcus pneumoniae ap200 12 .
ap200 12 , 13 ) or the genome of a phylogenetically close bacterium ( s. aureus col 14 ) were available , allowing the validation of the results obtained with enly .
each reads dataset was first assembled with newbler v. 2.6 , using default parameters , resulting in 719 , 254 and 124 contigs for e. coli ko11 , s. aureus 649 and s. pneumoniae ap200 , respectively .
contigs obtained from de novo assembly were , then , used , together with the corresponding reads , as input for the enly pipeline .
parameter values ( and an evaluation of their effects on the genome assembly ) used during these tests are reported in additional file 1 : supplementary figure 1 , together with introduced mismatch rates for different reads alignment thresholds ( -s option , additional file 1 : supplementary figure 2 and 3 ) . in all cases , enly was able to improve the de novo assembly ( table 1 ) . in detail , the number of closed gaps ranged from 16% to 19.1% of all the gaps present in each input draft genome .
most of the closed gaps resulted from merging two contigs although , in some cases ( e.g. e. coli ko11 drafts assembly ) , up to 5 different contigs were merged in a single ( sub ) scaffold .
importantly , after running enly on the de novo assemblies , an increase in the n50 value for each of the genome was observed , accounting for the overall improvement of the input draft assemblies .
interestingly , enly showed to perform reasonably good also when non-454 reads were used as input for the pipeline .
results obtained with reads obtained from iontorrent , miseq and pacbio sequencing runs are reported in additional file 1 : supplementary table 2 and 3 . the computational time required by enly correlates with the amount of contigs
/ reads embedded in the input files and with the number of cycles required by the user . on a machine with eight 3.1 ghz processors , the computational time required for the tests performed in this work ( and according to the parameters specified in additional file 1 : supplementary table 1 ) ranged from 2 up to 5 hours depending on the size of the reads datasets and parameters used ( see additional file 1 : supplementary figure 4 ) .
recently , two scaffolding approaches apparently similar to the one presented here have been developed .
both of them take advantage of the additional information that is present on a typical illumina paired - end sequencing run 15 , 16 .
currently , enly does not use paired - end information , being able to process single end sequencing reads ( as long as they are provided in fasta format ) for contigs extension ; moreover , by implementing the possibility to guide contigs extension / merging through the use of scaffold information ( independently generated by the user or obtained through the implemented contiguator tool ) , our pipeline reduces the probability of chimeric scaffolds . in conclusion , enly is a simple , cross - platform and parallelizable tool allowing the improvement of draft genomes resulting from de novo - assembled high - throughput sequencing reads .
it is based on the iterative mapping of reads at contigs ends and it is also able to use ( and generate ) scaffold information to guide contigs merging , reducing the probability of chimeric scaffolding .
testing it on a set of de novo draft genomes led to the closure of up to 20% of the gaps originally present , thus resulting particularly helpful during genome finishing procedures . | the reconstruction of the complete genome sequence of an organism is an important point for comparative , functional and evolutionary genomics . nevertheless , overcoming the problems encountered while completing the sequence of an entire genome can still be demanding in terms of time and resources .
we have developed enly , a simple tool based on the iterative mapping of sequence reads at contig edges , capable to extend the genomic contigs deriving from high - throughput sequencing , especially those deriving by newbler - like assemblies .
testing it on a set of de novo draft genomes led to the closure of up to 20% of the gaps originally present .
enly is cross - platform and most of the steps of its pipeline are parallelizable , making easy and fast to improve a draft genome resulting from a de novo assembly . | Introduction
Results and Discussion
Supplementary Material |
PMC1160193 | a constant flood of new genomic information has brought a new age of discovery to biology .
unfortunately , this deluge of new data is rarely fully utilized , in part because bench scientists find it increasingly challenging to maintain a current , integrated picture of the latest data .
this problem is particularly pronounced for scientists who study large numbers of potentially interesting molecules , a common result of microarray - based or proteome - based experiments .
regularly updating such data by hand can be extremely burdensome and is rarely done . as a result , many scientists work unaware of newly discovered annotation , homologs , clones or protein domains that could further their research .
current comparative table , or cct , is a web - based application designed to solve this problem by displaying the most up - to - date results of customized genomic searches in a convenient table .
it can be easily configured to automatically download new versions of databases and to run any number of bioinformatics searches on the new data .
cct then organizes the results in a table containing hyperlinks color - coded by result age , making it simple to pick out recently changed results .
this allows scientists to continuously harvest potentially useful data about any set of sequences of importance to their research .
cct joins a number of other excellent servers whose aim is to manage data overload ( 1,2 ) , but cct has unique strengths .
the most widely used of these other services , pubcrawler , searches for text and literature matches in pubmed , genbank or both but does not perform actual sequence searching ( 3 ) .
servers that do allow sequence searches ( e.g. and ) allow the searching of prescribed subsets of public databases and/or a single private database of each data type that is uploaded manually ( 2 ) .
like myhits and swiss - shop , cct performs sequence - based searches , but it uniquely offers a local installation , allowing independence from remote servers .
other novel features of cct include automated highlighting of even small changes in data files , a simple interface for scientists interested in multiple sequences and the ability to monitor any number of databases to which the user has access .
the computer has a 3 ghz processor , 200 gb of hard drive space and 2 gb of ram .
mac os x compatibility was tested on a dual 2 ghz g5 tower with 1.5 gb of ram , a 160 gb hard drive and mac os x 10.3.4 .
cct is implemented in perl and makes heavy use of the bioperl toolkit ( 4 ) .
a demonstration of cct and an installation guide are available at , and the software is freely available from .
a scientist typically begins using cct by adding three types of data through a web interface : ( i ) a file containing sequences of interest , ( ii ) database location(s ) to monitor for updates and ( iii ) searches to perform .
although cct can be run at will , it is typically run automatically . in this mode ,
cct periodically ( e.g. once per day , such as at 1 a.m. ) checks user - selected databases and downloads updated versions as they become available .
cct then runs user - selected searches on these data and builds a table with one row for each sequence and one column for each database searched ( figure 1 ) .
each cell contains links to the results of each search , color - coded by the length of time since a change in data has affected the search results .
in addition , when a result is updated , the new result is compared with the previous one , and differences are highlighted in the new data file ( figure 2 ) . taken together , link coloring and difference highlighting allow numerous search results to be quickly scanned and evaluated for novelty ( or for stability over time ) .
this feature is valuable for scientists engaged in ongoing projects as well as those deciding when to commit limited laboratory resources to the characterization of a set of interesting but preliminary sequences .
cct can be run locally or remotely via the internet and comes bundled with wrappers for six different bioinformatics search programs .
hmmpfam is a wrapper for the hmmpfam tool and is useful for automating searches of sequences against ever changing protein domain databases such as pfam ( 5 ) .
tblastn and blastp are the software 's wrappers for ncbi 's blast searches ( 6 ) .
the seq program is specific to cct and is useful for isolating regions of the genome , e.g. for finding genes and open reading frames .
seq takes tblastn or blastp output , captures the sequences for the target blast high - scoring pairs and extends them out to a user - specified delimiter .
for example , if a stop codon were selected , seq would capture the sequence of the flanking open reading frame .
revblast uses seq output as a query to blast search another database , permitting reciprocal blasting , a common method for finding ortholog pairs ( 7 ) .
finally , the homolog program takes revblast output and uses clustal - w ( 8) to generate a pairwise alignment if sequence pairs meet user - specified parameters .
when used together , these programs are an effective tool for finding orthologs and can be customized in a way that sets cct apart from other comparative genomics tools ( 9 ) .
the mac os x version of cct can be installed in 60 s in a few simple steps from a double clickable install package .
it is fully self - contained and includes code for bioperl , clustal - w , blastall and hmmpfam .
the linux version does not include this code in the expectation that linux users may want to integrate cct into existing bioinformatics resources on their servers .
cct 's user manual is part of every installation and can also be found at the software 's demo site .
the user manual contains an installation guide , a beginner 's guide , the addresses of sample databases , screenshots and other useful information .
in addition , cct is installed with a link to extensive code documentation to make its customization as easy as possible for users with any level of programming experience .
programmers can construct new program modules to interact seamlessly with cct using an included template file .
the design of cct falls into two main parts : the web interface and the script runcct.pl .
users can add and delete tables , searches and databases , and can view their data by browsing a cct web page .
runcct.pl controls most of cct 's daily work , such as downloading databases , running searches and updating tables .
this script can also be called manually from the command line and can be manipulated to perform only certain steps of its process or run only specified searches .
cct is freely available to all and it will continue to be developed ( ) .
cct can be a very useful tool for scientists who study large sets of genes in today 's evolving genomic landscape . by empowering non - bioinformaticians to automate custom searches and examine current results at a glance
, cct allows a remote database submission in the evening to influence the next morning 's bench experiment .
the molecule and description columns contain information supplied by the user ; the other columns contain data generated by automated searches against user - specified databases . in these database columns ,
links are color - coded to reflect the length of time since the search result last changed . to generate the table shown ,
cct was given a set of 48 genes in the mycobacterium tuberculosis ( mtb ) dormancy regulon ( 10 ) and directed to monitor four remote databases ( pfam , the mtb proteome and two incomplete genomes , m.smegmatis and m.marinum ) .
the pattern of red indicates that a new release of the m.marinum genome was downloaded in the past 24 h ( generating many new red tblastn links ) , and that a new sequence ( seq program ) of an ortholog ( homolog program ) was found for the tb protein rv1812c .
the predicted ortholog for rv0079 , in contrast , did not change as a result of the update and the corresponding links remain blue .
more detail concerning any result can be viewed by clicking on the corresponding link(s ) .
example of a result file revised to reflect a new release of the pfam database .
the regions above and below the horizontal line show different parts of the same result file .
this hmmpfam search result shows that the conserved hypothetical protein rv2030c matches a new domain in pfam , specifically the
phosphoribosyl domain has been changed subtly ( note the changed amino acids in the subject line ) . taken together with the change in database size for the new release ,
the e - value for this search has changed somewhat . to view the newest results only ( without highlighting to show changes ) | the current comparative table ( cct ) software program enables working biologists to automate customized bioinformatics searches , typically of remote sequence or hmm ( hidden markov model ) databases .
cct currently supports blast , hmmpfam and other programs useful for gene and ortholog identification .
the software is web based , has a bioperl core and can be used remotely via a browser or locally on mac os x or linux machines .
cct is particularly useful to scientists who study large sets of molecules in today 's evolving information landscape because it color - codes all result files by age and highlights even tiny changes in sequence or annotation . by empowering non - bioinformaticians to automate custom searches and examine current results in context at a glance
, cct allows a remote database submission in the evening to influence the next morning 's bench experiment .
a demonstration of cct is available at and the open source software is freely available from . | INTRODUCTION
MATERIALS AND METHODS
USAGE
Figures and Tables |
PMC5088337 | the drosophila enhancer of rudimentary , e(r ) , gene encodes enhancer of rudimentary homolog ( erh ) , a small protein of 104 amino acids [ 1 , 2 ] .
erh is a highly conserved protein that has been found in plants , animals , and protists .
drosophila erh is 76% identical to the vertebrate erh , 49% identical to the c. elegans erh , and 40% identical to the plant ( arabidopsis ) erh .
the vertebrate erh homologues are very highly conserved . the human , mouse , and xenopus proteins are identical and differ from the zebrafish erh by a single conservative amino - acid change .
the mouse and human proteins have been synthesized and purified from e. coli and the crystal structures determined [ 3 , 4 ] .
the protein contains three -helices and a -sheet that fold into a novel three - dimensional structure that comprises a single domain .
human erh exists as a dimer and the dimerization occurs via binding between the -sheets of the two monomers .
the binding is accomplished through primarily seven highly conserved amino acids ( figure 1 ) .
these include a transcription factor , dcoh / pcd in xenopus , a protein involved in the regulation of dna replication in humans , pdip46/skar , and ciz1 , a nuclear zinc - finger protein that may regulate dna synthesis and cyclin dependent kinases in humans [ 7 , 8 ] .
erh has been shown to coimmunoprecipitate with spt5 , a transcription elongation factor , and with fcp1 , a tfiif - associating component of ctd phosphatase .
erh has also been shown to interact with the spliceosome protein snrpd3 and to be required for the splicing of the mrna of cenp - e , a mitotic motor protein .
this diverse set of nuclear interactions suggests that erh may be involved in a number of general nuclear functions involving dna replication , transcription , and mrna splicing .
this prediction is supported by the nuclear localization of an egfp - erh fusion protein in transfected nih/3t3 cells .
further studies with antibodies to drosophila erh showed that the endogenous erh is localized to the nucleus in drosophila schneider cell lines and in cells of the early drosophila embryo .
searches for potential nuclear localization signals in drosophila and human erh by computer programs designed to identify nuclear localization signals ( predictprotein , wolf psort , and cnls mapper ) fail to identify any .
we define the sequence needed for the nuclear localization of erh and describe the identification of new potential nuclear binding partners of erh .
the amino - acid sequences of erh that are necessary for binding to its partners and for its nuclear localization are the same .
this suggests that erh is nuclearly localized through binding to these proteins or other binding partners .
for these procedures , all of the yeast strains , plasmids , and cdna prey libraries were obtained from the labs of dr .
the host saccharomyces cerevisiae strain egy191 is a his3 , ura3 , trp1 , and leu2 auxotroph carrying a chromosomal insertion of lexa binding sites upstream of the endogenous leu2 gene .
this strain also contained the plasmid prb1840 , which contains lexa dna - binding sites in front of a lacz reporter gene .
egy191/prb1840 was then transformed with the bait plasmid peg202 containing the appropriate lexa - e(r ) fusion gene to be used in the interaction - trap screens or the interaction assays .
the d. melanogaster e(r ) coding region was amplified from an e(r ) cdna via pcr using the primers 5 ggggatccccatgtcgcacaccatcct 3 and 5 ggctcgagttaggtattg gaactaa 3 ( table 1 ) , digested with bamhi and xhoi , and ligated into the polylinker of the parent bait plasmid peg202 as a bamh1-xho1 fragment .
the human e(r ) coding region was amplified via pcr using the primers 5 tggaattcatgtctcacaccattttg 3 and 5 ctcgagttatttcccagcctgtt 3 ( table 1 ) , digested with ecori and xhoi , and ligated into the polylinker of peg202 as an ecor1-xho1 fragment .
these procedures fuse the e(r ) genes in frame with the lexa dna - binding domain .
egy191/prb1840 was transformed with either the drosophila e(r ) as the bait , peg202-der , or the human e(r ) as the bait , peg202-her .
bait proteins expressed from peg202 will enter the yeast nucleus and bind lexa operators upstream of the two reporter genes leu2 and lacz but , alone , will fail to activate their transcription .
a drosophila 012-hour embryo cdna library was screened for genes encoding erh - interacting proteins .
the library was created in the plasmid pjg4 - 5 and contains 4 10 independent clones .
the prey genes in the library consist of a transcription activation domain fused to a random cdna .
if the coding region encodes an erh - binding protein , then it will bind to lexa - erh at the lexa promoters of the leu2 and lacz genes .
this will result in the activation of both genes resulting in a leu2 lacz colony .
another requirement of the screen is that the leu2 lacz phenotypes be galactose - dependent , since the prey genes are under the control of a gal1 promoter .
the egy191/prb1840/peg202-der strain was first transformed with the pjg4 - 5 cdna library , and a total of 102,000 transformants were isolated .
a total of 5213 colonies were isolated as putative galactose - dependent leu2 colonies . upon retesting ,
of the 172 colonies , 12 were galactose - dependent lacz . finally , the library plasmid from each of these colonies was isolated and retested for its dependence on the bait plasmid , peg202-der , for producing the galactose - dependent leu2 lacz phenotype .
eight of these clones proved to be bait - dependent and were saved for further analysis .
a hela cell cdna library was screened to isolate genes encoding human proteins that interact with human erh . a similar procedure to the drosophila screen
was performed except that peg202-her was used . a total of 73,000 individual colonies transformed with the hela cdna library .
from these five bait - dependent colonies were identified for further analysis . plasmid dna from the positive clones was isolated and used as the template for pcr .
two pjg4 - 5 specific primers 5 ccagcctcttgctgagtggagatg 3 and 5 gacaagccgacaaccttgattgga 3 were used .
these primers flank the ecori and xhoi sites in pjg4 - 5 and can be used to amplify the inserted cdna .
the amplified dna was sequenced and homology searches using blast ( https://blast.ncbi.nlm.nih.gov/blast.cgi ) were performed to identify the clones .
yeast expressing lexa - der and ha - rps3 was grown in 50 ml minimal galactose medium at 30 with shaking for 48 to 72 hours . to verify the expression of lexa - erh
the cells were pelleted by centrifugation in a microcentrifuge for 5 min at 12,000 rpm , the supernatant was removed , and the pellet was resuspended in 40 l laemmli loading buffer ( bio - rad laboratories , inc . ) with -me .
after resuspension , the sample was stored at 20 until being analyzed by sds - page .
the remaining culture was centrifuged at 3000 rpm for 15 min to pellet the cells .
the pellet was resuspended in 5 ml cip buffer ( 50 mm tris - hcl ph 7.5 , 100 mm nacl , and 5 mm edta 0.1% triton x-100 ) and protease inhibitor and cooled on ice
. a volume of glass beads ( sigma - aldrich ) equal to the volume of the cell pellet was added , and the sample was vortexed 5 times at 30 seconds bursts to shear the cells , with cooling on ice between vortexes .
cell debris was removed from the sample by centrifugation at 3,000 rpm for 5 minutes at 4. the protein containing supernatant was removed and kept on ice .
a rabbit polyclonal anti - ha antibody ( santa cruz biotechnology ) was added ( 1 : 500 ) to the sample and incubated on ice for 1 hour , with brief mixing in between
. 100 l of protein a beads ( pierce chemical co. ) was added and allowed to incubate for 1 hour on ice .
brief mixing of the sample was done throughout the 1-hour incubation to keep the beads in suspension .
the sample was centrifuged for 3 minutes at 1,500 rpm to pellet the beads and the supernatant was removed .
the final bead pellet was resuspended in 40 l laemmli loading buffer with -me and boiled for 1 minute . as a negative control ,
yeast expressing lexa and ha - rps3 was treated in parallel with the sample above .
also , yeast expressing ha and lexa - der was treated in the same manner to insure der was not sticking to the protein a beads .
40 l of each sample was loaded in the order : crude yeast extract expressing lexa / ha - rps3 , yeast extract expressing lexa / ha - rps3 treated with anti - ha , crude yeast extract expressing lexa - der / ha - rps3 , yeast extract expressing lexa - der / ha - rps3 treated with anti - ha , crude yeast extract expressing lexa - der / ha , yeast extract expressing lexa - der / ha treated with anti - ha , and marker .
the samples were run on a 10% polyacrylamide gel ( invitrogen nupage , np0341box ) in 1x invitrogen nupage mes sds running buffer ( np0002 ) and transferred to a nitrocellulose membrane ( pierce chemical co. ) using invitrogen nupage transfer buffer ( np0006 ) via electroblotting using a bio - rad trans - blot sd semi - dry transfer cell and an ec150 ( e - c apparatus corporation ) power source at 20v for one hour . after transfer , total protein was detected using a reversible membrane protein staining solution ( pierce chemical co. ) .
the membrane was blocked in blocking buffer ( 5% fat - free powdered milk in ttbs : 9.68 g tris buffer , 116.96 g nacl , 1760 l tween-20 in 4 l dh2o , ph to 7.5 ) for one hour before probing following standard western techniques .
the membrane was probed with a goat anti - lexa antibody ( 1 : 1000 ) in blocking buffer for one hour with gentle shaking .
the goat anti - lexa antibody was detected with a mouse anti - goat igg - hrp conjugated secondary antibody ( 1 : 5000 ) for one hour with gentle shaking .
the membrane was rinsed and the conjugate was detected using supersignal west femto substrate ( pierce chemical co. ) and the blot exposed on film . to map the regions of the human and drosophila erh that were necessary for interacting with their partners , sets of primers
were used to amplify different regions of the coding sequence for ligating to the lexa coding region in peg202 ( table 1 ) .
for the drosophila erh these primers were used to amplify coding sequences for amino acids 151 , 52104 , 124 , and 2551 .
for the human erh , coding sequences for amino acids 151 and 52104 were amplified , digested with ecori and xhoi , and ligated into peg202 .
the newly constructed baits were transformed into egy191/prb1840/pjg4 - 5-rps3 or egy191/prb1840/pjg4 - 5-rpl19 and tested for the galactose - dependent leu2 lacz phenotype .
gfp - erh constructs were generated by cloning the human and drosophila e(r ) fragments encoding amino acids 1104 , 151 , and 52104 and the drosophila erh fragments 124 and 2551 into the expression vector pegfp - c1 .
specific primer pairs ( table 1 ) were used to amplify the sequences , which were digested with bamhi and xhoi and ligated into pegfp - ci .
sk - hep cells were grown in emem plus 10% calf serum for two days to allow them to adhere to round microscope slide covers .
barry bode , department of biology , saint louis university ( current address department of biological sciences , northern illinois university ) .
prior to transfecting the cells , the transfection mix was prepared as follows : 1 g dna was added to 389 l eagle 's minimal essential medium , emem ( serum - free ) , and vortexed . to this mix
, 9 l of tfx-20 reagent ( promega corporation ) was added , and the mix was vortexed and allowed to incubate for 1015 min at room temp . after the incubation period , the growth medium was aspirated off the cells , and
the cells were returned to the 37 incubator for 2 hours . at the end of the incubation period , 300 l emem with 10% calf serum
the cells were returned to the incubator and allowed to grow for another two days before viewing with a fluorescent compound microscope with a gfp filter .
to identify possible binding partners for drosophila erh , a yeast two - hybrid screen was performed using drosophila lexa - erh as bait to screen a prey library consisting of drosophila embryo cdna clones fused to the transcription activation domain of gal4 . to construct the lexa - erh fusion gene ,
pcr was performed to amplify the e(r ) coding region with restriction sites for cloning into peg202 ( table 1 ) .
the prey plasmid from each positive clone was isolated and a partial sequence of the cdna insert was determined to identify the putative erh - binding partner .
five of the clones were of rps3 ( ribosomal protein s3 ) and three of the clones were of rpl19 ( ribosomal protein l19 ) .
another yeast two - hybrid screen was performed using the human erh as bait and a human hela cell cdna prey library .
three of the clones were human rps3 and two were human ddit4 ( dna damage inducible transcript 4 ) .
all of the putative binding partners of dmerh and hserh are known nuclear proteins , which is consistent with previous studies that localized erh to the nucleus [ 6 , 12 ] .
the fact that rps3 was identified as a binding partner of erh in both drosophila and humans suggests a conserved function of erh in both humans and drosophila . the yeast two - hybrid screen is a genetic test to show protein - protein interactions . to verify that drosophila erh and rps3 actually bind to each other ,
the first experiment was designed to show the requirement for the presence of rps3 for the precipitation of erh .
yeast cells were grown which expressed lexa - erh and either ha or ha - rps3 .
lexa - erh was not present in the immunoprecipitate from the cells expressing ha ( figure 2(a ) , lane 5 ) but was present in the immunoprecipitate from the cells expressing ha - rps3 ( figure 2(a ) , lane 3 ) .
controls showed that both cell types expressed lexa - erh ( figure 2(a ) , lanes 2 and 4 ) .
while these experiments showed that rps3 is necessary for the immunoprecipitation of lexa - erh , rps3 could conceivably bind to either lexa or erh . to show that rps3 was not binding to lexa
lexa did not coprecipitate , demonstrating the necessity for erh in the coimmunoprecipitation ( figure 2(b ) , lane 2 ) .
structural studies on purified human erh reveal that the protein likely forms a single domain with three -helices and one -sheet composed of four antiparallel -strands [ 3 , 4 ] .
the crystal structure also suggests that the -sheet serves as a surface for protein - protein binding .
given this structural information , it was of interest to determine the amino - acid sequences of erh that are necessary for its binding to its partners , rps3 and rpl19 .
the yeast two - hybrid interaction assay was used to map the erh sequences necessary for binding .
pcr was used to amplify specific regions of the erh coding region , which were then ligated into the lexa bait plasmid , peg202 ( table 1 ) . in this way ,
lexa - erh baits containing drosophila erh amino acids 151 , 52104 , 124 , and 2551 were generated and tested for their ability to interact with rps3 and rpl19 .
the results are summarized in table 2 . as a positive control , the full - length erh ( 1104 )
it also binds to the human rps3 , indicating that this protein - protein interaction is conserved .
amino acids 151 were shown to bind to rps3 and rpl19 , whereas amino acids 52104 did not .
amino acids 124 bind to rps3 and rpl19 , while amino acids 2551 do not .
the human erh was subdivided into amino acids 151 and 52104 and examined for their ability to bind to hsrps3 .
similar to the results with dmerh , amino acids 151 bind to hsrps3 , while amino acids 52104 do not ( table 3 ) .
previous studies on the immunolocalization of erh in drosophila s2 cells and early embryos demonstrated that in drosophila melanogaster endogenous erh is nuclearly localized .
those results corroborate studies in human cell lines in which egfp - hserh fusion proteins were localized to the nucleus .
these data argue that erh must have a nuclear localization signal ; however , scans of the drosophila and human erh amino - acid sequences using predictprotein ( http://predictprotein.org/ ) , wolf psort ( http://www.genscript.com/wolf-psort.html/ ) , and cnls mapper ( http://nls-mapper.iab.keio.ac.jp/cgi-bin/nls_mapper_form.cgi/ ) have not revealed any nuclear localization signals .
this leaves unanswered the questions as to how erh is nuclearly localized and what amino - acid sequences are necessary for nuclear localization .
we decided to utilize the gfp system to identify the sequences necessary for the nuclear localization of erh .
pcr was used to amplify all or part of the coding regions for ligation into the plasmid , pegfp - c1 , for the creation of gfp - dmerh and gfp - hserh fusions ( table 1 ) .
plasmids containing the fusion genes were transfected into human sk - hep cells and cellular localization was visualized with gfp .
the results are summarized in table 4 . for both erh and herh , the full - length protein localizes to the nucleus ( figures 3(a ) and 4(a ) ) .
the n - terminal half of each protein ( amino acids 151 ) can also direct nuclear localization ( figures 3(b ) and 4(b ) ) , while the c - terminal half of each protein ( amino acids 52104 ) does not ( figures 3(c ) and 4(c ) ) . for drosophila erh
, the n - terminal half was further subdivided into amino acids 124 and 2551 .
amino acids 124 direct the localization of gfp to the nucleus ( figure 3(d ) ) , although there appeared to be more cytoplasmic expression of gfp than what was seen when amino acids 151 ( figure 3(b ) ) or the entire protein ( figure 3(a ) ) was used .
these data localize the minimal sequence needed for nuclear localization to amino acids 124 . as a negative control , egfp by itself
the data argue that the first 24 amino acids are sufficient for the nuclear localization of erh .
there are two arginines within this region of drosophila erh , but nothing looks like a nuclear localization signal .
interestingly , this 24-amino - acid region is the same region that is necessary for erh to bind to its binding partners , rps3 and rpl19 ( table 2 ) .
the drosophila rps3 in particular has a strong nls starting at amino acid 6 ( piskkrk__fv ) .
the human rps3 has a similar nls starting at amino acid 4 ( qiskkrk__fv ) .
in addition , rps3 has been shown to localize to the nucleus in both drosophila melanogaster and humans .
together these data suggest that erh may be entering the nucleus through piggy backing onto one of its binding partners , such as rps3 .
the n - terminal 24-amino - acid region , which is necessary for both the nuclear localization of erh and its binding to rps3 and rpl19 , is comprised of two antiparallel -strands that are part of a four - strand -sheet . this -sheet serves as a hydrophobic surface in protein binding [ 3 , 4 ] .
five out of the seven residues within the -sheet , which have been identified as important in the dimerization of erh , are contained within the first two -strands .
these residues are conserved between human and drosophila erh ( figure 1 ) , suggesting their importance in protein binding in both species , and may explain how drosophila erh can bind to both drosophila and human rps3 . our current results also indicate that while the four strands of the -sheet form a face for protein binding , the first two -strands are necessary and sufficient for protein binding and nuclear localization .
although amino acids 124 are sufficient for nuclear localization , this sequence appears not to localize gfp to the nucleus as strongly as do amino acids 152 and 1104 ( figure 3 ) .
these additional amino acids ( 25104 ) are not sufficient for nuclear localization , but they may help stabilize the structure of amino acids 124 for optimal protein binding .
amino acids 2552 comprise a major -helix that is positioned behind the two -strands of amino acids 124 .
amino acids 6682 comprise the last two -strands that , along with amino acids 124 , form the -sheet that is necessary for the binding of erh to its partners .
these -strands , while helping to stabilize the -sheet , also contain two amino acids that have been shown to be contact points in protein - protein binding of erh .
these amino acids should strengthen the interactions and thus strengthen the nuclear localization of erh .
the present studies identified three new putative binding partners for erh , rps3 , and rpl19 in drosophila and rps3 and ddit4 in humans .
rps3 , besides being a ribosomal protein , is a dna repair enzyme [ 16 , 18 ] and ddit4 is a protein whose expression is induced in response to dna damage .
also , human rps3 has been shown to translocate from the cytoplasm to the nucleus in response to genotoxic stress treatment with hydrogen peroxide or dna alkylating agents .
these findings suggest the possibility that erh has a role in dna damage repair and that this function can be regulated by regulating its nuclear localization through its binding to rps3
both are 104 amino acids in length and can be lined up without gaps along their entire lengths .
the tertiary structure or the drosophila erh , predicted by swiss - model , is nearly identical to the solved tertiary structure of the human / mouse erh [ 3 , 4 ] . for the human / mouse erh ,
seven amino acids were identified as critical in protein binding ( 5i , 7l , 17r , 19y , 21d , 70l , and 79y ) .
the one difference is a conservation amino acid substitution at residue 70 ( m for l ) .
the first five of these amino acids are contained within the n - terminal 24 amino acids that this study has shown to be important for both binding of erh to rps3 and rpl19 and the nuclear localization of erh .
this high structural conservation suggests that the human and drosophila erh may be functionally equivalent .
the drosophila erh coding region was replaced with the human erh coding region in the drosophila genome without any noticeable effect on the fly .
all of the known mutant phenotypes associated with the deletion of the drosophila e(r ) gene were rescued by the human e(r ) gene , indicating that the human erh can functionally replace the drosophila erh .
the evolutionary conservation in the structure and function of the human and drosophila erh suggest that its roles in the cell may also be conserved between humans and drosophila .
given this possibility , the fact that the present study identified rps3 as a possible binding partner for both the human and drosophila erh is intriguing .
they share an 83% amino acid identity and an 89% amino acid similarity and both have been shown to be dna repair proteins [ 16 , 18 ] .
it may be that the roles and mechanisms of erh and rps3 in growth and dna repair are conserved between humans and drosophila .
rpl19 and rps3 are both upregulated in high growth situations , specifically in certain cancer cells [ 2427 ] .
rps3 has also been shown to be a transcription factor . as a subunit of nf-b , it is responsible for the activation of specific nf-b target genes .
these nuclear functions , along with previously identified nuclear functions , implicate erh in transcription initiation , transcription elongation , mrna splicing , dna synthesis , and dna repair .
erh is a very small protein and to date the only biochemical function that has been attributed to it is protein binding .
it may be that erh is modulating the activity of a number of different nuclear complexes purely through binding to specific subunits of the complexes .
yeast two - hybrid interaction trap assays identified rps3 , rpl19 , and ddit4 as binding partners of erh .
similarities in the function of these proteins suggest a role of erh in dna repair and cell growth .
amino acids 124 of erh were shown to be sufficient and necessary for the binding to rps3 and rpl19 .
the role of this region in these two properties of erh suggests a model for its nuclear localization .
erh , which appears not to have an nls , may be localizing to the nucleus via binding to one of its partners which has an nls . | the protein enhancer of rudimentary homolog , erh , is a small , highly conserved protein that has been found in animals , plants , and protists .
genetic and biochemical interactions have implicated erh in the regulation of pyrimidine biosynthesis , dna replication , transcription , mrna splicing , cellular proliferation , tumorigenesis , and the notch signaling pathway . in vertebrates and insects , erh is nuclearly localized ; however , an examination of the erh amino - acid sequence does not reveal any nuclear localization signals . in this paper
we show that the first 24 amino acids contain sequences necessary and sufficient for nuclear localization . through yeast two - hybrid screens , three new binding partners of erh , rps3 , rpl19 , and ddit4 ,
were identified .
rps3 was isolated from both human and drosophila screens .
these interactions suggest functions of erh in cell growth , cancer , and dna repair .
the erh sequences necessary for the interactions between erh and rps3 and rpl19 are mapped onto the same 24-amino - acid region in erh which are necessary for nuclear localization , suggesting that erh is localizing to the nucleus through binding to one of its dna - binding partners , such as rps3 or rpl19 . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions |
PMC4837798 | the potential benefits for clinical pathology of adopting well - designed digital technologies have been solidly established .
essentially , there are strong arguments pointing to improved patient care and more efficient care pathways in the digital era . the reality , however , often is that the path toward realizing this potential is not straightforward .
the clinical adoption is multi - faceted and for each domain , customized solutions may be required .
there are many efforts around the world aiming to pave the way for digital pathology into routine clinical use , and a particular concentration of development work is happening in the nordic countries ( sweden , denmark , norway , finland , and iceland ) .
for example , full histology scanning and extensive digital primary review are a reality since several years in the hospitals of linkping and kalmar .
recently , gvle hospital started a digital review for parts of the clinical routine , and concrete implementation plans are being carried out in many more institutions .
the nordic symposium on digital pathology ( ndp ) was created in 2013 to promote global exchange of state - of - the - art knowledge , taking advantage of this nordic digital greenhouse .
the specific focus of ndp is advancing toward the clinical adoption of whole - slide imaging ( wsi ) and other digital technologies in pathology .
in particular , a founding principle is the insight that effective progress requires strong collaboration among health care , industry , and academia , and ndp is designed to be a forum where professionals from all these domains can meet .
this third installment of ndp marks a continued evolution from the two previous events in 2013 and 2014 , in terms of participant numbers ( 190 compared to 125 and 144 , respectively ) and depth of issues discussed .
below we provide a summary of ndp 2015 , in the form of a meeting overview , results from the symposium 's validation workshop , speaker contributions , and finally brief conclusions .
a total of 190 attendees gathered , of which 43% listed health care as the primary affiliation , 40% industry , and 16% academia .
the health care representatives were dominated by pathologists , but also managers , laboratory technologists , and it staff were in significant numbers .
the participants represented 15 different countries with the nordic attendees making up the majority ( 82% ) , but less so than in the 2014 symposium ( 87% ) . invited talks
constituted the backbone of the program , together with a collaborative workshop on validation aspects of digital pathology .
the contents of these sessions will be outlined in the sections below . in the science and innovation session ,
this resulted in three jpi papers published alongside this editorial : consultation of urological specimens using real - time digital microscopy : optimizing the workflow for referred cancer patients ( henrik holten - rossing et al . ) , improving the creation and reporting of structured findings during digital pathology review
( ida cervin et al . ) , and feature - based analysis of mouse prostatic intraepithelial neoplasia in histological tissue sections ( pekka ruusuvuori et al . ) .
in addition , the ndp included an industrial exhibition consisting of 13 vendors , showing a wide range of products and services related to digital pathology operations .
figure 1 shows a snapshot from the symposium , and the program details are available at the ndp website www.ndp2015.se . the nordic symposium on digital pathology 2015 audience gathered for the keynote presentation of prof paul j. van diest a key part of the ndp program was the workshop discussing validation of digital pathology .
the workshop was organized as an open floor discussion with broad participation from the attendees . as an introduction
, a systematic review of previous digital pathology validation research was presented by bethany williams , leeds university . moreover ,
a survey among the health care attendees themselves , distributed in advance of the symposium , served as fuel for debate .
it is important to acknowledge that the survey respondents represent a biased selection among the pathology community .
since only ndp participants were asked , this means that respondents are likely to be among the most positive to digital pathology and also among the most experienced .
there is also a strong geographical dominance from the nordics and , in particular , sweden . of 83
it is likely that the pathologist dominance was even higher for some questions that require deep knowledge of clinical practice .
role distribution of survey respondents the survey first asked : today , to what degree do you use digital images of histology slides in your practice ?
the results are shown in figure 3 , showing moderate levels of adoption but no use in about 50% of respondents .
( another bias to note for these questions is that several people from the same institution may have responded ) .
current use of digital pathology among survey respondents the same question was asked for the predicted situation at the end of 2016 . as the 2016 prediction was also asked in the ndp2014 survey ,
as in the previous year , a vast majority expects to soon do digital to some degree , but the difference in predicted adoption rate indicates that the plans for digitization are progressing slightly slower than anticipated .
this development pace moderation is in line with reports from the symposium attendees that both funding allotment and procurement processes are causing delays in their digitization efforts . predicted use of digital pathology for primary review at the end of 2016 .
right : prediction from nordic symposium on digital pathology 2015 survey validation of digital pathology means controlling potential risks of the digitization step , i.e. , scanning and reproduction on a computer screen . in the college of american pathologists ( cap ) guidelines ,
wsi validation is defined as a process that aims to demonstrate that the new method performs as expected for its intended use and environment prior to its application for patient care .
an important aspect is to decide on an acceptable level of risk . while it is attractive to adopt zero tolerance for errors due to digitization , this may lead to an unbalanced distribution of efforts .
if the risks in other parts of the diagnostic chain are much larger than the ones in the digitization step , the mitigation and control efforts in those areas should come first .
therefore , to understand the perceived relative risk of digitization , the survey asked : compared with other parts of the sample preparation and diagnostic process , how big do you think the added safety risk of the digitization step is ? the results , presented in figure 5 , show that health care ndp attendees clearly consider the added risk from digitization to be smaller than risks in the diagnostic review , staining , sectioning , and grossing steps .
perceived added safety risk from digitization ( scanning and image reproduction on screen ) relative to risks stemming from other parts of the diagnostic pipeline .
overall , the results point to lower perceived risk , i.e. , that digitization is perceived as less error - prone there is an ongoing debate in the community regarding what type or scale of validation that is needed before adoption of digital pathology in diagnosis . to elicit the overall opinion ,
the survey asked : what 's your personal view on the need for validation efforts to increase the confidence for digital pathology in clinical practice ? for this group having substantial experience of clinical use of wsi , the dominant view ( 68% ) is that current knowledge is sufficient for going ahead with digital pathology adoption and that further validation is characterized as a continuous improvement effort .
this may again reflect the high proportion of early adopters in the audience . as seen in figure 6 ,
the majority sees further validation as a part of continued progress , but not as prohibitive for adoption in general validation can be performed , documented , and disseminated in many ways and by several types of stakeholders . as the decision to adopt digital pathology often boils down to the conviction of individuals
, it is interesting to know which sources of safety assurance that have the highest credibility .
the survey asked : assurance that digital pathology is safe for clinical use can come from many sources . for your own decision ,
if digital pathology is safe , how much confidence would you get from the following sources ? the results in figure 7 show that personal experience along with high - quality scientific trials is associated with the highest confidence , whereas ce marking and food and drug administration approval of systems are less assuring , relatively .
personal experience and high - quality trials are considered the most trustworthy sources after the survey results were presented , a guided discussion among all participants took place .
overall , the points of view shared demonstrated the many facets of validation to consider .
a fundamental prerequisite is that digital pathology does introduce new process steps that need to be scrutinized : the image digitization and the visualization ( display and interaction ) in a digital workstation .
there are now many concordance studies showing reassuring overall results . at the same time , potential concerns are that there are few well - designed large studies to date and that despite high overall concordance with light microscopy , this may conceal the possibility that for some case types , digitization might introduce problems not existing in microscope glass slide review . in line with the first statement of the cap guidelines , ndp attendees expressed the need for validation activities at each individual laboratory implementing wsi . in this preliminary stage , no clear consensus on scope and depth of such activities emerged from the discussion , but ongoing initiatives in sweden and the united kingdom are directed toward developing concrete support for validation work .
another aspect voiced was that even if agreeing that digital pathology adds a slight risk , this should be compared to the risk of nonadoption .
for example , an increased risk may be acceptable if the benefits of increased reproducibility or easier consultations across the healthcare enterprise clearly outweigh any additional risk .
a positive effect of the validation effort of digital pathology can also be that components of current practice , actually having evolved with a low level of scrutiny , are revisited with a critical eye .
the conclusion from the discussion is that there is a broad consensus in this group that continued efforts to scrutinize digital pathology are needed , whereas the views differ as to what degree this affects the pace of adoption .
paul j. van diest , professor and head of pathology at the university medical center utrecht , the netherlands , presented their digital pathology program , including both past efforts and future plans .
the experience includes many years of scanning all slides , working integration with their report system , and validation efforts with positive results .
currently , utrecht is implementing new systems and workflows to take the step to digital primary review , and prof .
van diest emphasized the need for establishing a solid and continuous change management process in the department , in contrast to a one - off implementation activity .
nasir rajpoot , associate professor at qatar university , qatar , and university of warwick , uk , gave a comprehensive overview of opportunities and challenges in image analysis for pathology .
he presented a wide range of methods developed in his research groups constituting a toolbox for detection and classification on several levels : nucleus , cell , gland , and entire tissue .
rajpoot also stressed the benefits of an open exchange of methods and data for the scientific community to effectively make advances in the field .
petra lindstedt , former head of the diagnostic division at region gvleborg , sweden , spoke to the benefits that had been the main motivation for their decision to go fully digital , a process now underway with several pathologists doing their main primary review on digital slides .
a benefit particular to smaller , rural hospitals is that a digital environment opens new possibilities for specialist careers based on a consultation volume from other providers .
johnny eriksson , cto at telemedicine clinic , spain , and cto radiology it at the university hospital rebro county , sweden , described the opportunities stemming from enterprise - wide digital image management .
the experience from radiology , including services such as running off - hour diagnostic routine for european hospitals from australia , demonstrates the potential but also the need to assiduously address the technical challenges involved .
anna bodn , pathologist and digitization project manager at linkping university hospital , sweden , presented the experiences from implementing a second generation of large - scale digital pathology in their clinic .
issues given increased consideration this time around included achieving highly stable and fast viewing , running scanners from multiple vendors in parallel , careful selection of monitors , and support for large slides .
manuel salto - tellez , professor and molecular pathologist at queen 's university belfast , uk , provided a compelling vision of improved health care , thanks to advances within reach for joint efforts in pathology and genomics .
several examples of such efforts from his research group were presented , including testing for braf , kras , and egfr mutations in cancer .
salto - tellez strongly emphasized the need for effectively combining the two disciplines of molecular and digital pathology , since the genomic findings require a context of phenotype and morphometry .
andr homeyer , scientist at the fraunhofer mevis research institute in bremen , germany , presented their group 's software framework for image analysis in pathology .
key objectives are to exploit image features across several scales and to meet high interactivity requirements by carefully designed feature extraction in the preprocessing stage .
simon hger of sectra , linkping , sweden , talked about how to construct a business case for digital pathology .
yee - wah tsang from coventry university , uk , presented the large validation study recently performed in coventry .
visiopharm arranged a talk from henrik holten - rossing from rigshospitalet copenhagen , denmark , presenting studies and experiences of advanced quantitative image analysis .
finally , a special session on education and collegial exchange was held , led by dr .
mats wolving , head of quality development at the swedish society of pathology and pathologist at sahlgrenska university hospital , gothenburg , sweden , and martin svenson of sectra .
they presented ongoing innovation efforts to develop new tools and workflows in this domain , and the audience contributed in an enlightening discussion regarding possibilities and obstacles with regards to this area of clinical practice .
a key part of the ndp program was the workshop discussing validation of digital pathology .
the workshop was organized as an open floor discussion with broad participation from the attendees . as an introduction
, a systematic review of previous digital pathology validation research was presented by bethany williams , leeds university . moreover ,
a survey among the health care attendees themselves , distributed in advance of the symposium , served as fuel for debate .
it is important to acknowledge that the survey respondents represent a biased selection among the pathology community .
since only ndp participants were asked , this means that respondents are likely to be among the most positive to digital pathology and also among the most experienced .
there is also a strong geographical dominance from the nordics and , in particular , sweden .
it is likely that the pathologist dominance was even higher for some questions that require deep knowledge of clinical practice .
role distribution of survey respondents the survey first asked : today , to what degree do you use digital images of histology slides in your practice ? ( in percentage of all histology cases ) .
the results are shown in figure 3 , showing moderate levels of adoption but no use in about 50% of respondents .
( another bias to note for these questions is that several people from the same institution may have responded ) .
current use of digital pathology among survey respondents the same question was asked for the predicted situation at the end of 2016 . as the 2016 prediction was also asked in the ndp2014 survey ,
, a vast majority expects to soon do digital to some degree , but the difference in predicted adoption rate indicates that the plans for digitization are progressing slightly slower than anticipated .
this development pace moderation is in line with reports from the symposium attendees that both funding allotment and procurement processes are causing delays in their digitization efforts . predicted use of digital pathology for primary review at the end of 2016 .
right : prediction from nordic symposium on digital pathology 2015 survey validation of digital pathology means controlling potential risks of the digitization step , i.e. , scanning and reproduction on a computer screen . in the college of american pathologists ( cap ) guidelines ,
wsi validation is defined as a process that aims to demonstrate that the new method performs as expected for its intended use and environment prior to its application for patient care .
while it is attractive to adopt zero tolerance for errors due to digitization , this may lead to an unbalanced distribution of efforts .
if the risks in other parts of the diagnostic chain are much larger than the ones in the digitization step , the mitigation and control efforts in those areas should come first .
therefore , to understand the perceived relative risk of digitization , the survey asked : compared with other parts of the sample preparation and diagnostic process , how big do you think the added safety risk of the digitization step is ? the results , presented in figure 5 , show that health care ndp attendees clearly consider the added risk from digitization to be smaller than risks in the diagnostic review , staining , sectioning , and grossing steps .
perceived added safety risk from digitization ( scanning and image reproduction on screen ) relative to risks stemming from other parts of the diagnostic pipeline .
overall , the results point to lower perceived risk , i.e. , that digitization is perceived as less error - prone there is an ongoing debate in the community regarding what type or scale of validation that is needed before adoption of digital pathology in diagnosis . to elicit the overall opinion ,
what 's your personal view on the need for validation efforts to increase the confidence for digital pathology in clinical practice ? for this group having substantial experience of clinical use of wsi , the dominant view ( 68% ) is that current knowledge is sufficient for going ahead with digital pathology adoption and that further validation is characterized as a continuous improvement effort .
as seen in figure 6 , about 20% are more hesitant and require further validation as a condition for adoption .
the majority sees further validation as a part of continued progress , but not as prohibitive for adoption in general validation can be performed , documented , and disseminated in many ways and by several types of stakeholders . as the decision to adopt digital pathology often boils down to the conviction of individuals
, it is interesting to know which sources of safety assurance that have the highest credibility .
the survey asked : assurance that digital pathology is safe for clinical use can come from many sources . for your own decision ,
if digital pathology is safe , how much confidence would you get from the following sources ?
the results in figure 7 show that personal experience along with high - quality scientific trials is associated with the highest confidence , whereas ce marking and food and drug administration approval of systems are less assuring , relatively .
personal experience and high - quality trials are considered the most trustworthy sources after the survey results were presented , a guided discussion among all participants took place .
overall , the points of view shared demonstrated the many facets of validation to consider .
a fundamental prerequisite is that digital pathology does introduce new process steps that need to be scrutinized : the image digitization and the visualization ( display and interaction ) in a digital workstation .
there are now many concordance studies showing reassuring overall results . at the same time , potential concerns are that there are few well - designed large studies to date and that despite high overall concordance with light microscopy , this may conceal the possibility that for some case types , digitization might introduce problems not existing in microscope glass slide review . in line with the first statement of the cap guidelines , ndp attendees expressed the need for validation activities at each individual laboratory implementing wsi . in this preliminary stage
, no clear consensus on scope and depth of such activities emerged from the discussion , but ongoing initiatives in sweden and the united kingdom are directed toward developing concrete support for validation work .
another aspect voiced was that even if agreeing that digital pathology adds a slight risk , this should be compared to the risk of nonadoption .
for example , an increased risk may be acceptable if the benefits of increased reproducibility or easier consultations across the healthcare enterprise clearly outweigh any additional risk .
a positive effect of the validation effort of digital pathology can also be that components of current practice , actually having evolved with a low level of scrutiny , are revisited with a critical eye .
the conclusion from the discussion is that there is a broad consensus in this group that continued efforts to scrutinize digital pathology are needed , whereas the views differ as to what degree this affects the pace of adoption .
paul j. van diest , professor and head of pathology at the university medical center utrecht , the netherlands , presented their digital pathology program , including both past efforts and future plans .
the experience includes many years of scanning all slides , working integration with their report system , and validation efforts with positive results .
currently , utrecht is implementing new systems and workflows to take the step to digital primary review , and prof .
van diest emphasized the need for establishing a solid and continuous change management process in the department , in contrast to a one - off implementation activity .
nasir rajpoot , associate professor at qatar university , qatar , and university of warwick , uk , gave a comprehensive overview of opportunities and challenges in image analysis for pathology .
he presented a wide range of methods developed in his research groups constituting a toolbox for detection and classification on several levels : nucleus , cell , gland , and entire tissue .
dr . rajpoot also stressed the benefits of an open exchange of methods and data for the scientific community to effectively make advances in the field .
petra lindstedt , former head of the diagnostic division at region gvleborg , sweden , spoke to the benefits that had been the main motivation for their decision to go fully digital , a process now underway with several pathologists doing their main primary review on digital slides .
a benefit particular to smaller , rural hospitals is that a digital environment opens new possibilities for specialist careers based on a consultation volume from other providers .
johnny eriksson , cto at telemedicine clinic , spain , and cto radiology it at the university hospital rebro county , sweden , described the opportunities stemming from enterprise - wide digital image management .
the experience from radiology , including services such as running off - hour diagnostic routine for european hospitals from australia , demonstrates the potential but also the need to assiduously address the technical challenges involved .
anna bodn , pathologist and digitization project manager at linkping university hospital , sweden , presented the experiences from implementing a second generation of large - scale digital pathology in their clinic .
issues given increased consideration this time around included achieving highly stable and fast viewing , running scanners from multiple vendors in parallel , careful selection of monitors , and support for large slides .
manuel salto - tellez , professor and molecular pathologist at queen 's university belfast , uk , provided a compelling vision of improved health care , thanks to advances within reach for joint efforts in pathology and genomics .
several examples of such efforts from his research group were presented , including testing for braf , kras , and egfr mutations in cancer .
salto - tellez strongly emphasized the need for effectively combining the two disciplines of molecular and digital pathology , since the genomic findings require a context of phenotype and morphometry .
andr homeyer , scientist at the fraunhofer mevis research institute in bremen , germany , presented their group 's software framework for image analysis in pathology .
key objectives are to exploit image features across several scales and to meet high interactivity requirements by carefully designed feature extraction in the preprocessing stage .
simon hger of sectra , linkping , sweden , talked about how to construct a business case for digital pathology .
yee - wah tsang from coventry university , uk , presented the large validation study recently performed in coventry .
visiopharm arranged a talk from henrik holten - rossing from rigshospitalet copenhagen , denmark , presenting studies and experiences of advanced quantitative image analysis .
finally , a special session on education and collegial exchange was held , led by dr .
mats wolving , head of quality development at the swedish society of pathology and pathologist at sahlgrenska university hospital , gothenburg , sweden , and martin svenson of sectra .
they presented ongoing innovation efforts to develop new tools and workflows in this domain , and the audience contributed in an enlightening discussion regarding possibilities and obstacles with regards to this area of clinical practice .
the need for , and interest in , knowledge exchange with regards to the clinical routine use of wsi and related it tools continues to be strong , as demonstrated by the increasing attendance at this third installment of the ndp . as digital pathology evolves and initial hurdles are managed ,
the symposium touched on many topics of digital pathology , some of which are growing in importance with increased digital maturity .
the feedback from the attendees is that this type of experience sharing across organizations , disciplines , and sectors is important for the progress in the field . as organizers , we are happy to conclude that ndp 2015 has made a substantial contribution to this end . finally , we gladly note the increasing ratio of non - nordic attendees and encourage colleagues from all over the world to come and benefit from future ndp symposia .
darren treanor is a member of the aperio / leica advisory board and sectra advisory board .
darren treanor is a member of the aperio / leica advisory board and sectra advisory board . | cross - disciplinary and cross - sectorial collaboration is a key success factor for turning the promise of digital pathology into actual clinical benefits .
the nordic symposium on digital pathology ( ndp ) was created to promote knowledge exchange in this area , among stakeholders in health care , industry , and academia .
this article is a summary of the third ndp symposium in linkping , sweden .
the nordic experiences , including several hospitals using whole - slide imaging for substantial parts of their primary reviews , formed a fertile base for discussions among the 190 ndp attendees originating from 15 different countries .
this summary also contains results from a survey on adoption and validation aspects of clinical digital pathology use . | INTRODUCTION
MEETING OVERVIEW
Workshop on Validation of Digital Pathology
Speaker Contributions
CONCLUSIONS
Financial Support and Sponsorship
Conflicts of Interest |
PMC3592597 | both the med associates and the coulbourn instruments systems can monitor multiple inputs and control multiple outputs . both can run multiple experiments concurrently with ease .
both require a windows - based system and really should be run on a dedicated computer that is not burdened by being connected to a network .
both offer a runtime screen that provides information about the status of the current experiment .
both systems are designed to be used with proprietary interfacing equipment available from the manufacturer .
the reader is urged to consult the manufacturers web sites ( www.coulinst.com and www.med-associates.com ) for detailed descriptions of the products .
i should note that i have received timely and helpful assistance from both companies when it was needed .
the user creates a text file consisting of statements written in medstate notation ( easily learned by a pascal programmer , challenging for the user with no programming experience ) .
this can be done using the editor provided with the system or with any editor or word processor capable of producing plain - text output .
the program can contain as many as 32 distinct state sets , each acting as an independent mini - program .
prior to use the program must be compiled , a process that will reveal coding errors that must be corrected before compilation can succeed .
once a program is successfully compiled it is available to be run from a pull - down menu within a separate med associates program .
medstate notation allows data to be stored in 26 variables ( labelled a to z ) , each of which can take the form of either a single variable or a 1-dimensional array .
these variables can store any data that the user specifies when the program is written .
for example , one might contain the total number of times that a lever was pressed , another might contain a list of inter - response latencies , and a third might indicate the number of correct responses .
although limiting the total number of variables to 26 might seem constraining , i have yet to find this too restrictive , especially because arrays are allowed .
the data are written to disk as a plain text file ; several formats are available for the data file , and the one selected will probably be determined by whether or not the file will be imported into a spreadsheet .
the programming window offers a picture of the front panel of the hardware that interfaces with the behavioral equipment . by pointing and clicking within this window
the user can can specify the stimuli to be turned on during each state , the responses or time events that cause the state to terminate , and the target state(s ) to be activated upon termination of the state .
for example , to turn on the houselight for 10 s , the programmer would click on the houselight button to toggle it on , and specify that the state should end after 10 s. the program must be written via the graphic state 2 programming interface , and the program is saved in a proprietary format that requires it to be viewed via graphic state 2 . because of this it is impossible to print the program for archival purposes .
once the program is constructed , gs2 performs a check to ensure that it contains no states that are not called by at least one other state or that fail to call another state a check that helps to ensure that the program is at least likely to terminate . because the user writes no code , errors in programming syntax are far less likely than in med associates system .
graphic state 2 consists of a single state set ; that is , only a single state can be active at any given time , as compared to medstate notation s 32 concurrent states . usually counters and timers are reset when a new state is entered , but the program allows them to retain their values across states or throughout an entire session .
graphic state 2 logs all events while a session is being run , even if they have no effect on the program .
if a state is ended by a response on lever 1 , for example , the program will also log responses on lever 2 even if they do nothing .
the data file that is created contains a record of every response or state change that occurred during the session , with a record of the time during the session when the event occurred and an indication of the cause of each state change .
instead a record of the entire session is available at its conclusion for analysis by graphic state 2 s extensive library of analysis routines or for exporting as a text file .
without question , the easier of the two systems to learn to program is graphic state 2 . the point - and - click interface obviates the need to learn seemingly obscure programming language , and the fact that the program records everything that occurs during a session means that the user does not have to program variables to track relevant information .
indeed , a student with no programming experience watched me write a simple program one morning , and by the afternoon had mastered graphic state 2 well enough to write a program to control a two - lever autoshaping procedure . in comparison ,
it is true that the commands are reasonably intuitive ( e.g. , on turns on a stimulus ) , but they must be learned and they must be entered correctly . furthermore , the user must specify and track all variables of interest ; the program will automatically record the values of variables a through z at the end of the session , but these values will be 0 unless the program contains code to update them ( e.g. , count responses in variable r , record number of trials in variable t , etc . ) .
the availability of only one state set within a graphic state 2 program makes some tasks either very difficult or perhaps impossible to program , especially those tasks that involve independent timing of various stimuli .
for example , consider the truly random control condition in a pavlovian conditioning study ( rescorla , 1967 ) .
the investigator wants a tone to turn on for 5 s and a feeder to operate for 0.1 s ; the two should occur perhaps 20 times each during a 30-min session , but totally randomly with respect to each other . in medstate notation
each could be controlled by its own state set , without any reference to or input from the other ( as if each stimulus was controlled by its own 1960s - era tape timer ) . in gs2 , because a stimulus can be turned on only by entry into a state , and because only a single state set is available , the two stimuli must of necessity be controlled together , and a complex program must be established consisting of states in which none is on , one is on , or both are on ; the durations of these states will be varied and complicated .
i think that the only way to implement this program in gs2 would be to create a fixed schedule of the two stimuli that simulates the random occurrence of both , then to program this fixed schedule .
a program in which the two stimuli occur truly randomly with respect to each other might well be impossible in gs2 .
gs2 creates a log of a session that contains every transition from one state to another ( because stimuli are tied to states this corresponds to a record of stimulus presentations ) and every response onset and offset ( whether the responses cause state transitions or not ) .
the result is a complete record of the session , available for export as a text file or for analysis within gs2 itself .
because the record of the session contains all responses that were available to be monitored the researcher can ask questions after the fact that were not anticipated at the time the session was run .
a large number of routines are provided by gs2 that allow extensive analysis of the data , including frequency of entry into each state , number of responses within each state , inter - response intervals , etc .
unfortunately the explanations of these routines are far from intuitive , and the routines seem geared toward free - operant schedules .
i run trial - based studies , often in runways or mazes , and the analyses have never been useful to me except as quick checks at the end of a session to ensure that data were recorded .
i have relied on the exported raw data text files , which i then filter extensively and analyze with a spreadsheet program ( e.g. , excel or gnumeric ) .
medstate notation s approach to data is far different : only the variables specified by the programmer are recorded ; there will be no record of the number of responses if the program does not contain a counter that is incremented with each occurrence of the response .
medstate notation allows data to be saved in text files in a limited number of formats , some human friendly and some ( supposedly ) spreadsheet friendly .
the text - files must be manipulated extensively with a spreadsheet before useful analysis can occur .
med associates offer an additional program called mpc2xl to ease translation from their data files to a spreadsheet ; i have no experience with it .
although both medstate notation and gs2 data files could be made far more user - friendly in terms of how data are formatted for subsequent use by spreadsheet programs , i find medstate notation s files easier to use .
they are inherently smaller , because they contain only those data that i have requested .
because i can specify counters , timers , and other variables in the program , the data file contains the information that i want with very little further analysis , for example , some of my behavioral routines allow rats to control the onset of their own pavlovian trials , and the number of trials initiated is a critical dependent variable .
a variable within a medstate notation program can record this number as the session runs , display it on the screen , and save it in the data file . in gs2
this variable would correspond to the number of times a particular state is entered ; there is no way to display this , but instead an analysis must be run on the data at the conclusion of the session . the ability to create user - defined variables that track meaningful information , and to display these variables as they are updated during the session , make a medstate notation session far more informative than a session run in gs2 .
a well - designed medstate notation screen can provide all of the data of interest at the end of a session .
gs2 sessions are likely to require post - session analysis in order to get even the most rudimentary of data .
med associates medstate notation and coulbourn instruments graphic state 2 both offer the scientist useful tools for behavioral research .
gs2 s strengths lie in its user - friendly programming interface and its many analytical routines . for the non - programming scientist
medstate notation , with the availability of multiple concurrent state sets , offers a far more powerful programming environment . the time spent in mastering medstate notation
i believe that the serious behavioral scientist is better served by the med associates product . | two of the most widely used programs for the control of behavioral experiments are med associates medstate notation and coulbourn instruments graphic state 2 .
the two systems vary considerably in their approach to programming and data recording , with graphic state 2 using a point - and - click interface that appeals to non - programmers while medstate notation requires the typing of programming code .
graphic state 2 provides many data analysis routines , while medstate notation allows the user to embed simple data analysis within the behavioral protocol .
graphic state 2 is simpler to use , but medstate notation is more versatile . | OVERVIEW OF THE SYSTEMS
PROGRAMMING EASE AND VERSATILLITY
DATA FILES AND ANALYSIS
CONCLUSION |
PMC3758988 | between december 2010 and october 2011 , 98 patients who were diagnosed with isolated acl ruptures underwent primary acl reconstruction using single bundle method with modified transtibial technique .
informed consent was obtained from all patients prior to receiving 3d - ct and radiograph for analyzing the femoral tunnel position after the operation . the patient pool consisted of 81 men and 17 women with a mean age of 27.3 years ( range , 15 to 60 years ) .
1c ) by two orthopedic surgeons on two separate occasions at an interval of 2 weeks to assess the interobserver and intraobserver reliability .
the quadrant method used to measure the postoperative femoral tunnel position by 3d - ct is as follows : 1 ) a true lateral view cutting at the middle of the intercondylar notch of the proximal femur by 3d - ct was attained through the picture of archiving and communication systems ( pacs , marotech , seoul , korea ) ( fig .
2 ) the image was enclosed with a rectangular measurement frame that was either parallel or perpendicular to the blumensaat line on power point ver .
2007 ( microsoft co. , redmond , wa , usa ) as shown in fig .
t calculated the deep to shallow distances from the center of the femoral tunnel parallel to the blumensaat line , and h calculated the high to low distances perpendicular to the blumensaat line .
in addition , the anatomical femoral attachment site of acl was shown on table 1 using the previous quadrant method .
after thorough arthroscopic evaluations of the acl , a 3 cm longitudinal skin incision at 2 cm medial to the tibial tuberosity was made .
the graft was cut in half and folded into half to produce a quadruple semitendinous graft .
if the harvested graft was shorter than 24 cm , a quadruple semitendinosus / gracilis graft was prepared by harvesting both the semitendinosus tendon and the gracilis tendon .
the intercondylar ridge of the lateral femoral condyle , and the bifurcate ridge that divides the am bundle and the posterolateral ( pl ) bundle were confirmed by removing the soft tissues at the femoral insertion of acl .
a bony mark at the anatomic center of acl footprint ( just posterior to the bifurcate ridge and center of anteroposterior [ ap ] width of footprint ) was made by passing a microfracture awl through the anteromedial portal as in fig .
2a . this center was deepened and widened to allow space for the guided pin through the tibial tunnel .
since the anatomical femoral tunnel is prepared by the trans - am portal , the transtibial guide pin tends to be located more to the anterior and proximal position of anatomic center than anticipated .
the ' triangular , funnel - shaped bony trough ' was employed to slip the eccentrically positioned guide into the anticipated anatomic center , which is part of our modified transtibial technique ( fig .
the starting point of the tibial edge / periphery was superior to the pes anserinus and the anterior margin of medial collateral ligament ( mcl ) ( fig .
the intra - articular site is positioned immediately lateral to the medial tibial spine and the extension of the inner margin of anterior horn of lateral meniscus .
the reference point of tibial tunnel is a little posterior region of the acl footprint .
the angle of the acl guide ( linvatec , largo , fl , usa ) was set at 47.5. the tibial tunnel was created using a reamer with diameter 1 mm smaller than the prepared acl graft . with a free hand technique , a guide pin was passed through the tibial tunnel towards the bony trough .
generally , the guide pin was located distal and anterior to the anatomical center , and extension of the knee nudged the guide pin towards the proximal and posterior region of the anatomic center , but was being bent at the intra - articular orifice of the tibial tunnel ( fig .
femoral reaming was performed with knee flexion which reduces the chances of blowing out the posterior wall and increases the femoral tunnel length .
when the reamer passes over the bending portion of the guide pin , the knee should be extended . after passing through the bending point
after the graft passage , femoral fixation was done with endobutton and bio - cross pin ( rigidfix system , mitek , johnson & johnson , norwood , ma , usa ) .
the graft was tensioned and fixed to the tibia by placing the ha screw ( bioscrew poly l - lactic acid , linvatec
linvatec ) .
we regularly performed follow - ups on the patients in the out - patient clinic and established a protocol for rehabilitation .
partial weight bearing with crutches for 6 weeks was mandated for patients who underwent meniscal repairs .
the treatment purpose of the patients was to gain a full range of motions at 2 to 6 weeks after surgery .
running and side - cutting activities were allowed at 3 months , with a return to sports activities at 6 months after surgery .
after thorough arthroscopic evaluations of the acl , a 3 cm longitudinal skin incision at 2 cm medial to the tibial tuberosity was made .
the graft was cut in half and folded into half to produce a quadruple semitendinous graft .
if the harvested graft was shorter than 24 cm , a quadruple semitendinosus / gracilis graft was prepared by harvesting both the semitendinosus tendon and the gracilis tendon .
the intercondylar ridge of the lateral femoral condyle , and the bifurcate ridge that divides the am bundle and the posterolateral ( pl ) bundle were confirmed by removing the soft tissues at the femoral insertion of acl .
a bony mark at the anatomic center of acl footprint ( just posterior to the bifurcate ridge and center of anteroposterior [ ap ] width of footprint ) was made by passing a microfracture awl through the anteromedial portal as in fig .
2a . this center was deepened and widened to allow space for the guided pin through the tibial tunnel .
since the anatomical femoral tunnel is prepared by the trans - am portal , the transtibial guide pin tends to be located more to the anterior and proximal position of anatomic center than anticipated .
the ' triangular , funnel - shaped bony trough ' was employed to slip the eccentrically positioned guide into the anticipated anatomic center , which is part of our modified transtibial technique ( fig .
the starting point of the tibial edge / periphery was superior to the pes anserinus and the anterior margin of medial collateral ligament ( mcl ) ( fig . 2c ) .
the intra - articular site is positioned immediately lateral to the medial tibial spine and the extension of the inner margin of anterior horn of lateral meniscus .
the reference point of tibial tunnel is a little posterior region of the acl footprint .
the angle of the acl guide ( linvatec , largo , fl , usa ) was set at 47.5. the tibial tunnel was created using a reamer with diameter 1 mm smaller than the prepared acl graft . with a free hand technique ,
generally , the guide pin was located distal and anterior to the anatomical center , and extension of the knee nudged the guide pin towards the proximal and posterior region of the anatomic center , but was being bent at the intra - articular orifice of the tibial tunnel ( fig .
femoral reaming was performed with knee flexion which reduces the chances of blowing out the posterior wall and increases the femoral tunnel length .
when the reamer passes over the bending portion of the guide pin , the knee should be extended . after passing through the bending point
after the graft passage , femoral fixation was done with endobutton and bio - cross pin ( rigidfix system , mitek , johnson & johnson , norwood , ma , usa ) . if femoral tunnel was short , the graft was press fitted with only endobutton .
the graft was tensioned and fixed to the tibia by placing the ha screw ( bioscrew poly l - lactic acid , linvatec
linvatec ) .
we regularly performed follow - ups on the patients in the out - patient clinic and established a protocol for rehabilitation .
partial weight bearing with crutches for 6 weeks was mandated for patients who underwent meniscal repairs .
the treatment purpose of the patients was to gain a full range of motions at 2 to 6 weeks after surgery .
a perturbation training program began at 6 weeks after surgery . running and side - cutting activities were allowed at 3 months , with a return to sports activities at 6 months after surgery .
the position of the femoral tunnel was measured using the quadrant method by 3d - ct,8 ) where t was 32.94% 5.16% , and h was 41.89% 5.58% ( fig .
these values were more distal and middle as compared with the previous data ( mean t , 29.85% ; h , 33.2% ) measured by quadrant method using cadaveric knees for validating anatomical acl footprints ( p < 0.05).9,10 ) the interobserver intraclass correlation coefficients ( iccs ) were 0.766 and 0.793 , whereas the intraobserver iccs were 0.875 and 0.893 .
the femoral tunnel angle on the ap view was 50.43 7.04 , for which the interobserver iccs were 0.783 and 0.795 , and the intraobserver iccs were 0.911 and 0.923 . on the process of optimizing this modified transtibial technique ,
mishaps such as breakage of the bended wire occurred on 3 occasions as the reamer passed through the tibial tunnel , and a shallow tibial tunnel was formed on another occasion , which all occurred during the initial series .
however , as the examiners became more experienced in executing this modified technique and when the safe zone of the bone stock was adjusted to the entry point of the tibia , these complications were avoided .
the purpose of this study is to describe a single bundle acl reconstruction performed with modified transtibial technique for positioning the femoral tunnel to anatomical footprint .
the femoral tunnel location was being evaluated with 3d - ct scan reconstructions with the quadrant method as described by bernard et al.,8 ) and the results of these scans were compared with those derived from arthroscopic identification . the results of this study showed that the t - value was 32.94% 5.16% , and the h - value was 41.89% 5.58% .
abebe et al.11 ) and kaseta et al.12 ) reported that the femoral tunnels were placed " deeper or more proximal " and " higher or more anterior " than the native acl insertions of the femur when the transtibial technique was used , which could result in increased laxity of the graft .
contrary to these results , the femoral tunnel in this study was located " shallower or more distal " and " lower or more posterior " than the mean value from multiple measurements of the anatomical acl footprints in cadaveric knees ( t , 29.85% ; h , 33.2% ) from previous studies that also used the quadrant method for calculation .
the mean values of the femoral tunnel created by the authors were placed between the am and pl bundle position , which were calculated as described by kopf et al.13 ) and tsukada et al.10 ) rue et al.6 ) reported that a laterally - oriented transtibial drilling femoral tunnel effectively overlapped approximately half the am bundle and half the pl bundle .
miller et al.14 ) proved that the intra - articular aperture of the femoral tunnel in all cases formed an ellipse , with the long and short axis averaging 13.9 1.6 mm and 9.4 0.8 mm in the trans - am method , and 12.1 0.8 mm and 10.6 0.6 mm respectively in the transtibial group .
accordingly , if the center of the femoral tunnel is relatively " distal " and " posterior , " the ellipse - shaped tunnel would allow the incorporation of original portions of the am and pl bundles leading to a more anatomical reconstruction .
hence , this modified transtibial technique has advantages over the traditional technique ( t , 37.2% 5.5% ; h , 11.3% 6.6% ) for being closer to its anatomical location.10,13 ) ferretti et al.15 ) reported the presence of a lateral intercondylar ridge in all patients who underwent acl reconstruction and the bifurcate ridge in 82% of 60 knees .
van eck et al.16 ) described 88% and 48% of these landmarks , respectively for their patients at arthroscopy .
however , to arthroscopically identify these landmarks on the medial wall of the lateral femoral condyle is difficult .
therefore , postoperative 3d - ct scans were used to validate whether this modified technique can reproducibly be used for the placement of the anatomic femoral tunnel .
our angle of the femoral tunnel in the coronal plane was on average 50.43 7.04 , when viewed by ap radiography , and it was lower than the results ( 61.7 5.5 , 58.8 8.3 ) from previous transtibial techniques , even when compared with the anteromedial portal technique ( 55.9 4.7 , 50.9 8.3 ) , which were measured by chang et al.17 ) and dargel et al.18 ) in addition , bedi et al.4 ) reported that the coronal angles of the oblique femoral tunnels from either the transtibial or the trans am portal techniques were 54.1 7.17 and 45.9 6.9 , respectively .
therefore , the rate of the posterior wall blowout was 50% with the latter technique .
thus , not only is the modified transtibial technique more reproducible , but it is also safer when such complications of the am portal technique is taken into consideration . there was no significant posterior wall blowout in our case series .
the entrance of the tibial tunnel is essential for the placement of the anatomic femoral tunnel when using the transtibial technique for acl reconstruction , especially if we want to prevent graft failures.19 ) recent studies have tried to accurately drill the tibial tunnel in relation to the native acl footprint of the femur , but without success.20,21 ) however , heming et al.7 ) reported the plausibility of the anatomical footprint using the transtibial technique , but commented that the starting point should be closer to the joint line , and the tibial tunnel length must be shortened for an accurately positioned footprint .
arnold et al.22 ) attempted to produce anatomical tunnels by transtibial drilling just proximal to the pes anserinus and ventral to the mcl , but in their case , the guide pin was projected too high in the notch and missed the femoral insertion . in this study ,
the authors followed arnold 's method to avoid creating shallow tibial tunnels , and furthermore , used a new modified method to prevent over - projecting the guide pin in the notch into a non - anatomical position .
there are some advantages of this modified transtibial technique in which the femoral tunnel is drilled appropriately through the tibial tunnel .
first , it is similar to the conventional method and easily replicated by surgeons who are familiar with the transtibial technique .
second , it is possible to create the femoral tunnel in an anatomic position through a relatively longer and deeper tibial tunnel with only a few additional and simple techniques ; triangular , funnel - shaped bony trough and by changing knee flexion angles during femoral tunneling .
third , it permits early rehabilitation through firm graft fixation resulting from tunnel length - graft length matching .
the limitations of the study may be that the authors used the mean values of the native acl insertions derived from multiple previous studies for comparison .
although the methods used in this paper are the same as those in previous studies , mean variations in the experimental instruments do not reflect the differences that arise , and , are therefore , a crude averaging of values . in addition , the icc was lower for the interobserver correlation coefficient in two observers , which may reflect the difficulty of selecting the true lateral view on the magnetic resonance imaging sagittal image , and measures the center of the femoral tunnel ( forming an ellipse interobservers ) . finally , there was a lack of full considerations for the length of the femoral tunnel and the characteristics of the tibial tunnel .
further studies to overcome these limitations are required , and essential evaluations of clinical outcomes for modified transtibial technique are necessary to prove its superiority over the traditional techniques . in conclusions ,
our modified transtibial technique is anticipated to provide a true anatomical placement of the femoral tunnel during acl reconstruction than the previous traditional transtibial techniques . | backgroundthe purpose of this study is to report a modified transtibial technique to approach the center of anatomical femoral footprint in anterior cruciate ligament ( acl ) reconstruction and to investigate the accurate femoral tunnel position with 3-dimensional computed tomography ( 3d - ct ) and radiography after reconstruction.methodsfrom december 2010 to october 2011 , we evaluated 98 patients who underwent primary acl reconstruction using a modified transtibial technique to approach the center of anatomical femoral footprint in single bundle acl reconstruction with hamstring autograft .
their femoral tunnel positions were investigated with 3d - ct and radiography postoperatively .
femoral tunnel angle was measured on the postoperative anteroposterior ( ap ) radiograph and the center of the femoral tunnel aperture on the lateral femoral condyle was assessed with 3d - ct according to the quadrant method by two orthopedic surgeons.resultsaccording to the quadrant method with 3d - ct , the femoral tunnel was measured at a mean of 32.94% 5.16% from the proximal condylar surface ( parallel to the blumensaat line ) and 41.89% 5.58% from the notch roof ( perpendicular to the blumensaat line ) with good interobserver ( intraclass correlation coefficients [ icc ] , 0.766 and 0.793 , respectively ) and intraobserver reliability ( icc , 0.875 and 0.893 , respectively ) . according to the radiographic measurement on the ap view , the femoral tunnel angles averaged 50.43 7.04 ( icc , 0.783 and 0.911 , respectively).conclusionsour modified transtibial technique is anticipated to provide more anatomical placement of the femoral tunnel during acl reconstruction than the former traditional transtibial techniques . | METHODS
Surgical Techniques
Postoperative Rehabilitation
RESULTS
DISCUSSION |
PMC4052202 |
a definite diagnosis of alzheimer 's disease ( ad ) can be accomplished only post - mortem by histopathology of the autopsied brain so as to reveal amyloid beta protein ( a ) in neuritic plaques and intraneuronal neurofibrillary tangles [ 1 , 2 ] . in past years
several alterations in peripheral cells and biological fluids of ad patients have been proposed as potential antemortem ad biomarkers [ 36 ] . however , none have met the criteria established for an ideal biomarker [ 7 , 8 ] , capable of assessing whether a mild cognitive impairment ( mci ) might reveal early stages of disease or should rather be linked to normal aging . in fact , advances in our knowledge of ad have shown that symptoms usually develop after a long preclinical pathogenetic process , making early detection of ad in asymptomatic subjects of great interest . indeed , the assessment of presymptomatic subject cohorts regardless of genetic predisposition [ 10 , 11 ] , including relatives of ad patients , could be highly useful in determining the effectiveness of pharmacological intervention in slowing ad onset and/or cognitive decline in ad patients .
clinical criteria for ad diagnosis have recently been revised and the terms possible and probable ad have been redefined by including some biomarkers for the pathophysiological process in support of ad diagnosis . however , their use has been limited to research settings due to the fact that these procedures are not easily available to all patients .
the combination of multiple parameters [ 5 , 13 ] obtained through neuropsychological testing and conventional and functional imaging [ 14 , 15 ] , together with the relatively low accuracy attainable in discriminating ad from other dementia , requires elaborate , serial , and costly procedures , available only in the best academic centers and for a limited number of patients .
therefore , there is a great need of reliable and low - cost peripheral markers to identify persons with incipient ad and complement clinical ad outcomes in a large number of subjects .
the national institute on aging and alzheimer 's association international workgroup recently reviewed the state - of - the - art in this field to develop recommendations to determine which factors best predict the risk of progression from
a combination of csf t - tau and a42/p - tau ratio has shown to be useful for defining mci patients at high risk of developing ad , yielding a positive predictive value of 81% .
the various constituents of blood , including plasma , platelets , and cellular fractions , are now being systematically explored as a pool of putative peripheral biomarkers for ad for the ease of sampling and repeatability of measures [ 15 , 19 ] . in this context ,
disease - specific autoantibody profiles , increased glutaminyl cyclase expression , and angiopoietin-1 levels have also been described in human sera . on the whole , all these findings need verification in further studies [ 23 , 24 ] ; nevertheless they lend further support to the hypothesis that ad patients suffer from a systemic metabolic dysfunction that , in addition to the brain , also affects peripheral tissues .
fibroblasts have also often been employed as an in vitro model for neurological diseases and , particularly , for ad [ 4 , 25 , 27 ] .
for instance , ad fibroblasts , unlike their normal counterpart , display a variety of alterations related to gm1 ganglioside catabolism , the function of k+ channel and ca++ homeostasis , erk1/2 levels in response to bradykinin , cholesterol ester cycle , and , eventually , dysregulated proteolysis [ 3236 ] .
recently , a fibroblast biomarker profile was proposed to identify accurately ad patients for therapeutic intervention .
a very attractive approach could be that of using patient fibroblasts reprogrammed to neurons and exhibiting ad phenotype as a model .
herein , we report that early prediction and diagnosis of ad might be improved by measuring typical proteolytic alterations that we formerly found in cultured ad fibroblasts .
these alterations involve transketolase ( tk ) , a thiamine - dependent enzyme that undergoes limited degradation by the enhanced activities of cysteine proteinases released upon cell extraction [ 32 , 33 ] , to yield isoforms with unusually high alkaline pi ( tk - alkaline bands , tk - ab ) . in this paper , for the first time subjects from 36 families of probable late - onset ad patients were analyzed for the frequency and intensity of the tk - ab signal , and for the apoe genetic asset .
then , statistic analyses were carried out to correlate tk - ab positivity and the presence of apoe 4 allele .
remarkably , asymptomatic first - degree relatives were recruited and identified as subjects at greater risk of developing ad , independently of their apoe genetic asset .
these subjects are often overlooked in most studies focused on detection of disease biomarkers without considering that ad relatives would probably be the most cooperative and interested individuals for investigation with noninvasive analyses .
healthy subjects , patients with other non - ad neuropathologies , and early - onset ad patients were also analyzed .
our results reveal tk - ab as a steady indicator of cultured ad fibroblasts , that is , associated significantly with both late- and early - onset forms of ad regardless of age - of - onset or stage of disease .
it could also be of prognostic value , helpful in assessing ad risk in single individuals and applicable to asymptomatic relatives of ad patients and apoe-4 carriers .
bace1 is the key rate - limiting enzyme for the production of the a peptide and its activity has been found to increase under starvation in vitro and after energy inhibition in app transgenic mice .
the amount of active bace1 increased significantly in extracts of cultured fibroblasts positive to the tk - ab test , supporting the usefulness of cultured fibroblasts as an excellent in vitro model for the study of the pathogenetic process of ad .
individuals who entered the study have been enrolled in the geriatric unit of prato hospital ( italy ) and included ( a ) elderly healthy subjects ( controls , n = 29 , mean age sd = 66.8 11.1 , age range : 5086 ) ; ( b ) patients with a clinical diagnosis of probable late - onset or sporadic ad ( n = 36 , mean age sd = 70.8 7.3 , age range : 6186 ) ; ( c ) asymptomatic first - degree relatives of ad patients ( n = 38 , mean age sd = 46.7 10.9 , age range : 3268 ) ; and ( d ) neurological non - ad patients ( n = 12 , mean age sd = 67.2 10.5 , age range : 5084 ) including 4 patients with vascular dementia , 3 patients with frontotemporal dementia , 2 patients with parkinson 's disease , 1 patient with severe neurosis , 1 patient with intermittent ataxia , and 1 patient with olivopontocerebellar ataxia .
clinical diagnosis of probable ad was made according to the criteria established by the diagnostic and statistical manual of mental disorders ( 4th edition , dsm iv ) , the national institute of neurological and communicative disorders and stroke , and the alzheimer 's disease and related disorders association ( nincdsadrda ) and reevaluated according to the nia - alzheimer 's association workgroups on diagnostic guidelines for ad .
medical examinations by neurologists with the aid of neuropsychological and laboratory tests and sometimes computed tomography or magnetic resonance of the brain were performed . the mini - mental state examination ( mmse ) and global deterioration scale ( gds )
informed written consent to use blood and dermal cells for analysis was obtained from the subjects or , when necessary , from their legal guardians under local institutional review board supervision and approval .
further , we analyzed fibroblasts ( kindly provided by s. sorbi , department of neurology , university of florence ) from early - onset ad patients ( n = 5 , mean age sd = 48.0 6.9 , age range : 3855 ) carrying mutations of app ( val717ile , 1 case ) , presenilin 1 ( ps1 ) ( met146leu , 2 cases ) , and presenilin 2 ( ps2 ) ( met239val , 2 cases ) .
skin biopsies were obtained from the forearm of subjects by a 2 mm punch , under local anesthesia with 2% xylocaine ; healing was usually complete within a week .
tissue fragments were placed in culture and fibroblasts were then propagated under culture conditions favoring tk - ab expression .
briefly , cells were seeded using a medium at ph 7.8 and maintained for 2 weeks without medium changes . then fibroblasts were harvested and lysed by sonication and cell extracts were separated by isoelectric focusing ( ief ) as previously reported .
separated proteins were transferred to a nitrocellulose membrane and then probed with rabbit polyclonal igg raised against human tk .
louis , mo ) or ecl procedure ( amersham biosciences ) [ 25 , 33 ] and quantified by densitometric analysis with the aid of imagej software ( see section 3 and legend in figure 1 ) .
peripheral blood mononuclear cells were isolated by centrifugation at 1,700 g on lymphoprep ( eurobio , les ulis cedex b , france ) .
genomic dna was then extracted by using the nucleospin tissue kit ( macherey - nagel gmbh & co. kg , dren , germany ) .
dna was amplified by pcr in a dna thermal cycler ( geneamp pcr system 2700 , applied biosystems , nj ) as reported by hixson and vernier .
fibroblasts from two healthy aging control subjects ( cont 1 and cont 2 ) , from six probable ad patients ( ad1 - 6 ) , and from two asymptomatic first - grade ad relatives ( rel 1 and rel 2 ) were analyzed .
cells were seeded and maintained in parallel under conditions favoring tk - ab production ( see above ) , called positive , and under normal conditions ( with normal medium ph and detached at confluence ) , called negative .
harvested fibroblasts were extracted with ripa buffer ( r0278 , sigma , 0.1 ml/1,000,000 cells ) and protein concentration was assessed by the bicinchoninic acid protein determination kit ( sigma ) . for bace1 determination , aliquots ( 20 g ) of total cell extracts were separated on 412% nupage bis - tris gels with mops - sds running buffer ( novex , invitrogen , carlsbad , ca ) and blots were probed with anti - bace1 antibody ( nb120 - 10716 , novus biologicals , littleton , co ) .
after incubation with hrp - conjugated secondary antibodies , specific bands corresponding to bace1 proteins were detected by the ecl procedure ( amersham , freiburg , germany ) and quantified by densitometric analysis with the aid of imagej software .
anti - gapdh antibodies ( cell signaling , beverly , ma ) were used for detection of the housekeeping protein .
tk - ab expression and apoe genotype frequencies in different groups of subjects were compared by means of fisher 's exact test .
the analysis of variance test ( anova ) was used to establish whether the degree of ad severity or patient age differed significantly between tk - ab - positive and -negative cases .
sensitivity , specificity , accuracy , and predictive values ( pv ) of tk - ab expression were reported together with 95% confidence intervals ( ci ) .
altered tk - isoforms ( tk - ab ) have frequently been observed in extracts of cultured fibroblasts from ad patients and proposed as potential indicators of disease ( see section 1 and [ 25 , 32 , 34 ] ) .
the conditions for optimal expression and detection of tk - ab following ief separation have been reported elsewhere [ 32 , 47 ] ; however , for the sake of clarity , a panel of the typical and of the most representative altered tk profiles as well as the densitometric criteria used to evaluate results of the analysis were reported ( figures 1(a ) and 1(b ) , resp . ) .
the protein band migrating at ph 8.4 was the major tk isoform in control samples , together with at least three minor and less basic bands .
instead , tk - ab exhibited a more alkaline pi and migrated within a ph range of 8.59.4 .
the presence of tk - ab was usually assessed by visual inspection ; however , to decide on uncertain cases a densitometric analysis of immunostained bands was employed .
intensity values of tk - ab ( ph 8.5 ) were calculated and divided by those of the normal tk band ( ph = 8.4 ) ; the border between negative and positive samples was arbitrarily set at a ratio of 0.4 . of note ,
a strong positive tk - ab signal ( ph 8.5 ) as observed for many of the ad patients was usually accompanied by a decrease in size and staining of the major tk band ( ph = 8.4 ) .
tk profiles were analyzed in fibroblasts derived from 5 distinct groups of individuals and results are reported in figure 2 ( for details see legend and section 2 ) .
the number of tk - ab - positive cases was markedly high among probable ad patients while both healthy controls and neurological non - ad patients ( neurol .
controls ) were all tk - ab negative . based on these findings , tk - ab could be validated as a potential peripheral ad signature , showing a sensitivity = 69.4% ( ci = 51.983.6 ) , a specificity = 100% ( ci = 91.4100 ) , and an accuracy = 85.7% ( ci = 75.992.6 ) .
the negative or positive predictive value ( pv ) was 78.8% ( ci = 65.388.9 ) or 100% ( ci = 86.3100 ) , respectively .
tk - ab - positive and -negative cases did not differ significantly with regard to age - at - onset ( analysis of variance : p = 0.76 ) or disease severity as determined by gds and mmse clinical tests ( p = 0.20 and p = 0.26 , resp . ) .
notably , early - onset ad patients ( dominant ad ) carrying app , ps1 , and ps2 mutations were all tk - ab positive .
nearly half ( 47.4% ) of asymptomatic first - degree ad relatives ( mean age < 50 ) turned out to be tk - ab - positive .
there was a striking statistically significant difference in tk - ab expression in either possible ad patients or ad relatives ( p < 0.0005 for both subsets ) versus healthy controls .
this result indicated a clear inclination of ad relatives toward the distribution pattern of ad patients and supported the view that individuals who have had an ad patient in the family run a major risk of developing ad .
in particular , we found the percentage of tk - ab positivity in our group of relatives at a level very near to that expected for the dominantly inherited ad . in this
regard , our cohort of patients had only a clinical diagnosis of probable late - onset or sporadic ad , but we are inclined to exclude the presence of inherited dominant mutations in relatives , given the high level of age - at - onset of the disease ( age range 6582 ) in patients .
the tk - ab positive relatives could conceivably have a predisposing genetic and/or environmental asset ; this might require further analyses .
although our sample size is small , we have attempted to establish whether the apoe-4 genotype , a well - known risk factor for ad , was potentially related to tk - ab expression .
the answer to this question is negative ; no statistically significant correlation was found between tk - ab expression and apoe genotype in either ad relatives or ad patients ( p = 0.746 and p = 0.159 , resp . ) which would infer that these parameters address distinct pathogenetic mechanisms .
the cross - evaluation of tk - ab positivity and apoe genotype was carried out within ad relatives to assess whether it might improve ad prediction within these relatively young and asymptomatic subjects .
results showed that the two homozygous 4/4 carriers were both tk - ab - positive . among heterozygous 3/4 carriers
there were 5 out of 10 tk - ab - positive cases ; finally , tk - ab - positive cases were highly represented among 3/3 ( 11 out of 21 ) and also one 2/3 carrier was positive .
an increased number of positive cases within 3/3 relatives of ad patients could have been expected , and tk - ab positivity did not improve the prediction of ad risk based on 4 allele frequency at the population level , but it might be crucial to identifying those subjects who , although asymptomatic , express tk - ab at early ages and are , conceivably , at higher risk of progressing to ad .
the combined results of the clinical diagnosis and genetic and biochemical tests in members of 6 out of the 36 families examined are shown in figure 3 , where each family is represented by a probable ad patient and 2 to 3 relatives ( r ) .
( ad , female , 70 years , onset at 67 ; r1 , female , 43 years ; r2 , male , 41 years ) r2 son , for its positivity to tk - ab test , could be considered at higher risk of ad , despite the fact he had a more protective apoe genetic asset than r1 .
( ad , female , 74 years , onset at 72 ; r1 , female , 71 years ; r2 , female , 70 years ) represented by a possible ad patient and two sisters : it was confirmed that 4/4 subjects were always positive to the tk - ab test whether they were clinically diagnosed as ad patients or still unrecognized as in the case of r1 who , due to 4/4 homozygosity , will conceivably develop ad in subsequent years .
( ad , female , 86 years , onset at 78 ; r1 , male , 58 years ; r2 , male , 54 years ; r3 , female , 50 years ) .
all three sons were tk - ab positive , indicating that the presence of the marker was a constant trait in this family ; evidently , the whole genetic asset conferred the same risk related to altered proteolysis to the three rs irrespective of 4 allele frequency that might play a role in determining the age of onset .
( ad , female , 81 years , onset at 79 ; r1 , female , 55 years ; r2 , male , 51 years ; r3 , male , 39 years ) .
the positivity to tk - ab test addressed r2 as the subject with highest probability of developing late - onset ad , that in principle should have been equally shared by the two sons ( r2 and r3 ) , both carrying the apoe 3/4 genetic asset .
( ad , male , 81 years , onset at 77 ; r1 , female , 49 years ; r2 , male , 53 years ) : as in family 4 , our results indicate r2 as the subject with the highest risk of developing late - onset ad
( ad , female , 67 years , onset at 66 ; r1 , female , 40 years ; r2 , female , 36 years ; r3 , male , 34 years ) . the ad patient , for both the apoe genetic asset and the absence of tk - ab trait , should be clinically reevaluated for other neuropathologies ; r2 seems to be the only subject with the highest probability of developing late - onset ad irrespective of the absence of 4 allele .
bace1 is the key rate - limiting enzyme for the production of the a peptide and its activity has been found to increase under starvation in vitro and after energy inhibition in app transgenic mice . in order to assess whether our fibroblast samples might show other well - known ad metabolic alterations , that could be related to tk - ab or not , bace1 activity was determined in extracts of cultured fibroblasts which were maintained in parallel under culture conditions positive and negative for tk - ab production ( see section 2 ) .
the results of a preliminary experiment are shown in figure 4 : two healthy aging control subjects ( cont 1 and cont 2 ) , two ad relatives ( rel 1 and rel 2 ) , and 6 probable ad patients ( ad1 - 6 ) were analyzed for bace1 electrophoretic pattern .
three immunoreactive bands of bace1 protein were detected ( see insert ) : ( i ) one indicated the precursor protein ( immature bace1 , ~50 kda ) and
( ii ) two forms corresponded to the glycosylated active enzyme ( mature bace1 , within the range of 7080 kda ) [ 51 , 52 ] .
we found that the amount of the active enzyme increased significantly under positive culture conditions in comparison to negative ones ( ratio > 1 ) , in fibroblast extracts which contemporarily yielded a positive tk - ab signal .
we propose that the tk - ab test may be used as a peripheral indicator of disease in fibroblasts from ad patients in this study . the sensitivity of this test ( as opposed to specificity , accuracy , and predictive values ) was not extraordinarily high but it should be remembered that these individuals were diagnosed as probable ad patients only by standard clinical criteria .
there are , however , some aspects concerning the significance and applications of tk analysis that deserve further comments .
first , the tk - ab signature seems to be independent of age - of - onset , severity , and form of ad .
such a steady expression from the early to the late clinical stages of disease is a distinctive feature of tk - ab as compared to other peripheral ad indicators .
in fact , even a peptides , the most sensitive and direct hallmark of ad , increase in plasma of rare dominant ad patients but not in most common late - onset ad patients who actually show a decline in a-42 levels [ 9 , 55 ] . secondly
, tk - ab could be detected even in the absence of clinical signs such as in ( i ) approximately half of the subjects younger than 50 who , irrespective of 4 frequency , have had an ad patient in the family and run a major risk of developing ad later in life and ( ii ) in asymptomatic 4/4 carriers who have a high probability of developing ad with age .
the identification of the tk - ab signature in combination with genetic profile in relatives of ad patients but it might also be worth for subjects with mci or less clearly determined cognitive deficits could provide early recognition of at - risk subjects and allow targeted intervention to delay neurodegeneration .
we can not say that all asymptomatic individuals with a single apoe 4 gene dose and positive for tk - ab will necessarily develop the disease at advanced age but , at least , it could be possible to identify those subjects who should enter a follow - up study .
thirdly , our results suggest that mechanisms underlying tk - ab production and bace1 activation might be related .
in addition , the fact that the amount of active bace1 increased significantly in extracts of cultured tk - ab - positive fibroblasts reinforces the specificity of our test . on the whole
, this study supports the usefulness of cultured fibroblasts as an excellent in vitro model for the study of the pathogenetic process of ad and for preliminary tests of toxicity and efficacy of agents capable of reestablishing the control of intracellular proteolysis , including bace1 activation . | there is great interest in developing reliable biomarkers to support antemortem diagnosis of late - onset alzheimer 's disease ( ad ) .
early prediction and diagnosis of ad might be improved by the detection of a proteolytic dysfunction in extracts from cultured ad fibroblasts , producing altered isoelectrophoretic forms of the enzyme transketolase ( tk - alkaline bands ) .
the tk profile and apolipoprotein e ( apoe ) genotype were examined in fibroblasts from 36 clinically diagnosed probable late - onset sporadic ad patients and 38 of their asymptomatic relatives , 29 elderly healthy individuals , 12 neurological non - ad patients , and 5 early - onset ad patients .
tk alterations occurred in ( i ) several probable ad patients regardless of age - of - onset and severity of disease ; ( ii ) all early - onset ad patients and apoe 4/4 carriers ; and ( iii ) nearly half of asymptomatic ad relatives .
normal subjects and non - ad patients were all negative . notably , culture conditions promoting tk alterations were also effective in increasing active bace1 levels .
overall , the tk assay might represent a low - cost laboratory tool useful for supporting ad differential diagnosis and identifying asymptomatic subjects who are at greater risk of ad and who should enter a follow - up study .
moreover , the cultured fibroblasts were confirmed as a useful in vitro model for further studies on the pathogenetic process of ad . | 1. Introduction
2. Patients and Methods
3. Results and Discussion
4. Conclusions |
PMC3424938 | citius , altius , forties which is latin for faster , higher , and stronger . with this basis , we too have adopted the same in our quest for a decalcifying agent .
lesions affecting hard tissues need intricate , technique - sensitive methodology for interpretation and diagnosis .
the pulpal soft tissue can only be assessed in decalcified sections which otherwise in ground section is not possible as it is lost .
the aim of decalcification is to remove calcium salts from mineralized tissue using chemical solutions like acids and chelating agents , while preserving the organic portions .
so , an ideal decalcifying agent should
be fast;be good and;do good .
though some agents remove the calcium ions completely and rapidly
so , we present here the comparative evaluation of different decalcifying agents with respect to rate of decalcification , the effect of decalcifying agents on the dental tissue , and its influence on the staining characteristics . the quest for at most near ideal decalcifying agent begins .
the aims and objectives of the study were :
to evaluate the fastest decalcifying agent;to evaluate the decalcifying agents based on its effect on the organic content and integrity of the soft tissue;to evaluate the staining characteristics of the teeth decalcified in the different decalcifying agents;to compare and contrast between the decalcifying agents .
to evaluate the fastest decalcifying agent ; to evaluate the decalcifying agents based on its effect on the organic content and integrity of the soft tissue ; to evaluate the staining characteristics of the teeth decalcified in the different decalcifying agents ; to compare and contrast between the decalcifying agents .
freshly extracted , noncarious , not attrited , 24 natural teeth were obtained from patients aged 4045 years.[35 ] the teeth , fixed in 10% formalin , included incisors , canines , premolars , and molars , and were used to analyze the six different decalcifying agents namely 5% nitric acid , perenyi 's fluid , formalin - nitric acid solution , neutral ethylene diamine tetra acetic acid ( edta ) decalcifying solution , 10% formic acid , and 5% trichloroacetic acid .
each decalcifying agent was used to decalcify an incisor , a canine , a premolar , and a molar each .
decalcifying agents were subjected to repeated agitation and replaced by freshly prepared agents as indicated by the chemical method of end point of decalcification for 5% nitric acid , perenyi 's fluid , formalin nitric acid solution , and 5% trichloroacetic acid . for both neutral edta and 10% formic acid ,
strong liquor ammonia is added drop by drop to 5 cm of decalcifying agent until it turns alkaline to litmus.if solution went cloudy , it indicated presence of calcium and the solution was replaced with fresh solutionif solution does not turn cloudy , 5 cm of saturated ammonium oxalate is added to the solution.if solution remains clear for 30 min , it was concluded that end point of decalcification was reached and the procedure is completed .
strong liquor ammonia is added drop by drop to 5 cm of decalcifying agent until it turns alkaline to litmus .
if solution went cloudy , it indicated presence of calcium and the solution was replaced with fresh solution if solution does not turn cloudy , 5 cm of saturated ammonium oxalate is added to the solution . if solution remains clear for 30 min , it was concluded that end point of decalcification was reached and the procedure is completed .
end point of neutral edta was assessed radiographically wherein the opacity suggested incomplete decalcification [ figure 1 ] .
the physical method of probing the teeth subjected to neutral edta with a needle was also suggestive of incomplete decalcification .
radiograph of a molar decalcified using neutral edta at the end of 91 days , before processing the speed of decalcification was graded from 15 [ 1-slowest and 5-fastest].[35 ] all the teeth were washed under running tap water for 10 min ( neutral edta decalcified teeth was washed for 2 h ) and continued with routine processing , paraffin wax infiltration , and embedding ; sectioning and staining with hematoxylin and eosin .
the stained sections were observed under the microscope and were graded from 14 [ 1-poor , 2-fair , 3-good , and 4-excellent ] based on the following criteria :
ease of sectioning;hard - tissue staining;soft - tissue staining both cytoplasmic and nuclear staining;soft - tissue attachment;soft - tissue shrinkage and;pulpal organization .
hard - tissue staining ; soft - tissue staining both cytoplasmic and nuclear staining ; soft - tissue attachment ; soft - tissue shrinkage and ;
parameter 1 : speed of decalcification : 5% nitric acid decalcified teeth the fastest and neutral edta was the slowest [ graph 1].parameter 2 : soft - tissue integrity : with respect to soft - tissue integrity , features like soft - tissue attachment , soft - tissue shrinkage , and pulpal organization , teeth decalcified with neutral edta exhibited excellent results , and the ones decalcified with 5% nitric acid scored the least [ graph 2 , figures 2 and 3].parameter 3 : staining quality : neutral edta and 5% trichloroacetic acid decalcified teeth stained the best , and 5% nitric acid and perenyi 's fluid stained the worst [ graph 3 , figures 4 and 5].overall , neutral edta scored over the other agents and perenyi 's fluid scored the least [ table 1 ] .
parameter 1 : speed of decalcification : 5% nitric acid decalcified teeth the fastest and neutral edta was the slowest [ graph 1 ] .
parameter 2 : soft - tissue integrity : with respect to soft - tissue integrity , features like soft - tissue attachment , soft - tissue shrinkage , and pulpal organization , teeth decalcified with neutral edta exhibited excellent results , and the ones decalcified with 5% nitric acid scored the least [ graph 2 , figures 2 and 3 ] .
parameter 3 : staining quality : neutral edta and 5% trichloroacetic acid decalcified teeth stained the best , and 5% nitric acid and perenyi 's fluid stained the worst [ graph 3 , figures 4 and 5 ] .
overall , neutral edta scored over the other agents and perenyi 's fluid scored the least [ table 1 ] .
time in days taken for decalcification soft - tissue integrity of the various decalcifying agents tooth decalcified using neutral edta decalcifying solution under high power 5% nitric acid decalcified tooth in low power staining characteristics of the different decalcifying agents high - power view of a tooth decalcified in 5% trichloroacetic acid tooth decalcified using perenyi 's fluid under low power cumulative scores of the decalcifying agents based on the various parameters
the process of decalcification is done to study the structure of tooth , pulp calcifications , and also to evaluate the biological response of dental pulp to restorative materials . for many years , scientists have tried to introduce new decalcifying substances or tried to modify known decalcification agents , in order to meet the criteria of a good decalcifying agent which
ensures complete removal of calcium;causes minimal damage to cells and tissues;causes non impairment to subsequent staining and;decalcifies at reasonable speed
.
ensures complete removal of calcium ; causes minimal damage to cells and tissues ; causes non impairment to subsequent staining and ; decalcifies at reasonable speed .
most authors have compared two to four decalcifying agents , sometimes varying the methods used and by employing a lot of permutations and combinations of the methods and agents , mainly to decalcify bone.[2810 ] in the present study , we attempted to compare the efficacy of six decalcifying agents , its rate of decalcification , its effect on organic and inorganic components of teeth , and staining characteristics .
the speed factor of the decalcifying agents was the highest with 5% nitric acid and lowest by neutral edta decalcifying solution , which was in accordance with literature
. also noted , based on chalky white appearance of enamel , was the initial rate of decalcification 4 days after the start of the procedure .
contrary to the overall increased time taken by neutral edta , it was noted that teeth decalcified in it had lost its enamel faster than 5% nitric acid . when sectioning it was noted that there was crumbling of tissue decalcified in 5% nitric acid and perenyi 's fluid which also contains nitric acid . also , tissue was indistinct when observed under microscope as also noted by zappa et al . with respect to 7% nitric acid .
teeth decalcified with neutral edta responded the best to microtome knife , hence deceiving the physical and radiological methods of testing end point of decalcification with respect to neutral edta . in terms of efficacy of agents with respect to soft - tissue integrity and hard- and soft - tissue staining , excellent results were actually obtained with the slowest decalcifying agent , i.e. , neutral edta .
soft - tissue attachment and soft - tissue shrinkage , as reported by zappa et al .
, suggest that formic acid and nitric acid produce worst results in contrast to the results obtained from our study , wherein formic acid gave good results as it showed minimal soft - tissue shrinkage and minimal loss of tissue [ figure 6 ] .
the pulp organization with its extra - cellular matrix and histological zones were clearly distinct and excellent in teeth decalcified with neutral edta and 5% trichloroacetic acid .
10% formic acid decalcified tooth in low power the overall superior results obtained with neutral edta may be attributed to the mechanism of capturing metallic ions like calcium which binds to the chelating agent .
this means that the calcium ions from the external layer of the apatite crystals will be removed .
when all calcium ions from the outer layer of apatite crystals are removed , they will be replaced by ions from the deeper layers . in this way , the crystal size decreases gradually , producing an excellent preservation of tissue components.[46 ] the quality of decalcified sections and rate of decalcification depends on factors like fixation concentration of decalcifying agent used , temperature , pressure , agitation , electric current , microwave radiation , tissue suspension , and size and type of tissue.[469 ] in a study by waerhaug , bone and teeth were decalcified rapidly under vacuum . the time taken for decalcification was reduced to one - tenth .
changes in temperature at which decalcification occurs also varies the time taken for complete decalcification .
vongsavan et al . , in his study on cat and rat teeth , reported a faster process of decalcification in microwave oven at 37 2c than in room temperature or conventional oven .
another study by pitol et al . showed that microwave - aided decalcification showed to be more effective than the traditional methods in aspects like reduction of time period for decalcification , good morphology of bone tissue , and an increase of calcium release using microwaving .
warshawsky and moore studied the effects of decalcifying agents , namely nitric acid , formic acid , and edta , on number and stainabilty of gram - positive bacteria .
the number of studies using edta was greater than the other decalcifying agents , especially when the need was for academic interests and research.[1619 ] to conclude , harder the structure , hardest to decalcify .
we propose , in cases of urgent requirement , use of acids may be employed although with poor tissue integrity . for preservation and presentation , when time is not a factor , use of neutral edta may be advocated for its excellent soft - tissue integrity and quality of staining .
an agent that seemed to balance both of the factors of time and tissue integrity was formalin - nitric acid solution [ figure 7 ] .
low - power view of a tooth decalcified in formic acid - nitric acid solution regardless of the solution used , methods of decalcification share their characteristic of being accelerated when additional factors are employed .
the present study was done with respect to different decalcifying agents only and none of the factors were employed , thus standardizing the procedure .
future studies in which the factors can be varied and decalcifying agent can be kept constant , might bring us to the near ideal decalcifying agent . | context : in routine histopathology , decalcification of bone and teeth is often an essential and important step during tissue processing .
various decalcifying agents have been used in the past .
the rate of decalcification and the effect of decalcifying agents on the tissue and its staining characteristics are two important parameters which influence the selection of decalcifying solutions . though some agents remove the calcium ions completely and rapidly , they adversely affect the staining characteristics and may also damage the organic components .
there have been very few studies which have systematically evaluated the efficacy of these agents in decalcifying dental hard tissues.aims:the present study was done to evaluate the rate of decalcification of six different decalcifying agents and also their effect on staining characteristics on dental hard tissues.materials and methods : six decalcifying agents namely , neutral ethylene diamine tetra acetic acid ( edta ) decalcifying solution , 5% nitric acid , perenyi 's fluid , formalin nitric acid , 5% trichloracetic acid , and 10% formic acid were used to decalcify 24 natural teeth ( four in each solution ) .
the endpoint of decalcification was evaluated by radiographic and chemical methods .
the decalcified teeth were then routinely processed , sectioned , and stained with hematoxylin and eosin stains.results:neutral edta was the most considerate to the soft and hard tissues and 5% nitric acid was the least considerate to the tooth structure.conclusions:neutral edta , though being the slowest decalcifying agent among the six agents used in the study , gave excellent results for soft - tissue integrity , and best quality of both soft - tissue and hard - tissue stainings . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION |
PMC419380 | this cancer is widespread in males with 1/5 of men being affected throughout life . every year
however , only 20% of them will lead to demise of the patient with an approximately 9-year reduction in expected survival .
thus , prostate cancer remains the second cause of oncologic death in males in europe and in the usa .
because of patient - to - patient heterogeneity in the clinical behavior of this disease , prognostic markers that may help tailor therapeutic strategies to individual clinical situations are continuously re - assessed .
this frequent reassessment leads to modifications of clinical criteria utilized for the selection of therapeutic strategies which , in turn , make difficult the comparison and interpretation of clinical results among different therapeutic strategies . in particular , there is a well known discrepancy between post - surgical progression after radical prostatectomy and pathological staging .
often local or systemic progression is observed in patients theoretically at low risk such those staged as pt2n0 .
thus , current pathological staging can not comprehensively evaluate neoplasm extent , aggressiveness and risk of progression .
clinical recurrence after surgical excision could be ascribed to microscopic nodal metastases and/or micro - invasion at the margins of resection missed by pathological staging . to complement pathological staging , new diagnostic and prognostic markers ( shbg , p53 , bcl2 human callicrein-2 , psma , and so forth ) are under evaluation .
reverse tanscriptase polymerase chain reaction ( rt - pcr ) for determination of prostate - specific antigen ( psa ) can detect a single prostate cancer cell in a tissue sample .
the high sensitivity of this technique may increase the accuracy of staging in low tumor burden cases and , consequently , be a more appropriate molecular marker for the determination of the effectiveness of a neoadjuvant therapy such as maximum androgenic blockage ( mab ) before radical prostatectomy .
in fact , this therapeutic approach is believed to induce a down staging of the disease .
however , it has been questioned whether pre - operative hormonal blockade may introduce a bias in pathological staging due to the iatrogenically induced tissue changes . in this study
, we assessed the incidence of micro - metastases at surgical margins and pelvic lymph nodes in patients submitted to radical prostatectomy after neoadjuvant mab or to radical prostatectomy alone .
we compared traditional histopathological staging to molecular detection of psa using taqman - based quantitative real - time pcr ( qrt - pcr ) never used before for this purpose .
this is a report about an open randomized monocentric pilot study with parallel arms , approved by the ethics committee of our institution .
thirty - three < 80 year - old patients were accrued from july 2000 to june 2002 .
all were clinically staged as having a previously untreated , histologically confirmed t1-t2 prostate cancer .
patients were randomly assigned to neoadjuvant hormonal therapy plus radical prostatectomy ( arm a ) or radical prostatectomy alone ( arm b ) . clinical and pathological stages were determined according to the 1997 american joint committee - international union against cancer tnm system .
pre - treatment evaluation included clinical history , digital rectal examination , serum psa determination using hybritech technique , routine blood tests , trans - rectal ultrasound , confirmation of out - side diagnosis , abdominal computerized tomography and radioisotope bone scan .
patients in both arms underwent pelvic lymphadenectomy and radical retro - pubic prostatectomy 55 to 70 days from randomization .
neoadjuvant therapy consisted of : nilutamide 150 mg daily until surgery and subcutaneous administration of buserelin depot 9,9 mg on the day of randomization .
all patients underwent radical retro - pubic bladder neck sparing prostatectomy with or without nerve sparing .
bilateral pelvic ( iliac and obturator ) node dissection was consistently performed and one right and one left node were taken proximal to the iliac bifurcation , and preserved in liquid nitrogen for subsequent qrt - pcr analysis .
in addition , the right and left lateral margins of resection were submitted for pathological evaluation after dissection of a small wedge for cryostatic preservation ( at least 0,5 cm of lenght ) . in all cases surgeons and pathologists have checked the integrity of prostatic capsule in order to be sure that these strips had been removed outside the capsule ( non containig prostatic tissue ) .
in addition , the expression of the tumor associated antigen psa was quantified by means of qrt - pcr in surgical margins and lymph nodes .
total rna was then isolated using rneasy mini kits ( qiagen , santa clarita , ca ) following manufacturer 's guidelines .
one g of total rna was reverse transcribed into cdna using 1 l of oligo - dt primers ( 1 g/l , gibcobrl ) , 5 l first strand buffer ( gibcobrl ) , 2 l 0.1 m dtt , 2 l magnesium sulfate ( gibcobrl ) , 1 l of 10 mm dntp and 1 l superscript - ii ( ss - ii , gibcobrl ) .
the reaction mixture was heated to 65c for 10 minutes before adding ss - ii , then synthesis was continued with 1 hour at 42c .
the expression of psa and of a housekeeping gene ( i.e. , -actin ) was assessed by qrt - pcr as performed by the abi prism 7700 sequence detection system ( perkin elmer , foster city , ca ) .
probes and primers ( see table 1 ) were designed to span intron - exon junctions in order to avoid genomic dna amplification .
probes and primers used to generate a standard curve for each gene , 1 l of cdna obtained from normal prostate was added to the following reagents : 1 l taqman primers ( 10 m , perkin elmer ) , 5 l 10 pcr buffer ( perkin elmer ) , 11 ml mgcl2 ( 25 mm ) , 1 ml dnpt ( 10 mm ) and 0.5 l amplitaq gold ( perkin elmer ) in a final volume of 50 l .
the thermal cycler conditions for this pcr reaction were as follows : after 10 minutes at 95c , 15 seconds at 95c followed by 1 minute at 60c were repeated 40 times .
the equation describing the standard curves was used to extrapolate the absolute copy number of psa and -actin .
quantitative real time pcr of sample cdna and standard amplified cdna was conducted in a 25 l final volume mixture containing primers , probe and 1 taqman master mix ( perkin elmer ) at optimized concentrations ( probe : 200 nm , primers : 400 nm ) .
the taqman abi prism 7700 sequence detection system allows running 96 samples at a time .
thermal cycler parameters included 2 minutes at 50c , 10 minutes at 95c and 40 cycles involving denaturation at 95c for 15 seconds and annealing / extension at 60c for 1 minute . to ensure that probes and primers in use did not amplify genomic dna , we extracted genomic dna from a normal prostate biopsy and tested it with the taqman primers .
given the small size of the amplicons ( < 100 bp ) , a fluorimager 595 ( molecolar dynamics , sunnydale , ca ) scanning system was used to detect the fluorescence of the vistra green fluorescent intercalator , which was adopted instead of ethidium bromide to visualize the bands of the agarose gel after electrophoresis . an argon laser for excitation ( 488 nm ) and an emission band - pass filter 530 df30 were applied . for qrt - pcr purposes ,
probes were labeled with a fluorescent reporter dye at the 5'-end ( 6-carboxyfluorescein , 6-fam ; emission max = 518 nm ) and a quencher dye at the 3'-end ( 6-carboxytetramethylrhodamine , tamra ; emission max = 582 nm ) .
probe and primers were designed to obtain amplicons less then 100 bp in length in order to enhance pcr efficiency .
furthermore , they were designed to span an intro - exon junction in order to avoid genomic dna amplification .
since both the amount of total rna added to each reverse transcription reaction tube ( based on waive length absorbance ) and its quality ( i.e. degradation ) are not reliable parameters as measures of starting material , transcripts of a housekeeping gene were quantified as endogenous control .
beta - actin ( -actin ) is one of the most used aspecific housekeeping genes , but according to the experimental conditions , other internal references can be adopted ( e.g. leukocyte markers ) . for each experimental sample
the value of both the target and the housekeeping gene are extrapolated from the respective standard curve .
the target value is then divided by the endogenous reference value to obtain a normalized target value independent from the starting material amount .
patients were seen for follow up 50 days after surgery and every 3 months thereafter during the first year , every 6 months during the subsequent four years and then annually . at each visit serum psa
statistical analysis was performed on 29 valuable patients randomized and included in the study . for
each patients ( arm a or b ) qrt - pcr results from lateral surgical margins and pelvic nodes were analyzed .
statistical evaluation and data elaboration was made using sas 's software ( statistical analysis system ) .
continuity adjust chi - squared test was used to assess differences in qrt - pcr results between arm a and b. p values of = 0.05 were considered statistically significant .
this is a report about an open randomized monocentric pilot study with parallel arms , approved by the ethics committee of our institution .
thirty - three < 80 year - old patients were accrued from july 2000 to june 2002 .
all were clinically staged as having a previously untreated , histologically confirmed t1-t2 prostate cancer .
patients were randomly assigned to neoadjuvant hormonal therapy plus radical prostatectomy ( arm a ) or radical prostatectomy alone ( arm b ) . clinical and pathological stages were determined according to the 1997 american joint committee - international union against cancer tnm system .
pre - treatment evaluation included clinical history , digital rectal examination , serum psa determination using hybritech technique , routine blood tests , trans - rectal ultrasound , confirmation of out - side diagnosis , abdominal computerized tomography and radioisotope bone scan .
patients in both arms underwent pelvic lymphadenectomy and radical retro - pubic prostatectomy 55 to 70 days from randomization .
neoadjuvant therapy consisted of : nilutamide 150 mg daily until surgery and subcutaneous administration of buserelin depot 9,9 mg on the day of randomization .
all patients underwent radical retro - pubic bladder neck sparing prostatectomy with or without nerve sparing . bilateral pelvic ( iliac and obturator ) node dissection was consistently performed and one right and one left node were taken proximal to the iliac bifurcation , and preserved in liquid nitrogen for subsequent qrt - pcr analysis .
in addition , the right and left lateral margins of resection were submitted for pathological evaluation after dissection of a small wedge for cryostatic preservation ( at least 0,5 cm of lenght ) . in all cases surgeons and pathologists have checked the integrity of prostatic capsule in order to be sure that these strips had been removed outside the capsule ( non containig prostatic tissue ) .
in addition , the expression of the tumor associated antigen psa was quantified by means of qrt - pcr in surgical margins and lymph nodes .
total rna was then isolated using rneasy mini kits ( qiagen , santa clarita , ca ) following manufacturer 's guidelines .
one g of total rna was reverse transcribed into cdna using 1 l of oligo - dt primers ( 1 g/l , gibcobrl ) , 5 l first strand buffer ( gibcobrl ) , 2 l 0.1 m dtt , 2 l magnesium sulfate ( gibcobrl ) , 1 l of 10 mm dntp and 1 l superscript - ii ( ss - ii , gibcobrl ) .
the reaction mixture was heated to 65c for 10 minutes before adding ss - ii , then synthesis was continued with 1 hour at 42c .
the expression of psa and of a housekeeping gene ( i.e. , -actin ) was assessed by qrt - pcr as performed by the abi prism 7700 sequence detection system ( perkin elmer , foster city , ca ) .
probes and primers ( see table 1 ) were designed to span intron - exon junctions in order to avoid genomic dna amplification .
probes and primers used to generate a standard curve for each gene , 1 l of cdna obtained from normal prostate was added to the following reagents : 1 l taqman primers ( 10 m , perkin elmer ) , 5 l 10 pcr buffer ( perkin elmer ) , 11 ml mgcl2 ( 25 mm ) , 1 ml dnpt ( 10 mm ) and 0.5 l amplitaq gold ( perkin elmer ) in a final volume of 50 l .
the thermal cycler conditions for this pcr reaction were as follows : after 10 minutes at 95c , 15 seconds at 95c followed by 1 minute at 60c were repeated 40 times .
the equation describing the standard curves was used to extrapolate the absolute copy number of psa and -actin .
quantitative real time pcr of sample cdna and standard amplified cdna was conducted in a 25 l final volume mixture containing primers , probe and 1 taqman master mix ( perkin elmer ) at optimized concentrations ( probe : 200 nm , primers : 400 nm ) .
the taqman abi prism 7700 sequence detection system allows running 96 samples at a time .
thermal cycler parameters included 2 minutes at 50c , 10 minutes at 95c and 40 cycles involving denaturation at 95c for 15 seconds and annealing / extension at 60c for 1 minute . to ensure that probes and primers in use did not amplify genomic dna , we extracted genomic dna from a normal prostate biopsy and tested it with the taqman primers .
given the small size of the amplicons ( < 100 bp ) , a fluorimager 595 ( molecolar dynamics , sunnydale , ca ) scanning system was used to detect the fluorescence of the vistra green fluorescent intercalator , which was adopted instead of ethidium bromide to visualize the bands of the agarose gel after electrophoresis .
an argon laser for excitation ( 488 nm ) and an emission band - pass filter 530 df30 were applied . for qrt - pcr purposes ,
probes were labeled with a fluorescent reporter dye at the 5'-end ( 6-carboxyfluorescein , 6-fam ; emission max = 518 nm ) and a quencher dye at the 3'-end ( 6-carboxytetramethylrhodamine , tamra ; emission max = 582 nm ) .
probe and primers were designed to obtain amplicons less then 100 bp in length in order to enhance pcr efficiency .
furthermore , they were designed to span an intro - exon junction in order to avoid genomic dna amplification .
since both the amount of total rna added to each reverse transcription reaction tube ( based on waive length absorbance ) and its quality ( i.e. degradation ) are not reliable parameters as measures of starting material , transcripts of a housekeeping gene were quantified as endogenous control .
beta - actin ( -actin ) is one of the most used aspecific housekeeping genes , but according to the experimental conditions , other internal references can be adopted ( e.g. leukocyte markers ) . for each experimental sample
the value of both the target and the housekeeping gene are extrapolated from the respective standard curve .
the target value is then divided by the endogenous reference value to obtain a normalized target value independent from the starting material amount .
patients were seen for follow up 50 days after surgery and every 3 months thereafter during the first year , every 6 months during the subsequent four years and then annually . at each visit serum psa
statistical analysis was performed on 29 valuable patients randomized and included in the study . for each
patients ( arm a or b ) qrt - pcr results from lateral surgical margins and pelvic nodes were analyzed . statistical evaluation and data elaboration
continuity adjust chi - squared test was used to assess differences in qrt - pcr results between arm a and b. p values of = 0.05 were considered statistically significant .
one dropped out for noncompliance , one for pneumonia , one for allergy to nilutamide and one because the sample was inadequate to perform qrt - pcr analysis .
of 29 patients , 15 ( 51,7% ) underwent neo - adjuvant mab treatment ( arm a ) and 14 ( 48,3% ) received prostatectomy alone ( arm b ) .
five of the 29 patients ( 17,2% ) experienced biochemical recurrence after a mean follow - up of 19 months , all of these patients belonged to arm b. in the neo - adjuvant arm a 5/15 ( 33,3% ) patients were staged as pt2a - g2 .
one of 15 patients ( 6,6% ) was staged as pt2a - g3 and resulted positive at qrt - pcr .
four of the 15 patients ( 26,8% ) were pt2b - g2 : 2 resulted negative and 2 positive at qrt - pcr .
five of the 15 ( 33,3% ) patients were pt3b - g2 and 4 resulted negative and 1 positive to qrt - pcr ( see table 2 ) .
the ratio of psa mrna expressing patients according to qrt - pcr in arm a was of 26,6% ( 4/15 ) . in arm b , 4/14 ( 28,6% )
patients were pt2a - g2 of this 3 negative and one positive at qrt - pcr analysis ; 6/14 ( 42,8% ) patients were pt2b - g2 , one negative and 5 positive to qrt - pcr ; 4/14 ( 28,6% ) patients were pt3b - g2 , one negative and 3 positive for psa expression in periprostatic tissues ( see table 3 ) pathological and qrt - pcr data of arm b patients .
overall 64,3% ( 9/14 ) of the patients had evidence of psa mrna expression in arm b , while 26.6% ( 4/15 ) resulted molecularly positive in arm a ( fisher exact test = 0.04 ) .
pathological and molecular finding of the patients who had biochemical recurrence are reported in table 4 .
analysis was performed on patients from arm a or b whether they had or had not cumulative evidence of psa mrna expression by qrt - pcr in lymph nodes and/or lateral surgical margins .
nine of 14 ( 64,29% ) patients expressed psa in arm b and four of 15 ( 26,67% ) in arm a ( see table 5 ) .
continuity adjust chi - square ( a chi - square modified for not continuous variable ) was calculated and resulted in 0.0965 , this value trend to statistical significance .
this results suggests a difference between arm a and b , although not statistically significant ( see table 6 ) . in table 7
value founded show the risk to be positive if not treated with neoadjuvant therapy respect to treated arm ; the risk to have positive surgical margins or nodes to qrt - pcr is 2,41 higher in not treated patients ( arm b ) .
in the neo - adjuvant arm a 5/15 ( 33,3% ) patients were staged as pt2a - g2 .
one of 15 patients ( 6,6% ) was staged as pt2a - g3 and resulted positive at qrt - pcr .
four of the 15 patients ( 26,8% ) were pt2b - g2 : 2 resulted negative and 2 positive at qrt - pcr .
five of the 15 ( 33,3% ) patients were pt3b - g2 and 4 resulted negative and 1 positive to qrt - pcr ( see table 2 ) .
the ratio of psa mrna expressing patients according to qrt - pcr in arm a was of 26,6% ( 4/15 ) . in arm b , 4/14 ( 28,6% )
patients were pt2a - g2 of this 3 negative and one positive at qrt - pcr analysis ; 6/14 ( 42,8% ) patients were pt2b - g2 , one negative and 5 positive to qrt - pcr ; 4/14 ( 28,6% ) patients were pt3b - g2 , one negative and 3 positive for psa expression in periprostatic tissues ( see table 3 ) pathological and qrt - pcr data of arm b patients .
overall 64,3% ( 9/14 ) of the patients had evidence of psa mrna expression in arm b , while 26.6% ( 4/15 ) resulted molecularly positive in arm a ( fisher exact test = 0.04 ) .
pathological and molecular finding of the patients who had biochemical recurrence are reported in table 4 .
analysis was performed on patients from arm a or b whether they had or had not cumulative evidence of psa mrna expression by qrt - pcr in lymph nodes and/or lateral surgical margins .
nine of 14 ( 64,29% ) patients expressed psa in arm b and four of 15 ( 26,67% ) in arm a ( see table 5 ) .
continuity adjust chi - square ( a chi - square modified for not continuous variable ) was calculated and resulted in 0.0965 , this value trend to statistical significance .
this results suggests a difference between arm a and b , although not statistically significant ( see table 6 ) . in table 7
value founded show the risk to be positive if not treated with neoadjuvant therapy respect to treated arm ; the risk to have positive surgical margins or nodes to qrt - pcr is 2,41 higher in not treated patients ( arm b ) .
radical prostatectomy is widely performed in early prostate cancer and it may lead to cure as long as the cancer is confined to the prostate and all neoplastic cells are removed . clinical staging is inaccurate and a significative number of patients with prostate cancer defined as ct1 - 2 are found to have positive surgical margins .
this in turn is associated with increased chances of cancer recurrence and progression within 5 years from the surgical resection of the primary tumor .
neoadjuvant hormonal therapy may reduce the rate of margin positiveness , therefore , decreasing the chance of observing a local recurrence after radical prostatectomy .
however , it has been suggested that after neoadjuvant hormonal therapy residual foci of atrophic glands can be difficult to identify with hematoxylin and eosin staining , increasing the possibility that pt may be under staged [ 13 - 15 ] .
clinical randomized studies adopting neoadjuvant therapy before radical prostatectomy have demonstrated a reduction of the risk of leaving positive surgical margins in pt1 - 2 patients at the time of the surgical procedure and this reduced risk is proportional to the length of treatment .
the primary end point of these studies was to assess whether neoadjuvant therapy can increase overall survival .
indeed , there is no published evidence that patients undergoing mab before radical prostatectomy have a clear benefit . at the same time
the use of pcr methods for determination of psa gene expression in surgical periprostatic samples has suggested new staging strategies based on higher sensitivity .
it is now possible to obtain information about presence of prostatic tissue in extra glandular structures with more sensitivity compared with conventional histology .
traditional methods to study protein or gene expression in tumor biopsies include mainly immunohistochemistry and polymerase chain reaction ( pcr ) .
these techniques are not quantitative and allow only a semi quantitative evaluation based on the subjective judgment of the observer , which makes the comparison between samples inaccurate ( unreliable ) .
flow cytometry is highly reliable to determine the levels of expression of a given antigen .
however , it requires tissue desegregation procedures , which can damage cell surface antigens , and can not be performed in case of hypo cellular specimens . to measure of the amount of gene expression
these techniques are cumbersome , time consuming and require multiple manipulations of the samples , increasing the risk of carrying over contamination .
quantitative real - time pcr ( qrtpcr ) allows a highly sensitive measurement of the transcriptional levels of the gene of interest in a few hours with minimal handling of the samples .
in addition , qrt - pcr is stringently sequence dependent because of the labelled probe . therefore , it yields indirect sequence confirmation that the amplified product is the gene of interest .
finally , the utilization of intro - exon junction spanning probes avoids mis - calculation of gene level expression due to genomic dna contamination .
this technique has been mainly used for viral load measurement , but recently it has been employed for eukaryotic studies and in particular analysis of human mrna expression in various clinical and therapeutic conditions . in this study , we adopted qrt - pcr for the sensitive identification of psa mrna level in areas where it should not be present such as positive surgical margins and/or draining lymph nodes .
in our experience , neoadjuvant therapy for 3 months before surgical intervention appears to affect disease staging .
in fact , according to qrt - pcr results , the rate of lymph nodes and/or surgical margins in which psa mrna could be detected was significantly lower ( 26.6% ) in arm a ( mab plus surgery ) as compared to that found in arm b ( surgery alone ) ( 64,3% ) .
all the 5 patients that experienced biochemical evidence of recurrence ( 17,2% ) belonged to arm b. although , psa mrna was detected by qrt - pcr in 60% of patients who experienced biochemical recurrence six of the remaining 8 patients in arm b had also detectable psa mrna .
this finding suggests that identification of psa mrna expression at the surgical margin or draining lymph - nodes is more a marker of the ability of the neoadjuvant therapy to sterilize the patient before surgical intervention than an independent predictor of recurrence .
in fact , the most significant difference in psa mrna expression was noted between the two randomization arms where in the neo - adjuvant protocol only 4 of 15 patients were positive while 9 of 14 were positive in the surgical treatment alone arm ( fisher test p2 value = 0.02 ) .
thus , qrt - pcr appears to yield useful information about the effectiveness of a given therapy and it may be more sensitive , compared to conventional histology , in identifying presence of viable prostate carcinoma cells in surgical resection margins and draining lymph nodes . finally , this study seems to confirm that neoadjuvant therapy with nilutamide 150 mg + buserelin depot for 3 months before surgical intervention induces clinically relevant down staging , at least in the short term . if these results were confirmed in a larger series of patients , we believe that gene expression of psa in surgical periprostatic samples might be taken into consideration as a novel and reliable indicator of minimal residual disease after neoadjuvant therapies . in this case
, the molecular detection of cancer cells might represent an indication to adjuvant treatments in order to sterilize microscopic foci of tumor possibly responsible for local recurrence . | backgroundwe assessed the incidence of micro - metastases at surgical margins ( sm ) and pelvic lymph nodes ( ln ) in patients submitted to radical retropubic prostatectomy ( rp ) after neoadjuvant therapy ( nt ) or to rp alone .
we compared traditional staging to molecular detection of psa using taqman - based quantitative real - time pcr ( qrt - pcr ) never used before for this purpose.methods29 patients were assigned to nt plus rp ( arm a ) or rp alone ( arm b ) .
pelvic ln were dissected for qrt - pcr analysis , together with right and left lateral sm.results64,3% patients of arm b and 26.6% of arm a had evidence of psa mrna expression in ln and/or sm .
17,2% patients , all of arm b , had biochemical recurrence.conclusionsqrt-pcr may be more sensitive , compared to conventional histology , in identifying presence of viable prostate carcinoma cells in sm and ln .
gene expression of psa in surgical periprostatic samples might be considered as a novel and reliable indicator of minimal residual disease after nt . | Background
Materials and methods
Study design and patients' population
Surgical procedure
Pathological staging and molecular analysis
Follow up
Statistical analysis
Results
Pathological and molecular findings
Statistical analysis
Discussion
Conclusions |
PMC4613567 | authors do not report any financial or personal links with other persons or organizations , which might affect negatively the content of this publication and/or claim authorship rights to this publication . | authors discuss methods of echocardiographic diagnosis of the pulmonary sling with stenosis and hypoplasia of the left pulmonary artery and patent arterial duct with massive left - to - right shunt , based on a case of the newborn with resistant to treatment heart failure , with initial diagnosis of patent ductus arteriosus , referred to surgical treatment .
the optimal echocardiographic views permitting establish diagnosis of the pulmonary sling were suggested .
the special attention was paid to high parasternal and suprasternal views visualizing vessels of the upper mediastinum as well as characteristic differences between the normal and pathologic picture .
the typical features of the echocardiogram suggesting pulmonary sling , like the lack of the left pulmonary artery in its expected position , and the abnormal branching pattern of the right pulmonary artery were indicated .
the greatest diagnostic difficulties in visualization of the abnormal route of the left pulmonary artery were related to the presence of air - containing tissues , like lungs and central airways between the ultrasound probe and area of interest .
the other was the masking influence of the large patent arterial duct , that may mimic the left pulmonary artery arising from the pulmonary trunk .
the other entities requiring differentiation with sling , like aplasia of the left lung , the direct or indirect aortic origin of the left pulmonary artery , were discussed .
the role of other visualization technics , like computed 3d tomography , and magnetic nuclear resonance , as well as direct visualization of central airways with bronchoscopy in establishing precise diagnosis were stressed . | Conflict of interest |
PMC4259517 | group ii metabotropic glutamate receptors ( mglu2 encoded by grm2 and mglu3 encoded by grm3 ) are g - protein coupled receptors that inhibit adenylate cyclase ( niswender and conn , 2010 )
. a key function of group ii mglu receptors is to act pre - synaptically to inhibit neurotransmitter release , and mglu2 receptors act primarily as autoreceptors to modulate the release of glutamate ( anwyl , 1999 ; cartmell and schoepp , 2000 ; schoepp , 2001 ) , whilst mglu3 are also glial .
group ii mglu receptors have been implicated in both cognitive and emotional processes , and have been linked with a number of neuropsychiatric conditions , including anxiety , stress - related disorders , schizophrenia and substance misuse ( harrison et al . , 2008 ; markou , 2007 ; moghaddam , 2004 ; swanson et al . ,
nevertheless , the precise role that these receptors play in cognition and emotion remains to be fully established .
genetically modified mice represent an important tool for investigating the role of different receptor subtypes in behaviour ( bannerman , 2009 ) . in order to elucidate the role of group ii mglu receptors in cognition we have studied grm2/3 double ko mice , lacking both mglu2 and mglu3 ( lyon et al . , 2011 ) .
by studying these double ko mice we aimed to circumvent any possible compensatory changes and/or redundancy of function between these two homologous receptor subtypes ( lyon et al . , 2008 ) .
we previously reported that grm2/3 mice exhibit a distinct pattern of cognitive impairments across a range of hippocampus - dependent spatial memory tests ( lyon et al . ,
grm2/3 mice were impaired on appetitively motivated spatial memory tests ( e.g. spatial working and reference memory on the radial maze ) , and on tests which rely on spontaneous exploratory behaviours ( e.g. spatial novelty preference in a y - maze ) , but in contrast they performed as well as wild - type ( wt ) controls on aversively motivated spatial memory paradigms like the morris water maze .
indeed , grm2/3 mice were impaired on an appetitive version of a y - maze spatial reference memory task but normal on a swim - escape version of the same task .
grm2/3 mice were hypoactive when tested in photocell activity cages , leading us to suggest reduced levels of arousal .
furthermore , while injection stress impaired spatial working memory performance on an appetitive task in wts , it actually improved the performance of the grm2/3 mice , consistent with an altered arousal
an important question that remains concerns the relative contributions of mglu2 and mglu3 to these processes .
a number of lines of evidence , mainly from pharmacological studies , have suggested a role for group ii mglu receptors in anxiety ( harvey and shahid , 2012 ; swanson et al . , 2005 ) .
an anxiolytic effect of mglu2/3 agonists has been demonstrated in a number of rodent anxiety paradigms [ e.g. elevated plus maze ( linden et al . , 2005 ) ; fear potentiated startle ( helton et al . , 1998 ) ;
lactate - induced panic ( shekhar and keim , 2000 ) ] , and confirmed in healthy human volunteers in the fear potentiated startle test ( grillon et al . , 2003 ) , in panic disorder patients exposed to co2-induced anxiety ( schoepp et al . , 2003 ) and in patients with general anxiety disorder ( dunayevich et al .
furthermore , mglu2 selective positive allosteric modulators are now being developed which also display anxiolytic / antidepressant properties ( fell et al . , 2011 ) .
interestingly ( although somewhat counter - intuitively ) , mglu2/3 antagonists have also been suggested for the treatment of anxiety ( iijima et al . , 2007 ;
, 2006 ) , although results are more equivocal . against this , studies in genetically modified mice lacking either mglu2
( grm2 mice ) or mglu3 ( grm3 mice ) have reported no anxiety phenotype ( fell et al .
, 2011 ; linden et al . , 2005 ; morishima et al . ,
one possible explanation for these null results is compensatory changes in gene expression and/or redundancy of function between these two homologous receptor subtypes for the anxiety phenotype in the single ko lines ( lyon et al . , 2008 ) , whereas pharmacological ligands will activate or inhibit both mglu2 and mglu3 .
therefore , in the present study we assessed anxiety in grm2/3 mice ( thus circumventing the possibility of compensatory changes / redundancy of function ) , using ethologically based , unconditioned tests , as part of a more extensive test battery investigating sensorimotor , motivational and emotional behaviours .
this builds upon and extends our previous work characterising hippocampus - dependent cognitive behaviours in these mice ( lyon et al . , 2011 ) . for comparison
, we also evaluated each of the single kos ( grm2 and grm3 mice ) against their respective wt littermate controls , using the same battery of tests .
in addition , we assessed the grm2 and grm3 mice on key spatial memory tests employed in our previous studies with grm2/3 mice ( lyon et al . , 2011 ) .
thus , spatial working / short - term memory was assessed in grm2 and grm3 mice using both appetitively motivated , rewarded alternation on the t - maze and a spontaneous , exploratory driven spatial novelty preference task in the y - maze .
long - term spatial memory was assessed using both appetitive and aversive / swim - escape versions of the y - maze reference memory task .
adult male mice ( > 2.5 months ) on a c57bl/6j background were obtained from glaxosmithkline , harlow , uk .
single grm2 and grm3 mice , and their respective wt littermate controls , were generated as previously described ( corti et al .
separate lines of wt and grm2/3 mice were produced as in lyon et al .
group sizes varied between experiments and lines , and are given in the relevant tables and figures .
mice were 2.54 months old at the start of the anxiety / sensorimotor test battery , and 45.5 months old at the end of this test battery .
they then began spatial memory testing , and were 78 months old at the end of the spatial memory tests .
animals were housed in groups of 24 and kept on a 12-h light dark cycle ( lights on at 07:00 and off at 19:00 ) , with all testing conducted during the light phase . for the battery of anxiety and motor tests , and for the aversively motivated tasks ,
for all appetitively motivated tasks , mice were maintained on a restricted feeding schedule at not less than 90% of their free - feeding weight . for several days
prior to the start of each appetitive test , mice were habituated to the maze and to drinking the sweetened condensed milk ( diluted 50:50 with water ) that was used as a reward .
home office project and personal licenses held by the authors , and the study was approved by the local ethics committee .
genotypes of all animals were confirmed by the mouse genotyping group at gsk , harlow .
grm2 fragments were amplified using forward primer ( ctg tct ctc tat ctc tct gc ) and reverse primer ( tgt gtg tgt gta aca tga tgg ) .
pcrs were performed with a denaturing step at 95 c ( 15 min ) then 94 c ( 30 s ) , followed by annealing at 60 c ( 90 s ) and extension at 72 c ( 1 min ) .
after 35 cycles , the reaction was maintained at 72 c for a further 10 min , and the products resolved on a 2% agarose gel .
the wt product was a single 900 bp band , and the ko product a 450 bp band .
grm3 genotyping yielded a 2 kbp product in wt and a 500 bp product in ko .
the disparity in size prevented the two fragments from being amplified in a single multiplex pcr .
two separate pcrs were therefore conducted , one for the wt product ( forward primer : gtt tct agg act tcc tat gg ; reverse primer : aac gat gct ctg aca aac tcc ) and a second for the ko product ( forward primer : cgt acg tcg gtt gct atg g ; reverse primer : gtc aga tat agt gag agc agg ) .
both pcrs were performed with a denaturing step at 95 c ( 15 min ) then 94 c ( 30 s ) , followed by annealing at 56 c ( 90 s ) and extension at 72 c ( 150 s ) .
after 35 cycles , the reaction was maintained at 72 c for 10 min .
the absence of grm2 mrna in founding members of the grm2 population was confirmed by the use of in situ hybridisation histochemistry ( yokoi et al . , 1996 ) , as was the absence of grm3 mrna in the founding members of the grm3 population ( corti et al . , 2007 ) .
the order of testing was as follows : black & white alley ; neophagia ( first version ) ; open field ; horizontal bar ; rotarod test ; multiple static rods ; elevated plus maze , neophagia ( second version ) ; spatial novelty preference test ; locomotor activity ; appetitive spatial working memory t - maze , appetitive / aversive spatial reference memory ( srm ) y - maze ( with test order counterbalanced for the two versions of the reference memory y - maze task ) .
comparisons of grm2/3 , grm2 and grm3 mice against their respective wt control groups were conducted separately , and by different experimenters .
all behavioural testing took place during the light phase of the day between 9.00 am and 6.00 pm .
data from the grm2/3 mice on spontaneous locomotor activity and the spatial memory tests has been published previously ( lyon et al . , 2011 ) .
anxiety was assessed using a number of ethological , unconditioned tests , which assessed approach / avoidance conflict in these mice . the black & white alley ( 120 cm 9 cm , 29 cm high walls ) was painted black from one end to the middle , and white from the other end to the middle .
the mouse was placed into the alley at the end of the black arm and observed for two minutes .
variables measured were latency to enter the white arm ( which is regarded as more anxiogenic than the black arm ) , total amount of time spent in the white arm , and number of crossings between the two arms .
this test exploits the natural wariness of rodents to consume novel foodstuffs . to overcome potential floor / ceiling effects , two versions of the task
were performed with increasing anxiogenic character . in each , the mouse was placed in a novel environment ( underneath an upturned plastic jug / in a closed arm on an elevated y - maze ) and presented with a completely novel food ( sweetened condensed milk / noyes reward pellets ) .
all mice were food deprived the night before for approximately 15 h. variables measured were latency to contact , and then to consume , the novel food .
a maximum of 3 min was allowed for each trial . the elevated plus maze consisted of four 27 cm long , 8 cm wide alleys connected by a central platform to form a plus - shape .
two of the alleys had high ( 30 cm ) walls , and two had low ( 0.5 cm ) walls .
the high walled alleys ( closed arms ) are presumed to be less anxiogenic than the low walled ones ( open arms ) , while the central platform ( junction ) ( 12.5 cm by 6 cm ) remains relatively neutral .
variables measured were latency to enter a closed arm , latency to enter an open arm , percentage of time spent in each area of the maze ( open vs closed vs junction ) , and total distance covered within the maze .
the open field comprised a brightly illuminated circular arena ( a white metallic drum ) , with diameter 60 cm .
the centre of the open field was defined as a circular area with an outer edge 19 cm from the edge of the arena .
the mouse was placed inside the arena facing the sidewall and electronically tracked for five minutes .
variables measured were percentage of time in the central area versus the outer area , and total distance covered in the arena .
the horizontal bar was 0.2 cm in diameter , 38 cm long , and made of metal .
it was attached to two wooden supports , and positioned 49 cm above the padded bench surface .
the mouse was placed with its front paws touching the bar and then quickly released , so that it grasped the bar at its central point .
performance was scored based on the length of time that the mouse held onto the bar : a score of 5 was given for holding on for 30 s or for traversing the bar to reach one of the wooden supports ; 4 for holding on for 2130 s ; 3 for 1120 s ; 2 for 610 s ; and 1 for 05 s. the rotarod was a 3.5 cm wide ( diameter ) , 4.5 cm long knurled rod , attached at each end to 30 cm diameter flanges .
if the mouse was still in place after ten seconds , the rotation of the rod was gradually accelerated at a rate of 20 rpm .
the latency to fall from the rod and the speed of rotation at which the mouse fell were recorded .
the multiple static rods consisted of five 60 cm long wooden rods attached perpendicularly to a supporting beam .
the rods were arranged in order of decreasing diameter : rod 1 was 3.3 cm wide ; rod 2 , 2.7 cm ; rod 3 , 2.1 cm ; rod 4 , 1.4 cm ; rod 5 , 0.8 cm .
the mouse was placed 2 cm from the distal end of the rod , facing away from the supporting beam , and the time taken for the mouse to turn 180 to face the supporting beam , recorded .
these orientation times are sensitive to small deficits in motor co - ordination . the time taken for the mouse to run 60 cm along the beam to reach the refuge of the support ( transit time
the mouse was placed on the largest diameter rod first , continuing with the next largest on each subsequent trial .
spontaneous locomotor activity was measured during a two - hour period in the light phase ( 12 pm2 pm ) .
all mice were placed singly into a transparent plastic cage ( 26 cm 16 cm 17 cm ) with a ventilated lid .
two infrared photocell beams crossed the cage 1.5 cm above the floor , with each beam 7 cm from the centre of the cage .
mice were left in a quiet room with the lights on for 2 h. the number of beam breaks made by each mouse was recorded in 24 bins of 5 min .
single ko and wt mice were compared on a spontaneous , spatial novelty preference task in which behaviour is driven , not by an overt unconditioned stimulus ( us ; e.g. a food reward ) , but instead relies upon animals ' natural exploratory drive .
this task therefore provides a non - aversive experimental context but performance does not rely on the motivating or rewarding effects of food .
we previously showed that grm2/3 double ko mice exhibit a reduced spatial novelty preference on this task ( lyon et al . , 2011 ) , and we repeated the test in grm2/3 mice here .
the y - maze was made from transparent perspex , and consisted of three 30 cm long , 8 cm wide arms with 20 cm high walls , connected by a central junction .
each mouse was assigned two arms ( the start arm and the other arm ) to which they were exposed during the first phase of the task ( the exposure phase ) .
allocation of arms to specific spatial locations was counterbalanced within each genotype . during the 5-min exposure phase , the entrance to the third , novel , arm was closed off by the presence of a large perspex block .
the mouse was placed at the end of the start arm , facing the experimenter , and allowed to explore the start arm and the other arm freely for five minutes , beginning as soon as the mouse left the start arm .
the number of entries into each arm and the length of time spent there were recorded . at the end of the five minutes
, the mouse was removed from the maze and returned to the home cage for one minute . during this time
, the perspex block closing off the novel arm was removed and the sawdust redistributed throughout the maze to minimise the use of odour cues .
the mouse was then returned immediately to the start arm , facing the experimenter , for the 2-min test phase .
this consisted of two minutes free exploration during which the mouse could enter all 3 arms , beginning as soon as the mouse left the start arm .
the amount of time that the mouse spent in each arm , and the number of entries into each arm , were recorded , during both the exposure and the test phase . for the test phase ,
a discrimination ratio [ ( novel arm)/(novel + other arm ) ] was calculated both for number of arm entries and time spent in each arm .
previous work in this laboratory has demonstrated that wt mice display a marked preference for the novel arm during the test phase , and that this preference relies on extramaze cues .
this preference for the novel arm is abolished in mice with cytotoxic hippocampal lesions ( sanderson et al . , 2007 ) .
our previous study demonstrated that spatial working memory on the elevated t - maze is impaired in grm2/3 mice ( lyon et al . , 2011 ) .
the t - maze consisted of a wooden start arm ( 47 10 cm ) and two identical goal arms ( 35 10 cm ) , surrounded by a 10 cm high wall .
a food well was positioned 3 cm from the end of each goal arm , and the whole maze was surrounded by prominent distal extramaze cues .
mice received five trials per day for six days , with an iti of approximately forty minutes .
for analysis and presentation , data are presented as 3 blocks of 10 trials , having combined data from consecutive days .
each trial consisted of a sample run followed by a choice run . on the sample run
, mice were forced either left or right ( chosen pseudorandomly with equal numbers of left and right turns , and no more than three consecutive turns in any direction ) by the presence of a large wooden block , closing off one of the goal arms . at the end of the goal arm the mouse collected a reward of 0.1 ml sweetened condensed milk .
the block was then removed and the mouse placed back in the start arm , facing the experimenter , for the choice run .
the mouse could now select either goal arm but was rewarded only for choosing the arm that had not been visited on the sample run , i.e. , it was rewarded for alternating ( non - matching to place ) .
the interval between the sample run and the choice run was approximately 5 s. the number of correct alternations was recorded for each mouse . in addition , we recorded latencies for both the sample runs and the choice runs .
we recorded the latency of the mice to run ( i ) from the beginning of the start arm to the food well on the sample trial , and ( ii ) from the beginning of the start arm until making a choice into one of the goal arms on the choice trial .
spatial working memory performance on the t - maze is dependent on the hippocampus ( deacon et al . ,
the aim of the experiment was to compare performance of wt and single ko mice on appetitively and aversively motivated versions of the y - maze spatial reference memory task , with the order of testing and the experimental rooms in which the tests were performed ( and therefore the spatial cues available ) , fully counterbalanced in order to control for practice effects or differences in the salience of the available spatial cues ( see also lyon et al . , 2011 ) .
thus , 50% of wt and 50% of ko mice first performed the appetitive y - maze srm task in room a. these animals then performed an aversively motivated y - maze srm task in room b. conversely , the remaining 50% began with the aversively motivated y - maze task in room a , followed by the appetitively motivated y - maze task in room b. we have previously shown that the grm2/3 double ko mice are impaired on the appetitive , but not the aversive , version of the task ( lyon et al . , 2011 ) .
the elevated y - maze consisted of three identical wooden arms , each 50 cm long by 9 cm wide , with a low wall ( 0.5 cm ) , connected by a central polygonal platform ( 14 cm diameter ) .
each mouse was assigned a goal arm , defined by its position relative to extramaze spatial cues , which was baited with 0.1 ml sweetened condensed milk on all trials . on each trial ,
the mouse was placed at the end of one of the two non - baited arms ( the start arm ) , facing the experimenter ; 50% of trials began from the arm to the right of the goal arm , and 50% from the arm to the left .
having been placed at the end of the start arm , the mouse was allowed to choose one of the remaining arms .
if it chose the goal arm , it was allowed to consume the milk reward before being returned to the home cage .
mice that chose incorrectly were returned to the home cage immediately . to prevent the use of intramaze cues ,
mice received ten trials per day for 6 days , with an inter - trial interval ( iti ) of approximately five minutes .
the last block of ten trials was conducted using post - choice reinforcement : the condensed milk reward was added to the food well only after the mouse had made a choice , to ensure that the animals were not locating the milk by virtue of its odour .
previous work in this laboratory using the same maze has demonstrated that this task is hippocampus - dependent in mice ( deacon et al . , 2002 ) .
the y - maze was made from transparent perspex , and consisted of three 30 cm long , 8 cm wide arms with 20 cm high walls , connected by a central junction .
the maze was filled with water ( temperature 21 c 1 c ) to a depth of approximately 12 cm which obliged the mice to swim .
mice could escape from the water by climbing onto a platform ( 8 cm by 8 cm ) hidden approximately 1.5 cm below the water surface in one of the arms of the maze .
mice received five trials per day in this deep water escape y - maze task for six days . on each trial
the mouse was allowed 90 s to find the platform ; any that failed to do so were guided there by the experimenter .
mice were allowed to rest on the platform for 30 s before being transferred to a heated cage . on day seven ( 24 h after training trial 30 ) ,
a transfer test was performed , analogous to that used in the water maze , in order to assess the extent of any spatial memory for the platform location .
the platform was removed from the maze and the mouse allowed to swim freely for 30 s. time spent searching in each arm was recorded .
previous work in this laboratory has confirmed that the swimming y - maze task , like the appetitive y - maze , is hippocampus - dependent ( unpublished ) .
when comparing the performance of two groups of mice , parametric data were analysed using t - tests , or with a repeated measures anova if there was a within - subjects factor . where data violated the assumptions of normality or equality of variance , which are required for parametric analysis ( i.e. the data were non - parametric ) , then mann whitney u - tests were performed for simple group comparisons with no within - subjects factors . for non - parametric data sets which included both between and within - subjects factors ( e.g. multiple static rods data collected across different sized rods )
, the data were first transformed using a log10 transformation to satisfy the assumptions of normality and equality of variance , and then analysed using a two way , repeated measures anova .
all animals displayed normal appearance and no gross abnormalities in home - cage behaviours ( assessed during short , non - systematic observations by the experimenter during the light period ) .
body weight of grm2/3 and grm3 mice did not differ from their respective wt controls ( grm2/3 , n = 20 , 28.0 g 0.5 g vs. wt , n = 19 , 28.5 g 0.3 g ; t < 1 ; p > 0.20 ; grm3 , n = 15 , 26.7 g 0.3 g vs. wt ,
n = 15 , 27.1 g 0.5 g ; t < 1 ; p > 0.50 ) .
grm2 mice were slightly , but significantly , lighter than their wt littermates at the start of the test battery ( grm2 , n = 14 , 27.1 g 0.4 g , vs. wt , n = 15 , 28.8 g 0.6 g ; t(27 ) = 2.23 ; p < 0.05 ) .
however , this difference was short - lasting and no longer evident at the time of rotarod testing .
overall the tests revealed no major differences in emotionality between grm2/3 and wt mice ( tables 1 and 4 ) .
similarly , no consistent differences in anxiety - like behaviours were evident between either grm2 ( tables 2 and 5 ) or grm3 mice ( tables 3 and 6 ) , and their respective controls .
grm3 mice did demonstrate shorter latencies to enter the open arms in the elevated plus maze ( u = 58 ; p < 0.05 ; table 3 ) , and to approach ( u = 65.5 ; p < 0.05 ) and eat ( u = 39.0 ; p < 0.01 ) the novel food in the second neophagia test compared to controls ( table 6 ) , potentially consistent with reduced anxiety in these animals . a similar , but milder , profile was also evident in the open field test , in which there was a trend for grm3 mice to be faster to enter the central area of the arena ( t(28 ) = 1.89 ; p = 0.07 ; table 3 ) .
however , no differences were found in either % time in open arms ( elevated plus maze , table 3 ) , or latencies to consume after contact ( difference score ; latency to eat minus latency to contact ; neophagia tests , table 6 ) , or total time spent in the central area ( open field test , table 3 ) , which are more widely used measures of anxiety - like behaviours in these tests .
observation of the mice in their homecages revealed no noticeable differences in motor function between the groups .
however , grm2/3 mice were significantly impaired in tests assessing motor coordination ( table 7 ) . on the accelerating rotarod
, grm2/3 mice fell from the rod sooner ( t(30 ) = 2.33 ; p < 0.05 ) , while it was rotating at a lower speed ( t(30 ) = 2.29 ; p < 0.05 )
. this can not be attributed to differences in body weight ( see above ) .
grm2/3 mice were also impaired on the multiple static rods test of motor coordination , taking longer ( i ) to turn around on the rod and ( ii ) to traverse its length ( table 7 ) .
performance in the multiple static rods test was analysed using separate two - way rm - anova for orientation times ( ots ) and transit times ( tts ) .
average ots and tts for rods 13 were used in each analysis ( as values for individual rods were very similar ) together with the individual values for rods 4 and 5 . ot data
were subjected to a log10 transformation prior to analysis to ensure that the data satisfied the criteria for parametric analysis .
two - way rm - anova for the transformed ot data revealed an effect of group ( f(1,30 ) = 8.03 ; p < 0.01 ) and an effect of rod ( f(2,60 ) = 66.20 ; p
< 0.01 ) , plus a group by rod interaction ( f(2,60 ) = 7.76 ; p < 0.01 ) .
duncan 's multiple pairwise comparisons revealed that the performance of grm2/3 double ko and wt mice differed significantly on rod 5 ( p < 0.05 ) .
tt data were also log10 transformed , and two - way rm - anova again revealed an effect of group ( f(1,30 ) = 5.12 ; p < 0.05 ) , an effect of rod ( f(2,60 ) = 85.08 ; p
< 0.01 ) , and a group by rod interaction ( f(2,60 ) = 8.07 ; p < 0.01 ) .
duncan 's multiple pairwise comparisons confirmed that , in common with the ot data , the performance of the two groups differed significantly on rod 5 ( p < 0.05 ) .
overall , the motor tests reveal a relatively mild , but significant , motor deficit in grm2/3 mice .
in contrast , there were no differences between either grm2 or grm3 mice and their wt control mice on any of the motor tests ( tables 8 and 9 ) .
we have reported previously that spontaneous locomotor activity was reduced in the grm2/3 mice in photocell activity cages ( lyon et al . , 2011 ) .
assessment of grm2 mice in the same apparatus revealed an interesting pattern of results ( fig .
the grm2 mice were initially hyperactive but then their activity levels fell below those of the wt mice such that the kos were hypoactive during the second hour of recording .
anova revealed a significant genotype block interaction ( f(11,297 ) = 2.61 ; p < 0.01 ) .
simple main effects analysis revealed significant hyperactivity in the ko mice during the first 10 min time bin , but then hypoactivity in bins 79 , 11 and 12 ( 60120 min ) ( all p < 0.05 ) .
overall , grm3 mice did not differ significantly in activity levels from their wt littermates during the 2 h test ( main effect of genotype : f < 1 ; p > 0.50 ) .
however , there was a significant genotype by time bin interaction ( f(11,308 ) = 2.07 ; p < 0.05 ) , which seemed to reflect a slower decrease in activity levels in the grm3 mice compared to the controls during the second hour of testing , although simple main effects analysis showed that there were no significant effects of genotype for any time bin individually ( all p > 0.05 ) . in agreement with our previous study ( lyon et al . , 2011
) , we again found that grm2/3 mice exhibited impaired short - term spatial memory on the exploratory driven , spatial novelty preference task ( fig .
2a ) . during the exposure phase , both wt and grm2/3 mice spent a similar amount of time exploring the other
( to - be - familiar ) arm ( wt = 120.8 8.4 , grm2/3 mice = 114.4 5.2 s ; t <
however , grm2/3 mice made significantly fewer arm entries ( start and other arms combined ) during the exposure phase ( wt = 44.3 2.2 , grm2/3 mice = 30.5 2.5 ; t(20 ) = 4.13 ; p < 0.01 ) , thus confirming a hypoactive phenotype in these mice ( lyon et al .
during the subsequent test phase , both groups showed a significant preference for the previously unvisited ,
a discrimination ratio was calculated [ ( novel arm)/(novel + other arm ) ] for time spent in arms during the test phase , and statistical analysis revealed that the preference for the novel arm was significantly above chance for both genotypes ( one group t - tests conducted against a single value of 0.5 which corresponds to chance performance ; p 's < 0.01 ) .
importantly , the wt mice displayed a significantly greater novelty preference than the grm2/3 mice ( effect of genotype - t(20 ) = 2.23 ; p < 0.05 ) .
grm2/3 mice also made significantly fewer arms entries ( all three arms combined ) during the test phase ( wt = 17.2 1.1 ; grm2/3 mice = 12.1 0.9 ; t(20 ) = 3.46 ; p < 0.01 ) .
there was no difference between grm2 mice and their wt controls on the spatial novelty preference task ( fig .
arm did not differ between wt and grm2 mice ( wt = 85.1 5.3 s , grm2 mice = 93.8 3.9 s ; t(27 ) = 1.30 ; p = 0.20 ) .
a similar number of arm entries ( start and other arms combined ) was also evident during the exposure phase ( wt = 27.6 1.3 s , grm2 mice = 28.1 1.6 s ; t < 1 ; p > 0.80 ) . during the test phase
, both groups showed a significant preference for the novel arm ( one group t - tests conducted against a single value of 0.5 which corresponds to chance performance ; p 's < 0.01 ) , and this did not differ between the genotypes ( t
there was no effect of genotype on number of arm entries ( all three arms combined ) made during the test phase ( wt = 13.5 0.6 , grm2 mice = 14.5 0.8 ; t < 1 ; p > 0.30 ) .
similarly , the grm3 mice did not differ significantly from their wt littermates ( fig .
2c ) during the exposure phase in terms of time exploring the other arm ( wt = 100.3 6.5 s , grm3 = 100.5 6.8 s ; t < 1 ; p > 0.70 ) , and total number of arm entries ( wt = 24.7 7.5 s , grm3 = 30.9 9.5 s ; t(28 ) = 1.95 ; p = 0.06 ) . during the test phase , a significant preference for the novel arm ( one group t - tests conducted against a single value of 0.5 which corresponds to chance performance ;
p 's < 0.01 ) was again shown by both groups and the discrimination ratio for time in arms did not differ between genotypes ( t < 1 ; p > 0.80 ) .
the number of arm entries during the test phase was also comparable between genotypes ( wt = 13.7 3.1 , grm3 = 13.9
2.9 ; t < 1 ; p > 0.80 ) .
spatial working memory was assessed during non - matching to place testing ( rewarded alternation ) on the elevated t - maze .
we have previously shown that grm2/3 mice display a robust and enduring impairment on this task ( lyon et al . , 2011 ) .
in addition , these double ko mice exhibited shorter latencies to complete both the sample and the choice runs of the task .
single grm2 mice were impaired in terms of choice accuracy on the spatial working memory t - maze task across the three blocks of testing ( main effect of genotype
f(1,27 ) = 4.24 ; p < 0.05 ; fig .
furthermore , they also had shorter run latencies than the wt controls on both the sample ( wt = 14.9 2.9 s , grm2 mice = 3.6 0.3 s ; main effect of genotype
f(1,27 ) = 5.36 ; p < 0.05 ) and choice runs ( wt = 11.7 2.0 s , grm2 mice = 3.4 0.5 s ; main effect of genotype
grm3 mice were impaired on the first block of spatial working memory testing , but improved across the three blocks such that they were actually more accurate than wt by the end of testing ( fig .
there was a significant genotype by block interaction ( f(2,56 ) = 6.28 ; p < 0.01 ) .
simple main effects analysis revealed that grm3 mice were impaired compared to wt controls during the first 10 trial block , but exhibited better performance during the last block compared to their littermate controls ( all p < 0.05 ) .
grm3 deletion had no effect on the run latencies for either the sample ( f < 1 ; p > 0.90 ) or the choice runs ( f < 1 ; p > 0.80 ) .
we reported previously that grm2/3 mice were impaired on an appetitively motivated , spatial reference memory task on the elevated y - maze ( lyon et al .
, neither the single grm2 nor single grm3 mice were impaired on this task ( fig .
although there was some suggestion that the grm2 mice performed significantly less accurately on the first block of testing , anova revealed neither a main effect of genotype ( f < 1 ; p > 0.40 ) , nor a significant genotype by block interaction ( f(5,125 ) = 2.08 ; p = 0.07 ) , although there was a significant main effect of block ( f(5,125 ) = 55.71 ; p < 0.01 ; fig .
4a ) . because the test order by block interaction was also significant ( f(5,125 ) = 3.32
; p < 0.01 ) , a further analysis was performed , restricted to just those animals that were experimentally nave with respect to the appetitive y - maze ( i.e. those mice that had not previously been trained on the aversive , swim - escape task ) .
again , there was a main effect of block ( f(5,60 ) = 33,91 ; p < 0.01 ) , but neither a main effect of genotype ( f < 1 ; p > 0.50 ) , nor a genotype by block interaction ( f(5,60 ) = 1.11 ; p = 0.37 ) . for the grm3 mice , there was no sign of any spatial reference memory impairment .
anova revealed a main effect of block ( f(5,130 ) = 30.93 ; p < 0.01 ) , and a test order by block interaction ( f(5,130 ) = 3.05 ; p < 0.05 ) , but no effect of genotype ( f < 1 ; p > 0.80 ) , nor genotype by block interaction ( f < 1 ; p > 0.50 ; fig .
the same result was obtained when the analysis excluded those mice that had previously been trained on the aversive , swim - escape task ( main effect of block
f(5,70 ) = 13.28 ; p < 0.01 ; no effect of genotype
f(1,14 ) = 2.29 ; p = 0.15 ; no genotype by block interaction
the performance of the animals on the first block of testing in these experiments was better than expected , and better than we have experienced previously ( e.g. bannerman et al .
it did not reflect an innate bias to one of the arms of the maze .
target arm allocation between the three arms of the maze was counterbalanced within each genotype , and analysis of the data with target arm included as a between subjects factor generated no main effect of target arm ( grm3 cohort : f(2,24 ) < 1 ; p > 0.60 ; grm2 cohort : f(2,23 ) < 1 ; p > 0.80 ) nor genotype by target arm interactions ( grm3 cohort : f(2,24 ) < 1 ; p > 0.90 ; grm2 cohort : f(2,23 ) < 1 ; p > 0.60 ) .
it is also not the case that the mice were solving the task by smelling the food reward . during the sixth block of y - maze testing the milk reward
was only delivered after the mice had made a choice ( post - choice baiting ) .
performance remained at a high level on these trials , confirming that the mice were not solving the task using the smell of the reward .
furthermore , the entire maze was rotated periodically to prevent the mice from using intramaze cues .
initially high performance levels also did n't appear to reflect any effect of the prior training on the aversive swim / escape y - maze task , as performance levels in the two sub - populations of mice ( y - maze nave vs. y - maze experienced ) were very similar .
thus , it seems most likely that there was within - session learning during the first 10 trials of training .
importantly , analysis of just the very first training trial for each animal in the two studies was not significantly above chance ( 62.7% correct ; binomial test : p > 0.05 ) .
both grm2 and grm3 mice acquired the aversive , swim / escape y - maze reference memory task as well as their respective controls , consistent with our previous study in the grm2/3 mice which had also revealed no impairment ( lyon et al . , 2011 ) .
f < 1 ; p > 0.40 ; genotype by block interaction
4b ) , and showed an equivalent preference for the target arm during the 30 s probe test at the end of testing ( genotype comparison ; t(27 ) = 1.54 ; p = 0.14 ; % time in target arm : wt = 55.5 3.2 , one group t - test conducted against a single value of 33.3% which corresponds to chance performance ; t(14 ) = 6.91 ; p < 0.01 ; grm2 mice = 62.2 2.9 , one group t - test conducted against a single value of 33.3% which corresponds to chance performance ; t(13 ) = 9.96 ; p < 0.01 ; fig .
the same result was obtained when mice that had already performed the appetitive task were excluded ( data not shown ) . likewise , grm3 mice were normal during acquisition ( main effect of genotype
f < 1 ; p > 0.50 ; genotype by block interaction
4e ) , and spent a similar amount of time in the arm that had previously contained the platform during the probe test ( genotype comparison ; t < 1 ; p > 0.50 ; % time in target arm : wt = 57.3 2.7 , one group t - test conducted against a single value of 33.3% which corresponds to chance performance ;
t(14 ) = 8.99 ; p < 0.01 ; grm3 mice = 60.6 4.0 , one group t - test conducted against a single value of 33.3% which corresponds to chance performance ; t(14 ) = 6.86 ; p < 0.01 ; fig .
again , this was true irrespective of whether the analysis was limited to y - maze nave subjects or also included mice that had already completed the appetitive task ( data not shown ) .
in the present series of experiments we have extended our behavioural characterisation of grm2/3 mice ( lyon et al . , 2011 ) and , in addition , investigated the behavioural phenotype of single grm2 and grm3 mice to extend earlier work by other groups ( fell et al .
, 2011 ; fujioka et al . , 2014 ; lainiola et al . , 2014 ;
we found no consistent effect on anxiety in either the double or single ko mice .
the grm2/3 mice were impaired on a novelty preference test assessing short - term spatial memory , but this phenotype was absent in both grm2 and grm3 mice .
similarly , while we have shown previously that grm2/3 mice are impaired at acquiring an appetitively ( but not aversively ) motivated spatial reference memory y - maze task ( lyon et al . , 2011 ) , we found no such impairment in either of the single ko lines .
in contrast , spatial working memory ( rewarded alternation ) testing on the elevated t - maze revealed a clear impairment in the grm2 mice throughout testing ( although again the effect was not as big as in the double kos ) , whereas grm3 mice exhibited a biphasic effect ( initially impaired but performing better than controls by the end of training ) . a biphasic effect on activity levels
was also seen for the grm2 mice during testing in photocell activity cages . the grm2/3 mice
( but neither single ko line ) were also impaired on tests of motor co - ordination , demonstrating that their behavioural phenotype extends beyond the domain of hippocampus - dependent spatial memory ( see also morishima et al . , 2005 ) .
these findings have implications for the roles which the group ii metabotropic glutamate receptors , individually and collectively , play in anxiety , cognition , and their interaction .
a key finding from these studies is that the behavioural phenotype in the double ko mice is more pronounced than the phenotype in either of the single lines . in the present study
there was no effect on short - term spatial memory in this task in either single line ( see also lainiola et al .
, 2014 for grm3 mice ) , whereas there was a clear deficit in the double kos ( see also lyon et al . , 2011 )
similarly , whereas grm2/3 mice were impaired on tests of motor coordination such as the accelerating rotarod , there was no sign of a phenotype in the single ko lines in our studies .
the absence of a deficit on the accelerating rotarod test in the grm2 mice in the present study is at odds with a previous paper reporting a motor deficit in these animals ( morishima et al . , 2005 ) , although our demonstration of impaired motor performance in the grm2/3 mice is at least consistent with a role for group ii metabotropic receptors in this behaviour .
we can not at this stage determine whether this impairment in our grm2/3 mice is due to a fundamental problem with motor function ( e.g. motor coordination ) , or whether the deficit reflects an altered reaction to the novelty and/or arousal - inducing properties of the task in the ko mice ( see section 4.3 below for further discussion of a potential role for these receptors at the interface between arousal and behavioural performance ) .
the latter possibility could potentially explain the different outcomes with the grm2 mice reported here and elsewhere ( morishima et al . , 2005 ) .
future studies could examine rotarod performance over repeated testing sessions in order to examine motor learning , although it is worth pointing out that the previously reported deficit in the grm2 mice on the rotarod test was present throughout repeated testing , and both wt and kos appeared to improve their performance at a similar rate ( morishima et al . , 2005 ) .
furthermore , whereas we have shown previously that grm2/3 mice are robustly impaired on appetitively motivated tests of long - term , associative spatial memory ( e.g. spatial reference memory on the y - maze ( lyon et al . , 2011 ) ) , in the present study we found no such effects in either grm2 or grm3 mice . even when phenotypes were evident in the grm2 and grm3 mice ( e.g. appetitively motivated spatial working memory on the elevated t - maze ) , the magnitude of the effect was much smaller than that seen with the grm2/3 mice ( see lyon et al . , 2011 ) .
these results suggest that there is some redundancy of function between the two group ii metabotropic glutamate receptor subtypes .
it is also possible that there are compensatory changes in the respective single lines which mask the effects of genetically ablating one receptor subtype .
( 2008 ) showed that mrna expression of the remaining group ii receptor subtype is upregulated in the dentate gyrus subfield of the hippocampus of grm2 and grm3 mice .
moreover , recent data show that striatal dopamine is increased in grm2/3 mice but not in grm2 or grm3 mice ( lane et al . , 2013 ) , and it is tempting to speculate that this neurochemical difference may , at least in part , constitute a correlate of the emergent behavioural phenotype seen here in the double ko mice .
of course it is also important to point out that although there is some similarity and overlap between the expression profiles for grm2 and grm3 , there are also considerable differences in the regional , cellular and sub - cellular distributions of these two receptor subtypes ( ferraguti and shigemoto , 2006 ; harrison et al . , 2008 ; spooren et al . , 2003 ) .
this could explain some of the differences in the phenotypes of the single grm2 and grm3 mice , but it also begs the question as to how these distinct expression profiles could result in additive effects in behaviour ( and possible redundancy of function ) , and how they could support compensatory mechanisms leading to the rescue of behavioural phenotypes in the single ko lines .
the complex behaviours studied here are likely to be supported by neural circuits and networks encompassing a number of brain regions , and it is possible that grm2 and grm3 could contribute to the performance of a particular behaviour by acting at different loci .
moreover , it is not inconceivable that an increase in the expression of one receptor subtype at one locus could compensate for the absence of the other at a different site .
however , it is also possible that these compensatory changes could occur in a brain region where both receptor subtypes are expressed such as the hippocampus ( lyon et al . , 2008 ) . in this regard
it is worth pointing out that many of the behaviours that we investigated in the present study , including tests of both spatial memory and anxiety , depend on the hippocampus ( see bannerman et al . , 2004 for review ) . in agreement with previous reports ( fell et al .
, 2011 ; fujioka et al . , 2014 ; linden et al .
2005 ) , we found no consistent evidence for altered anxiety in mice lacking either mglu2 or mglu3 alone , suggesting that deletion of just one of the two receptor subtypes is not sufficient to produce robust effects on anxiety - like behaviours .
one possible explanation for these null results is either the compensatory changes and/or redundancy of function between these two homologous receptor subtypes in the single ko lines , as discussed in the previous paragraph .
a main aim of the present study therefore was to address this issue by assessing anxiety in the grm2/3 mice .
we assessed anxiety in grm2/3 mice using several different ethological , unconditioned tests of anxiety which generate an approach avoidance conflict ( barkus et al .
we found no effect on anxiety on the elevated plus maze , open field , black & white alley or neophagia ( novelty suppressed feeding ) tests .
this suggests that previous failures to see effects on anxiety with the single group ii mglu ko lines are not simply due to compensatory changes and/or redundancy of function between mglu2 and mglu3 in these lines .
notably , these null results for anxiety in the ko mice contrast with numerous studies which have reported anxiolytic effects with mglu2/3 agonists ( dunayevich et al .
, 2008 ; grillon et al . , 2003 ; helton et al . , 1998 ; linden et al . , 2005 ;
importantly , these anxiolytic effects of mglu2/3 agonists have been observed in both human and rodent studies , and so the lack of an anxiety phenotype in the ko mice is unlikely to be due simply to species differences .
furthermore , the anxiolytic effects of the group ii agonists are absent or reduced in single grm2 and grm3 mice ( linden et al . , 2005 ) , confirming that the drugs are indeed acting through both of the group ii metabotropic glutamate receptor subtypes to reduce anxiety .
there are a number of possible explanations for the lack of anxiety phenotypes in the present study despite the clear anxiolytic effects of the group ii metabotropic agonists
. it may simply be that under the conditions experienced by the mice in these anxiety tests , endogenous glutamate acting on group ii metabotropic receptors contributes little to these behaviours , which would be consistent with more recent studies which have failed to see effects of an mglu2/3 antagonist on anxiety tests like the elevated plus maze ( bespalov et al . , 2008 ) .
a related point concerns the inherent variability that is often observed in such ethological , unconditioned anxiety tests ( e.g. note the variability in the performance levels of the three wt cohorts in our study ) , and the possibility that this could lead to ceiling or floor effects which could mask any genotype effects .
for example , in the present studies , wt performance levels on the elevated plus maze varied quite considerably from one cohort to the next ( e.g. percent time in open arms ranged from 7.4 to 26.4% ) .
this is an important caveat and we can not exclude the possibility that fluctuations in wt performance , not only make comparisons across genotypes difficult , but could also obscure genotypic differences from wt controls .
however , it is worth pointing out that we saw no differences between the three separate ko lines and their respective wt controls across a number of different anxiety tests , with very different sensorimotor and motivational demands , and with a varying range of performance ( anxiety ) levels in the control animals .
furthermore , the lack of an anxiety phenotype in both of the single lines ( grm2 and grm3 mice ) is in agreement with previously published results ( fell et al . , 2011 ; fujioka et al . , 2014 ;
the absence of an anxiety phenotype was seen in several experiments in which the wt levels of performance would likely have allowed either increases or decreases in anxiety to be observed ( e.g. the percent time in the open arms of the elevated plus maze for these wt controls was 26.4% ; see table 1 ) .
nevertheless , we can not of course exclude the possibility that on any given test , a different level of performance in the wt controls could have revealed a genotypic difference .
alternatively , group ii mglu receptors could have an indirect or modulatory role on these emotional behaviours , with the potential for both anxiety promoting and anxiety reducing effects .
in genetically modified mice lacking these receptors , the balance between anxiolytic and anxiogenic phenotypes may be maintained , but the flexibility to respond to further perturbations to the system ( e.g. following drug administration ) may be lost .
of course we also can not rule out the possibility that developmental adaptations may occur in ko mice which could compensate in adulthood and ameliorate any potential effects on anxiety .
in contrast to our results , increased anxiety in both the open field and elevated plus maze has been reported in a sub strain of wistar rats ( b&k : wi ) which express substantially reduced levels of mglu2 protein ( ceolin et al . , 2011 ) .
it is possible that species differences ( rat vs. mouse ) could be an important factor .
mice may experience much higher levels of stress and arousal when being handled and assessed on tests of anxiety compared to rats , and this could subtly alter the balance between anxiety promoting and anxiety reducing effects , resulting in a different phenotype following mglu2 ablation in the two species .
however , further studies are needed to verify whether the anxiogenic phenotype can be specifically ascribed directly to mglu2 deficiency in these b&k : wi rats .
it is also worth noting that we did see some phenotypic differences in the grm3 mice on the anxiety tests .
they were faster to enter the open arms of the elevated plus maze and , to approach and eat the food in the second neophagia test .
there was also a trend for them to be faster to enter the central area of the open field .
notably , however , there were no group differences on the more commonly used measures of anxiety in these tests ( e.g. percent time in open arms of the elevated plus maze or in the central area of the open field , latency to eat minus latency to contact in the food neophagia test : barkus et al .
, 2010 ; gray and mcnaughton , 2000 ) . in a recent study , fujioka et al .
( 2014 ) also reported increased activity levels in grm3 mice ( e.g. in the open field and in the dark compartment of the light / dark transition test ) , but with no change in classic measures of anxiety [ time in centre of open field , time in the light compartment of the light / dark transition test ( fujioka et al . , 2014 ) ] .
one interpretation is that these subtle effects on behaviour in the grm3 mice reflect differences in arousal / activity levels which can influence performance on these ethological anxiety tests indirectly , rather than anxiolytic effects per se ( see below for further discussion ) .
( 2005 ) found a similar pattern of results for their grm2 mice , with increased locomotor activity observed in a number of different testing situations ( including the open field test , light / dark transition test , elevated plus maze , social interaction test ) , but no genotype differences in performance measures more directly related to anxiety ( although note that we did not replicate these effects in grm2 mice in the present study ) .
we previously demonstrated that grm2/3 mice show a unique fractionation within hippocampus - dependent spatial memory , leading us to suggest a role for group ii mglu receptors at the interface between arousal and cognition ( lyon et al .
our previous data suggested a possible role for these receptors in the well - established inverted u - shaped function that relates acute levels of stress and arousal with cognitive performance ( yerkes and dodson , 1908 ) .
this was , in part , based on the dissociation that we saw between ( i ) impaired performance on both appetitively motivated and spontaneous tests of spatial memory , and ( ii ) normal performance on aversively motivated versions of the tasks ( e.g. swim - escape tasks ) .
we did not observe such a clear dissociation between these tasks in the single ko lines in the present study .
for example , neither single grm2 nor grm3 mice were impaired on appetitively- or aversively - motivated spatial reference memory tasks , consistent with previous studies ( higgins et al . , 2004 ) . as previously discussed
, this may reflect the opportunity for compensation and/or redundancy of function in these single ko lines .
nevertheless , there is evidence from the grm2 and grm3 mice in the present study that is potentially consistent with our previous findings and speculations .
first , it is worth noting that where cognitive deficits are apparent in the single lines in this study , they are on appetitively motivated tasks rather than aversively motivated paradigms , as is the case in the grm2/3 mice .
second , the results with the grm3 mice on the appetitively motivated t - maze spatial working memory task revealed a biphasic effect on performance , in keeping with an inverted u - shaped function . while the grm3 mice were significantly impaired during the first block of 10 trials , they then performed significantly better than the wt mice during the third block of 10 trials .
two recent studies have reported short - term spatial ( working ) memory deficits in grm3 mice , although the effects were subtle and dependent , to some extent , on the precise experimental parameters being used ( fujioka et al . , 2014 ; lainiola et al . ,
indeed , fujioka et al . ( 2014 ) found a deficit in grm3 mice on a t - maze , forced alternation spatial working memory task , very similar to the spatial working memory t - maze task employed here , which was present during the initial training sessions , but was then absent during later testing when delays were introduced .
thus , cognitive performance in wt and grm3 mice may be differentially dependent on changes in stress / arousal levels as the animal 's experience with the task changes with training across time .
third , both grm2 and grm3 mice showed alterations in the patterns of reactivity towards environmental stimuli , in line with an altered interaction between arousal state and exploration .
the patterns of locomotor activity measured in photocell activity cages in both single ko lines were very different from what we have seen previously in the double ko mice , which displayed a pronounced reduction in locomotor activity during a 2 h test ( lyon et al . , 2011 ) .
in the present study , grm3 mice displayed similar activity levels to wt controls and , if anything , were slightly more active in the second hour of testing .
( 2014 ) also recently reported hyperactivity in grm3 mice in a home cage activity monitoring system ( fujioka et al . , 2014 ) .
the grm2 mice exhibited a biphasic effect , being hyperactive during the first 10 min bin , but exhibiting reduced activity levels as the test proceeded .
this behaviour during the first 10 min of the locomotor test is potentially consistent with previous demonstrations that grm2 mice exhibit hyperactivity in response to novel and potentially stressful environments such as in the open field or in other ethological , unconditioned tests of anxiety ( higgins et al .
2005 ) , albeit we did not see such effects in the anxiogenic open field in our present studies .
it is also notable that the grm2 mice displayed consistently faster running latencies ( sample and choice runs ) during the appetitively motivated , spatial working memory t - maze task , reminiscent of the previous result in the double kos ( lyon et al . , 2011 ) .
thus , although we see significant differences in activity levels between group ii metabotropic glutamate receptor ko mice and their controls , the pattern and direction of these effects can vary considerably from one situation to the next .
this also means comparisons across studies are difficult to make ( e.g. comparing single ko data from the present study with our previous data from the double ko line ) .
for example , although the different lines were tested in the same activity cages in the same testing room , the studies were conducted at different times ( and by different researchers ) making comparisons difficult .
these patterns of results reflect the likely complexity of the relationship between stress / arousal levels and behavioural activity , depending on the experimental settings .
the concepts of anxiety and arousal , although far from identical , are often closely coupled , although the interaction between them is likely to be complex .
although we have not seen clear effects on anxiety in any of the ko mice , it is known that mglu2/3 agonists and , in some studies , antagonists produce robust anxiolytic effects which are dependent on these receptors ( linden et al . , 2005 ) .
changes in stress / arousal levels have profound effects on anxiety , but increases in arousal are also an important component of the anxiety response .
neuropsychological accounts have suggested that anxiety is a response to situations of conflict or uncertainty , and that when such situations are detected then a constellation of behavioural responses are evoked , including increases in attention and arousal processes ( gray , 1982 ; gray and mcnaughton , 2000 ) .
thus , a more general account in which group ii metabotropic glutamate receptors play an important role in mediating the effects of changes in arousal on behaviour , could easily accommodate the role of these receptors in anxiety .
the behavioural consequences of deleting either mglu2 or mglu3 alone are less than those associated with deleting both receptors , consistent with the idea that there is redundancy of function and/or compensation in the single ko lines ( lyon et al . , 2008 ) .
although it is difficult to make comparisons across studies run at different times , and by different experimenters , the phenotypes of the single kos were less pronounced ( if present at all ) , across a number of different behavioural tasks .
it is also worth pointing out that the apparatus , testing rooms and test running orders were comparable for both the single and double ko lines .
generating sufficient numbers of double ko mice necessitated , for both financial and ethical reasons , the use of separate lines for double kos and controls .
thus , unlike the case for the grm2 and grm3 lines , the grm2/3 controls are non - littermate wts , although they were bred in the same holding room , in the same facility , at the same time .
this is a common practice with double ko mouse studies , but as a result we can not rule out the possibility that the grm2/3 mice results are affected by differences in genetic background , epigenetic effects or parental behaviour ( for discussion see lyon et al . , 2011 ) .
in the context of the present study , however , this may be less of an issue , as the major novel finding with these mice was a negative one : the lack of an anxiety phenotype .
therefore , any confounding effect resulting from the breeding strategy would have to be both opposite in direction and of equal magnitude in order to cancel out any putative effect of the double ko .
a further , additional caveat relates to the use of constitutive , permanent kos in which the gene ( or genes ) of interest are absent both throughout development and in adulthood . with these mice
it is impossible to separate effects arising from the absence of the gene on developmental processes , from effects arising due to the absence of the gene at the time of testing .
therefore , we can not exclude the possibility that when we do see effects in our group ii metabotropic glutamate receptor ko animals , these phenotypes could reflect a developmental disruption .
we also can not exclude the possibility that the absence of a gene ( or genes ) throughout the entire lifetime of the animal could result in effective compensatory changes which might produce a null phenotype ( e.g. for anxiety ) , despite a role for these receptors in normal animals .
inducible ko mice provide a possible future approach for resolving these issues . we have argued here and elsewhere ( lyon et al . , 2011
; see also marek , 2010 ) , that group ii metabotropic glutamate receptors may play an important role at the interface between arousal and behavioural performance , according to an inverted u - shaped function ( yerkes and dodson , 1908 ) .
this is potentially consistent with the biphasic effects on activity levels that are seen in the ko mice in different experimental settings throughout the present study ( see also lyon et al . , 2011 ) .
however , these studies illustrate the difficulties of studying experimental manipulations that affect the clearly non - linear interaction between arousal processes and behavioural performance . depending on the baseline levels of stress and/or arousal generated by the experimental situation
, it may be possible to generate any one of three results ( group a > group b , group a = group b , group a < group b ) .
therefore , although the inverted u - shaped function is an attractive post - hoc explanation for much of the data generated here and elsewhere ( lyon et al . , 2011 ) , it is very difficult in practice to predict a priori the direction and/or magnitude of an effect in any given experiment or , therefore , come up with a falsifiable hypothesis .
arousal / stress levels and hence performance could be affected by a whole host of factors ( e.g. age , gender , housing conditions , experimenter , time of day , prior history , experimental test conditions , behaviour of other animals , presence / absence of injection , level of food restriction ) , and this could be especially pertinent for hand run tests ( e.g. ethological anxiety tests , maze tests of learning and memory ) compared with operant tasks .
ultimately , independent physiological measures of stress / arousal levels ( e.g. corticosterone levels , indicators of sympathetic tone such as heart rate or blood pressure ) , taken during the behavioural test itself , may be necessary to understand fully the complex relationship between stress , arousal and cognitive performance , and the role which group ii mglus play in these processes . in summary ,
the behavioural phenotypes of the grm2 and grm3 mice were less pronounced than those of the double grm2/3 mice , consistent with the possibility of redundancy of function and/or compensation in the single ko lines .
however , there was no clear effect on measures of anxiety in either the single or double ko mice .
nevertheless , behavioural phenotypes were observed in these animals which are potentially consistent with a role for group ii metabotropic glutamate receptors at the interface between arousal processes and behavioural performance . | group ii metabotropic glutamate receptors ( mglu2 and mglu3 , encoded by grm2 and grm3 ) have been implicated in both cognitive and emotional processes , although their precise role remains to be established .
studies with knockout ( ko ) mice provide an important approach for investigating the role of specific receptor genes in behaviour . in the present series of experiments we extended our prior characterisation of grm2/3/ double ko mice and , in complementary experiments , investigated the behavioural phenotype of single grm2/ and grm3/ mice .
we found no consistent effect on anxiety in either the double or single ko mice .
the lack of an anxiety phenotype in any of the lines contrasts with the clear anxiolytic effects of mglu2/3 ligands .
motor co - ordination was impaired in grm2/3/ mice , but spared in single grm2/ and grm3/ mice . spatial working memory ( rewarded alternation ) testing on the elevated t - maze revealed a deficit in grm2/ mice throughout testing , whereas grm3/ mice exhibited a biphasic effect ( initially impaired , but performing better than controls by the end of training ) . a biphasic effect on activity levels
was seen for the grm2/ mice .
overall , the phenotype in both grm2/ and grm3/ mice was less pronounced if present at all compared to grm2/3/ mice , across the range of task domains .
this is consistent with possible redundancy of function and/or compensation in the single ko lines .
results are discussed with reference to a possible role for group ii metabotropic glutamate receptors at the interface between arousal and behavioural performance , according to an inverted u - shaped function . | Introduction
Materials and methods
Results
Discussion |
PMC1567469 | chloroform is a known contaminant of chlorinated drinking water and of swimming pool water disinfected with chlorine or one of its derivatives .
few data exist regarding the importance of dermal and inhalation exposure routes to the chloroform body burden resulting from domestic and recreational use of chlorinated water . in our experimental study involving 11 male swimmers
, we quantified the body burden resulting from exposure to various concentrations of chloroform in water and air of an indoor swimming pool , during a daily 55-min exercise period . from the first to the sixth exercise period , chcl3 mean concentration in water was increased from 159
micrograms / l to 553 micrograms / l . corresponding mean air chcl3 level ranged from 597 ppb to 1630 ppb . to dissociate the dermal exposure route from that of inhalation
, swimmers used scuba tanks during an additional exercise period .
chloroform concentrations were measured in alveolar air before and after each exercise period , as well as after 35 min of physical activity .
chloroform levels in water and air were measured every 10 min .
we examined the relationship between alveolar air concentration ( a measure of body burden ) at 35 and 55 min and environmental chloroform concentrations by using multiple regression models .
the natural logarithm of alveolar air concentration was strongly correlated with aqueous chloroform concentration both at 35 ( p2 < 0.001 , r2 = 0.75 ) and 55 min
( p < 0.001 , r2 = 0.86 ) .
the relationship with air concentrations was also statistically significant ( 35 min : p < 0.001 , r2 = 0.58 , 55 min : p < 0.001 , r2 = 0.63).(abstract truncated at 250 words)imagesfigure 1.figure 2 .
afigure 2 .
bfigure 3 .
afigure 3 .
b | Images |
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PMC4241936 | it is well established on the fact that regular exercise can help in treating various brain disorders such as depression , insomnia , parkinson disease , and alzheimer s disease .
however in case of the patients with intellectual disabilities , they lack determination to participate in any physical exercise mainly due to decreased motor nerves activity , behavioral disorder , lack of concentration and poor communication skills . additionally ,
since establishing voluntarily participations and self - motivations are difficult for the intellectual disabled patients , the effectiveness of the rehabilitation treatment is also very limited .
therefore there is a need for appropriate measure to be taken for the purpose of providing interest and motivation to the intellectually disabled patients .
considering these aspects , through unique and interesting characteristics of water , aquatic exercise could maximize the effectiveness of exercise .
since embarrassment of physical disabilities can be overcome in water , patients with intellectual disabilities can actively participate in exercising .
recently , as part of effective methods of improving the cognitive functions caused by the deficiency of neurotransmitters in depression , alzheimer s disease , stroke , and parkinson s disease , the ces ( cranial electrotherapy stimulation ) equipments are given attentions to .
the ces activates on the particular neurons of the electrical current in the brain stem , increasing the production of the neurotransmitters thereby increasing the electric and chemical activations .
the ces can be used as non - pharmaceutical intervention method for the purpose of chronic pain syndromes such as anxiety , depression , insomnia , stress relief , headache , backache , toothache , muscle pain that are reported to be largely effective in improving anxiety and pain prior to surgery .
importantly , the effect of ces is related to enhanced motion performance capability in stroke patients that can be used as a tool to enhance the effectiveness of exercise during the process of rehabilitation [ 4 - 6 ] .
therefore it is expected that the effect of the ces measure may enhances the cognitive function of the intellectually disabled .
on the other hand , the bdnf ( brain - derived neuropathic factor ) , the igf-1 ( insulin - like growth factor ) , and the vegf ( vascular endothelial growth factor ) that came into the recent spotlight , may increase the resistance on the death of the neurons cells and facilitates the production of cerebrovascular and nerve formations [ 7 - 9 ] .
it is being reported that the signal control of the nerve formation in the hippocampus affecting bdnf , igf-1 and vegf , strengthens the neural plasticity , learning and memory and enhances the cognitive function . in light of these facts ,
exercise of the brain with nerve disability , the ces interventions and the analysis of the bdnf , igf-1 and vegf could verify the rehabilitation methods and their effectiveness .
hence , in this experiment , the aquatic exercise was conducted to the mentally ill patient for the purpose of inducing interest in exercising , and the ces measures that have been known to be effective in improving cognitive function , concentration , and anxiety symptoms , were taken .
therefore the objective of this experiment is to prove the rehabilitation effect of the aquatic exercise and ces through 12 weeks of conducting aquatic exercises and by analyzing the ces measures such as cognitive function , bdnf , igf-1 , and vegf .
the participants of this experiment are 15 intellectually disabled patients , aged between 13~17 with second to third degree of mental disability ratings which included autistic patients .
they were from the disabled community c , students of the middle and high school swimming lessons doing as part of their special physical education program .
5 patients were allocated in each group of the control group , exercise group and exercise + ces group respectively .
the aquatic exercise program was produced through expert meeting between the special physical education instructors and swimming instructors . the aquatic exercise composed of 10 minutes of preparation exercise , 30 minutes of main exercise and 10 minutes of clean - up exercise which involved total of 50 minutes , running every three times a week .
putting focus on the actual swimming style , the aquatic program composed of leg posture , hand posture , respiration , freestyle , and backstroke . having regards on the intensity of exercise ,
individual differences on the physical functions of the disabled patients were considered thereby the duration and the levels of difficulty were appropriately chosen by the instructor .
the ces used in the experiment was alpha - stim 100 ( electromedical products international , usa ) , where the electrical current being 100~600 a , with the frequency value of 0.5 , 1.5 , 100 hz ( pulse / sec ) .
the ces measures were divided into three stages of introduction , development and ending stages that composed of approximately 25~35 minutes . in the ces manual , it is recommended to use within 600 a range in accordance to the individual response .
however considering the fact that the participants in the experiments were all intellectually disabled patients , from week 1~4 , low current of 100a was initiated , increasing to 200 a in week 5~8 , to the maximum current of 300 a in week 9~12 .
the cognitive function test used in the experiment was the k - wab ( korean western aphasia battery ) , the standardized version of wab ( western aphasia battery ) .
the cognitive function assessment provisions were the oral language test , written language test and the movement test .
oral language test comprised total of 420 points which consisted of speaking , comprehension , naming and speech repeating provisions .
the written language test comprised total of 200 points which consisted reading and writing provisions .
the movement test comprised total point of 180 , which included provisions such as action organization , space , calculation and drawing .
the method of measurement was carried out by professional assessor asking questions to the participants , and then asked to follow in accordance to the instruction given which was then assessed in points through observation and recording of the answers and conducts given by the participants . for blood analysis ,
10 ml of blood samples were taken from brachial vein at before and after training .
igf-1 was measured by the diasorin using a liaison igf-1 kit and analyzed by clia method .
using the spss 19.0 statistical software , mean and deviance were calculated , and mean difference test was conducted through the method of two - way anova with repeated measures . in case that correlation effects appear , one - way anova and paired t - test were conducted .
the participants of this experiment are 15 intellectually disabled patients , aged between 13~17 with second to third degree of mental disability ratings which included autistic patients .
they were from the disabled community c , students of the middle and high school swimming lessons doing as part of their special physical education program .
5 patients were allocated in each group of the control group , exercise group and exercise + ces group respectively .
the aquatic exercise program was produced through expert meeting between the special physical education instructors and swimming instructors . the aquatic exercise composed of 10 minutes of preparation exercise , 30 minutes of main exercise and 10 minutes of clean - up exercise which involved total of 50 minutes , running every three times a week .
putting focus on the actual swimming style , the aquatic program composed of leg posture , hand posture , respiration , freestyle , and backstroke . having regards on the intensity of exercise ,
individual differences on the physical functions of the disabled patients were considered thereby the duration and the levels of difficulty were appropriately chosen by the instructor .
the ces used in the experiment was alpha - stim 100 ( electromedical products international , usa ) , where the electrical current being 100~600 a , with the frequency value of 0.5 , 1.5 , 100 hz ( pulse / sec ) .
the ces measures were divided into three stages of introduction , development and ending stages that composed of approximately 25~35 minutes . in the ces manual , it is recommended to use within 600 a range in accordance to the individual response .
however considering the fact that the participants in the experiments were all intellectually disabled patients , from week 1~4 , low current of 100a was initiated , increasing to 200 a in week 5~8 , to the maximum current of 300 a in week 9~12 .
the cognitive function test used in the experiment was the k - wab ( korean western aphasia battery ) , the standardized version of wab ( western aphasia battery ) .
the cognitive function assessment provisions were the oral language test , written language test and the movement test .
oral language test comprised total of 420 points which consisted of speaking , comprehension , naming and speech repeating provisions .
the written language test comprised total of 200 points which consisted reading and writing provisions .
the movement test comprised total point of 180 , which included provisions such as action organization , space , calculation and drawing .
the method of measurement was carried out by professional assessor asking questions to the participants , and then asked to follow in accordance to the instruction given which was then assessed in points through observation and recording of the answers and conducts given by the participants .
10 ml of blood samples were taken from brachial vein at before and after training .
igf-1 was measured by the diasorin using a liaison igf-1 kit and analyzed by clia method .
using the spss 19.0 statistical software , mean and deviance were calculated , and mean difference test was conducted through the method of two - way anova with repeated measures . in case that correlation effects appear , one - way anova and paired t - test were conducted .
the changes in the cognitive function assessment data showed that there were significant differences in all groups , point in time , and cause - effect relationships ( p < 0.05 , p < 0.001 ) .
there was significant increase in cognitive function values in exercise and exercise + ces group than the controlled group ( p < 0.05 ) .
additionally , the exercise + ces group showed significant increase in the value when compared to the exercise along group ( p < 0.05 ) .
the results of the changes in the neurotransmitters between groups are listed in the table 4 below .
the changes in the bdnf assessment data showed that there were significant differences in all groups , point in time , and cause - effect relationships ( p < 0.05 , p < 0.001 ) .
although there were no significant differences within the groups before and after trainings when compared with the controlled group , there was a significant difference between the exercise group and the exercise + ces group ( p < 0.05 ) .
even the compa - rison data between each groups , despite the fact that the exercise and exercise + ces intervention groups showed significant increase than the controlled group ( p < 0.05 ) , there was no significant difference observed when compared between the exercise and exercise + ces groups ( p < 0.05 ) .
the changes in the igf-1 assessment data showed that there were no significant differences in all groups , point in time , and cause - effect relationships ( p > 0.05 ) .
the changes in the vegf assessment data showed that there were significant differences in all groups , point in time , and cause - effect relationships ( p < 0.05 , p < 0.001 ) . in another word , there were significant differences in before and after treatments between the exercise and exercise + ces groups ( p < 0.05 ) .
even the comparison data between each group , despite the fact that the exercise and exercise + ces intervention groups showed significant increase than the controlled group , there was no significant difference observed when compared between the exercise and exercise + ces groups .
the changes in the cognitive function assessment data showed that there were significant differences in all groups , point in time , and cause - effect relationships ( p < 0.05 , p < 0.001 ) .
there was significant increase in cognitive function values in exercise and exercise + ces group than the controlled group ( p < 0.05 ) .
additionally , the exercise + ces group showed significant increase in the value when compared to the exercise along group ( p < 0.05 ) .
the results of the changes in the neurotransmitters between groups are listed in the table 4 below .
the changes in the bdnf assessment data showed that there were significant differences in all groups , point in time , and cause - effect relationships ( p < 0.05 , p < 0.001 ) .
although there were no significant differences within the groups before and after trainings when compared with the controlled group , there was a significant difference between the exercise group and the exercise + ces group ( p < 0.05 ) .
even the compa - rison data between each groups , despite the fact that the exercise and exercise + ces intervention groups showed significant increase than the controlled group ( p < 0.05 ) , there was no significant difference observed when compared between the exercise and exercise + ces groups ( p < 0.05 ) .
the changes in the igf-1 assessment data showed that there were no significant differences in all groups , point in time , and cause - effect relationships ( p > 0.05 ) .
the changes in the vegf assessment data showed that there were significant differences in all groups , point in time , and cause - effect relationships ( p < 0.05 , p < 0.001 ) . in another word , there were significant differences in before and after treatments between the exercise and exercise + ces groups ( p < 0.05 ) .
even the comparison data between each group , despite the fact that the exercise and exercise + ces intervention groups showed significant increase than the controlled group , there was no significant difference observed when compared between the exercise and exercise + ces groups .
in this experiment , the effect of aquatic exercise and ces measures on the changes of cognitive functions , bdnf , igf-1 , vegf , and serotonin values were analyzed and discussed accordingly . according to the wilson et al .
, in relation to exercise and cognitive function , it has been reported that exercise stimulates the biochemical changes of the nerves in the brain which induce the activation of the neurotransmitters and formation of the genetics thereby affecting the proliferation of the neurons and their survival resulting in the enhancement of learning , memory and cognitive function skills .
however through which mechanism that exercising affects the brain and its cognitive function remains unknown . in this experiment , the cognitive functions of the exercise group and exercise + ces group increased after the treatment group than before treatment . according to the wilson et al .
, the reduction in the cognitive function was highly correlated to the reduction of the physical functions .
salmons & sahakian showed that low concentration and attention deficiency characteristics that are common in patients with brain disabilities improved through regular exercise . especially in the foster et al .
it has been shown that the cognitive function was greatly increased in the exercise + ces group than the exercise group alone hence additional ces intervention was identified to be helpful in enhancing the cognitive function of the patients with intellectual disabilities .
it is therefore estimated that the ces interventions stimulates the tiny current in the brain neurons which boost the electric and chemical activations thereby enhancing the cognitive function of the intellectually disabled .
the changes in the bdnf in the experiment showed significant increase by 52.6% in the exercise group , and 52.3% in the exercise + ces group .
however the ces measures were found to be ineffective as there was no significant difference identified between the exercise group and exercise + ces group . according to the seifert et al .
, it has been suggested that the bdnf concentration was increased in the brain after 3 month of exercising , and the ferris et al . and
hillman et al . studies have reported that the brain function was increased including cognitive function resulted by the bdnf increase through exercising . analyzing the correlation between the cognitive function and the bdnf from this experiment , static correlation ( r = 0.45 , p < 0.05 ) was identified where
it is thereby suggested that regular exercise increase the level of the bdnf , which enhances the cognitive function of the brain .
additionally , in the experiment , the changes in the igf-1 level in exercise group and exercise + ces group increased after treatment in comparison with the before treatment , yet the difference was insignificant .
eliakin have suggested that there was a sudden increase in the igf-1 level immediately after exercise , and berg & bang study have reported that the igf-1 concentration started to increase at 5 minutes of exercising where the peak concentration was reached at approximately 20 minutes . considering these aspects ,
it is suggested that the changes in the igf-1 level in one - off exercise reacts more vigorously in accordance with the intensity of exercise , than the level that has been stabilized before and after the treatment . on the other hand ,
the experiment showed significant increase in the vegf by 63.4% in the exercise group , and 71.4% in the exercise + ces group .
however , additional effect of ces measures could not be confirmed as there was no significant difference between the exercise and exercise + ces group . according to the franzoni et al . , regular exercise was found to be effective in increasing the function of the endotheliocytes by increasing the productivity of the vegf .
studies have identified that the increased level of vefg enhances the blood flow rate of the internal aorta and arteriole which would helps the blood supply and vascularization through peripheral reduction in the blood vessel resistance which is associated with the brain cognitive function . from the experiment ,
the aquatic exercise increased the vegf level in the brain that positively affected the brain vascular cells and blood flow rate , which could explain why there had been an increased cognitive function .
as follows , the aquatic exercise has been shown to be effective in improving cognitive function of the intellectually disabled patients and additional ces interventions was shown to improve even in greater effect in the cognitive function skills .
additionally , regular exercise increased the concentration of bdnf and vegf where as bdnf and vegf level increased , the cognitive function skill too increased .
the purpose of this study was to investigate the effects of aquatic exercise and ces treatment on the cognitive function by using k - wab and bdnf , igf-1 , and vegf of persons with intellectual disabilities .
cognitive function level were significantly increased in exercise + ces group than exercise group and control group .
the changes of blood bdnf and vegf concentration were significantly increased in exercise group and exercise + ces group than control group . as a result , it can be explain that exercise helps cognitive function of persons with intellectual disabilities and micro - current stimulation with ces treatment further improves cognitive function by activating nerve cell .
in addition , it can be suggested that the increment of bdnf by exercise stimulates the production of brain cells and the increase of vegf by exercise accelerates the production of endothelial cells . in conclusion , it can be concluded that ces treatment with exercise can amend cognitive function of persons with intellectual disabilities more effectively and increase of bdnf and vegf by exercise can explain the cognitive function improvement of persons with intellectual disabilities . | [ purpose]the purpose of this study was to investigate the effects of aquatic exercise and ces treatment on the cognitive function by using k - wab and bdnf , igf-1 , and vegf of persons with intellectual disabilities.[methods]all subjects were 15 male with intellectual disabilities who were participating in the aquatic training program and ces treatment during 12 weeks at rehabilitation center .
the subjects were divided into control group , exercise group , and exercise+ces group .
blood samples for bdnf , igf-1 , and vegf were taken from brachial vein at rest between before and after treatment.[results]the results are summarized as follows : cognitive function level increased significantly in the exercise+ces group compared to those in the exercise and control group .
the changes of blood igf-1 concentration were no significant difference among groups .
the changes of blood bdnf and vegf concentration were significantly increased in exercise group and exercise+ces group than control group .
however , blood bdnf and vegf concentration were significantly difference between exercise group and exercise+ces group.[conclusion]in conclusion , it can be concluded that ces treatment with exercise can amend cognitive function of persons with intellectual disabilities more effectively and increase of bdnf and vegf by exercise can explain the cognitive function improvement of persons with intellectual disabilities . | INTRODUCTION
RESEARCH METHODS
Subjects
Aquatic training program
CES treatment
Cognitive function test
Blood sampling and analysis
Statistics analysis
RESULTS
Changes in cognitive function
Changes in neurotransmitters
DISCUSSION
CONCLUSION |
PMC1538894 | the generation of torsion angle restraints is among the most critical steps in protein structure determination by nmr . in many cases ,
torsion angles also are used in lieu of other restraints ( i.e. noes ) when these data are missing .
the importance of torsion angle information tends to increase with the size of the protein being studied as the quality and quantity of other restraints , such as noes , deteriorate due to increased spectral overlap and reduced sensitivity . because of their importance in structure calculations , all commonly used software packages for nmr structure determination , such as cns ( 1 ) , xplor ( 2 ) , cyana ( 3 ) and amber ( 4 ) , accept torsion angles as restraints . in nmr , the information about torsion angles is commonly obtained from scalar couplings ( e.g. jhnh , jh-1n , jch ) and cross - correlated relaxation experiments ( 510 ) , and often involves comparing peak intensities or measuring peak splitting .
the accuracy of these measurements can be severely compromised by signal broadening and low signal - to - noise ratios , especially when dealing with larger ( > 150 residue ) proteins .
indeed , it is well known that h , c and co shifts are very sensitive to backbone / angles , while n shifts appear to be significantly influenced by side chain 1 angles of the preceding residue ( 11 ) .
chemical shift measurements are less affected by peak overlap or reduced spectral sensitivity than measurements of peak intensities and peak splittings .
furthermore , chemical shift measurements are routinely obtained for all peptides and proteins and often do not require additional nmr experiments beyond those needed for backbone assignments . indeed , chemical shift measurements are commonly done as the first step in the nmr - assisted determination of protein structures . the simplicity and accuracy of chemical shift measurements in combination with the public availability of thousands of protein chemical shift assignments has prompted the development of a number of protocols that use chemical shifts to predict backbone dihedral angles ( 1217 ) .
talos ( 17 ) is one such example and is currently one of the most commonly used programs that utilize chemical shifts to obtain dihedral angles .
talos predicts and torsion angles by comparing the chemical shifts and primary sequence of the protein being studied with a database of homologous polypeptides with known torsion angles and chemical shifts .
talos has made a profound impact on protein nmr , having appeared in more than 700 published works to date .
for instance , it does not predict side chain ( 1 ) or cis - trans peptide bond angles ( ) nor can it handle mis - referenced chemical shifts . furthermore , talos does not provide information about bounds of torsion angle restraints that are required for nmr structure calculations .
it also runs very slowly ( 22 min for a 150 residue protein on a 2.6 ghz processor ) .
the motivation for the current work was our belief that the prediction of torsion angles could be extended to angles other than and , that these predictions could be performed much faster , and that their accuracy could be significantly improved if the recent advances in our understanding of chemical shifts and the growing body of protein structural information could be implemented in a prediction protocol .
here we describe a program , called preditor ( prediction of torsion angles from chemical shift and homology ) , that is able to accurately predict a large number of protein torsion angles ( , , , ) using either h , c and n chemical shift assignments or protein sequence ( alone ) as input .
overall , the program is 35 faster and the accuracy ( for combined shift - based and homology - based prediction of / angles , using a 30 tolerance ) is 20% greater than talos .
the program can also predict g+ , g , trans states of 1 angles with 84% accuracy and angles are predicted with essentially 100% accuracy ( using a 2-state , cis / trans state prediction ) .
preditor is able to extend the limits of torsion angle prediction accuracy by ( i ) combining shift based and homology based predictions , ( ii ) taking advantage of better understanding the relationship between chemical shifts and torsion angles and ( iii ) utilizing recent advances in correcting mis - referenced nmr assignments and predicting of protein flexibility from chemical shifts .
preditor is composed of two parts , a front - end web interface ( figure 1 ) written in python and html and a back - end
calculator that consists of several programs including rci ( 18 ) , csi ( 19 ) , vadar ( 20 ) , blast ( 21 ) and refcor [ based on ( 22 ) ] as well as several parsing and conversion utilities for reading input files .
four of the programs blast , vadar , csi and the core of the preditor code are written in ansi standard c. several other programs including rci , refcor and most input parsing and conversion utilities are written in python .
preditor also accesses two databases , a local database of nmr assignments and torsion angles and the protein data bank ( 23 ) .
preditor accepts three kinds of input files : ( i ) chemical shift assignments in bmrb nmr - star format ( 24 ) , ( ii ) chemical shift assignments in shifty format ( 25 ) and ( iii ) raw protein sequence in fasta format ( 26 ) .
users can either upload an input file into the web server ( via a browse button ) or paste the data in a standard text box .
users are also offered several options to adjust program operations to suit their specific needs . by default
, preditor uses both chemical shifts ( via refdb ) and sequence homology ( to structural homologues in the pdb ) to predict torsion angles .
however , the reliance on homology - based predictions may not be desirable in some cases .
for example , a user may wish to assess the agreement between the shift - based ( nmr ) torsion angles and the torsion angles measured for an existing x - ray structure of the same protein .
in such cases , preditor allows users to select the radio - button option to predict dihedral angles from chemical shifts only .
in other cases , a user may only want to use homology - based predictions in his or her research .
for instance , a comparison of torsion angles predicted from chemical shifts with those predicted via homology may aid in resolving ambiguous cases during the nmr assignment process . in these cases
, users may select the radio - button option to predict dihedral angles via homology only .
preditor also offer users the flexibility to specify the pdb i d of the protein structure that should be used in homology - based predictions .
this feature is especially useful when the structure of the best - matching homologue was solved , say , under significantly different experimental conditions ( e.g. high urea concentration to partially unfold the protein ) or if the structure corresponds to a protein in a different functional form ( e.g. ligand - bound , post - translationally modified , etc . ) . to help users identify an appropriate homologue
, we offer an option to blast their protein against the pdb and to display the results so they are hyperlinked to the corresponding pages of the pdb .
if a fasta sequence only is used as input , torsion angles are predicted from a pdb homologue with the best blast e - value .
one of the more important advantages of preditor over existing programs is its capability to correct mis - referenced chemical shifts ( 17,27 ) .
chemical shift referencing , particularly for c and n shifts , continues to be a major problem in biomolecular nmr with about 20% of newly deposited assignments in the bmrb database being mis - referenced ( 28 ) .
mis - referenced shifts could substantially reduce the performance of chemical shift - based torsion angle predictions .
preditor 's reference correction option is always turned on by default and can be switched off if necessary .
an average preditor run takes about 38 cpu seconds on a 2.6 ghz processor equipped with 512 mbytes of ram .
as might be expected , preditor displays the name of the input file , the options selected , the pdb i d , the blast e - value and the level of identity of the pdb homologue ( if any ) used in predictions . below these data , the predicted torsion angles ( , , and 1 ) , their errors and confidence scores are shown in a tabular format .
in addition to these data , the output page contains hyperlinks to additional web pages with the results of the sequence alignment ( blast results ) , chemical shift reference corrections ( refcor results ) , the predictions of protein flexibility ( rci results ) and secondary structure predictions ( csi results ) derived from chemical shifts .
the preditor server also allows users to download the predicted / calculated dihedral angles restraints in cns , cyana , amber and xplor format .
in addition to its rich and extensive data output , preditor also offers a comprehensive list of help pages to assist users in preparing their input files , in understanding torsion angle prediction methods and in understanding the program output .
this information is provided to make the preditor protocol as transparent as possible and to facilitate any troubleshooting if necessary .
the basic preditor prediction algorithm has been described in much more detail elsewhere ( 27 ) .
preditor predicts protein torsion angles using a combined protocol that can be roughly divided into two components : shift - based predictions and homology - based predictions .
if a set of nmr assignments is submitted to the program , both types of predictions are run and their results are merged based on their respective errors or confidence levels , and on switch points as explained below . if a fasta sequence is supplied as an input file , only homology - based predictions are performed .
chemical shift - based predictions are initiated by re - referencing the nmr assignments ( if this option is not turned off by the user ) .
refcor applies a recently published protocol that uses h chemical shifts for the initial identification of secondary structure and the calculation of reference offsets ( 22 ) .
refcor can also go beyond the original procedure and properly predict secondary structure and correct referencing using other nuclei ( c , co and c ) or a consensus chemical shift index calculation ( 19 ) . these features enable preditor / refcor to re - reference shifts in the absence of h assignments .
a more detailed description of refcor features and performance will be published elsewhere . for shift - based predictions , preditor derives backbone torsion angles by comparing the chemical shifts of successive amino acid triplets from the query sequence with triplets contained in preditor 's own shift / structure database .
currently , the most recent version of this shift / structure database consists of 141 different protein entries obtained from refdb ( providing chemical shift data ) and the pdb ( providing torsion angle data )
the chemical shifts in the database were re - referenced via shiftcor ( 28 ) .
updates to the database and updates of preditor 's performance relative to its standard test sets will be posted on preditor 's help pages at regular intervals . to calculate the backbone torsion angles from chemical shifts ,
preditor calculates a sequence / shift similarity score [ s(i , j ) ] . for each query triplet
i and each database triplet j the similarity score s(i , j ) is calculated using the following equation .
1s(i , j)={0.5*kn1(seqsim)+kn2(|ci+ncj+n|)+kn3(|ci+ncj+n|)+kn4(|coi+ncoj+n|)+kn5(|hi+nhj+n|)+kn6(|ni+nnj+n|)+kn7(|hni+nhnj+n| )
where sums over the triplet of n = 1 to 1 , knm corresponds to empirically determined weighting coefficients ( table 1 on the preditor help page ) for each triplet n of each term m , seqsim represents the sequence similarity between each sequence triplet using the seqsee weight matrix ( 29 ) .
x is the secondary chemical shift ( 30 ) of nucleus x. the similarity score [ s(i , j ) ] between the query triplet and database triplet is calculated for all triplets in the database .
the ten triplets with the lowest scores are selected and torsion angles for central triplet residues are extracted .
the predicted torsion angles are clustered if the difference of the or angles is less than 15. the mean and angles of the cluster with the lowest overall s(i , j ) score are used as the predicted torsion angles for the central residue of the query triplet .
preditor uses probabilistic 1 hypersurfaces calculated by dunbrack ( 31 ) to generate an initial set of predicted 1 angles based on conformations of or angles .
each 1 angle is predicted to be in one of three states ( 60 , + 60and 180 ) and assigned a confidence score between 0 and 1 ( with 1 being most confident ) . by default , preditor assigns a trans conformation ( 180 ) to angles of all non - proline residues .
the presence of cis peptide bonds in proline residues is detected via comparison of the c and c shifts ( 32 ) . if the absolute difference between the c and c shifts is greater than 9 p.p.m . or if trp , tyr , phe , gly and cys are in either i 1 or i + 1 positions around proline ( 33 ) and c , c shift difference is between 8 and 9 p.p.m .
preditor 's homology - based predictions are initiated from a pairwise sequence alignment of the input sequence and all known pdb sequences using blast ( 21 ) .
the structure with the lowest e - value is retrieved from the pdb and its dihedral angles are calculated by vadar ( 20 ) .
torsion angles are mapped to the query sequence using the sequence alignment provided by blast .
in addition , a angle of 65 is mapped on to pro regardless of the values of homology - predicted angles . prior to merging shift - based and homology - based predictions , the error limits ( for / angles ) and confidence scores ( for , , 1 ) angles are calculated for each method as described below .
predictions with higher confidence scores or lower error limits are given precedence over predictions with lower confidence scores or higher error limits .
however , if shift - derived torsion angles of four or more consecutive residues are significantly different ( > 60 ) frompdb - derived values , the shift - based predictions are given precedence .
when homology - based predictions are not possible , the shift - derived predictions are used .
the selection of either homology - based predictions or shift - based predictions is also determined by
the rationale for adding the switch points was our observation that accuracy of predicted angles decreases significantly with increase of beta - sheet content in proteins and reduction of sequence identity of the homologue identified by blast ( 27 ) .
the switch points were obtained empirically by thorough analysis of these dependencies and are listed in web table 2 on preditor 's help page .
preditor assigns an error to each predicted / torsion angle by combining its confidence scores with predicted or identified secondary structures and local sequence identity .
the relationship to confidence scores and estimated / torsion angle errors are listed in web table 3 of preditor 's help page .
these error values were determined empirically by attempting to minimize both the size of the assigned error and the number of erroneous predictions with high ( 0.7 ) confidence values . generally speaking residues in helices have smaller errors than beta strands and angles have smaller errors than angles .
confidence scores in preditor are calculated using the following formula :
2c=2(3*s(i , j)/239)**2
where c is the confidence score and s(i , j ) is the similarity score calculated in equation 1 .
the confidence scores depend on both the sequence and chemical shift similarity of the query protein sequence triplets to the corresponding sequence triplets found in the preditor database .
this formula produces values that range from 0.0 to 1.0 ( with 1.0 having the highest confidence ) . roughly speaking a preditor confidence rating 0.7 corresponds to the talos rating of
ambiguous. with the homology search turned off , preditor predictions having a confidence score 0.7 , have an error rate that is slightly > 3% . typically 6065% of preditor predictions ( with the homology search turned off ) have a confidence score 0.7 .
with preditor 's pdb - homology search turned on , the error rate is similar ( 3% ) but the percentage of accepted predictions is 15% greater . likewise , for pdb - based predictions the error limits are generally several degrees smaller . note also that when pdb homologues are found the confidence scores vary with the local sequence identity of the pdb homologue ( see preditor 's help page for more details ) .
preditor uses a probabilistic hypersurface model ( 34 ) to estimate the confidence of 1 prediction from chemical shifts . as with
the / torsion angles the confidence values for 1 angle predictions vary between 0.0 and 1.0 , with 1.0 being the highest level .
confidence levels of homology - based predictions of 1 have been derived empirically from the dependence of prediction accuracy on the level homologue sequence identity .
confidence levels derived from pdb homologues are shown in web table 4 on preditor 's help page . when assignments for fewer than six nuclei ( c , c , co , h , n and hn ) are available , the predicted torsion angle error is multiplied by a scaling factor ( see web table 5 on preditor 's help page ) .
these scaling factors were determined for each of the 63 possible assignment combinations by measuring the average increase of preditor 's prediction errors for each of these combinations in the preditor database .
preditor was optimized , tested and evaluated on a training set of 141 proteins ( 20 489 residues ) , for which complete or nearly complete h , c and n chemical shifts were known and for which high quality pdb structures were available ( web table 6 on the preditor help page ) . to evaluate the performance of the algorithm for the training set
, we used a leave - one - out procedure by removing a query protein from preditor database prior to running the algorithm .
the accuracy of the predictions , (/ ) , was determined using the following equation :
3(/)i=(|obspred|)+(|obspred| )
where obs and obs are observed and pred and pred are predicted and angles , respectively . if the (/ )
value for residue i was < 30 ( denoted as / ) the prediction was considered
correct. to estimate accuracy 1 angles , the observed and predicted 1 angles were grouped into trans , gauche+ and gauche these three categories .
predictions are considered to be correct if both predicted and observed 1 angles belonged to the same rotamer group .
the overall performance for (/ ) or 1 was determined by calculating percentage of correctly identified torsion angles from their total number in a given protein .
the measurement of angle accuracy was based on a similar protocol and the performance of the program to predict and identify the 15 cis peptide bonds in the training set .
the average / accuracy for the full version of preditor on this 141 protein training set was 90.3% .
the average 1 accuracy was 83.8% ( versus 64.4% for shift - only derived predictions ) , while the accuracy was 99.98% for trans peptide bond identification and 93% for cis peptide bond identification . to assess preditor 's relative performance against talos a subset of 31 of the 141 proteins were also analyzed by talos .
the results of this comparison , both in terms of accuracy and computational speed are summarized in supplementary table a ( see the supplementary data and ) .
these data indicate that preditor is substantially faster ( 37 ) and 20.2% more accurate than talos . to test the program further and to ensure that the preditor algorithms had not been over - trained or become biased .
an independent test set of 15 randomly chosen proteins ( 2665 residues ) not found in either the talos or preditor training set was analyzed by both preditor and talos .
as seen in table 1 , preditor is 18% more accurate than talos for and predictions , when both pdb - derived and shift - based predictions are used .
the average preditor run takes 37.5 cpu seconds while the average talos run is 1305 cpu seconds on the same 2.6 ghz processor .
these results are essentially identical to the results seen in supplementary table a. in summary , preditor is a web server that is capable to rapidly obtain accurate estimates of , , 1 and torsion angles , including their error limits and confidence levels , using only chemical shift assignments or protein sequence as input data .
comparisons suggest that these estimates are as good or better than what can be obtained using existing methods and that the approach used here is generally applicable to any protein for which h , c and n shift assignments are available .
in addition to its speed and high level of performance preditor also offers other unique features including dihedral angle prediction via pdb structure mapping , automated chemical shift re - referencing ( to improve accuracy ) , prediction of proline cis / trans states , automated generation of xplor , cyana , amber or cns torsion angle restraint output and a simple - to - use web - based user interface .
because of its improved accuracy and extended capabilities , we believe preditor may lead to important changes in general nmr structure determination protocols , allowing more users to place tighter constraints and/or more weight on torsion angle restraint data .
preditor supports bmrb , shifty and fasta formatted input files performance using the / score of the full version of preditor , a disabled version of preditor ( shift - based predictions only ) and talos for the test set of 15 randomly chosen proteins | every year between 500 and 1000 peptide and protein structures are determined by nmr and deposited into the protein data bank .
however , the process of nmr structure determination continues to be a manually intensive and time - consuming task .
one of the most tedious and error - prone aspects of this process involves the determination of torsion angle restraints including phi , psi , omega and chi angles .
most methods require many days of additional experiments , painstaking measurements or complex calculations . here
we wish to describe a web server , called preditor , which greatly accelerates and simplifies this task .
preditor accepts sequence and/or chemical shift data as input and generates torsion angle predictions ( with predicted errors ) for phi , psi , omega and chi-1 angles .
preditor combines sequence alignment methods with advanced chemical shift analysis techniques to generate its torsion angle predictions .
the method is fast ( < 40 s per protein ) and accurate , with 88% of phi / psi predictions being within 30 of the correct values , 84% of chi-1 predictions being correct and 99.97% of omega angles being correct .
preditor is 35 times faster and up to 20% more accurate than any existing method .
preditor also provides accurate assessments of the torsion angle errors so that the torsion angle constraints can be readily fed into standard structure refinement programs , such as cns , xplor , amber and cyana .
other unique features to preditor include dihedral angle prediction via pdb structure mapping , automated chemical shift re - referencing ( to improve accuracy ) , prediction of proline cis / trans states and a simple user interface .
the preditor website is located at : . | INTRODUCTION
PROGRAM DESCRIPTION
VALIDATION
SUPPLEMENTARY DATA
Figures and Tables |
PMC1638278 | the 1990 clean air act mandated oxygenation of gasoline in regions where carbon monoxide standards were not met . to achieve this standard , methyl tertiary butyl ether ( mtbe )
was increased to 15% by volume during winter months in many locations .
subsequent to the increase of mtbe in gasoline , commuters reported increases in symptoms such as headache , nausea , and eye , nose , and throat irritation .
the present study compared 12 individuals selected based on self - report of symptoms ( self - reported sensitives ; srss ) associated with mtbe to 19 controls without self - reported sensitivities . in a double - blind , repeated measures , controlled exposure , subjects were exposed for 15 min to clean air , gasoline , gasoline with 11% mtbe , and gasoline with 15% mtbe .
symptoms , odor ratings , neurobehavioral performance on a task of driving simulation , and psychophysiologic responses ( heart and respiration rate , end - tidal co(2 ) , finger pulse volume , electromyograph , finger temperature ) were measured before , during , and immediately after exposure .
relative to controls , srss reported significantly more total symptoms when exposed to gasoline with 15% mtbe than when exposed to gasoline with 11% mtbe or to clean air .
however , these differences in symptoms were not accompanied by significant differences in neurobehavioral performance or psychophysiologic responses .
no significant differences in symptoms or neurobehavioral or psychophysiologic responses were observed when exposure to gasoline with 11% mtbe was compared to clean air or to gasoline .
thus , the present study , although showing increased total symptoms among srss when exposed to gasoline with 15% mtbe , did not support a dose - response relationship for mtbe exposure nor the symptom specificity associated with mtbe in epidemiologic studies.imagesfigure 1figure 2 | Images |
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PMC2603268 | there
has been large interest in speeding the acquisition of mri data by acquiring
fewer samples in k - space and resolving the artifacts .
recently , there have been
significant advances in applying inverse problem techniques to
reconstruct images from undersampled k - space mri data [ 16 ] .
the methods use nonuniform undersampling and
a nonlinear recovery scheme in which a constraint , such as a spatial total
variation ( tv ) constraint , is applied on the estimated solution , while preserving
fidelity to the acquired data in k - space .
it has been shown that using an l1
norm or a tv norm as constraint exploits the implicit sparsity in the data , and
can be used in both space and time dimensions .
the method is best known to
reconstruct piecewise constant or smoothly varying data from its undersampled
fourier samples ; but the application of the method to mr imaging techniques
like dynamic contrast enhanced myocardial perfusion imaging ( with respiratory
motion in the data ) and diffusion tensor imaging ( dti ) can be limited as these
images are often not piecewise constant . in
this paper , we propose a technique to improve the reconstruction of
general signals that may not fit the tv constraint well .
the technique uses
preprocessing of the measured undersampled data to determine an improved
ordering of the pixel intensities of the image estimate . if an ordering that
improves the match of the estimated images and the constraint being used within
the reconstruction can be
found , an improved reconstruction can result .
the image estimates are
reordered solely to be used with the constraint or regularization term in the
iterative reconstruction .
the reordering approach is general in its
applicability and can be used in contexts which are based on regularization
techniques and in which ordering of the image intensities can be determined a priori . in the next sections ,
we
give a brief overview of the compressed sampling or constrained reconstruction
method for mri from a regularization point of view and then present the theory
and applications of the reordering method .
the
compressed sampling method is described rigorously from a mathematical
standpoint recently in a series of papers [ 2 , 810 ] .
the method
is used to reconstruct a signal from a set of random fourier samples below the
nyquist rate by solving a convex optimization problem , in which fidelity to the
measured data is preserved at sample locations , while applying an l1
or a tv constraint on the estimated solution .
the method exploits the implicit
sparsity in the estimated solution or a transform of the estimated solution .
one useful transform for signals or images is finite differences as the signals
that are not directly sparse can be sparse in terms of finite differences
( especially piecewise constant or smoothly varying signals or images ) and hence
an l1
norm of finite differences ( tv norm ) is used .
although
the l1 norm of the signal or image estimate is not a direct measure
of sparsity in the data , it has been shown that for a wide variety of data
using an l1 norm is equivalent to using the l0
norm ( the
number of nonzero samples ) , which is a direct measure of sparsity .
solving
the optimization problem with an l1
norm is generally easier than
solving the problem with an l0
norm .
the
compressed sampling method when applied to mr image reconstruction can be
thought as a constrained reconstruction method in an inverse problem framework
[ 3 , 1114 ] . for the 1d case
, the relation between fourier data and the signal
space estimate can be represented as fm = d , where m is the signal of interest , d is the
fully sampled k - space data , and f represents the fourier transform ; but it often takes a long time to acquire
full k - space data and results in tradeoffs in image quality , resolution , and
coverage of the organ . to accelerate the data acquisitions , when full data are
not acquired in k - space , and only undersampled data are acquired , the relation
between the artifact - free signal estimate m and acquired data is given by ( 1)wfm = d , where w implements a binary undersampling pattern with ones ( where data are
acquired ) and zeros ( where data are missing ) , and d is the undersampled fourier data .
reconstructing the signal m directly using ( 1 ) is not feasible as w does not exist in general and hence
the solution is not unique .
the existence of the
solution is imposed by considering least - square solutions which minimize the functional wfm d22 ,
where ||||2 represents an l2
norm .
uniqueness
of the solution is imposed by using one or more constraints on the solution .
a
popular constraint used in the field of compressed sampling is the total
variation constraint given by m2 + 1 ,
where is the gradient of the estimated signal , is a small positive constant to avoid
singularities in the derivative of the functional , and ||||1 represents an l1
norm .
reconstruction
is performed by minimizing a convex cost function ( c ) ( 2)c = wfm d22 + m2 + 1 .
hence , a
solution which preserves fidelity to the acquired data and which has the
minimum total variation is chosen as the final solution . in ( 2 )
, is the regularization parameter which controls
the tradeoff between the fidelity and the constraint terms .
the total variation
constraint helps to resolve the artifacts while not penalizing the edges
heavily .
the
method can be extended to 2d and multi - image dimensions and it works very well
when the k - space data are undersampled in an irregular fashion and the
underlying complex images are smoothly varying or are piecewise constant .
when the images are not piecewise constant ( which is the case for most mr
images ) , the performance of the method can be affected .
we describe below a
reordering method to improve the performance of the constrained reconstruction
method when the data do not match the constraints well .
the method preprocesses
the signal to select a monotonic ordering of the estimated solution in space
and/or time and incorporates the reordering in the constraints to obtain better
reconstructions . for
clarity ,
the reordering method is first described for the 1d case and then the
method is extended to 2d and multidimension cases .
applications of the
reconstruction method with reordering for dynamic myocardial perfusion imaging
with respiratory motion and for brain dti data are presented .
when
the signal of interest is varying rapidly and is not smooth or the data are not
piecewise constant , the total variation of the signal is already high and hence
reconstruction from undersampled fourier domain samples can be inaccurate .
consider , for example , a smoothly varying 1d signal and a rapidly varying
signal that are labeled original full data as shown in figures 1(a ) and 1(b ) , respectively .
when the corresponding fourier samples ( k - space data ) of
the curves are undersampled by a factor of two in a pseudorandom fashion ( using
rand function in matlab ( the mathworks , natick , mass , usa ) ) and
reconstructed using the inverse fourier transform , the signals labeled undersampled
data r = 2 in figures 1(a ) and 1(b ) are obtained . when these undersampled
signals are reconstructed according to ( 2 ) ,
the original curves are overlaid in figures 1(c ) and 1(d ) for
reference .
reconstruction
using ( 2 ) is better for the smooth curve in figure 1 as compared to that for
the rapidly varying curve . to improve the reconstruction in the latter case ,
we
first reorder the estimated curve in the signal space according to an optimal
order , and then apply the total variation constraint .
the optimal ordering can
be determined as the ordering that makes the signal intensities in the curve
from the fully sampled dataset monotonic and smoothly varying . reordering the
estimated solution helps by reducing sudden variations in the curves and gives
a better match to the assumed constraint . in practice
, the curve or images from
fully sampled data will not be available to obtain the optimal ordering and
some sort of approximate reconstruction must be used to determine the ordering .
while this area needs more research , we show here that relatively simple
methods for determining reorderings can improve reconstructions of some types
of undersampled data .
better
reconstruction from the undersampled fourier samples is obtained when the
reordered curve is used in the constraint term , as the a priori assumption that the curve has lower variation is better
satisfied .
so the new reconstruction from undersampled data is performed
according to ( 3 ) in which the only difference is that the tv constraint is
applied on the reordered data as opposed to applying the constraint directly on
the given data .
reordering the estimated signal can also be thought as
multiplication of the signal with a reordering matrix p.
this matrix can be a permutation matrix of ones and zeros ( it
could also be a diagonal matrix for a 1d signal and it can be generalized for
multidimensional signals ) as follows : ( 3)m^ = minmwfm d22 + (pm)2 + 1 .
note
that the reordering in ( 3 ) is not directly based on the intensity values
obtained from the aliased signal from undersampled data in k - space ( or for
other applications , whatever domain the measurement data is obtained ) .
that is , p is determined once , and is fixed
while minimizing ( 3 ) . ordering the undersampled data according to
the optimal
order does not mean that the reordered undersampled image estimates are
monotonic , but means that
if this ordering is used , the original full data in the signal space will best
match the tv constraint .
consider figure 1(e ) which shows the sorted curve of
the original curve in figure 1(b ) .
the curve is monotonic and smoothly varying
and has lower total variation as compared to the original curve .
the ordering
in this case was chosen as the sorting order that made the original full curve
monotonic and smoothly varying .
although not shown
here , the reconstruction with reordering was comparable to that without
reordering for the case of the smooth curve in figure 1(a ) . from
the compressed sampling point of view , reordering the data can lead to sparser
representations of the data and hence higher acceleration factors .
alternatively , better reconstructions for a given acceleration factor can be
obtained .
figure 2 illustrates the point for the original full curve shown in
figure 1(b ) .
the figure compares the sparsity of the original curve and the
sorted curve in terms of finite differences .
the finite difference curve for
the sorted signal is sparser ( has fewer nonzero values ) as compared to that of the
original signal and hence using an l1
norm of the finite differences
with reordering leads to better reconstructions . choosing
the optimal regularization parameter
is important to obtain good
reconstructions .
the l - curve method is a popular technique for choosing
the optimal value .
the fidelity
norm is plotted against the constraint norm of the reordered data and the optimal
parameter is given by the corner of the l - curve .
figures 3(a ) and 3(b ) show the
l - curves obtained for reconstructions from undersampled data for the curve
shown in figure 1(b ) without and with reordering , respectively .
the l - curve in
figure 3(a ) is also overlaid on figure 3(b ) for direct comparison .
the l - curves
and the optimal parameters obtained are different for both cases . the optimal
parameter without reordering is higher than that with reordering . as in the iterative methods , the number of
iterations also plays the role of regularization parameter ; a fixed maximum
number of iterations which gave minimum rms reconstruction error for various
values was chosen in computing the l - curves . from
the above , it is apparent that correct reordering can help in better
reconstruction from undersampled fourier data when the signals are not smoothly
varying . in the above experiment , we used full data to determine the optimal
ordering . to be able to use the
reordering method , we need to have an ordering that makes the original signal best
match the constraint . in practice
, it is likely not possible to get the exact
ordering of the signal curves or images as that obtained using fully sampled
fourier data due to various factors like blurring of the prior signal , noise in
the prior signal , and so on . to simulate this case , we randomly perturbed the
exact sorting order to see the effect of having inexact ordering on the
performance of the algorithm . in figure 4 , the
x - axis represents the number of random perturbations , that is , the number of indices of
the exact sorting - order vector that are randomly perturbed .
consider s to be the sorting - order vector for the original signal . when there is one
random perturbation ,
( i ) a random number is generated between 1 and the length
of s denoted by r_a , then ( ii ) a second distinct random number between 1 and the
length of s denoted by r_b , and finally ( iii ) the value of the s at index r_a is exchanged with that at index r_b .
a value of 10 on the x - axis means that the
values of the exact sorting - order vector at 10 distinct randomly picked
indices ( out of 70 ) are exchanged with those at a different set of 10 distinct
randomly picked indices .
the y - axis represents the natural log of the total
absolute difference between original full data and the reconstructed signal .
we
can see that as the number of random perturbations increases , the total absolute error
using reordering gradually increases , but this number is still better or comparable to that
without the reordering except for a few perturbations toward the end of the
plot where the entire sorting - order vector was randomly perturbed .
the
reordering method described above for the 1d case can be extended to 2d and
applied in the context of images . as in the 1d case , reordering in 2d for
images helps in better reconstruction when the images of interest are nonsmooth
or are not piecewise constant . for
example , figure 5(a ) shows a simulated piecewise constant heart image with
blood pools and with an ischemic region in the myocardium .
when full fourier
data of the image are undersampled in a variable density ( vd ) cartesian fashion
( so that 5 lines in the center of k - space are fully sampled and the
remaining phase encodes are sampled in a pseudorandom fashion to give a net
reduction factor of ~6.5 ) , direct inverse fourier transform reconstruction
gives the image shown in figure 5(b ) .
when the constrained reconstruction
approach with a tv spatial constraint ( xm2 + ym2 + 1 ) without reordering is used , figure 5(c ) is
obtained .
we can see that figure 5(c ) matches well with figure 5(a ) ; but when the
image is not piecewise constant , the performance of the constrained
reconstruction can be affected .
figure 5(d ) shows an actual mr heart image from
a patient at a single time in a perfusion sequence .
the image was reconstructed
using a standard 2d inverse fourier transform of the fully acquired k - space
data .
figure 5(e ) shows the standard 2d inverse fourier transform
reconstruction of undersampled k - space data for the time frame , with zeros
inserted for the missing k - space data points .
the data were undersampled by a factor of three in
vd cartesian fashion ( 10 phase encodes fully sampled around the center and the
remaining ones in a pseudorandom fashion ) . figure 5(f )
shows the constrained reconstruction from the undersampled data using a spatial
tv constraint without any reordering which has a few residual artifacts . for
improving the reconstruction in this case , the image is reordered independently
in x and y directions before applying the 2d tv constraint ,
that is , reorderings
are determined separately for each row and each column . in practice , since the
data we deal with in mri are
complex , the optimal ordering is determined independently for the real and
imaginary components of the image and separately in x and y directions . a row - reordered real part
of the image of the original complex
mr image of the heart is shown in figure 5(g ) . the tv spatial constraint with
reorderings for a complex image
can be explicitly written as 1 , where = x(prxmreal)2 + x(pixmimag)2 + y(prymreal)2 + y(piymimag)2 + , in which mreal is the real part of the image estimate and mimag is the imaginary part of the image
estimate .
prx and pix denote the reordering matrices for ordering the signal in x dimension for the real and imaginary parts , respectively , while pry and piy are the corresponding reordering matrices in y dimension . for simplicity and
compactness , the above spatial constraint with reordering is referred to as x(pxm)2 + y(pym)2 + 1 , where pxm gives the image reordered for each row in the x direction and pym is the image reordered for each column in the y direction .
figure 5(h ) shows the
reconstruction from the undersampled data using a spatial tv constraint with
reordering .
the ordering of the data was
obtained using the image reconstructed from fully sampled data .
figure 6 shows a plot comparing the
reconstruction error with increasing number of perturbations in the exact
spatial ordering for the actual mr heart image in figure 5(d ) .
the
reconstruction error is calculated as the total absolute difference between the
full data reconstruction and the data reconstructed using the tv spatial constraint .
a value of 10 on the x - axis means that the sorting - order vectors for 10% of the
total number of rows ( rounded to nearest integer and randomly picked ) and those
for 10% of the total number of columns ( rounded to nearest integer and randomly
picked ) are randomly perturbed for both the real and imaginary parts of the
complex image data .
perturbation for
a given row or a column is done independently for the entire length of the
sorting - order vector as described for the 1d case in section 2.4 .
a value of
100 on the x - axis means that the sorting - order vectors for all the rows and all
the columns are completely perturbed ( randomly and independently ) for real and
imaginary parts of the image .
the error for the reconstruction with reordering
is gradually increasing with increasing perturbations and when the exact
sorting orders are severely modified , the error gets higher than that without
reordering .
the
reordering method described above can be extended to multi - image mr
acquisitions like dynamic myocardial perfusion imaging and brain dti . in
perfusion imaging , a series of images of the heart are acquired to track the
uptake and washout patterns of the contrast agent in the myocardium .
dti
requires the acquisition of multiple images with diffusion weightings in different
directions .
reordering can be done in the multi - image dimension in the time
dimension for the case of myocardial perfusion imaging and in the diffusion
encoding dimension for the case of dti . as in the 1d case , reordering in the multi - image dimension for the
images can give a better reconstruction when the signal changes in the
dimension are not smoothly varying which is the case for perfusion imaging with
respiratory motion and for dti .
the
constraint for the reordering in the multi - image dimension is represented as t(ptm)2 + 1 , where t represents the gradient operator in the
multi - image dimension and ptm is the data reordered in the corresponding
dimension .
the subscript t is used
because the multi - image dimension is analogous to the temporal dimension of
dynamic perfusion datasets . for a given image frame in the multi - image dataset ,
reconstruction
can then be performed by using tv constraints in both space and multi - image
dimensions and with reordering in the corresponding dimensions
as follows : ( 4)c = wfm d22 + 1t(ptm)2 + 1 + 2x(pxm)2 + y(pym)2 + 1 .
a similar
framework to ( 4 ) was proposed
in for reconstructing undersampled radial
myocardial perfusion data but without reordering and with a different temporal
constraint . as in , ( 4 )
the reordering method for
multi - image acquisitions ( 4 ) was tested using a dynamic phantom .
gd was slowly
injected into a tube running through a water phantom and fully sampled
cartesian k - space data were
acquired over time using an echoplanar imaging sequence on a siemens 3 t trio
scanner .
raw k - space data was then undersampled offline in a variable density
( vd ) pseudorandom fashion , in which 12 central low - resolution k - space lines
were sampled for all time frames , and the remaining phase encodes were sampled
in a pseudorandom fashion to give a net reduction factor of three . the acquisition
matrix for
reconstruction from the undersampled data was then performed according
to ( 4 ) in two steps . in the first step
,
the information about the reordering was obtained using images obtained using
the central low - resolution data from vd undersampling .
the image
estimates were reordered first in the time dimension only and the
reconstruction was performed . in this step ,
the real and imaginary parts of the
complex low - resolution image space data for each pixel were sorted
independently in the time dimension according to their intensity values .
the
corresponding sorting orders for the real and imaginary parts were used for
reordering the real and imaginary parts of the complex undersampled image space
data . after performing an initial reconstruction with only temporal reordering
results of the final reconstruction with reordering were compared to full data
reconstructions using the standard inverse fourier transform and to the
reconstructions without any reordering .
the reordering method was applied
on dynamic myocardial perfusion imaging with respiratory motion and on brain
dti data .
full cartesian raw k - space perfusion data were obtained using a siemens 3 t trio
scanner using a turboflash saturation recovery sequence .
the parameters for the
data acquisition were tr = 1.8 milliseconds , te = 1 millisecond , flip angle = 12 , gd dose = 0.025 mmol / kg , slice thickness = 6 mm , and
acquisition matrix = 192 96 .
the data were acquired with
informed consent in accordance with the university of utah institutional review board .
brain dti image data were
acquired on a ge 3 t scanner and full k - space data were generated from the magnitude image data by
applying 2d fourier transforms on each diffusion encoding direction .
full
k - space data for both perfusion and brain dti data were undersampled in a variable density
pseudorandom fashion outside the center and with the central 18 k - space lines
sampled for each time frame , and reconstruction was performed in two steps as
described in section 3.1 .
the net r value for the perfusion data was 2.5 while
that for the dti data was 3 .
the
results of the reordering method on the multi - image phantom data are shown in
figure 7 .
figure 7(a ) shows the image of a slice that was reconstructed from
full data using ift at single time point .
figure 7(b ) shows the corresponding
image reconstructed from r~3 data using stcr without any reordering .
figure 7(c )
shows the image reconstructed from r~3 data using stcr with reordering in both
temporal and spatial dimensions .
figure 7(d )
shows the absolute difference image between figures 7(a ) and 7(b ) and
figure 7(e ) shows the absolute difference image between figures 7(a ) and 7(c ) .
the images in figures 7(d ) and 7(e ) are scaled to the
same window level to highlight the differences .
the
results of the reordering method for dynamic myocardial perfusion imaging are
shown in figure 8 .
figure 8(a ) ( column ) shows images at two different time
points in a perfusion sequence reconstructed from full perfusion data using
standard inverse fourier transforms .
figure 8(b ) ( column ) shows the
corresponding stcr reconstructions from r~2.5 data without any reordering
in time or space dimensions .
figure 8(c ) ( column ) shows the corresponding stcr
reconstructions with reordering in both time and space dimensions .
the
reordering helps in reconstruction when there is a significant respiratory motion in the
data .
we previously reported higher acceleration
factors ( r~4 with interleaved undersampling and r~5 with variable density
sampling ) in for myocardial perfusion , when there was minimal or no
respiratory motion in the data . in the presence of significant respiratory
motion , the method in was not fully able to resolve the artifacts from
undersampling .
the current method was better able to reduce the artifacts even
in the presence of large respiratory motion .
the
results obtained by applying the reordering method on brain dti data are shown
in figure 9 .
figure 9(d ) compares the line intensity
profiles for full data reconstruction ( figure 9(a ) ) and the reconstruction
without reordering ( figure 9(b ) ) .
figure 9(e ) compares the intensity profiles
for full data reconstruction ( figure 9(a ) ) and the reconstruction with
reordering ( figure 9(c ) ) .
the signals in figure 9(e ) match better than those in
figure 9(d ) .
this
paper introduces a modified constraint term for compressed sampling and
constrained image reconstruction approaches . in general
, it is possible to
choose a regularization or constraint term which is a good model for the image
being reconstructed .
the basic idea of the reordering method is that it is
possible to tailor these regularization or constraint operators to improve the
reconstruction by reordering the signal . from a compressed sampling point of
view
reordering can be thought as a new set of data - specific transforms that
further improve the sparsity .
recently , a new method using a prior image
constraint was proposed to improve the constrained reconstruction of
dynamic ct images .
the additional prior image constraint minimizes the l1
distance between the estimated solution and the prior image .
the reordering
method proposed here is different in the sense that it does not directly use
the intensities in the prior image .
the method uses only the ordering
information from a prior image or set of images , which can be preserved if the
prior images are at a different and unknown intensity scale as compared to the
estimated solution .
reordering
can be done in multiple dimensions to improve the sparsity when the signals are
not smoothly varying . here , we used images from the central low - resolution data
to determine orderings initially in the multi - image dimension and then used the
resulting images to obtain the spatial reordering for each image separately .
this is because the central low - resolution data are more faithful in the multi - image dimension
than they are in
the spatial dimension .
reordering in the multi - image dimension offered more
significant improvements as compared to reordering in the spatial dimensions .
this is because the temporal constraint generally plays a more important role
in resolving the artifacts as compared to the spatial constraint for dynamic
imaging . to obtain significant improvements just using spatial reordering
improved ways of
obtaining reordering , like doing a separate training scan before the actual
acquisition , may help to achieve higher accelerations .
the
reordering method incorporates the ordering information of the signal to better
match the total variation constraint assumption and thus improves the
reconstruction from undersampled data .
methods like adaptive regularization
[ 18 , 19 ] were proposed to improve the performance of tv regularization - based denoising
techniques by using a priori
information about the signal . in these methods ,
the regularization parameter is
varied based on the a priori
knowledge of the locations of edges and smooth regions in a signal , so that
less regularization is done where strong edges are present in the signal .
while
this type of approach may be extended to tv constrained reconstruction ,
choosing the optimal amount of variation of the regularization parameter can be
complicated for rapidly varying signals .
an alternative method that can use
such a priori information was explored here with the reordering method that
uses only a single regularization parameter .
the
reordering method may not be appropriate when the ordering is incorrect in such
a way that the total variation of the reordered full image sets is increased as
compared to that of the original full data . in practice
in such cases , l - curves can be
used to determine to some extent if reordering is appropriate .
l - curves can be
computed for reconstructions with and without reordering and the tv norms
corresponding to the optimal regularization parameters can be compared . if the
ordering is appropriate , then the tv norm corresponding to the optimal
regularization parameter with reordering is lower than that for the
corresponding optimal regularization parameter without reordering .
consider
figure 10(a ) which shows the l - curve obtained for the 1d randomly varying curve
shown in figure 1(b ) , with a large number of random perturbations ( ~65% ) in the
exact ordering .
the tv norm corresponding to the optimal is 36.08 . when the number of random perturbations is decreased ( ~21% ) , the l - curve
in figure 10(b ) is obtained and the tv norm corresponding to the optimal is 5.02 .
the l - curve in figure 10(a ) is
overlaid in figure 10(b ) for reference .
the tv norm corresponding to the
l - curve obtained without reordering is 14.05 .
the
reconstruction time with image reordering was higher than the standard l1
norm
reconstruction , as in each iteration , the estimated signal is reordered before
computing the constraint update .
for the data reordering in 1d case , the
reconstruction time was 1.04 times slower , while that for the dynamic case with
reordering in both spatial and temporal dimensions was 2.8 times slower .
it took ~35 seconds
per iteration on a linux machine with an amd processor ( 2.5 ghz ) and 6 gb ram
for stcr with reordering in multiple dimensions .
the implementation was done in
matlab and a host of methods including the use of gpus are available to greatly
speed up reconstruction methods .
a
method involving reordering in time and space dimensions of the image estimates
to better match the chosen constraints of an inverse problem - type
reconstruction was presented .
the method uses non - reordered reconstructions to
obtain information about the signal to be reconstructed to determine the
orderings of the pixel intensities .
the orderings can be estimated from the low - resolution
images when a variable density undersampling scheme is used , and from
non - reordered constrained reconstructions .
the method can be forgiving to errors
in the images used to choose the orderings as the method does not use the data
directly but uses only its ordering information .
the method was shown to have
promise for cardiac perfusion imaging and offered some small improvements for
dti data .
future improvements in finding more optimal reorderings , perhaps as
part of the estimation process , may make the approach useful in a wide array of
applications . | recently , there has been a significant interest in applying reconstruction techniques , like constrained reconstruction or compressed sampling methods , to undersampled k - space data in mri . here
, we propose a novel reordering technique to improve these types of reconstruction methods . in this technique , the intensities of the signal estimate are reordered according to a preprocessing step when applying the constraints on the estimated solution within the iterative reconstruction .
the ordering of the intensities is such that it makes the original artifact - free signal monotonic and thus minimizes the finite differences norm if the correct image is estimated ; this ordering can be estimated based on the undersampled measured data .
theory and example applications of the method for accelerating myocardial perfusion imaging with respiratory motion and brain diffusion tensor imaging are presented . | 1. INTRODUCTION
2. THEORY
3. APPLICATIONS
4. RESULTS
5. DISCUSSION
6. CONCLUSION |
PMC4563282 | the term combat sports describes a group
of sports whose competitive essence consists of direct combat between two competing
athletes2 , 3 .
a
sports injury is defined as damage to part of the body resulting in inability to practice or
compete normally4 .
because combat sports
frequently involve striking , throwing , or immobilizing an opponent , some researchers suggest
that they are more dangerous for athletes than other sports5 . almost all injuries involved in combat sports are caused by
mechanical energy , and this manifests as musculoskeletal injuries .
musculoskeletal injuries
usually occur when the body experiences overload through accident or overuse5 , 6 .
many sporting events in korea revolve around combat sports such as judo , wrestling , kendo ,
boxing , taekwondo , and ssireum .
ssireum , also called korean wrestling , is a form of korean
martial arts in which two athletes use strength and various skills to throw their opponent
to the ground7 .
combat sports can be
classified into two types : strike and non - strike ( throwing or immobilizing an opponent ) .
strike sports involve striking an opponent directly by kicking or punching or by using a
weapon .
. the rules of taekwondo ,
or rather gyorugi , state that a competitor can only score by striking his opponent in the
chest using only his fists or by kicking his opponent s head and torso using only the part
of the foot below the ankle8 , 9 .
boxing is a contest that involves striking the body or head
of an opponent by means of a punch10
. the
rules of kendo state that the head , wrist , waist , or throat of an opponent can be struck
with a bamboo sword11,12,13 .
taekwondo athletes wear a guard on the head , torso ,
arms , legs , and groin , while kendo athletes are equipped with protective armor on the head ,
torso , and forearms .
conversely , direct striking is not
permitted in non - strike sports , so players do not generally wear protective equipment .
different rules apply in judo , wrestling , and ssireum , and the common goal of these sports
is to overpower an opponent through force and skill7 ,
14,15,16 .
every combat sports event has physical or
injury characteristics based on the combat method , rules , and skills used . even within the
same sport ,
combat sports are the
focus of much attention worldwide , and for this reason , there is a wealth of research
available regarding the injuries sustained by combat athletes in various competitions .
these
data are typically classified according to event , injured body part , and the epidemiology of
the injury .
however , little research has been carried out on the playing style of combat
athletes , especially in strike and non - strike sports .
our study aims to describe the
characteristics of injuries sustained in strike and non - strike combat sports so that the
data can be used in sports physiotherapy .
the participants
included elite college players of the following sports : judo ( 47 ) , ssireum ( 19 ) , wrestling
( 13 ) , kendo ( 30 ) , boxing ( 16 ) , and taekwondo ( 34 ) . of the participants ,
133 were male and 26
were female . in the case of ssireum and boxing ,
the
volunteers had no physical or psychological conditions , and all of them provided informed
written consent to participation in this study .
for the purposes of the study , we divided
the participants into two groups according to playing style : strike sports and non - strike
sports .
kendo , boxing , and taekwondo are strike sports , while judo , ssireum , and wrestling
are non - strike sports .
the questionnaire was filled out directly by the athletes under the supervision of a
researcher .
injury questionnaire , which contained three queries based
on the following information regarding the injury type : 1 .
type of injury ( muscular system ) 2 . injured body part ( cephalic , body , upper limb , lower limb ) 3 . where and when the injury occurred and the aftermath ( when the injury occurred , when the
athlete returned to exercise , recurrence of the injury , side effects , psychological reaction
after the injury ) in the case of queries 1 and 2 , more than one response was permitted in order to cover all
injuries experienced by the athletes .
injury in this study was defined as any bodily
damage that interfered with competition or training .
statistical analyses were conducted using the pasw statistics
software ( version 18.0 ) to calculate averages and standard deviations .
data relating to the
general characteristics of the athletes were expressed as the mean standard error ( se ) ,
and these data were assessed using the student s t - test .
we also analyzed the frequency of injury in each sport , and crosstabs were created
for each question using a test and fisher s exact test . in accordance with the
terms of resolution 5 - 1 - 20 , december 2006 , the protocol for the study was approved by the
committee of ethics in research of the university of yongin .
furthermore , all volunteers
provided informed consent for participation in the study7 , 8 , 10 .
the general characteristics of the participants are described in table 1table 1.general characteristics of the combat sports playersvariablejudossireumwrestlingkendoboxingtaekwondogender ( m / f)male ( % ) 34 ( 72.3)19 ( 100.0)9 ( 69.2)27 ( 90.0)16 ( 100.0)28 ( 82.4)female ( % ) 13 ( 27.7)-4 ( 30.8)3 ( 10.0)-6 ( 17.6)total ( % ) 47 ( 100.0)19 ( 100.0)13 ( 100.0)30 ( 100.0)16 ( 100.0)34 ( 100.0)age ( yrs)20.40.219.90.320.10.320.00.219.40.319.80.3height ( cm)173.01.3178.11.1169.34.5174.51.1173.42.9178.31.0weight ( kg)79.42.896.15.477.94.570.81.167.92.969.41.7bmi ( kg / cm)26.20.630.21.526.91.023.20.322.50.721.70.3career ( yrs)9.20.49.30.55.90.58.60.56.00.77.80.4training timerunning ( h / day)1.70.11.30.11.30.20.60.11.00.31.10.1major ( h / day)2.00.02.10.12.20.23.70.21.90.12.60.1wt ( h / day)1.40.10.80.11.10.10.40.11.00.10.80.1total ( h / day)5.2.0.84.30.24.60.44.70.24.10.24.60.2days / week ( days)6.00.55.10.15.30.15.60.15.50.16.00.1all data are presented as the meanse .
m : male player ; f : female player ; bmi : body
mass index ; wt : weight training . * # : p < 0.05 .. all combat athletes had experienced musculoskeletal injuries except for one
kendo athlete ( table 2table 2.crosstabs of the type of injury and sports playersvariablemusculoskeletal injuriesskin injuriesnerve injuriestooth injuriesetc.non-strike sportsjudo ( % ) 47 ( 100.0)9 ( 19.1)8 ( 17.0)3 ( 6.4)0 ( 0.0)ssireum ( % ) 19 ( 100.0)6 ( 31.6)4 ( 21.1)1 ( 5.3)0 ( 0.0)wrestling ( % ) 13 ( 100.0)5 ( 38.5)4 ( 30.8)2 ( 15.4)0 ( 0.0)strike sportskendo ( % ) 25 ( 96.2)5 ( 19.2)4 ( 15.4)0 ( 0.0)0 ( 0.0)boxing ( % ) 16 ( 100.0)4 ( 25.0)2 ( 12.5)3 ( 18.8)0 ( 0.0)taekwondo ( % ) 34 ( 100.0)6 ( 17.6)3 ( 8.8)4 ( 11.8)0 ( 0.0)non - strike sports ( % ) 79 ( 100.0)20 ( 25.3)16 ( 20.3)6 ( 7.6)0 ( 0.0)strike sports ( % ) 75 ( 98.7)15 ( 19.5)9 ( 11.7)7 ( 9.1)0 ( 0.0)x(p ) - ( 0.490)0.763 ( 0.382)2.126 ( 0.145)0.114 ( 0.735)-fisher s exact test . *
skin injuries were most frequent among wrestlers in the case of non - strike
sports and among boxers in the case of strike sports .
there was a high incidence of nerve
injuries such as neuralgia among the non - strike athletes ; however , this was not
statistically significant ( table 2 ) .
the order of frequency with regard to
injury types was musculoskeletal injuries followed by skin , nerve , and tooth injuries in
both groups ( table 2 ) . in terms of
musculoskeletal injuries , dislocation was the most frequent injury in non - strike athletes ,
and the difference between the groups was significant .
fracture was most common in the
taekwondo athletes , followed by the boxers and judo athletes ( table 3table 3.crosstabs of the type of musculoskeletal injury and sports playersvariabledislocationfracturerupture of ligamentrupture of musclesprain or strainbruiseherniated discetc.nssjudo ( % ) 9 ( 19.6)23 ( 50.0)27 ( 58.7)10 ( 21.7)22 ( 47.8)23 ( 50.0)7 ( 15.2)0 ( 0.0)ssireum ( % ) 1 ( 5.6)2 ( 11.1)13 ( 72.2)6 ( 33.3)8 ( 44.4)12 ( 66.7)4 ( 22.2)1 ( 5.6)wrestling ( % ) 3 ( 25.0)4 ( 33.3)4 ( 33.3)4 ( 33.3)11 ( 91.7)8 ( 66.7)5 ( 41.7)1 ( 8.3)sskendo ( % ) 0 ( 0.0)2 ( 8.0)7 ( 28.0)3 ( 12.0)15 ( 60.0)10 ( 40.0)7 ( 28.0)4 ( 16.0)boxing ( % ) 1 ( 6.3)9 ( 56.3)6 ( 37.5)3 ( 18.8)11 ( 68.8)12 ( 75.0)4 ( 25.0)0 ( 0.0)taekwondo ( % ) 2 ( 5.9)23 ( 67.6)19 ( 55.9)18 ( 52.9)20 ( 58.8)24 ( 70.6)6 ( 17.6)0 ( 0.0)nss ( % ) 13 ( 16.9)29 ( 37.7)44 ( 57.1)20 ( 26.0)41 ( 53.2)43 ( 55.8)16 ( 20.8)2 ( 2.6)ss ( % ) 3 ( 4.0)34 ( 45.3)32 ( 43.7)24 ( 32.0)46 ( 61.3)46 ( 61.3)17 ( 22.7)4 ( 5.3)x(p)6.695 ( 0.010)0.921 ( 0.337)3.185 ( 0.074)0.671 ( 0.413)1.015 ( 0.314)0.472 ( 0.492)0.080 ( 0.778)- ( 0.439)fisher s exact test .
. there was a higher incidence of ligament rupture among the ssireum athletes
than among athletes of any other sport .
although the non - strike sports showed a higher incidence of
ligament rupture than the strike sports , the difference was not statistically significant
( table 2 ) .
over half of all the athletes had experienced sprains , strains ,
and bruising , and most of the wrestlers had been injured in this way .
the incidence of
bruising was high in both the strike and the non - strike sports .
the boxers and taekwondo
athletes showed the most bruising injuries , followed by the ssireum and wrestling athletes .
the incidence of herniated disc was similar in both groups , with wrestlers among the most
affected ( table 3 ) .
there were differences in
terms of injuries to the cephalic body parts , and neck injuries in particular were more
frequent in the non - strike sports than in the strike sports .
however , the incidence of neck
injuries was similar in the kendo athletes and non - strike sports athletes .
boxers had
experienced more injuries to the face and head than the other athletes ( table 4table 4.crosstabs of region of cephalic injury and sports playersvariableneckfaceheadetc.non-strike sportsjudo ( % ) 8 ( 21.1)4 ( 10.5)2 ( 5.3)0 ( 0.0)ssireum ( % ) 7 ( 43.8)1 ( 6.3)1 ( 6.3)0 ( 0.0)wrestling ( % ) 4 ( 30.8)2 ( 15.4)7.1 ( 7.7)0 ( 0.0)strike sportskendo ( % ) 8 ( 29.6)0 ( 0.0)0 ( 0.0)0 ( 0.0)boxing ( % ) 3 ( 18.8)7 ( 43.8)3 ( 18.8)0 ( 0.0)taekwondo ( % ) 0 ( 0.0)4 ( 12.5)0 ( 0.0)0 ( 0.0)non - strike sports ( % ) 19 ( 28.4)7 ( 10.4)4 ( 6.0)0 ( 0.0)strike sports ( % ) 11 ( 14.7)11 ( 14.7)3 ( 4.0)0 ( 0.0)x(p)3.981 ( 0.046)0.569 ( 0.451)- ( 0.707)-fisher s exact test . *
body part injuries were less frequent than other injuries in all
sports , with the exception of lower back injuries .
the ssireum athletes showed the highest
frequency of lower back injuries , followed by the kendo and wrestling athletes ( table 5table 5.crosstabs of region of body injury and sports playersvariableabdomenchestlow backbacketc.non-strike sportsjudo ( % ) 1 ( 2.6)2 ( 5.3)11 ( 28.9)1 ( 2.6)1 ( 2.6)ssireum ( % ) 0 ( 0.0)1 ( 6.3)11 ( 68.8)2 ( 12.5)0 ( 0.0)wrestling ( % ) 0 ( 0.0)2 ( 15.4)7 ( 53.8)1 ( 7.7)1 ( 7.7)strike sportskendo ( % ) 1 ( 3.7)0 ( 0.0)15 ( 55.6)2 ( 7.4)0 ( 0.0)boxing ( % ) 0 ( 0.0)1 ( 6.3)6 ( 37.5)1 ( 6.3)0 ( 0.0)taekwondo ( % ) 0 ( 0.0)1 ( 3.1)9 ( 28.1)0 ( 0.0)1 ( 3.1)non - strike sports ( % ) 1 ( 1.5)5 ( 7.5)29 ( 43.3)4 ( 6.0)2 ( 3.0)strike sports ( % ) 1 ( 1.3)2 ( 2.7)30 ( 40.0)3 ( 4.0)1 ( 1.3)x(p)- ( 1.000)- ( 0.255)0.157 ( 0.692)- ( 0.707)- ( 0.602)fisher s exact test . *
. there were no significant differences between the two groups in terms of
body part injuries .
shoulder
and elbow injuries were frequent in the non - strike sports group , and injuries to the right
wrist and hands were less frequent in the strike sports group .
there was a lower incidence
of elbow injuries in the ssireum athletes than in the athletes pursuing other non - strike
sports ( left=12.5% ; right=6.3% ) .
wrist injuries were more common in the kendo ( left=55.6% ;
right=48.1% ) and boxing athletes ( left=50.0% ; right=50.0% ) than in the athletes pursuing
other sports , while hand injuries were highest in the boxers .
no major differences between
the groups were identified in terms of finger injuries , which were most frequent in judo ,
followed by boxing , taekwondo , and wrestling ( table
6table 6.crosstabs of region of upper limb injury and sports playersvariableleft shoulderright shoulderleft elbowright elbowleft wristright wristhandsfingersetc.nssjudo ( % ) 16 ( 42.1)17 ( 44.4)12 ( 31.6)17 ( 44.7)12 ( 31.6)8 ( 21.1)4 ( 10.5)25 ( 65.8)0 ( 0.0)ssireum ( % ) 5 ( 31.3)8 ( 50.0)2 ( 12.5)1 ( 6.3)7 ( 43.8)6 ( 37.5)0 ( 0.0)5 ( 31.3)0 ( 0.0)wrestling ( % ) 8 ( 61.5)7 ( 53.8)7 ( 53.8)7 ( 53.8)3 ( 25.0)3 ( 23.1)1 ( 7.7)6 ( 46.2)1 ( 7.7)sskendo ( % ) 2 ( 7.4)4 ( 14.8)5 ( 18.5)3 ( 11.1)15 ( 55.6)13 ( 48.1)3 ( 11.1)3 ( 11.1)0 ( 0.0)boxing ( % ) 5 ( 33.3)5 ( 31.3)4 ( 25.0)4 ( 25.0)8 ( 50.0)8 ( 50.0)7 ( 43.8)10 ( 62.5)0 ( 0.0)taekwondo ( % ) 2 ( 6.3)1 ( 3.1)2 ( 6.3)2 ( 6.3)10 ( 31.3)10 ( 31.3)9 ( 28.1)19 ( 59.4)1 ( 3.1)nss ( % ) 29 ( 43.3)32 ( 47.8)21 ( 31.3)25 ( 37.3)22 ( 33.3)17 ( 25.4)5 ( 7.5)36 ( 53.7)1 ( 1.5)ss ( % ) 9 ( 12.2)10 ( 13.3)11 ( 14.7)9 ( 12.0)33 ( 44.0)31 ( 41.3)19 ( 25.3)32 ( 42.7)1 ( 1.3)x(p)17.299 ( 0.000)20.137 ( 0.000)5.638 ( 0.018)12.452 ( 0.000)1.679 ( 0.195)4.028 ( 0.045)8.047 ( 0.005)1.736 ( 0.188)- ( 1.000)fisher s exact test . nss : non - strike sports ; ss : strike sports . * p <
0.05 ) . there was no significant difference between the groups in terms of lower
limb injuries .
lower limb injuries were the most common body part injury in both groups .
furthermore , there were no significant differences in terms of injuries to left and right
knees and toes , but the frequency was slightly higher in the non - strike sports group .
injuries to the left and right ankles were the most common lower limb injuries in both
groups .
hip joint injuries , on the other hand , were the least common lower limb injuries in
both groups .
overall , there was a higher incidence of lower limb injuries in taekwondo
athletes than in the other sports ( table
7table 7.crosstabs of region of lower limb injury and sports playersvariableleft kneeright kneeleft ankleright ankleleft hipright hipleft toesright toesetc.nssjudo ( % ) 20 ( 52.6)19 ( 50.0)24 ( 63.2)20 ( 52.6)4 ( 10.5)4 ( 10.5)15 ( 39.5)15 ( 39.5)0 ( 0.0)ssireum ( % ) 7 ( 43.8)8 ( 50.0)12 ( 75.0)10 ( 62.5)1 ( 6.3)1 ( 6.3)4 ( 25.0)5 ( 31.3)0 ( 0.0)wrestling ( % ) 7 ( 53.8)9 ( 69.2)7 ( 53.8)6 ( 46.2)1 ( 7.7)1 ( 7.7)0 ( 0.0)1 ( 7.7)2 ( 15.4)sskendo ( % ) 10 ( 37.0)12 ( 44.4)17 ( 63.0)13 ( 48.1)1 ( 3.7)1 ( 3.7)4 ( 14.8)4 ( 14.8)0 ( 0.0)boxing ( % ) 5 ( 31.3)6 ( 37.5)6 ( 37.5)8 ( 50.0)0 ( 0.0)0 ( 0.0)0 ( 0.0)1 ( 6.3)1 ( 6.3)taekwondo ( % ) 14 ( 43.8)17 ( 53.1)25 ( 78.1)24 ( 75.0)6 ( 18.8)5 ( 15.6)8 ( 25.0)9 ( 28.1)4 ( 12.5)nss ( % ) 34 ( 50.7)36 ( 53.7)43 ( 64.2)36 ( 53.7)6 ( 9.0)6 ( 9.0)19 ( 28.4)21 ( 31.3)2 ( 3.0)ss ( % ) 29 ( 38.7)35 ( 46.7)48 ( 64.0)45 ( 60.0)7 ( 9.3)6 ( 8.1)12 ( 16.0)14 ( 18.7)5 ( 6.7)x(p)2.092 ( 0.148)0.706 ( 0.401)0.000 ( 0.982)0.567 ( 0.451)0.006 ( 0.938)0.32 ( 0.857)3.167 ( 0.075)3.062 ( 0.080)- ( 0.447)fisher s exact test .
* p <
0.05 ) . in the responses regarding what the athletes were doing at the time the
injuries occurred , training fights ( 75.19% ) were cited first , followed by competition
( 12.40% ) , weight training ( 5.43% ) , running ( 3.10% ) , stretching ( 2.33% ) , and other ( 1.55% ) .
in terms of the causes of injury , violent training was the most common ( 45.37% ) , followed by
chronic fatigue ( 13.89% ) , lack of warm - up and cooldown exercises ( 13.89% ) , excessive desire
( 12.96% ) , too much stress ( 4.63% ) , other causes ( 3.70% ) , distraction ( 2.78% ) ,
excessive
weight reduction ( 0.93% ) , foul by an opponent ( 0.93% ) , and unreasonable demands by a coach
( 0.93% ) .
the responses regarding when the athletes returned to exercise after injury were as
follows : after partial treatment ( 56.00% ) , after complete recovery ( 19.33% ) , after a few
days of rest ( 14.67% ) , and immediately after receiving the injury ( 10.00% ) .
the responses
regarding why injured athletes returned early included greed ( 48.51% ) , walking on eggshells
around colleagues ( 20.15% ) , coach pressure ( 16.42% ) , and other ( 14.93% ) . in response to
whether they suffered side effects after injury , 85.81% of the athletes answered yes
similarly , in response to whether they relapsed after injury ,
84.62% of the athletes answered yes , while 15.38% answered no . with regard to the
athletes psychological reactions after injury , their main concern was relapse , followed by
difficulty engaging in exercise , loss of ability , other concerns , and concern that a coach
would react negatively .
m : male player ; f : female player ; bmi : body
mass index ; wt : weight training .
* # : p < 0.05 . fisher s exact test . * p < 0.05 fisher s exact test .
* p <
0.05 fisher s exact test . * p < 0.05 fisher s exact test . * p < 0.05 fisher s exact test .
nss : non - strike sports ; ss : strike sports . * p <
0.05 fisher s exact test .
in this study , we surveyed the characteristics of injuries in combat sports and found that
the physical characteristics differed among the athletes in each sport .
skin and nerve injuries were the next
most common , while tooth injuries occurred least frequently in the combat athletes .
we
compared combat sports events according to playing styles , and the results showed that there
were obvious differences between the two groups .
non - strike sports showed a greater
incidence of dislocation than strike sports , while sprains , strains , and bruises were common
in both groups . in terms of body parts
injured , there were major differences between the
groups regarding upper limb injuries . in the case of most upper limb injuries , the
differences were statistically significant .
finger injuries in the judo athletes generally
occurred when they tried to catch their opponents using skill .
injuries to the left and
right shoulders and elbows were more frequent in the non - strike sports , whereas the right
wrist and hands were more frequently injured in the strike sports .
these results can also be
seen in other studies . according to research on sports injuries in pediatrics , children
learning judo are more prone to upper limb injuries than other children , while children
learning taekwondo are more prone to lower limb injuries than other children5 , 17 .
some researchers suggest that judo and wrestling athletes are more likely to incur upper
limb injuries than lower limb injuries21 , 22 .
nevertheless , the same studies indicate
that elbow injuries are less common in ssireum athletes than in athletes pursuing other
sports because , unlike judo and wrestling , ssireum does not involve attempting to catch an
opponent during a match . also , ssireum athletes grab hold of the satba ( a cloth - sash tied
around the waist and thigh ) of their opponents before their matches begin7 . hand and finger injuries are more frequent
in boxing , judo , and taekwondo athletes than in those pursuing other sports .
we believe that
the fists of boxers sustain a direct impact when they strike their opponents . however , some
studies suggest that the reason for hand injuries in taekwondo athletes may be attributable
to their skill in protecting their heads and necks using their upper limbs23 . in this study ,
finger injuries were
particularly common in the judo athletes , which was in line with the results of other
studies on judo injuries4 , 21 .
finger injuries typically occur in judo athletes when
they try to catch opponents using skill .
for example , neck injuries were more frequent in the non - strike sports than in the
strike sports , but there were no differences between the groups in terms of cephalic
injuries .
some studies on combat sports injuries indicate that judo and boxing athletes
incur many severe head and neck injuries15 , 24 . however
, we did not conduct a survey on
severe injuries such as brain hemorrhage and spinal cord injury because the participants in
our study were young athletes and currently participating in their sports .
there was a relatively wide
weight range among the participants of the non - strike group , and injuries to both knees were
less frequent in this group , but the difference was not statistically significant .
more than
half of the judo and wrestling athletes had experienced knee injuries . in line with other
studies
, we found that the most frequent injury in athletes pursuing two events was injury
to the medial collateral ligament25 , 26 .
injuries in combat sports were most
likely to occur during training fights , and the athletes who had incurred injuries felt that
the most common cause was violent training . to prevent injury in combat sports , sports
physical therapists need to pay particular attention to athletes during training . of the
athletes who returned to training following injury
almost all of the athletes who
had been injured experienced side effects and relapse of their injuries .
this is why many
researchers emphasize the importance of injury prevention education for athletes18 , 27 . to summarize our study ,
the type and location of the injury differs
according to playing style in combat sports . in this
regard , dislocation and injuries to the
neck , shoulders , and elbows were found to be more frequent in non - strike sports , while
injuries to the wrists and hands were found to be more frequent in strike sports .
sprains ,
strains , bruises , and injuries to the lower limbs were very common in both groups .
our data
is based on a small sample size and therefore does not include all the injuries that may be
incurred in combat sports . however , we suggest that the characteristics of injuries in
combat sports could be classified according to playing style
. this would enable our study to
provide physical therapists and researchers with information on prevention of injury .
sports
physical therapists need to study precautions and interventions regarding sports injuries
according to the playing style of athletes .
in addition , we recommend further systematic
studies on the various playing styles in combat sports in order to assist sports physical
therapists in devising interventions28 , 29 . | [ purpose ] this study describes the characteristics of injuries in strike and non - strike
combat sports , and the results are intended for use in the area of sports physiotherapy
research .
[ subjects and methods ] the study was conducted on 159 athletes involved in a
variety of combat sports .
the participants included elite college players of the following
sports : judo ( 47 ) , ssireum ( 19 ) , wrestling ( 13 ) , kendo ( 30 ) , boxing ( 16 ) , and taekwondo
( 34 ) . of the participants ,
133 were male and 26 were female . in the case of ssireum and
boxing ,
all of the athletes were male .
[ results ] in the case of the combat sports , the
types of injury and injured regions differed according to playing style . dislocation and
injuries to the neck , shoulders , and elbows were more frequent in the non - strike sports ,
while injuries to the wrists and hands were more frequent in the strike sports .
there was
a high incidence of sprains , strains , bruises , and injuries to the lower limbs in both
groups .
[ conclusion ] we suggest that the characteristics of injuries in combat sports
differ according to playing style , and our study will therefore provide physical
therapists and researchers with information that can be used to prevent injury . | INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION |
PMC3483533 | mouse genome informatics ( mgi ) is the primary international database resource for the laboratory mouse , providing integrated genetic , genomic and biological data to facilitate the study of human health and disease .
the mgi team curates the biomedical literature ( 11 000 publications per year ) and normalizes and integrates sequence and functional data about mouse genetics and genomics from almost 50 other external database and informatics resources .
mgi organizes curation teams around particular types of data including sequence data , phenotypes , embryonic expression data , comparative and functional information , mouse tumorigenesis and mouse models for human diseases .
mgi utilizes multiple bio - ontologies and is the authority for mouse gene and strain nomenclature .
the mouse genome database ( 1 ) includes data on gene characterization , nomenclature , mapping , gene homologies among mammals , sequence links , phenotypes , disease models , allelic variants and mutants and strain data .
the gene expression database ( 2 ) integrates different types of gene expression information from the mouse and provides a searchable index of published experiments on endogenous gene expression during development .
the mouse tumor biology ( 3 ) database provides data on the frequency , incidence , genetics and pathology of neoplastic disorders , emphasizing data on tumors that develop characteristically in different genetically defined strains of mice .
the mgi group is a founding member of the gene ontology consortium ( go , www.geneontology.org , ( 4 ) ) .
mgi fully incorporates the go in the database and provides a go browser for access to mouse functional annotation . finally , the mousecyc database ( 5 ) focuses on mus musculus metabolism and includes cell level processes such as biosynthesis , degradation , energy production and detoxification .
it is part of the biocyc collection of pathway databases created at sri international ( 6 ) .
here , we outline the workflow process for one component of the mgi data acquisition and integration process that associated with the gene ontology project at mgi ( figure 1). mgi assigns functional annotations ( go terms ) to genes and protein products through semi - automated methods and manual curation .
semi - automated annotation strategies include mapping and translating data from the enzyme commission , swiss - prot , interpro ( see http://www.geneontology.org/go.indices.shtml ) , rat and human ortholog experimental data and others .
curation of these data sets includes review and resolution of quality control reports generated through the process of data loading and comparisons to existing data in mgi . for the purpose of this paper
, we will not discuss these semi - automated integration methods , but rather concentrate on the manual literature curation , which is a vital source of experimental mouse functional data .
while we focus on the go component of mgi in this description , the literature curation process is very similar for other mgi components that curate literature .
figure 1.go curation workflow : papers of interest are identified and entered into the database system ( triage ) and associated with genes ( indexed ) .
go curation workflow : papers of interest are identified and entered into the database system ( triage ) and associated with genes ( indexed ) .
the subcomponents of the literature curation process include the following :
( a)literature triage : identifying and obtaining relevant scientific literatureeach curator is assigned a specific subset of journals from a set of 160 relevant journals out of 650 subscriptions carried by the jackson laboratory .
journals are chosen for triage based on the numbers of articles that have been identified and curated for that journal over the previous years .
others are selected on the basis of particular annotation processes such as full curation of the wnt family of proteins , or in another example , full curation of genes implicated in human diseases . on a yearly basis
, the number of papers selected from all journals is tabulated , and the selection of which journals to regularly curate the next year is determined based on the relevancy of the journal publications during the previous year and the number of full time equivalents available for this task .
typically , a few journals are dropped and a few are added each year to the formal triage process .
the quosa application ( http://www.quosa.com ) is used as an aid to access and identify recent papers as represented in pubmed containing data about the mouse and determining which component of the database will be curated from the paper ( go , expression , mutant alleles , phenotypes , mapping , tumor ) .
quosa is used to retrieve full text pdfs for a journal issue or time frame of interest . because the experimental organism mouse is often not mentioned in the abstract ( 334% depending on the journal )
, a curator selects an issue of a journal and then searches the full text of papers containing the keywords
mice. the application highlights these terms showing the context of the search keywords , which enables curators to quickly determine whether the experiments described are of a suitable nature to be used for go annotation ( i.e. do the experiments aid in determining the normal function of the gene ? ) ( figure 2 ) . the number of papers examined and the number selected are somewhat journal dependent .
for example , out of 8090 papers per each weekly issue of journal of biological chemistry , roughly 60 contain one or more of the three keywords , and of those , the curator may select 1015 as being relevant for some area of the database . for an issue of nature , which may have 1520 research articles per issue , up to 5 may have the keywords and
papers selected are uploaded to an in - house server for the next steps.(b)adding the publications to the mgi system through the editorial interfacepapers selected as containing data appropriate for go annotation are entered into a master bibliography module .
each record is tagged for the area of use of the database for which it has information .
information about journal , volume , pages and the abstract are automatically obtained nightly from pubmed using the pmid .
used box would then have an x added automatically.(c)indexing the papers to determine the genes being studieda paper is then associated or indexed to the genes discussed within by adding the paper to each gene s detail module ( figure 3b ) .
prominer , a natural language processing ( nlp ) application , is used to assist in the gene indexing process ( 7 ) .
utilizing official nomenclatures and synonym lists for mouse / rat / human gene names and gene symbols , prominer marks up papers for review by a curator , who then associates genes - to - papers in the mgi editorial interface ( ei ) ( figure 4 ) .
figure 2.quosa information manager showing part of a paper from an issue of jbc with mouse , murine or mice highlighted .
curator quickly looks at context to select appropriate area of mgi that the paper best fits
figure 3.(a ) mgi master bib ei , showing a record for a paper that has been selected for alleles / phenotypes and go .
the paper has not yet been curated for go ( indicated by an x in the selected and x in not used ) .
references to be associated with this gene are entered into the lower left hand panel .
if a paper is associated with multiple genes , the same paper is entered for each gene .
literature triage : identifying and obtaining relevant scientific literature each curator is assigned a specific subset of journals from a set of 160 relevant journals out of 650 subscriptions carried by the jackson laboratory .
journals are chosen for triage based on the numbers of articles that have been identified and curated for that journal over the previous years .
others are selected on the basis of particular annotation processes such as full curation of the wnt family of proteins , or in another example , full curation of genes implicated in human diseases . on a yearly basis
, the number of papers selected from all journals is tabulated , and the selection of which journals to regularly curate the next year is determined based on the relevancy of the journal publications during the previous year and the number of full time equivalents available for this task .
typically , a few journals are dropped and a few are added each year to the formal triage process .
the quosa application ( http://www.quosa.com ) is used as an aid to access and identify recent papers as represented in pubmed containing data about the mouse and determining which component of the database will be curated from the paper ( go , expression , mutant alleles , phenotypes , mapping , tumor ) .
quosa is used to retrieve full text pdfs for a journal issue or time frame of interest .
because the experimental organism mouse is often not mentioned in the abstract ( 334% depending on the journal ) , a curator selects an issue of a journal and then searches the full text of papers containing the keywords
mice. the application highlights these terms showing the context of the search keywords , which enables curators to quickly determine whether the experiments described are of a suitable nature to be used for go annotation ( i.e. do the experiments aid in determining the normal function of the gene ? ) ( figure 2 ) .
for example , out of 8090 papers per each weekly issue of journal of biological chemistry , roughly 60 contain one or more of the three keywords , and of those , the curator may select 1015 as being relevant for some area of the database . for an issue of nature , which may have 1520 research articles per issue , up to 5 may have the keywords and
adding the publications to the mgi system through the editorial interface papers selected as containing data appropriate for go annotation are entered into a master bibliography module .
each record is tagged for the area of use of the database for which it has information .
information about journal , volume , pages and the abstract are automatically obtained nightly from pubmed using the pmid .
for example , if this paper was used for a go annotation , the used box would then have an x added automatically .
indexing the papers to determine the genes being studied a paper is then associated or indexed to the genes discussed within by adding the paper to each gene s detail module ( figure 3b ) .
prominer , a natural language processing ( nlp ) application , is used to assist in the gene indexing process ( 7 ) . utilizing official nomenclatures and synonym lists for mouse / rat / human gene names and gene symbols , prominer marks up papers for review by a curator , who then associates genes - to - papers in the mgi editorial interface ( ei ) ( figure 4 ) .
quosa information manager showing part of a paper from an issue of jbc with mouse , murine or mice highlighted .
curator quickly looks at context to select appropriate area of mgi that the paper best fits .
( a ) mgi master bib ei , showing a record for a paper that has been selected for alleles / phenotypes and go .
the paper has not yet been curated for go ( indicated by an x in the selected and x in not used ) .
references to be associated with this gene are entered into the lower left hand panel . if a paper is associated with multiple genes , the same paper is entered for each gene .
output from prominer : prominer is used to assist in the gene indexing process . utilizing official nomenclatures and synonym lists for mouse / rat / human gene names and gene symbols , prominer marks up papers for review by a curator , who then associates genes - to - papers in the mgi ei .
sometimes a paper discusses several genes , but not all of them may be objects for direct go annotation .
for example , a paper describing the effects of a knock out of a particular gene may use analysis of other gene products to analyze the particular processes being affected , but the annotation to involvement in the process would only be made to the gene being knocked out .
currently , the topical areas selected for each paper are not directly tied to the genes associated with the paper .
thus , a paper selected for go for one of the genes will appear in the go ei interface of unused papers for each of the genes indexed to the paper .
ideally , all papers would be immediately used for go annotation , but on average 300 new papers are added to the database each week , only less than half of these are curated each week due to resource limitations .
therefore , various priority selection criteria are used to choose which genes and papers warrant immediate attention
. reports of interest are generated , such as genes with no go annotation but have new indexed literature selected for go or genes with mutant alleles that have literature selected for go ( figure 5 ) .
additionally , participation in various collaborative projects , such as the reference genome project ( 8) , or the protein ontology ( 8) defines primary sets of genes to work based on community input .
a curator at mgi uses the go ei module to enter annotations ( figure 6 ) .
curators use the annotation guidelines set forth by the gene ontology consortium ( http://www.geneontology.org/go.annotation.shtml ) .
it is divided into two main data entry sections : the annotation area and the annotation properties area .
a list of papers selected for go that are associated with this gene is shown in the lower right panel .
individual protein isoforms or modified forms can be indicated using annotation properties where an i d for a specific isoform can be indicated . after reading a paper
next , the reference number is added ( 2 ) , as well as the evidence code ( 3 ) .
if required by the type of evidence code , additional information is added to the inferred from column ( 4 ) .
once the annotation is saved , information about the cell type that the experiment was done in , or the specific isoform , or tissue , is entered into the annotation properties section ( 5 ) .
additional ontologies such as the cell ontology ( 9 ) , mouse adult ( 10 ) and embryonic anatomies ( 11 ) , protein ontology ( 12 ) , and psi - mod ( 13 ) are used in the properties fields .
several of these are used to supply an extension to the annotation which are used in the gene association file ( gaf ) ( 5 ) .
curators can use the obo - edit tool ( 14 ) to load multiple ontologies to aid in searching for appropriate terms , as well as viewing the chosen term in the context of the rest of the ontology ( figure 7 ) .
the go vocabulary is refreshed daily from the go site , and only current go terms can be used , otherwise data entry is prohibited . only reference identifiers previously entered into
there are other mechanisms , such as data loads , that provide go annotation . in all cases , provenance is provided .
the go ei also has a built - in report generator that highlights words matching go terms found in the abstracts of papers selected for go as an aid to suggesting the type of information and evidence present in a paper ( figure 8) .
figure 6.mgi go data entry module : the interface is divided into three main sections : go annotation , annotation properties and search and reference tracking .
drop - down menus display pick - lists of allowed entries in various fields ( evidence property , go qualifier and evidence codes ) . numbered areas : 1 ,
go i d entry ; 2 , reference entry ; 3 , evidence code entry ; 4 , inferred_from entry required for certain evidence codes and 5 , annotation properties entry .
the far left panel shows the vocabularies that have been loaded for searching and viewing .
the right panel displays the terms in all of the vocabularies that contain the word
kidney. the go term kidney mesenchyme morphogenesis is selected and is visible as a tree view showing its children ( middle panel ) , and as a graphical view showing its parents ( lower left ) .
figure 8.report generated using the abstracts of papers selected for go for the gene being annotated within the go ei .
mgi go data entry module : the interface is divided into three main sections : go annotation , annotation properties and search and reference tracking .
drop - down menus display pick - lists of allowed entries in various fields ( evidence property , go qualifier and evidence codes ) . numbered areas : 1 ,
go i d entry ; 2 , reference entry ; 3 , evidence code entry ; 4 , inferred_from entry required for certain evidence codes and 5 , annotation properties entry .
the far left panel shows the vocabularies that have been loaded for searching and viewing .
the right panel displays the terms in all of the vocabularies that contain the word
kidney. the go term kidney mesenchyme morphogenesis is selected and is visible as a tree view showing its children ( middle panel ) , and as a graphical view showing its parents ( lower left ) .
report generated using the abstracts of papers selected for go for the gene being annotated within the go ei .
annotations are tracked based on a variety of criteria such as annotation source ( mgi curation or data load ) and evidence ( experimental or predictive , such as through orthology or functional domain ) .
scripts review changes to the go structure and provide qc reports for curators noting genes whose annotations may be affected by these changes ( figure 9 ) .
additionally , we use the master bibliography tables and the go annotations to keep track of various areas that need focus , such as genes with no go annotation but have papers that are selected for go but not used.
figure 9.go change log report showing changes to the go and genes with annotations using the term that may need to be looked at .
go change log report showing changes to the go and genes with annotations using the term that may need to be looked at .
all data assertions in mgi are supported by evidence and citation to the source of the information . for assertions that are associated with controlled vocabularies such as the go ,
links are provided to vocabulary browsers that provide the relationships between the assertion and other knowledge in that area of the ontology . using the table and associated information
mgi also provides a graphical display of go annotations from the go detail page for each gene .
figure 10.the go annotation details for drd2 displayed as summary text ( a ) , table ( b ) or graph ( c ) .
the go annotation details for drd2 displayed as summary text ( a ) , table ( b ) or graph ( c ) .
go annotations are also shared with the go consortium ( goc ) through a gaf .
this is a tab - delimited file that contains most of the elements of a go annotation as outlined in the go ei section above .
presently , only the cell type and gene product annotation properties are included in the gaf .
this file is available on either the goc web site or along with other data sets , from the mgi ftp site ( ftp://ftp.informatics.jax.org/pub/reports/index.html ) .
the gaf and the go vocabulary file are used as input for many analytical tools such as go termfinder ( 15 ) .
instructions for construction of a go gaf file are found in go documentation at http://www.geneontology.org/go.format.annotation.shtml .
although some groups have used nlp to expedite the curation of literature ( 16 ) , this can be especially difficult to do when applied to mouse biology because of the integration of human and mouse studies within the same description of results .
the concepts captured by the go can not be gleaned just from simple text matching of terms , but must also take into account inferences that reflect a given context .
additionally , an understanding of the nature of an experimental assay is important to correctly use the information as evidence of a particular result . while we continue to work with nlp developers to design a system to automate identification and tagging of papers ( 17 ) ,
it is clear that the complexity of understanding the information in a biomedical publication requires the intervention of an experienced biologist - curator in the process .
funding for open access charge : mgi database resources are funded by grants from the national human genome research institute ( hg00330 , hg02273 ) , national institutes of health / national institute of child health and human development ( hd062499 ) and the national cancer institute ( ca89713 ) . | the mouse genome database , the gene expression database and the mouse tumor biology database are integrated components of the mouse genome informatics ( mgi ) resource ( http://www.informatics.jax.org ) .
the mgi system presents both a consensus view and an experimental view of the knowledge concerning the genetics and genomics of the laboratory mouse . from genotype to phenotype , this information resource integrates information about genes , sequences , maps , expression analyses , alleles , strains and mutant phenotypes .
comparative mammalian data are also presented particularly in regards to the use of the mouse as a model for the investigation of molecular and genetic components of human diseases .
these data are collected from literature curation as well as downloads of large datasets ( swissprot , locuslink , etc . ) .
mgi is one of the founding members of the gene ontology ( go ) and uses the go for functional annotation of genes . here
, we discuss the workflow associated with manual go annotation at mgi , from literature collection to display of the annotations .
peer - reviewed literature is collected mostly from a set of journals available electronically .
selected articles are entered into a master bibliography and indexed to one of eight areas of interest such as go or homology or
phenotype. each article is then either indexed to a gene already contained in the database or funneled through a separate nomenclature database to add genes .
the master bibliography and associated indexing provide information for various curator - reports such as papers selected for go that refer to genes with no go annotation. once indexed , curators who have expertise in appropriate disciplines enter pertinent information .
mgi makes use of several controlled vocabularies that ensure uniform data encoding , enable robust analysis and support the construction of complex queries .
these vocabularies range from pick - lists to structured vocabularies such as the go .
all data associations are supported with statements of evidence as well as access to source publications . | Introduction
Curation triage: selecting what genes to annotate
Data entry: creating a GO annotation using the EI module for GO
Tracking metrics and quality control measures to set priorities for upcoming work
Annotation presentation and usage
Information access: NLP and beyond
Funding |
PMC2815291 | natural orifice surgery has evolved from a preclinical setting into a common occurrence at the university of california san diego ( ucsd ) . with close to 40 transvaginal cases ,
the laparoscopic repair of recurrent umbilical defects , or defects greater than 3 cm in size , has been shown to decrease recurrence rates .
we have chosen to further investigate the benefits of laparoscopy by applying a natural orifice approach .
one of the perceived advantages in natural orifice surgery is the potential reduction in the incidence of hernia formation .
patients with abdominal wall hernias may be at increased risk of forming additional hernias at incision sites .
in addition , patients with recurrent incisional hernias may , likewise , be at increased risk .
we believe that reducing or eliminating abdominal wall incisions may be of benefit in the repair of abdominal wall hernias . to this end ,
a preclinical research protocol in transvaginal incisional herniorrhaphy was undertaken in a porcine model . after obtaining comfort with the technical procedure ,
here , we describe what we believe to be the first natural orifice transluminal endoscopic surgical ( notes ) approach to the repair of an abdominal wall hernia .
the patient is a 38-year - old female with a painful recurrent umbilical hernia who was repaired 8 years prior with a polypropylene - based mesh .
the patient has had three children since the time of her repair and had a recurrence of her umbilical hernia , which was approximately 2 2 cm in size .
the patient denied any obstructive signs or symptoms , but rated her daily pain at the site as 5/10 .
her past medical and surgical history is only pertinent for a cesarean section and the aforementioned hernia repair . the risks , benefits , and alternatives to a transvaginal incisional hernia repair
she was consented under an approved ucsd transvaginal incisional hernia repair institutional review board ( irb ) protocol .
importantly , all current ucsd notes protocols stipulate that an abdominal port be utilized for visualization of the transvaginal access .
the patient was brought to the operating room , sequential compression devices applied , 1 g of cefazolin was given , and general anesthesia induced without complication .
the patient was placed in the lithotomy position and prepped and draped in standard sterile fashion .
a lower left quadrant 5-mm trocar was placed below the pubic hairline using optical assistance .
a well incorporated mesh was noted in the umbilical region with a small herniation of falciform material through a 2-cm defect superiorly .
given that the mesh was well incorporated and peritonealized , it was left in place .
a uterine retractor was then placed and a 15-mm dual - lumen trocar ( applied medical ) was placed through the retrocervical cul - de - sac .
the dual - lumen trocar allows for placement of the scope , as well as another 5-mm rigid or flexible instrument .
the herniated falciform was then dissected free of the fascial edges using a combination of endoscopic and flexible instrumentation delivered through the vaginal port .
next , the peritoneum was cleared superiorly approximately 4 cm using the same method .
having dissected the facial edges free , the defect was then re - approximated primarily in the manner of franklin et al . .
this was done transcutaneously using a suture passer through the center of the skin overlying the defect , and sequentially passing an 0-ethibond suture approximately 1 cm lateral to each fascial edge .
next , a 7 7-cm flex hd acellular human dermis mesh ( johnson & johnson ) was inserted through the vaginal trocar once four anchoring ethibond sutures were tied to the corners .
a needle passer was passed in four quadrants , tacking the mesh in a transfascial manner .
lastly , a protack ( autosuture ) device was used to anchor the remainder of the material to the patient s anterior abdominal wall at approximately 1-cm intervals circumferentially ( fig .
2final view of the hernia repair entrance of the vaginal trocar final view of the hernia repair the abdomen was inspected and had good hemostasis ; the 5-mm abdominal port was removed .
the gynecology team then closed the patient s vaginal access site with a 2 - 0 vicryl suture in a running fashion .
the 5-mm trocar was removed and the skin closed with a 4 - 0 monocryl suture .
the patient was admitted to the hospital for observation as per the irb and was discharged on postoperative day 2 in good health . at 6 weeks
she was then seen at 3 and 5 months and is still completely satisfied with her repair .
the repair of primary and incisional hernias of the ventral abdominal wall via a transvaginal approach is technically feasible , and the result of our initial case was exceptional .
however , there are still significant obstacles which must be addressed before this approach can be widely utilized .
some have suggested that blind or primary access of the vagina for natural orifice surgery is safe [ 3 , 4 ] .
we do not agree . at this time , we still believe that visualization of the trocar insertion through the vagina is necessary .
though all of the key steps of the described operation were performed utilizing the vaginal access port , we still believe that blind access to the abdomen with a trocar through the vagina may put the patient at risk for visceral or vascular injury .
an additional concern in the application of natural orifice surgery to the repair of ventral hernias is the selection of the proper material for the hernia repair .
several authors have described the preclinical technical aspects of notes hernia repair in a cadaver or animal model , and most have questioned whether a prosthetic material can be used in regard to potential mesh infection [ 5 , 6 ] .
we believe this to be a very valid point . additionally , many recurrent ventral hernias have also had prior implantation of prosthetic materials , as did this patient .
her mesh was firmly incorporated and peritonealized . given this , we decided to progress with the operation . had the initial mesh been exposed or not incorporated , we would have abandoned the notes approach in favor of a more traditional method .
we do not believe that there is sufficient preclinical data from our own laboratory , or others , to advocate the placement of a prosthetic device in the face of transvaginal or transgastric abdominal access .
therefore , we used a biologic material for the repair . the use of biologic materials for abdominal wall reconstruction in contaminated or infected fields is well established . however , there is increasing concern that the re - herniation rate will be unacceptably high if the material is used to bridge the defect , rather than to buttress an appropriately re - approximated fascia .
fascial re - approximation was fairly simple with this small defect , but this severely limits the patient population which may benefit from a natural orifice approach to ventral hernias .
for the procedure to have widespread utilization , prosthetic devices will have to be used .
one of the main benefits that we have seen in our transvaginal cholecystectomy population has been the reduction of postoperative pain . while our patient was fairly comfortable postoperatively , she still experienced some pain ( 5/10 ) at the transfacial fixation sutures on postoperative day 1 . whether or not these sutures are still necessary when using a material that is ultimately absorbed or incorporated is questionable .
there are no large case series from which to draw observations in regard to the laparoscopic placement of biologic materials for the repair of ventral hernias .
given the minimal penetration of the abdominal tacks into the posterior fascia once passed through biologic material , we still believe them to be necessary .
the initial application of natural orifice techniques to the repair of an abdominal wall defect was technically feasible and clinically successful .
the patient presented here did very well postoperatively , with minimal complaints of pain or any signs of infection .
the outcome from this operation was encouraging and we look toward further investigation to answer the aforementioned questions noted in this paper . | introductionnatural orifice surgery has evolved from a preclinical setting into a common occurrence at the university of california san diego ( ucsd ) . with close to 40 transvaginal cases ,
we have become comfortable with this technique and are exploring other indications .
one of the perceived advantages in natural orifice surgery is the potential reduction in the incidence of hernia formation .
patients with abdominal wall hernias may be at increased risk of forming additional hernias at incision sites .
in addition , patients with recurrent incisional hernias may , likewise , be at increased risk .
we believe that reducing or eliminating abdominal wall incisions may be of benefit in the repair of abdominal wall hernias . here , we describe what we believe to be the first natural orifice transluminal endoscopic surgical ( notes ) approach to the repair of an abdominal wall hernia.methodsthe patient is a 38-year - old female with a painful recurrent umbilical hernia , previously repaired 8 years prior with a polypropylene - based mesh .
the patient underwent a transvaginal recurrent umbilical hernia repair with one other 5-mm port in the abdomen for safety.resultsthe patient had no intraoperative or postoperative complications . at 5 months
follow up , the patient had no complaints , no evidence of hernia recurrence , and was very pleased with her result.conclusionsthe repair of primary and incisional hernias of the ventral abdominal wall via a transvaginal approach is technically feasible , and the result of our initial case was exceptional
. however , there are still significant obstacles which must be addressed before this approach can be widely utilized .
these obstacles include safe entrance into the abdominal cavity via a transvaginal approach , the proper mesh to be placed during the repair , and the risk of infection . | Introduction
Methods
Results
Discussion
Conclusion |
PMC3204056 | from 20022004 , i had the privilege of working with a diverse group of patient advocates , communications professionals , political consultants , scientists , and professional politicians to conceive , nurture , design , and secure the passage of california 's landmark proposition 71 ballot initiative , which established the california institute for regenerative medicine ( cirm ) .
cirm was designed to award $ 3 billion in competitive , peer - reviewed grant funds to stem cell research running from basic science to clinical application .
the proposition 71 ballot initiative that created cirm passed with almost 60% of the voters supporting it , primarily because of the medical promise of stem cell research , the unique political pressures and ethical debate surrounding stem cell research in the united states , and the potential and recognized benefit that biomedical research brings to california 's biotechnology industry and economy .
much has been said and written about the merits and challenges of this landmark effort and its current function .
i want to focus this essay , however , on a number of valuable lessons i learned about the role that scientists and rigorous scientific advice can play in the political process , in discussions with the public and patient advocates , and in the crucible of a high - visibility political and scientific initiative .
in particular , i learned that , while the public political process can be messy , practicing research scientists not only can provide valuable information and advice , but can gain a great deal of valuable experience .
i also learned that , while the world of science is not the only data - driven group , it nonetheless contributes a much longer - term perspective on issues than do other groups .
do n't crush our hopes ! overhype ! too much pessimism ! i heard all of these conflicting statements and more from my patient advocate friends and colleagues during the proposition 71 campaign .
the lesson i learned is that one has to walk a fine line between too much pessimism , which some patient advocates will tell you has a negative impact , because it kills their hope , as opposed to too much optimism , which contributes to the problem of overhyping or promising more than can be delivered in the time frame that you or someone else imagines . in short , i learned that i should paint a reasonable picture of what success would bring , while describing reasonable expectations and likely obstacles .
for example , it is and was clear that success with stem cell approaches for type 1 diabetes will potentially bring complete insulin independence . but a number of important technical and safety problems will have to be solved on the path to achieving that goal , which leads to an uncertain time frame .
i have often been asked , both during the proposition 71 process and since then , how long it will take a particular area of research to reach clinical application or , even harder to answer , when will my wife 's , child 's , or parent 's disease be cured ? these are difficult , if not impossible , questions to answer , because the point of research is to find answers to unanswered questions , often with little accurate information available at the outset .
the answers one gives thus require an explanation , not a number , since one can not possibly give an accurate number .
i also learned that it is appropriate to describe what success might look like , to talk about past experience with success in generating new therapies or discoveries , and to describe how broad portfolios of parallel opportunities will yield some that will proceed more quickly and some that will go more slowly .
i would describe being a scientist in this ( and other ) fields as being like an explorer at the edge of a new continent , where i could see mountains and forests in the distance and knew it was likely that there were riches to be had .
thus i could argue it was worth proceeding and there would be great rewards , but predicting exactly how long it would take to yield results would be problematic .
but i also learned that it is compelling to note that not starting a long journey leads to no benefit , or adds delay to what will be a long journey , no matter what .
nonetheless , the issue of time is hard and can also be difficult to communicate .
thus i also learned one can talk about short - term and long - term returns and describe what the process will look like .
finally , i learned that telling people that scientists are working as rapidly as possible to find better therapies is helpful .
many people respect this honesty and find it reassuring that the scientific community does care about trying to find therapies for people 's diseases , even if success will take time .
the media and the communications industry are essential if scientists are to communicate with the public .
in fact , the media is a megaphone that allows us to talk to more than just a few people at a time . in that context
, the media is an essential part of how scientists work with other groups in political settings .
but i often hear my colleagues say that reporters never get it right or that some reporter did n't quote them accurately or that they
talked to a reporter for half an hour or more and they only used a one - sentence quote or a 15- to 20-s segment in the final radio or tv report .
first , my experience is that the vast majority of people i interacted with in the media are intelligent , hard - working professionals with little scientific background , who are nonetheless trying very hard to get the facts straight and to be as objective as possible .
second , my experience is that the point of a 20- or 30-min interview is that this is time a scientist can use to educate a reporter about the topic being covered , so the overall story is accurate , including the parts not directly based on the interview .
furthermore , my experience is that the journalist will be more likely to choose the right quote and/or get it right if the scientist has done a good job of providing an understandable explanation of the topic under discussion . to achieve this goal ,
however , requires that scientists learn to explain complex topics in plain everyday english with a minimum of jargon or latinate language , which of course , is the same as good teaching .
for example , i learned to say blood - forming stem cell instead of hematopoietic stem cell to nonscientific audiences .
any coordinated action of a group with media and the public will inevitably require teamwork , as well as consolidation and discipline around shared messages .
this can get tricky when the shared messages fail to agree with any one scientist 's view regarding correctness of information imparted or focus on the proper priorities .
but if every member of a group chooses to broadcast his or her own version of a message , instead of uniting around common themes , what will emerge is cacophony , confusion , and likely failure .
the question is how to balance scientific and personal integrity with teamwork , shared goals , and shared messages
. my own solution was to work vigorously in private to ensure that the common message themes were rigorously correct , so that i , and my colleagues on the team , were always saying things that to the best of our knowledge were scientifically accurate and defensible .
but i also chose to agree to focus on shared message points publicly , even if my own view was that these points were not as important scientifically as other messages .
similarly , i had to accept that the public at large and the scientific community sometimes use the same words differently .
my personal and scientific view is that few diseases other than infectious diseases are ever truly cured and that what we offer are therapies that relieve , reduce , or manage a disease . in the public arena , however , what i regard as short- to medium - term therapies seem to be thought about as cures . in this case , my solution was to speak about therapies in my own interviews and public talks , but not to publicly debate the issues around the word cure with my own team .
in this situation , the crucial role of a scientist is to work as hard as possible privately to get the substance of the messages absolutely correct , and then to work with the team on promoting them in a coordinated and agreed - upon way .
any coordinated action of a group with media and the public will inevitably require teamwork , as well as consolidation and discipline around shared messages .
this can get tricky when the shared messages fail to agree with any one scientist 's view regarding correctness of information imparted or focus on the proper priorities .
but if every member of a group chooses to broadcast his or her own version of a message , instead of uniting around common themes , what will emerge is cacophony , confusion , and likely failure .
the question is how to balance scientific and personal integrity with teamwork , shared goals , and shared messages .
my own solution was to work vigorously in private to ensure that the common message themes were rigorously correct , so that i , and my colleagues on the team , were always saying things that to the best of our knowledge were scientifically accurate and defensible .
but i also chose to agree to focus on shared message points publicly , even if my own view was that these points were not as important scientifically as other messages .
similarly , i had to accept that the public at large and the scientific community sometimes use the same words differently .
my personal and scientific view is that few diseases other than infectious diseases are ever truly cured and that what we offer are therapies that relieve , reduce , or manage a disease . in the public arena , however ,
what i regard as short- to medium - term therapies seem to be thought about as cures . in this case , my solution was to speak about therapies in my own interviews and public talks , but not to publicly debate the issues around the word cure with my own team .
in this situation , the crucial role of a scientist is to work as hard as possible privately to get the substance of the messages absolutely correct , and then to work with the team on promoting them in a coordinated and agreed - upon way .
building broad coalitions to support biomedical and other fields of scientific research is clearly an area where scientists , politicians , and other interested groups will have to work together more than ever to protect publicly funded science in the coming years .
there is increasing scrutiny by politicians and the public , who want to know how the investment of their funds benefits them . responding to that legitimate concern , and finding ways to continue to expand scientific research that supports healthy vigorous societies , will require scientists to be part of these teams of diverse interest groups .
i can virtually guarantee from my experiences that decisions about funding and science policy made in the absence of scientific input will likely be unpalatable to scientists and ultimately not in the best interest of our broader societies .
thus a key lesson from the proposition 71 experience is that engagement of scientists with diverse nonscientific groups can make a big difference and that scientists must actively engage with the public in the future if we are to contribute robustly to the medical and economic health of our communities . | i describe a number of valuable lessons i learned from participating in california 's proposition 71 effort about the role that scientists and rigorous scientific advice can play in a public political process .
i describe how scientists can provide valuable information and advice and how they can also gain a great deal from the experience that is valuable to a practicing research scientist .
finally , i argue that in the future , building similar broad coalitions to support biomedical and other areas of scientific research will be essential to protect publicly funded science .
thus , a key lesson from the proposition 71 experience is that engagement of scientists with diverse nonscientific groups can make a big difference and that scientists must actively engage with the public in the future if we are to contribute robustly to the medical and economic health of our communities . | INTRODUCTION
FINDING A HAPPY MEDIUM
THE PROBLEM OF TIME
MEDIA AND SOUND BITES
THE PROBLEMS WITH TEAMWORK
APPLYING PROPOSITION 71 LESSONS TO OTHER INITIATIVES? |
PMC3182334 | brain - derived neurotrophic factor ( bdnf ) is a member of the neurotrophin family , a group of structurally related polypeptide growth factors .
the family also includes nerve growth factor ( ngf ) , neurotrophin-3 ( nt-3 ) , and neurotrophin-4 ( nt-4/5 ) .
other neurotrophins have also been identified , nt-6 and nt-7 ; however , these likely do not exist in mammals [ 24 ] .
neurotrophins activate one or more of the high - affinity tropomyosin - receptor kinase ( trk ) receptor family [ 1 , 5 , 6 ] , as well as the low - affinity p75 neurotrophin receptor ( p75ntr ) . neurotrophins direct growth and differentiation in the developing nervous system [ 1 , 3 , 6 , 8 ] .
levels of the different neurotrophins relate in a predictable pattern to stages of embryonic development .
infusion of bdnf into the adult brain promotes neurogenesis [ 9 , 10 ] , and dendritic spine reorganization in the rat hippocampal formation .
bdnf gene transfection triggers dendritic and axonal branching in dentate gyrus ( dg ) granule cell cultures while mice with a targeted gene deletion show substantial impairment in normal cerebellar development , among other developmental and behavioral deficits .
trk receptors precipitate the activation of many signaling cascades , including phospholipase c ( plc ) , phosphoinositide 3-kinase ( pi3k ) , and ras [ 14 , 15 ] , which are involved in different aspects of neuronal survival and neurite outgrowth [ 1719 ] .
the p75ntr is implicated in both pro- and antitrophic processes , such as neurite outgrowth and apoptosis [ 14 , 21 , 22 ] .
bdnf regulates synaptic transmission and activity - dependent plasticity [ 23 , 24 ] and promotes long - term potentiation ( ltp ) .
bdnf exerts presynaptic effects in a retrograde or paracrine fashion [ 27 , 28 ] and postsynaptic effects by modulating nmda receptor subunit expression .
experimental reductions in bdnr or trkb activity significantly impair ltp [ 25 , 30 , 31 ] .
previous research has demonstrated that exercise increases hippocampal bdnf mrna [ 32 , 33 ] and protein [ 34 , 35 ] .
the effect of exercise to augment bdnf activity depends on camp - response - element binding protein ( creb ) activation , as creb suppression mitigates this effect .
increases in hippocampal bdnf mrna have been detected after a period of running as short as 6 hours , to as long as two months , signifying a sensitive yet sustainable effect .
physical activity leads to greater activation of cellular signaling pathways associated with survival and neurogenesis , and can enhance hippocampal ltp induction , all of which strongly implicate bdnf in their effects .
this review will describe how bdnf and neurotrophins are involved in nervous system development , the signal transduction mechanisms associated with neurotrophin activity , and the modulating role bdnf plays in synaptic plasticity and ltp .
the influence of exercise on bdnf activity , especially in the hippocampus , and the relationship between bdnf and excitotoxicity will also be examined .
bdnf and nt-4/5 bind to trkb receptors [ 41 , 42 ] , as does nt-3 to a lesser extent .
when a ligand binds to a trk receptor ( see figure 1 ) , the receptor dimerizes and autophosphorylates tyrosine residues to yield docking sites for the src homology 2 domain - containing adapter protein ( shc ) and phospholipase c- ( plc- ) , which are coupled to intracellular signaling cascades such as ras , pi3k [ 45 , 46 ] , and , as mentioned , plc- [ 14 , 15 , 47 , 48 ] .
shc regulates protein interactions by binding , with a high - affinity , tyrosine - phosphorylated sites , and in this way regulates intracellular signaling such as ras .
once shc is docked with the receptor and bound to the adapter protein grb2 , ras promotes activation of the mitogen - activated protein kinase ( mapk)/extracellular signal regulated kinase ( erk ) cascade , as well as the pi3k cascade [ 3 , 15 ] .
mapk / erk is essential for neurogenesis and promotes survival by two ways , by induction of prosurvival genes and inhibition of proapoptotic proteins .
ras also suppresses apoptosis via pi3k ; pi3k activates akt , which sequesters pro - apoptotic proteins in the cytoplasm away from their transcriptional targets .
the pi3k cascade can be activated two ways by trk receptors , through ras as well as through adapter proteins shc , grb2 , and grb2-associated binder-1 ( gab1 ) [ 15 , 45 ] . in some neurons , bdnf phosphorylates the insulin receptor substrate , which can activate pi3k as well .
vaillant and colleagues demonstrated that inhibition of any portion of the ras - pi3k - akt pathway significantly reduced survival of sympathetic neurons in culture in the presence of ngf .
the adapter proteins shc and plc- are phosphorylated once they dock with the trk receptor [ 3 , 14 , 47 ] .
once activated , plc- then catalyzes the breakdown of lipids to inositol 1,4,5 triphosphate ( ip3 ) , which promotes the release of calcium from intracellular stores , and a subsequent increase in intracellular calcium concentration and diacylglycerol ( dag ) occurs .
dag regulates protein kinase c ( pkc- ) , which may be required for the mapk / erk signal , and both dag and mapk / erk have been implicated in neurite outgrowth [ 18 , 19 ] .
interestingly , ltp is inhibited when plc- signaling is blocked , which may be related to reduced ip3 formation and subsequent reduction in intracellular calcium release .
the other neurotrophin - responsive receptor , the p75ntr , can bind to each factor and regulate the trk receptor affinity for its ligand .
the trk receptors and the p75ntr are often present in close proximity on the cellular membrane .
while trkb responds to nt-3 , nt-4/5 , and bdnf , the receptor selectively binds only bdnf when colocalized with p75ntr .
the largely presynaptic p75ntr serves dual roles in that it modulates trk receptor binding and is involved in prolonged ras - mediated activation of erk and neurite outgrowth , though p75ntr - induced neurite outgrowth ceases after prolonged p75ntr activation .
the p75ntr also activates c - jun n - terminal kinase ( jnk ) and causes apoptosis in a variety of neurons [ 14 , 21 , 22 ] .
although all trk receptors are assumed to behave in much the same fashion , differences do exist .
kaplan and miller demonstrated , using trkb receptors with mutations at different binding sites , that trkb receptors use both pi3k and mapk cascades for cell survival while trka receptors depend mainly on pi3k .
this demonstrates some comparative flexibility for trkb receptors with regard to pro - survival function .
importantly , two truncated forms of trkb receptors exist that lack tyrosine kinase function and bind bdnf to negatively modulate trkb activity .
given that bdnf - associated signaling modulates neuron survival and apoptosis , much attention has been focused on the role of bdnf ( and other neurotrophins ) in development .
indeed , the critical involvement of trk receptors in neuronal survival [ 16 , 5052 ] , neurogenesis , and neurite outgrowth [ 12 , 18 , 19 ] implicates bdnf in developmental processes . throughout development , neurotrophins influence and guide neuronal differentiation in the central nervous system ( cns ) [ 6 , 8 ] and peripheral nervous system ( pns ) [ 1 , 3 ] .
barnab - heider and miller demonstrated that cortical progenitor cells express bdnf and nt-3 and their associated trkb and trkc receptors , and that these neurotrophins were essential to the survival of the progenitor cells .
bdnf mrna expression is lowest in the brains of early rat embryos and increases into adulthood .
it demonstrates a reciprocal relationship with nt-3 mrna , which is at the highest levels in early embryos .
maisonpierre and colleagues characterized distribution of bdnf , nt-3 , and ngf mrna during rat development and found that the amount of all three dramatically increased between embryonic days 11 and 12 , with transcripts widely distributed by embryonic day 13 .
this timing corresponds directly to the onset of robust neurogenesis in the peripheral and central nervous systems [ 60 , 61 ] .
examined bdnf protein concentration during postnatal development in rats and found that levels in the septum , cerebellum , and hippocampus increased over time and then stabilized ( although hippocampal protein levels took much longer to plateau ) while hypothalamic bdnf protein levels increased for a period following birth and then decreased .
kim et al . further investigated bdnf - immunoreactivity ( ir ) in the rat forebrain and upper brainstem during postnatal development .
the investigators defined three groups of bdnf cells : those that are detected early , increase in number , and remain stable during adulthood ( e.g. , piriform cortex , cingulate gyrus ) , those that increase with age then decrease in adulthood ( e.g. , basolateral nucleus of the amygdala ) , and those that appear briefly before dramatic reductions in adulthood ( e.g. , substantia nigra , various hypothalamic nuclei ) . in the hippocampus
, bdnf - ir cells appeared in the pyramidal layer of ca2 and ca3 early on ( postnatal day 7 ) and staining increased through 28 days following parturition but then decreased , with comparatively reduced cell staining detected in 56-day - old adults .
the bdnf - ir cell decrease detected in ca2 and ca3 in adulthood was completely absent in colchicine - treated adults , which instead showed an increase in bdnf - ir cells in these areas , possibly reflecting increased accumulation of bdnf in the cell body associated with inhibited axonal transport .
bdnf - ir fibers were detected in the stratum lucidum layer of ca2 and ca3 and the polymorph layer of the dg also early on during the intial 28-day postnatal period , and these bdnf - ir fibers actually continued to increase into adulthood .
bdnf serves a unique role among neurotrophins in development , especially with regard to the cerebellum .
an examination of the cerebella of mice with a targeted bdnf gene deletion ( bndf mice ) revealed that bdnf is essential for normal development and function of the cerebellar cortex .
the study showed reduced trk activation in purkinje cell bodies and dendrites in the mutant mice , indicating a lack of redundancy among neurotrophins with regard to bdnf in these cells . in bndf mice
, there was a dramatic increase in cell death among developing granule cells , impaired development of the layers of the cerebellar cortex , and irregular foliation in the middle and posterior cerebellum ; defects were apparent in the declival sulcus ( absent ) , the intercrural fissure ( absent ) , the prepyramidal fissure ( nearly absent ) , and uvular sulcus ( nearly absent ) .
bndf is crucial to postnatal survival , as most bndf mice die shortly after birth , but some do survive for a month or more [ 64 , 65 ] .
mice that lack bdnf demonstrate severe deficiencies in pns development , especially with regard to afferent neurons , but exhibit comparatively mild deficiencies in cns development [ 64 , 66 ] .
bndf mice show an abnormal gait with uncoordinated movements , and their stance is substantially wider ( distance between left and right paws ) , despite their smaller size compared to wildtype mice .
this evidence further implicates the importance of bdnf not just to neuron development in the pns but also to cerebellar development and function .
intraventricular bdnf application encourages neurogenesis in several parts of the rat brain , such as striatum , septum , thalamus , and hypothalamus , and infusion directly into the hippocampus increases the number of granule cells in the dg .
danzer and colleagues transfected hippocampal cells in culture with either bdnf or ngf ; dg granule cells exhibited considerable axonal and dendritic branching following bdnf , but not ngf transfection .
this effect was abolished with the application of a trk receptor tyrosine kinase inhibitor , demonstrating that bdnf and trk signaling promote neurogenesis both within and outside of the context of development .
the involvement of p75ntr in refining trk receptor selectivity may serve to modulate targeting of growing neurons ( see figure 2 ) , with neurotrophic factors acting as markers .
target tissues for growing pns neurons ( e.g. , skeletal muscle ) express neurotrophins , and bdnf is released postsynaptically to influence cns neurons in a retrograde fashion [ 27 , 69 ] . in a study of rat
sympathetic neurons that largely express ngf and trka receptors , bdnf activated p75ntr - mediated apoptosis in conditions with low available ngf .
for example , if a predominantly ngf / trka - associated neuron were to grow to network with a predominantly bdnf / trkb - associated neuron , the growing neuron would quickly become apoptotic .
postsynaptic bdnf release and activation of the presynaptic p75ntr , coupled with the lack of ngf / trka signaling , would reduce the survivability of the growing neuron .
thus p75ntr serves a regulatory role with regard to trk receptor behavior and neuronal proliferation . in summary ,
neurotrophins influence neuronal differentiation and are important to neuronal survival [ 16 , 5052 ] .
bdnf mrna is present in relatively low levels early in life and generally increases through to adulthood [ 59 , 62 ] , especially in certain hippocampal neurons .
studies of mice lacking bdnf demonstrate how it is essential for proper pns [ 64 , 66 ] and cerebellar development .
bdnf is directly involved in the regulation of synaptic transmission and activity - dependent synaptic plasticity by both pre- and postsynaptic mechanisms [ 1 , 23 ] ( see figure 3 ) .
these mechanisms underlie the critical role of bdnf in ltp [ 2426 , 29 , 65 ] . in a study with cultured hippocampal and cerebellar granule cells , caldeira and
colleagues demonstrated that bdnf increased the levels of nmda receptor subunits in the plasma membrane of hippocampal cells .
they recorded a correlated increase in intracellular calcium concentration and described an increased calcium entry through the additional nmda receptors .
interestingly , hartmann and colleagues stimulated cultured hippocampal neurons presynaptically and detected a postsynaptic bdnf release , dependent upon calcium influx , thereby suggesting a bdnf - dependent positive feedback loop between presynaptic and postsynaptic signaling in the generation of ltp .
. found that bdnf is essential for activity - induced potentiation of presynaptic vesicle cycling in cultured embryonic neocortical neurons of bdnf - knockout mice .
this phenomenon was dependent upon nmda receptor activation , and was significantly reduced in bdnf - deficient neurons , indicating a possible retrograde messenger or paracrine role for bdnf . in support of these findings ,
previous work in bdnf knockout mice showed a pronounced impairment of vesicle docking in hippocampal synapses while direct application of bdnf reversed the deficits .
estrogen promotes hippocampal plasticity , and in ovariectomized rats , the loss of estrogen reduces cytoskeletal reorganization in hippocampal dendritic spines and produces deficits in ltp .
kramr and colleagues recently demonstrated that application of bdnf to hippocampal sections restored spine actin polymerization and ltp stability in these rats .
backpropagating ( retrograde ) action potentials modulate short- and long - term changes to synaptic activity and also serve to relieve the mg block on nmda receptors .
the extent of backpropagation in a neuron is dependent upon dendritic morphology , ion channel distribution , and synaptic input .
importantly , application of an endoplasmic reticulum ca - atpase inhibitor stops this dendritic bdnf release , suggesting a calcium - dependent mechanism for retrograde release .
these findings thus reveal several mechanisms for bdnf - mediated promotion of ltp in the hippocampus .
bdnf is released in an activity - dependent manner , as demonstrated in both hippocampal neurons and peripheral neurons . while it may not be the only mediator of ltp
, mice lacking bdnf demonstrated reduced tetanus - induced ltp , a deficit that disappeared with the return of bdnf function .
as described above , bdnf works through both pre- and postsynaptic mechanisms to support ltp , and a simultaneous block of pre- and postsynaptic plc- activity , but not either by itself , is required to reduce ltp in mouse hippocampal cultures .
interestingly , the involvement of trkb in ltp is not completely dependent on bdnf signaling ; nmda receptors can activate trkb by way of zinc influx , and adenosine 2a receptors can also influence trkb . in brief ,
bdnf is involved in trafficking nmda receptor subunits to the membrane , thereby increasing the potential for calcium influx .
a sufficient local buildup of calcium causes postsynaptic bdnf release , which then leads to an increase in presynaptic vesicle cycling .
exercise leads to substantial changes in bdnf and nmda receptor activity in the hippocampus , and these changes in large part underlie the effects of exercise on learning and synaptic plasticity . in rats with free access to activity wheels for one to six weeks , increases in hippocampal bdnf mrna [ 32 , 33 ] and bdnf protein [ 34 , 35 ] have been detected .
berchtold , castello , and cotman demonstrated that hippocampal bdnf protein remained elevated for two weeks following three weeks of access to cage wheels ( protein levels were 186% of controls at the end of exercise , 137% one week followup , and 133% at two weeks ) .
physical activity increases hippocampal bdnf expression after a relatively short amount of time , as voluntary wheel running induced bdnf mrna after only 6 hours of running .
a substantial increase in bdnf mrna in the dg has also been described after 2 weeks , 3 weeks , 4 weeks , and 2 months of voluntary running .
bdnf mrna and protein induction due to exercise varies over the life - span , however .
younger mice ( 2 months ) exhibit a larger increase compared to older mice ( 15 and 24 months ) over four weeks of running . forced treadmill running for 30 minutes per day for six weeks increased hippocampal bdnf protein and brdu positive proliferating cells in younger rats ( 5 months ) , an effect that was less robust but still significant in older rats ( 24 months ) .
exercise influences bdnf even under conditions of homeostatic perturbations such as stress or dysfunction in energy metabolism . in
chronically stressed sprague - dawley rats , access to cage wheels for six weeks only during dark cycle ( active period ) yielded increased levels of bdnf protein in the striatum compared to sedentary rats .
the characteristic lower levels of hippocampal bdnf protein and dendritic spine density in type ii diabetic mice ( db / db ) were significantly enhanced with free access to cage wheels . under a condition of food deprivation
every other day , six weeks of wheel running still yielded significantly increased hippocampal bdnf protein compared to normally fed and food - deprived sedentary wistar rats , similar in magnitude to the levels observed in normally fed exercising rats .
running is associated with a robust activation of survival pathways and vesicular proteins , effects that are linked to bdnf .
shen and colleagues showed prolonged hippocampal mapk signaling following only a week of voluntary running .
chen and russo - neustadt demonstrated a significant increase in hippocampal pi3k protein in rats after two weeks of wheel running . in another study , chen , and
russo - neustadt found a similar consequence of running on akt protein , an effect dependent upon creb signaling ; in fact , the researchers showed that exercise - induced increases in hippocampal bdnf protein and mrna are also creb activation dependent , indicating the essential nature of creb signaling to exercise and bdnf - dependent cellular survival effects .
these pathways are likely directly associated with the neurogenesis detected in the dg of exercising rats [ 80 , 81 ] .
voluntary exercise selectively increases hippocampal synapsin i and synaptophysin mrna [ 35 , 82 ] , a phenomenon blocked by a recombinant human trkb - igg chimera , a highly specific antagonist of bdnf .
synapsin i tethers vesicles to the cytoskeleton while synaptophysin may modulate vesicle recycling during high - intensity activity . indeed ,
this bdnf - mediated increase in synapsin following exercise may in part explain how exercise enhances ltp , along with the other mechanisms for bdnf - facilitated ltp described above .
voluntary running also yielded better performance on the morris water maze test , an ltp - associated effect abolished with the selective blocking of bdnf .
importantly , farmer and colleagues also identified an increase in nmda receptor subunit nr2b mrna in the dg following activity wheel running , which may represent yet another mechanism explaining how exercise promotes ltp and learning and memory .
a complicated relationship exists between bdnf and epileptogenic hyperexcitability , but this relationship may be fundamentally characterized as a positive feedback loop . in general , enhanced bdnf signaling is associated with increased excitability , presumably through increased synaptic plasticity .
for example , mrna and protein levels for bdnf are increased in the hippocampus after seizure [ 66 , 87 ] , and bdnf knockout mice demonstrate reduced seizure behavior in response to kindling . in transgenic mice with an overexpression of the truncated trkb receptor , the reduced bdnf signaling led to reduced seizure activity after systemic kainic acid administration .
. demonstrated development of hyperexcitable circuits due to abnormal mossy fiber sprouting , in part caused by activity - induced bdnf release and trk receptor activity .
this sprouting did not occur in the presence of a trk receptor tyrosine kinase inhibitor or anti - bdnf antibody .
such abnormal remodeling is , in part , responsible for dentate hyperexcitability [ 89 , 90 ] .
glutamatergic excitotoxicity , thought to result from excessive calcium influx via nmda receptors , is a major contributor to neuronal damage associated with anoxia , ischemia , and seizure . in cultured cerebellar granule cells , high glutamate concentrations cause immediate necrosis . following withdrawal to normal medium ,
hippocampal cell cultures , a high concentration of glutamate caused mainly necrosis while a lower concentration of glutamate led almost exclusively to delayed apoptosis .
glazner and mattzon evaluated the effect of bdnf application on nmda receptor subunits , and found that nr1 and nr2a protein levels are increased whereas nr2b levels are decreased ; associated effects included increased calcium response to nmda activation , increased necrosis ( cell swelling ) , and decreased apoptosis ( cell shrinkage and chromatin fragmentation ) .
bdnf - mediated enhancement of nmda receptor functions are consistent with the effects of exercise on increasing nmda receptor expression described above , further supporting the role of bdnf in exercise - enhanced synaptic plasticity .
these results further illustrate how bdnf overexpression may lead to hyperexcitable circuitry through multiple mechanisms .
bdnf thus encourages neurogenesis [ 9 , 10 ] , axonal , and dendritic sprouting , and nmda receptor - mediated transmission in a trk - b - dependent manner , which are generally considered to be beneficial effects on neuronal function .
however , these same mechanisms may lead to hyperexcitable circuits in the dg [ 89 , 90 , 97 ] , which may eventually lead to spreading excitotoxicity .
this condition can therefore become a self - sustaining cycle of sprouting and hyperexcitability , fueled in part by activity - dependent bdnf release [ 69 , 73 , 74 ] . in a kainic acid model of temporal lobe epilepsy utilizing transgenic mice , increased trkb signaling facilitated significantly shorter time to epileptogenesis and more severe epileptiform electroencephalogram activity compared to wild - type mice , and epileptogenesis was delayed in reduced trkb ( truncated form ) mice . based on this evidence for a prolonged state of hyperexcitability coupled with a shorter facilitation resulting from enhanced expression of bdnf or trkb signaling in the hippocampus
, it follows that exercise - induced plasticity could leave the brain more vulnerable to epileptic seizures and excitotoxicity .
microinjections of kainic acid into the hippocampi of anaesthetized rats produce more damage in subjects that were provided with running wheels for several weeks compared to sedentary controls .
this enhanced excitotoxicity in exercising rats was interpreted by the investigators as a consequence of exercise - induced upregulation of hippocampal bdnf expression . on the other hand ,
a large body of evidence from both clinical studies in humans and basic research in animal models reveals that physical exercise produces a wide range of neuroprotective effects [ 100103 ] .
for example , exercise in humans decreases risk of neurological disease and stroke , and it may improve recovery after brain trauma .
the brain damage caused by experimental infarct is diminished in exercising rats compared to sedentary controls .
exercise also protects against oxidative stress , and the neurotoxic effects of 6-ohda [ 104 , 105 ] and mptp . since
neural hyperexcitability is a mechanism for the injury and death that occurs in all of these neurological insults and/or disorders [ 107 , 108 ] , determining how exercise protects against excitotoxicity should have broad implications for understanding its beneficial effects on the brain .
nonetheless , these conditions are only indirectly linked to excitotoxicity , and a more direct examination of the influence of exercise on hyperexcitability is required to understand the mechanisms for its neuroprotective effects .
in contrast to the findings of ramsden and colleagues described above , in which exercise led to increased excitotoxicty when kainic acid was injected into the hippocampus of anaesthetized rats , some evidence suggests that exercise may protect against hyperexcitability .
experiments specifically designed to this hypothesis have revealed that exercise protects against kainic acid - induced seizures when studied in awake , freely behaving rats .
reiss et al . reported that three weeks of voluntary exercise decreases the effects of kainic acid on seizure behaviors and hippocampal c - fos expression . further supporting the hypothesis that exercise diminishes hyperexcitability
are similar reports of exercise protecting against experimentally induced seizures using other convulsant manipulations [ 110 , 111 ] .
exercise also diminishes the excitotoxic effects of systemic domoic acid in mice and reduces the learning deficits caused by systemic kainic acid administration in rats . taken together , the conflicting findings of exercise effects on hyperexcitability and excitotoxicity suggest that an exercise - induced compensatory mechanism may counter the hyperexcitable state associated with upregulation of bdnf .
importantly , this compensatory mechanism may only function in awake , freely behaving subjects . in the report by ramsden and colleagues , the exercise - associated increase in neurotoxicity caused by kainic acid injected directly into the hippocampi
if such a mechanism depended on extrahippocampal circuitry , then it is reasonable to assume that it may not engage in an anaesthetized state .
though it is likely that a variety of mechanisms exist to counter the hyperexcitable state in the hippocampus caused by bdnf , elucidation of such a mechanism related to exercise should be based on specific criteria .
this mechanism should involve some neural factor that ( 1 ) is upregulated by exercise , ( 2 ) diminishes neural hyperexcitability acutely , and ( 3 ) exerts latent neuroprotective effects .
research from this laboratory suggests that hippocampal afferents originating from the noradrenergic locus coeruleus may be a critical component of this mechanism .
more specifically , experimental evidence points to the neuropeptide galanin as a critical factor in dampening hippocampal excitability .
galanin is a 29 - 30 amino acid neurotransmitter and putative trophic factor that regulates neural activity in several brain structures , most notably the hypothalamus and hippocampus .
retrograde tracing / double labeling experiments reveal that the hippocampus receives galaninergic innervation via projections from the locus coeruleus .
at least three g - protein coupled galanin receptor subtypes have been confirmed , designated gal r1 - 3 .
galaninergic transmission is predominantly inhibitory , producing hyperpolarization through increased k or decreased ca conductance .
several lines of evidence reveal that galanin functions as an endogenous neuroprotective factor for hippocampal neurons [ 116118 ] .
infusion of galanin into the hippocampus inhibits seizures provoked by a variety of methods , and the galanin receptor antagonists m-35- and m-40 block the antiseizure effects of galanin .
transgenic galanin knock - out mice show increased susceptibility to kainic acid - induced seizures whereas transgenic mice overexpressing galanin display a resistance to these seizures .
hippocampal electrophysiological activity is similarly exaggerated in the galanin knockouts and decreased in galanin overexpressers . in vitro studies in hippocampal cultures from transgenic mice
previous work from this laboratory has demonstrated that either three weeks of voluntary wheel running or treadmill training increases galanin gene expression in locus coeruleus neurons in rats [ 120123 ] .
no exercise - induced changes in neuropeptide - y or tyrosine hydroxylase were observed in these studies , suggesting that locus coeruleus neurons respond to exercise by specifically upregulating galanin . the critical role of galanin in mediating the protective effects of exercise against convulsant - induced seizures was demonstrated by the administration of the galanin receptor antagonist m-40 , which largely blocked the neuroprotection as measured by behavioral seizure severity .
these results suggest that exercise - induced upregulation of galanin mediates the enhanced inhibitory tone that may counteract bndf - mediated hyperexcitability .
to conclude , bdnf receptor activity encourages neurogenesis [ 9 , 10 ] , suppresses apoptosis , and modulates synaptic activity via a variety of signaling cascades [ 45 , 48 , 50 ] .
these effects are beneficial with regard to development [ 1 , 8 ] and synaptic plasticity [ 25 , 26 , 29 , 65 ] , but can be harmful in conditions sensitive to hyperexcitability .
dendritic bdnf release is activity - dependent , based on calcium influx [ 27 , 69 ] .
this release causes an increase in presynaptic vesicle cycling , which further stimulates synaptic activity .
the bdnf - induced increase in nmda receptor subunits creates more opportunity for calcium influx , supporting additional bdnf release .
the increased trkb signaling boosts ip3 activity and intracellular calcium release , contributing to the enhancement of the cycle even further .
these processes feed off of one another to overwhelm any modulatory effects of truncated trkb receptors ( which bind bdnf but lack tyrosine kinase function ) or p75ntr , the latter of which will act more to refine the actions of the increased available bdnf to yet further potentiate the cycle .
since bdnf encourages neurogenesis [ 9 , 10 ] and neurite outgrowth , the effects of these processes are further compounded with the establishment of additional excitatory synapses ( see figure 4 ) .
thus hyperexcitable circuits in the dg can develop due to excessive bdnf and nmda receptor activity [ 89 , 90 ] , activity that maintains a self - supporting cycle of neurotransmitter release in the hippocampus that can be overwhelmed to promote excitotoxic vulnerability .
considering the maladaptive nature of this condition , selection for hippocampal circuitry that regulates this hyperexcitability would provide clear evolutionary benefits .
furthermore , environmental stimuli that promote hippocampal bdnf expression , such as exercise , would be expected to engage this compensatory mechanism .
evidence suggests that a circuit including galaninergic projections from the lc to the hippocampus provides this function [ 116 , 118 , 120 , 121 , 123 ] . | the present paper examines the nature and function of brain - derived neurotrophic factor ( bdnf ) in the hippocampal formation and the consequences of changes in its expression .
the paper focuses on literature describing the role of bdnf in hippocampal development and neuroplasticity .
bdnf expression is highly sensitive to developmental and environmental factors , and increased bdnf signaling enhances neurogenesis , neurite sprouting , electrophysiological activity , and other processes reflective of a general enhancement of hippocampal function .
such increases in activity may mediate beneficial effects such as enhanced learning and memory .
however , the increased activity also comes at a cost : bdnf plasticity renders the hippocampus more vulnerable to hyperexcitability and/or excitotoxic damage .
exercise dramatically increases hippocampal bdnf levels and produces behavioral effects consistent with this phenomenon . in analyzing the literature regarding exercise - induced regulation of bdnf
, this paper provides a theoretical model for how the potentially deleterious consequences of bdnf plasticity may be modulated by other endogenous factors .
the peptide galanin may play such a role by regulating hippocampal excitability . | 1. Brain-Derived Neurotrophic Factor Overview
2. Signal Transduction
3. Development
4. Synaptic Plasticity
5. Exercise
6. Excitotoxicity
7. Summary and Conclusion |
PMC5403115 | myf5 and mrf4 ( also known as myf6 ) are upstream of myod in muscle precursor differentiation ; alternate tbx1 and pax3 pathways can initiate myod expression in pharyngeal muscles and in body and limb skeletal muscles , respectively , but not in eoms .
regulatory elements for mrf4 reside within the myf5 locus , and myf5-targeted alleles have variable eom hypoplasia depending on the degree of residual mrf4 expression , with complete loss of both myf5 and myf4 resulting in a specific loss of eoms .
we purchased myf5 mice from jackson laboratory ( b6.129s4-myf5/j , stock no . 007893 ; bar harbor , me , usa ) .
this myf5 strain expresses cre recombinase at the endogenous myf5 locus , disrupting the myf5 basic helix - loop - helix domain , and is also predicted to disrupt transcription of the adjacent mrf4 gene because the myf5 strain , created by using a similar targeting vector , disrupts expression of both genes and lacks eoms ( but not other muscle types ) .
myf5 mice were crossed to transgenic isl : gfp reporter mice ( mgi : j:132726 ; gift of sam pfaff , salk institute , san diego , ca , usa ) , which contain a farnesylated green fluorescent protein ( gfp ) that localizes to the membrane of motor neurons and axons and allows for visualization of the nerves during development .
we refer to myf5::isl : gfp embryos as wild type and myf5::isl : gfp embryos as myf5 . all animal work was approved and performed in compliance with boston children 's hospital institutional animal care and use committee protocols and the arvo statement for the use of animals in ophthalmic and vision research .
wild - type and myf5 embryos were taken at embryonic day ( e ) 11.5 and fixed in 4% paraformaldehyde ( pfa ) overnight ( o / n ) , washed , dehydrated , and placed in dent 's fixative ( 20% dmso , 80% methanol ) o / n , rehydrated , and placed in blocking solution ( 5% heat - inactivated goat serum , 20% dmso , 75% 1 pbs ) o / n .
actin -smooth muscle - cy3 antibody ( anti-sma ; sigma - aldrich corp . , st .
louis , mo , usa ) and anti - gfp ( invitrogen , carlsbad , ca , usa ) for 5 days at room temperature ( rt ) , washed , incubated in secondary antibody ( 488 goat anti - rabbit igg ; alexa fluor , life technologies , carlsbad , ca , usa ) for 3 days at rt , washed , dehydrated , and cleared o / n in babb ( one part benzyl alcohol and two parts benzyl benzoate ) .
images were acquired with a zeiss lsm 710 laser scanning confocal microscope and zen software ( zeiss , oberkochen , germany ) and processed with imaris software ( bitplane , zurich , switzerland ) .
onward , orbital dissections provide superior image detail and quality as compared to whole mount preparations .
wild - type and myf5 embryos were taken at the appropriate age and fixed in 4% pfa o /
cortex and olfactory bulbs above the orbit were removed under a dissecting microscope , leaving the distal cranial nerves and eoms ( if present ) intact .
orbits were incubated with anti-sma for 3 days at rt or 4c , washed , and incubated in secondary antibody for 3 days at rt . after washing with pbs
, orbits were further dissected sagitally at e12.5 or , from e13.5 onward , either from the superior aspect , looking down on the orbit from above ( superior view ) , or from the inferior aspect , looking up at the orbit from the palate ( inferior view ) .
the oculomotor nerve was measured at its widest diameter , which occurs where the superior division of cn3 forms in control embryos , adjacent to where cn4 crosses above cn3 .
the length of the cn3 branch to the io muscle was measured along its trajectory from the distal decision region to the distal nerve tip .
statistical analyses of significance were performed by using unpaired t - tests in graphpad prism ( graph pad , san diego , ca , usa ) .
whole embryos ( e11.5 ) or brain and brainstem ( e18.5 ) were fixed in 4% pfa o /
n , washed in pbs , equilibrated in 30% sucrose , and frozen in optimal cutting temperature ( oct ) media .
serial coronal sections of 20-m thickness were cut on a cryostat and stained with rabbit anti - islet1 ( isl1 ; abcam , cambridge , ma , usa ) .
after blinding as to genotype , isl - positive motor neuron nuclei were counted in every fifth section through the rostral - caudal extent of the oculomotor and trochlear nuclei by using the cell - counter plug - in in fiji , and the total number of motor neurons counted was then multiplied by 5 .
there were n = 3 mutant and n = 3 control embryos at each of the two ages .
myf5 and mrf4 ( also known as myf6 ) are upstream of myod in muscle precursor differentiation ; alternate tbx1 and pax3 pathways can initiate myod expression in pharyngeal muscles and in body and limb skeletal muscles , respectively , but not in eoms .
regulatory elements for mrf4 reside within the myf5 locus , and myf5-targeted alleles have variable eom hypoplasia depending on the degree of residual mrf4 expression , with complete loss of both myf5 and myf4 resulting in a specific loss of eoms .
we purchased myf5 mice from jackson laboratory ( b6.129s4-myf5/j , stock no . 007893 ; bar harbor , me , usa ) .
this myf5 strain expresses cre recombinase at the endogenous myf5 locus , disrupting the myf5 basic helix - loop - helix domain , and is also predicted to disrupt transcription of the adjacent mrf4 gene because the myf5 strain , created by using a similar targeting vector , disrupts expression of both genes and lacks eoms ( but not other muscle types ) .
myf5 mice were crossed to transgenic isl : gfp reporter mice ( mgi : j:132726 ; gift of sam pfaff , salk institute , san diego , ca , usa ) , which contain a farnesylated green fluorescent protein ( gfp ) that localizes to the membrane of motor neurons and axons and allows for visualization of the nerves during development .
we refer to myf5::isl : gfp embryos as wild type and myf5::isl : gfp embryos as myf5 . all animal work was approved and performed in compliance with boston children 's hospital institutional animal care and use committee protocols and the arvo statement for the use of animals in ophthalmic and vision research .
wild - type and myf5 embryos were taken at embryonic day ( e ) 11.5 and fixed in 4% paraformaldehyde ( pfa ) overnight ( o / n ) , washed , dehydrated , and placed in dent 's fixative ( 20% dmso , 80% methanol ) o / n , rehydrated , and placed in blocking solution ( 5% heat - inactivated goat serum , 20% dmso , 75% 1 pbs ) o / n .
actin -smooth muscle - cy3 antibody ( anti-sma ; sigma - aldrich corp . , st .
louis , mo , usa ) and anti - gfp ( invitrogen , carlsbad , ca , usa ) for 5 days at room temperature ( rt ) , washed , incubated in secondary antibody ( 488 goat anti - rabbit igg ; alexa fluor , life technologies , carlsbad , ca , usa ) for 3 days at rt , washed , dehydrated , and cleared o / n in babb ( one part benzyl alcohol and two parts benzyl benzoate ) .
images were acquired with a zeiss lsm 710 laser scanning confocal microscope and zen software ( zeiss , oberkochen , germany ) and processed with imaris software ( bitplane , zurich , switzerland ) .
from e12.5 onward , orbital dissections provide superior image detail and quality as compared to whole mount preparations .
wild - type and myf5 embryos were taken at the appropriate age and fixed in 4% pfa o /
cortex and olfactory bulbs above the orbit were removed under a dissecting microscope , leaving the distal cranial nerves and eoms ( if present ) intact .
orbits were incubated with anti-sma for 3 days at rt or 4c , washed , and incubated in secondary antibody for 3 days at rt .
after washing with pbs , orbits were further dissected sagitally at e12.5 or , from e13.5 onward , either from the superior aspect , looking down on the orbit from above ( superior view ) , or from the inferior aspect , looking up at the orbit from the palate ( inferior view ) .
the oculomotor nerve was measured at its widest diameter , which occurs where the superior division of cn3 forms in control embryos , adjacent to where cn4 crosses above cn3 .
the length of the cn3 branch to the io muscle was measured along its trajectory from the distal decision region to the distal nerve tip .
statistical analyses of significance were performed by using unpaired t - tests in graphpad prism ( graph pad , san diego , ca , usa ) .
whole embryos ( e11.5 ) or brain and brainstem ( e18.5 ) were fixed in 4% pfa o /
n , washed in pbs , equilibrated in 30% sucrose , and frozen in optimal cutting temperature ( oct ) media .
serial coronal sections of 20-m thickness were cut on a cryostat and stained with rabbit anti - islet1 ( isl1 ; abcam , cambridge , ma , usa ) .
after blinding as to genotype , isl - positive motor neuron nuclei were counted in every fifth section through the rostral - caudal extent of the oculomotor and trochlear nuclei by using the cell - counter plug - in in fiji , and the total number of motor neurons counted was then multiplied by 5 .
there were n = 3 mutant and n = 3 control embryos at each of the two ages .
in whole mount views of e11.5 wild - type embryos , the eom anlage can be seen adjacent to the globe ( figs . 1a , 1c ) and by e12.5
in contrast , homozygous myf5 embryos lack eoms but develop facial and skeletal muscles ( figs . 1b , 1d ) .
sagittal views of cranial nerve and extraocular muscle development in e11.5 and e12.5 myf5::isl : gfp and myf5::isl : gfp embryos .
ocular motor nerves are genetically labeled ( green ) , and eom and arteries are antibody labeled with -sma ( red ) .
extraocular muscles are absent in myf5 embryos ( b , d , f ) , and thus the red muscle channel has been turned off in wild - type images ( a , c , e ) , to permit equivalent comparison of nerve trajectories in the presence and absence of eoms .
( a , a ) whole mount wild - type embryo and ( b ) whole mount myf5 embryo at e11.5 .
the trajectories of the nerves from the brainstem appear nearly identical in mutant and wild - type embryos at e11.5 .
the dotted line denotes the area of the orbit imaged in greater detail in ( c ) , ( c ) , and ( d ) .
cn4 is defasciculated , while cn6 is fasciculated along their nerve trunks , and distal cn3 has formed a decision region .
the developing muscle anlage stained with -sma can be seen in the wild type ( c ) , and an artery within the orbit stained with -sma is visible in both mutant and wild - type ( c , d ) .
( e , e , f ) orbital imaging at e12.5 . in the myf5 orbit
, distinct extraocular muscles have formed and share a common origin at the annulus of zinn ( e , asterisk ) .
the trunk of cn4 is now fasciculated , while the distal end forms a decision region adjacent to the so and sends terminal branches into the body of the muscle ( e , e , arrowhead ) . by contrast
, the distal end of cn4 in the myf5 orbit fails to form an equivalent distal decision region and has no terminal branches ( f , arrowhead ) . in the wild - type orbit ,
the inferior division decision region has become more compact ( e , long arrow ) and extends a branch to the io muscle ( e , caret ) .
the myf5 cn3 terminal decision region is less compacted ( f , long arrow ) and the branch to the io muscle appears hypoplastic ( f , caret ) .
arrow in the lower left corner indicates direction relative to the eye . a , anterior ( toward front of eye )
; s , superior ; isl , islet1 ; -sma , -smooth muscle actin . in e11.5 wild - type embryos
, the ocular motor nerves follow stereotypical trajectories to reach the developing orbit ( figs .
the main trunk of cn3 is fasciculated , and its distal end has formed a decision region ( figs .
despite the absence of the eom anlage in the e11.5 myf5 embryos , the ocular motor nerves appear similar to wild - type ( figs .
1e , 1e ) ; the four rectus muscles and the so muscle ( whose tendon travels through the trochlea before forming its muscle body ) share a common origin at the annulus of zinn ( denoted by an asterisk in fig .
1e ) , while the io muscle is located nearer the front of the orbit ( see fig .
the main trunk of cn4 is now fasciculated , and terminal axons are fanning out into the so muscle .
the cn3 inferior division decision region is nestled adjacent to the mr and ir muscles , and the fasciculated branch to the io muscle is extending from or through it .
thin branches extend from the cn3 superior and inferior divisions into the sr , mr , and ir muscles .
the distal end of the abducens nerve crosses inferiorly to cn3 and extends toward the lr muscle .
( a ) schematic diagram showing semisagittal lateral view of mature mouse eye and orbital muscles .
( b ) horizontal top - down view showing orientation of mature mouse orbits relative to the head ; this orientation shifts during early development .
( c ) schematic of boxed area in ( b ) , showing the muscles and nerves visible in a top - down , superior view .
( d ) schematic showing the muscles and nerves visible in a bottom - up , inferior view .
the views in ( c ) and ( d ) are those used in figure 3 .
( mr , ir , so , io ) are green , cn4 and the muscle it innervates ( so ) are blue , and cn6 and the muscles it innervates ( lr and rb ) are pink . beginning at e12.5 , differences are noted in development of ocular motor nerves in the presence and absence of eoms ( fig .
distal cn4 does not form the extensive fan - like terminal branches seen in wild - type embryos .
the cn3 inferior division decision region remains large ; its appearance is similar to wild - type at e11.5 .
a branch of cn3 extends toward where the io muscle should be located , but it appears thinner , straighter , and blunter than in wild - type embryos ( fig . 1f , caret ) . in mouse , the orbits are oriented slightly laterally ( fig .
2b ) , and the position changes slightly as the embryo grows , so further anatomic descriptions are in reference to the eye . by e13.5 , orbit growth requires that orbital dissections be performed from either the superior ( looking down from above ) or inferior view ( looking up from below ; see schematic figs .
the distal aspect of cn4 widens considerably , and terminal branches emanate into the so muscle from one - half of the decision region , while branches from the other half are oriented away from the muscle ( figs .
multiple branches of the cn3 superior division extend from the main trunk and travel along and into the sr muscle ( fig .
the cn3 inferior division decision region contains multiple nerve branches extending directly into the ir and mr muscles , while a long fasciculated branch extends to and sends terminal branches into the io muscle .
cn6 crosses inferiorly to cn3 , extends along the lr muscle , and sends terminal branches into it ( figs .
orbital dissections of e13.5 to e15.5 myf5:isl : gfp and myf5:isl : gfp embryos . nerves and muscles
orbits are imaged from a superior ( a , a , b , e , e , f , i , i , j ) or inferior ( c , c , d , g , g , h , k , k , l ) view , as per figures 2b through 2d schematics .
( a d ) at e13.5 , myf5 eoms share a common origin at the annulus of zinn ( a , asterisk ; c ) . from the superior view ( a , a ) the
so muscle begins to curve at its insertion onto the globe , and the rectus muscles have further differentiated from each other .
cn3 superior division has multiple branches extending to the sr muscle ( a , triple - headed arrow ) .
cn6 has crossed inferiorly to cn3 and extends terminal branches into the lr muscle . from the inferior view ( c , c ) ,
cn3 inferior division sends terminal branches to the ir and mr muscles ( c , long arrow ) , and a well - formed branch to the io muscle ( c , caret ) . at e13.5 ,
the myf5 orbit is devoid of eoms ( b , d ) . from the superior view ( b ) , cn4 has fasciculated but lacks an extensive distal decision region and terminal branches ( arrowhead ) , and cn3 superior division is not visible . from the inferior view ( d ) , cn3 distal decision region ( d , long arrow ) and the branch to the io muscle ( d , caret ) are hypoplastic and misoriented , and cn6 is stunted and barely crosses over cn3 ( d , double - headed arrow ) .
( e h ) at e14.5 , the myf5 eoms continue to grow and change in orientation ( e , g ) : the sr muscle is now a broad band inserting on the top of the globe , the lps muscle has appeared between the sr and so muscles , and the so muscle has obtained its stereotypic curved shape .
there is extensive terminal branching of cn3 superior division into the sr muscle ( e , e ) , and cn4 has developed a blunt - ended branch that is not in association with an eom ( e , e ) .
cn3 inferior division branch has continued extending terminal branches to the mr and ir muscles , and the branch to the io muscle extends into the muscle ( g , g ) . in the absence of eom , the myf5 cn4 appears thin and has developed multiple aberrant branches ( f ) , cn3 main trunk widens ( h ) where the absent superior branch should have formed ( h , triple arrowhead ) , the inferior division decision region is small , with no visible branches to the ir and mr muscles , and the branch to the io muscle is now hypoplastic and aberrant ( h ) .
cn6 trajectory is aberrantly parallel to cn3 and its growth cones pathologically wander ( h , double arrowhead ) .
l ) at e15.5 , myf5 orbits reveal the near - final ocular motor innervation patterns , and each nerve sends terminal and intramuscular branches into the appropriate eom .
cn3 superior division branches have coalesced into a single trunk , and the blunted branch of cn4 is retracting ( i , i , k , k ) . by contrast , in myf5 orbits the nerves have failed to develop further since e14.5 and appear to be regressing , while the abducens nerve wanders ( double arrowhead ) .
although more of the oculomotor nerve appears visible in figure 2j than in figure 2f , this is a staining and imaging artifact . in both images ,
the oculomotor nerve can be seen traveling inferiorly to the trochlear nerve and descending out of view near the stained artery ; the embryo in ( j ) was imaged slightly deeper .
cn4 terminates in a single blunt end adjacent to where the so muscle should be ( fig .
the cn3 superior division has not formed , and the nerve trunk is significantly wider where it should have formed , averaging 62.05 8.17 m in myf5 embryos and 35.92 8.43 m in wild type ( p = 0.0052 ) ( figs .
the cn3 inferior division decision region is small and no terminal branches extend toward the region where the ir and mr should be .
the branch extending toward where the io muscle should be is short , thin , and straight rather than arching as seen in wild - type embryos ( figs .
cn6 appears thinner and shorter than in wild type and ends bluntly just after passing inferiorly to cn3 ( fig .
the myf5 cn3 widens at the origin of the absent superior division and has a shorter branch to the io muscle as compared to the myf5 orbit .
( a ) orbit of an e13.5 myf5 from the inferior view as pictured in figure 2c with measurements illustrated .
the diameter of cn3 was measured at the location where the superior division normally forms in control embryos .
the length of the inferior division branch to the io muscle was defined as the distance from the termination of the distal decision region to the distal tip of the nerve and was measured in three dimensions by using two to three straight lines that approximated the curvature of the nerve .
( b ) cn3 superior division branch point is significantly wider in myf5 embryos from e13.5 to e15.5 ( average from 62.05 8.17 m at e13.5 to 63.96 2.08 m at e15.5 ) than in the wild - type embryos ( average 35.92 8.43 m at e13.5 to 38.26 2.89 m at e15.5 ) .
this difference in width was significant at each age measured ( e13.5 : p = 0.0052 ; e14.5 : p < 0.0001 ; e15.5 : p < 0.0001 ) .
( c ) at e13.5 , cn3 inferior division branch to the io muscle is significantly longer in myf5 ( 373.8 138.7 m ) than in myf5 embryos ( 138.7 56.6 m ; p = 0.0017 ) .
while this branch continues to lengthen in wt orbits , reaching an average length of 412.8 25.5 m at e14.5 and 477.6 57.9 m at e15.5 , it fails to lengthen substantially in mutant orbits ( 155.3 87.7 m at e14.5 and 106.7 44.0 m at e15.5 ) .
the branch to the io muscle is significantly shorter in the mutant at each age ( e14.5 : p = 0.0007 ; e15.5 : p < 0.0001 ) .
a second branch oriented away from the so terminates in a blunt end without visible muscle nearby ( figs .
the cn3 superior division branches have grown in length and are running along the body of the sr muscle ( figs . 3e , 3e ) .
the cn3 inferior division continues to send terminal branches into the mr and ir muscles , and the branch to the io muscle extends further into the muscle ( figs .
cn4 is thin and forms multiple aberrant distal nerve branches that appear incorrectly oriented relative to the artery visible within the orbit ( fig .
the cn3 superior division has not formed , and the nerve trunk widens where the superior division normally forms ( figs . 3h , 4b ) .
the cn3 inferior division does not extend branches to the area of the ir or mr muscles .
the branch to the io muscle is only a thin wisp extending toward where the muscle would normally be located ( figs .
cn6 , which had crossed inferiorly to the oculomotor nerve at e13.5 , now has an aberrant trajectory parallel to the oculomotor nerve . at e15.5 ,
the ocular motor nerves in wild - type embryos are close to their mature configuration ( figs .
so muscle has defined terminal branches associated with the muscle , while the second blunted branch appears thinner and less developed than at e14.5 ( figs .
the cn3 superior division has formed a single fasciculated main trunk and developed more extensive intramuscular nerve branches into the sr ( figs .
the cn3 inferior division sends longer terminal branches to the ir and mr muscles , and the io nerve branch has continued to elongate with its terminal branches in close association with the io muscle ( figs .
cn6 continues to expand into the lr muscle , and branches to the rb muscle are now visible ( figs .
cn4 maintains a few wandering branches that are mislocalized relative to the normal location of the so muscle ( fig .
the cn3 trunk where the cn3 superior division should have formed remains significantly wider in myf5 embryos ( 63.96 2.08 m ) than in wild - type embryos ( 38.26 2.89 m , p < 0.001 ) ( figs .
cn3 inferior division branches to the mr and ir muscles are not visible and the branch to the io muscle has failed to elongate ( 155.3 87.7 m at e14.5 , 106.7 44.0 m at e15.5 ; figs .
this branch is significantly shorter than that in the wild type at e15.5 ( 477.6 57.9 m ; p < 0.001 ) .
the cn6 trajectory remains aberrantly parallel to cn3 , and its terminal axons are defasciculated ( fig .
3l , double - headed arrow ) . to determine if eoms are required for motor neuron survival , we examined the number of motor neurons in oculomotor and trochlear nuclei at e11.5 and e18.5 . at e11.5 , myf5 and control embryos had a similar number of isl1-positive motor neurons in the oculomotor and trochlear nuclei ( 4285 214 control , 4477 182 myf5 , p = 0.84 ; figs . 5a
c ) , indicating that there is normal initial proliferation of motor neurons . by e18.5
, there is substantial normal developmental cell death in the control embryos , as reported previously .
myf5 embryos had greater levels of cell death , however , and have significantly fewer oculomotor and trochlear neurons than control embryos ( 2465 271 vs. 868.2 81.7 , p = 0.00049 , figs .
f ) . myf5 oculomotor and trochlear neuron number and position are similar to control at e11.5 , but are significantly reduced in number by e18.5 .
( a , b ) oculomotor nucleus motor neurons were labeled with anti - isl1 in control ( a ) or myf5 ( b ) brainstems at e11.5 .
( c ) the number of motor neurons in the oculomotor and trochlear nuclei is equal in myf5 and control embryos at e11.5 ( 4285 214 , n = 3 , vs. 4477 182 , n = 3 , p = 0.84 ) , indicating that motor neurons are formed in equal numbers .
( d , e ) oculomotor nucleus motor neurons were labeled with anti - isl1 in control ( d ) or myf5 ( e ) brainstems at e18.5 .
( f ) the number of motor neurons is reduced by 2/3 in myf5 oculomotor nucleus ( 2465 271 , n = 3 , vs. 868.2 81.7 , n = 3 , p = 0.00049 ) , indicating that extraocular muscles are necessary for oculomotor neuron survival .
in whole mount views of e11.5 wild - type embryos , the eom anlage can be seen adjacent to the globe ( figs . 1a , 1c ) and by e12.5
in contrast , homozygous myf5 embryos lack eoms but develop facial and skeletal muscles ( figs . 1b , 1d ) .
sagittal views of cranial nerve and extraocular muscle development in e11.5 and e12.5 myf5::isl : gfp and myf5::isl : gfp embryos .
ocular motor nerves are genetically labeled ( green ) , and eom and arteries are antibody labeled with -sma ( red ) .
extraocular muscles are absent in myf5 embryos ( b , d , f ) , and thus the red muscle channel has been turned off in wild - type images ( a , c , e ) , to permit equivalent comparison of nerve trajectories in the presence and absence of eoms .
( a , a ) whole mount wild - type embryo and ( b ) whole mount myf5 embryo at e11.5 .
the trajectories of the nerves from the brainstem appear nearly identical in mutant and wild - type embryos at e11.5 .
the dotted line denotes the area of the orbit imaged in greater detail in ( c ) , ( c ) , and ( d ) .
cn4 is defasciculated , while cn6 is fasciculated along their nerve trunks , and distal cn3 has formed a decision region .
the developing muscle anlage stained with -sma can be seen in the wild type ( c ) , and an artery within the orbit stained with -sma is visible in both mutant and wild - type ( c , d ) .
( e , e , f ) orbital imaging at e12.5 . in the myf5 orbit
, distinct extraocular muscles have formed and share a common origin at the annulus of zinn ( e , asterisk ) .
the trunk of cn4 is now fasciculated , while the distal end forms a decision region adjacent to the so and sends terminal branches into the body of the muscle ( e , e , arrowhead ) . by contrast
, the distal end of cn4 in the myf5 orbit fails to form an equivalent distal decision region and has no terminal branches ( f , arrowhead ) . in the wild - type orbit ,
the inferior division decision region has become more compact ( e , long arrow ) and extends a branch to the io muscle ( e , caret ) .
the myf5 cn3 terminal decision region is less compacted ( f , long arrow ) and the branch to the io muscle appears hypoplastic ( f , caret ) .
arrow in the lower left corner indicates direction relative to the eye . a , anterior ( toward front of eye )
in e11.5 wild - type embryos , the ocular motor nerves follow stereotypical trajectories to reach the developing orbit ( figs . 1a , 1c ) .
the main trunk of cn3 is fasciculated , and its distal end has formed a decision region ( figs .
despite the absence of the eom anlage in the e11.5 myf5 embryos , the ocular motor nerves appear similar to wild - type ( figs .
by e12.5 , distinct eoms are recognizable ( figs . 1e , 1e ) ; the four rectus muscles and the so muscle ( whose tendon travels through the trochlea before forming its muscle body ) share a common origin at the annulus of zinn ( denoted by an asterisk in fig .
1e ) , while the io muscle is located nearer the front of the orbit ( see fig .
the main trunk of cn4 is now fasciculated , and terminal axons are fanning out into the so muscle .
the cn3 inferior division decision region is nestled adjacent to the mr and ir muscles , and the fasciculated branch to the io muscle is extending from or through it .
thin branches extend from the cn3 superior and inferior divisions into the sr , mr , and ir muscles .
the distal end of the abducens nerve crosses inferiorly to cn3 and extends toward the lr muscle .
schematic diagrams of orbital anatomy . ( a ) schematic diagram showing semisagittal lateral view of mature mouse eye and orbital muscles .
( b ) horizontal top - down view showing orientation of mature mouse orbits relative to the head ; this orientation shifts during early development .
( c ) schematic of boxed area in ( b ) , showing the muscles and nerves visible in a top - down , superior view .
( d ) schematic showing the muscles and nerves visible in a bottom - up , inferior view .
the views in ( c ) and ( d ) are those used in figure 3 .
color coding : cn3 and the muscles it innervates ( mr , ir , so , io ) are green , cn4 and the muscle it innervates ( so ) are blue , and cn6 and the muscles it innervates ( lr and rb ) are pink . beginning at e12.5 , differences are noted in development of ocular motor nerves in the presence and absence of eoms ( fig .
distal cn4 does not form the extensive fan - like terminal branches seen in wild - type embryos .
the cn3 inferior division decision region remains large ; its appearance is similar to wild - type at e11.5 .
a branch of cn3 extends toward where the io muscle should be located , but it appears thinner , straighter , and blunter than in wild - type embryos ( fig . 1f , caret ) .
2b ) , and the position changes slightly as the embryo grows , so further anatomic descriptions are in reference to the eye . by e13.5 , orbit growth requires that orbital dissections be performed from either the superior ( looking down from above ) or inferior view ( looking up from below ; see schematic figs .
2c , 2d ) . in e13.5 wild - type embryos , the eoms continue to grow and separate .
the distal aspect of cn4 widens considerably , and terminal branches emanate into the so muscle from one - half of the decision region , while branches from the other half are oriented away from the muscle ( figs .
multiple branches of the cn3 superior division extend from the main trunk and travel along and into the sr muscle ( fig .
the cn3 inferior division decision region contains multiple nerve branches extending directly into the ir and mr muscles , while a long fasciculated branch extends to and sends terminal branches into the io muscle .
cn6 crosses inferiorly to cn3 , extends along the lr muscle , and sends terminal branches into it ( figs .
orbits are imaged from a superior ( a , a , b , e , e , f , i , i , j ) or inferior ( c , c , d , g , g , h , k , k , l ) view , as per figures 2b through 2d schematics .
( a d ) at e13.5 , myf5 eoms share a common origin at the annulus of zinn ( a , asterisk ; c ) . from the superior view ( a , a ) the
so muscle begins to curve at its insertion onto the globe , and the rectus muscles have further differentiated from each other .
cn3 superior division has multiple branches extending to the sr muscle ( a , triple - headed arrow ) .
cn6 has crossed inferiorly to cn3 and extends terminal branches into the lr muscle . from the inferior view ( c , c )
, cn3 inferior division sends terminal branches to the ir and mr muscles ( c , long arrow ) , and a well - formed branch to the io muscle ( c , caret ) . at e13.5 ,
the myf5 orbit is devoid of eoms ( b , d ) . from the superior view ( b ) , cn4 has fasciculated but lacks an extensive distal decision region and terminal branches ( arrowhead ) , and cn3 superior division is not visible . from the inferior view ( d ) , cn3 distal decision region ( d , long arrow ) and the branch to the io muscle ( d , caret ) are hypoplastic and misoriented , and cn6 is stunted and barely crosses over cn3 ( d , double - headed arrow ) .
( e h ) at e14.5 , the myf5 eoms continue to grow and change in orientation ( e , g ) : the sr muscle is now a broad band inserting on the top of the globe , the lps muscle has appeared between the sr and so muscles , and the so muscle has obtained its stereotypic curved shape .
there is extensive terminal branching of cn3 superior division into the sr muscle ( e , e ) , and cn4 has developed a blunt - ended branch that is not in association with an eom ( e , e ) .
cn3 inferior division branch has continued extending terminal branches to the mr and ir muscles , and the branch to the io muscle extends into the muscle ( g , g ) . in the absence of eom , the myf5 cn4 appears thin and has developed multiple aberrant branches ( f ) , cn3 main trunk widens ( h ) where the absent superior branch should have formed ( h , triple arrowhead ) , the inferior division decision region is small , with no visible branches to the ir and mr muscles , and the branch to the io muscle is now hypoplastic and aberrant ( h ) .
cn6 trajectory is aberrantly parallel to cn3 and its growth cones pathologically wander ( h , double arrowhead ) .
( i l ) at e15.5 , myf5 orbits reveal the near - final ocular motor innervation patterns , and each nerve sends terminal and intramuscular branches into the appropriate eom . cn3
superior division branches have coalesced into a single trunk , and the blunted branch of cn4 is retracting ( i , i , k , k ) . by contrast , in myf5 orbits the nerves have failed to develop further since e14.5 and appear to be regressing , while the abducens nerve wanders ( double arrowhead ) .
although more of the oculomotor nerve appears visible in figure 2j than in figure 2f , this is a staining and imaging artifact . in both images ,
the oculomotor nerve can be seen traveling inferiorly to the trochlear nerve and descending out of view near the stained artery ; the embryo in ( j ) was imaged slightly deeper .
cn4 terminates in a single blunt end adjacent to where the so muscle should be ( fig .
the cn3 superior division has not formed , and the nerve trunk is significantly wider where it should have formed , averaging 62.05 8.17 m in myf5 embryos and 35.92 8.43 m in wild type ( p = 0.0052 ) ( figs . 4a , 4b ) .
the cn3 inferior division decision region is small and no terminal branches extend toward the region where the ir and mr should be .
the branch extending toward where the io muscle should be is short , thin , and straight rather than arching as seen in wild - type embryos ( figs .
cn6 appears thinner and shorter than in wild type and ends bluntly just after passing inferiorly to cn3 ( fig .
the myf5 cn3 widens at the origin of the absent superior division and has a shorter branch to the io muscle as compared to the myf5 orbit .
( a ) orbit of an e13.5 myf5 from the inferior view as pictured in figure 2c with measurements illustrated .
the diameter of cn3 was measured at the location where the superior division normally forms in control embryos .
the length of the inferior division branch to the io muscle was defined as the distance from the termination of the distal decision region to the distal tip of the nerve and was measured in three dimensions by using two to three straight lines that approximated the curvature of the nerve .
( b ) cn3 superior division branch point is significantly wider in myf5 embryos from e13.5 to e15.5 ( average from 62.05 8.17 m at e13.5 to 63.96 2.08 m at e15.5 ) than in the wild - type embryos ( average 35.92 8.43 m at e13.5 to 38.26 2.89 m at e15.5 ) .
this difference in width was significant at each age measured ( e13.5 : p = 0.0052 ; e14.5 : p < 0.0001 ; e15.5 : p < 0.0001 ) .
( c ) at e13.5 , cn3 inferior division branch to the io muscle is significantly longer in myf5 ( 373.8 138.7 m ) than in myf5 embryos ( 138.7 56.6 m ; p = 0.0017 ) . while this branch continues to lengthen in wt orbits ,
reaching an average length of 412.8 25.5 m at e14.5 and 477.6 57.9 m at e15.5 , it fails to lengthen substantially in mutant orbits ( 155.3 87.7 m at e14.5 and 106.7 44.0 m at e15.5 ) .
the branch to the io muscle is significantly shorter in the mutant at each age ( e14.5 : p = 0.0007 ; e15.5 : p < 0.0001 ) .
, cn4 provides terminal innervation to the so muscle . a second branch oriented away from
the cn3 superior division branches have grown in length and are running along the body of the sr muscle ( figs .
the cn3 inferior division continues to send terminal branches into the mr and ir muscles , and the branch to the io muscle extends further into the muscle ( figs .
cn6 crosses inferiorly to cn3 and extends terminal branches into the lr muscle ( figs .
3 g , 3g ) . in e14.5 myf5 embryos , eoms remain absent ( figs . 3f , 3h ) .
cn4 is thin and forms multiple aberrant distal nerve branches that appear incorrectly oriented relative to the artery visible within the orbit ( fig .
the cn3 superior division has not formed , and the nerve trunk widens where the superior division normally forms ( figs . 3h , 4b ) .
the cn3 inferior division does not extend branches to the area of the ir or mr muscles .
the branch to the io muscle is only a thin wisp extending toward where the muscle would normally be located ( figs .
cn6 , which had crossed inferiorly to the oculomotor nerve at e13.5 , now has an aberrant trajectory parallel to the oculomotor nerve . at e15.5 ,
the ocular motor nerves in wild - type embryos are close to their mature configuration ( figs .
so muscle has defined terminal branches associated with the muscle , while the second blunted branch appears thinner and less developed than at e14.5 ( figs .
the cn3 superior division has formed a single fasciculated main trunk and developed more extensive intramuscular nerve branches into the sr ( figs .
the cn3 inferior division sends longer terminal branches to the ir and mr muscles , and the io nerve branch has continued to elongate with its terminal branches in close association with the io muscle ( figs . 3k , 3k , 4c ) .
cn6 continues to expand into the lr muscle , and branches to the rb muscle are now visible ( figs .
cn4 maintains a few wandering branches that are mislocalized relative to the normal location of the so muscle ( fig .
the cn3 trunk where the cn3 superior division should have formed remains significantly wider in myf5 embryos ( 63.96 2.08 m ) than in wild - type embryos ( 38.26 2.89 m , p < 0.001 ) ( figs .
cn3 inferior division branches to the mr and ir muscles are not visible and the branch to the io muscle has failed to elongate ( 155.3 87.7 m at e14.5 , 106.7 44.0 m at e15.5 ; figs .
this branch is significantly shorter than that in the wild type at e15.5 ( 477.6 57.9 m ; p < 0.001 ) .
the cn6 trajectory remains aberrantly parallel to cn3 , and its terminal axons are defasciculated ( fig .
to determine if eoms are required for motor neuron survival , we examined the number of motor neurons in oculomotor and trochlear nuclei at e11.5 and e18.5 . at e11.5 , myf5 and control embryos had a similar number of isl1-positive motor neurons in the oculomotor and trochlear nuclei ( 4285 214 control , 4477 182 myf5 , p = 0.84 ; figs .
c ) , indicating that there is normal initial proliferation of motor neurons . by e18.5 ,
there is substantial normal developmental cell death in the control embryos , as reported previously .
myf5 embryos had greater levels of cell death , however , and have significantly fewer oculomotor and trochlear neurons than control embryos ( 2465 271 vs. 868.2 81.7 , p = 0.00049 , figs .
myf5 oculomotor and trochlear neuron number and position are similar to control at e11.5 , but are significantly reduced in number by e18.5 .
( a , b ) oculomotor nucleus motor neurons were labeled with anti - isl1 in control ( a ) or myf5 ( b ) brainstems at e11.5 .
( c ) the number of motor neurons in the oculomotor and trochlear nuclei is equal in myf5 and control embryos at e11.5 ( 4285 214 , n = 3 , vs. 4477 182 , n = 3 , p = 0.84 ) , indicating that motor neurons are formed in equal numbers .
( d , e ) oculomotor nucleus motor neurons were labeled with anti - isl1 in control ( d ) or myf5 ( e ) brainstems at e18.5 .
( f ) the number of motor neurons is reduced by 2/3 in myf5 oculomotor nucleus ( 2465 271 , n = 3 , vs. 868.2 81.7 , n = 3 , p = 0.00049 ) , indicating that extraocular muscles are necessary for oculomotor neuron survival .
most prior studies of ocular cranial nerve development in mouse have relied on dii labeling or neurofilament staining , highlighting the initial nerve trajectories but not permitting visualization of their terminal processes in the orbit . by dissecting and visualizing the mouse orbit from timed embryos with genetically labeled motor axons , we characterized in greater detail the development of mouse oculomotor , trochlear , and abducens nerves , and showed that formation of the initial nerve trajectory does not require signals from the eoms , but formation of terminal branching does .
ocular motor nerves arrive to the orbit and form distal decision regions in a spatially and temporally similar fashion in myf5 and wild - type embryos , demonstrating that axon growth and guidance from the brainstem to the proper position within the orbit is independent of eoms and likely occurs through cues from the mesenchyme and/or through cell autonomous processes .
subsequent full nerve extension and terminal branching into the eom targets , maintenance of correct nerve position as the orbit expands , and survival of axons and their motor neurons , however , are dependent on the presence of eoms .
the role of eoms in ocular motor nerve development is similar to the findings from previous reports of the contribution of skeletal muscle to spinal nerve development and survival .
silver stains and histologic sectioning following surgical ablation or irradiation of developing chick limb muscles reveal normal development of the main motor nerve trunks and distal nerve branches adjacent to the missing muscles , but failure of the formation of terminal branches that normally would innervate the missing individual muscles .
similarly , myod::myf5 mouse embryos lack all skeletal muscle and have normal initial growth and guidance of spinal motor nerves but fail to develop terminal nerve branches , followed by motor neuron apoptosis .
together , these data support the influence of locally acting mesenchymal guidance cues on proximal nerve patterning and locally acting eom cues on terminal nerve branching into target muscles .
we found minor differences in the relative timing of ocular motor nerve development in mouse compared to chick and zebrafish . in mouse
we found that cn6 and cn3 reach the orbit simultaneously and before cn4 , which is similar to zebrafish but different from chick , in which the arrival of cn6 is followed by cn3 and then cn4 . in both mouse and chick , cn3 projects an initial nerve branch to the io muscle , followed by branches to the mr , ir , and sr muscles .
by contrast , in zebrafish a branch is sent to the sr muscle many hours after branches are sent to inferior division muscles , correlating with the temporal development of the cn3 nucleus , in which zebrafish sr motor neurons are born after mr and ir motor neurons .
first , we identified a trochlear branch in developing wild - type embryos that is not associated with a muscle , forms a blunt end , and subsequently retracts .
consistent with its apparent lack of muscle target , the growth and regression of this wild - type trochlear branch resembles the growth and regression of ocular nerves in myf5 embryos .
second , we found that the cn3 superior division , which in the mature state forms a single branch that then divides distally to innervate both the sr and lps muscles , fails to form in myf5 embryos .
this suggests that , while axons within the main trunk of cn3 are guided in a cell - autonomous manner , by axon
axon interactions , or by mesenchymal cues , the superior division axons exit from the main cn3 trunk in response to muscle cues .
we have long been puzzled by the observation that the cn3 superior division first appears as multiple small branches extending away from the main cn3 trunk , which subsequently coalesce and elongate .
our current imaging approaches now reveal that , at e11.5 to e12.5 , the sr / lps muscle anlage lies in close juxtaposition to the main trunk of cn3 , and these multiple small branches extend directly along and into the muscle anlage .
as the orbital size expands over subsequent days , the distance between the origin and eom targets of these small branches increases and , by e15.5 , the branches have fasciculated into the superior division .
myf5 embryos do not form these multiple small branches , and , at the position where they normally form , the cn3 main trunk is wider than in wild - type embryos .
this is reminiscent of the pathology of kif21a mice . in these mutant mice that recapitulate a human ccdd
, the superior division also fails to form , and a widened bulge is visible along cn3 that , by electron microscopy , contains convoluted axon trajectories and stalled growth cones . the kif21a bulge is located far proximally to the normal superior division branch - point and forms secondarily to attenuated kif21a autoinhibition . by contrast , we found the myf5 widening to be located at the position of the normal distal superior division branch - point .
the parallels , however , suggest that the myf5 nerve widening may result from paused growth cones that fail to exit the main cn3 trunk secondary to the absence of eoms .
ocular motor neurons , like other motor neurons , receive trophic support from their target muscles , and are responsive to neurotrophic factors , particularly bdnf and gdnf ( see ref .
it is therefore not surprising that survival of ocular motor neurons depends on the presence of eoms .
it is remarkable , however , that in the absence of target muscles , more than one - third of the oculomotor and trochlear neurons remain at e18.5 .
by contrast , in myod::myf5 embryos , which lack all skeletal muscles , all somatic motor neurons in the spinal cord die by e17.5 , and most motor neurons in the facial nucleus are dead or dying .
the ocular motor nerves are selectively spared until late in the course of motor neuron degenerative disorders such as amyotrophic lateral sclerosis and spinal motor atrophy .
this selective sparing is theorized to result from increased resistance of the ocular motor neurons to environmental stressors or excitotoxicity .
the retention of some motor neurons in cn3 and cn4 as late as e18.5 without trophic support from their target muscles may be another example of the intrinsic relative resistance of oculomotor neurons to pathologic conditions .
using genetic tools , orbital dissection , and a developmental framework in timed mouse embryos , our findings catalogued the development of the ocular motor nerves and they implicate the eoms in the control of their terminal branching .
the identities of the signals provided by eoms , whether they differ among different eoms , and whether they are mediated through short - range diffusible cues , direct contact , or both , remain to be determined . | purposeto spatially and temporally define ocular motor nerve development in the presence and absence of extraocular muscles ( eoms).methodsmyf5cre mice , which in the homozygous state lack eoms , were crossed to an islmn : gfp reporter line to fluorescently label motor neuron cell bodies and axons . embryonic day ( e ) 11.5 to e15.5 wild - type and myf5cre / cre : islmn : gfp whole mount embryos and dissected orbits were imaged by confocal microscopy to visualize the developing oculomotor , trochlear , and abducens nerves in the presence and absence of eoms . e11.5 and
e18.5 brainstems were serially sectioned and stained for islet1 to determine the fate of ocular motor neurons.resultsat e11.5 , all three ocular motor nerves in mutant embryos approached the orbit with a trajectory similar to that of wild - type . subsequently , while wild - type nerves send terminal branches that contact target eoms in a stereotypical pattern , the myf5cre / cre ocular motor nerves failed to form terminal branches , regressed , and by e18.5 two - thirds of their corresponding motor neurons died .
comparisons between mutant and wild - type embryos revealed novel aspects of trochlear and oculomotor nerve development.conclusionswe delineated mouse ocular motor nerve spatial and temporal development in unprecedented detail .
moreover , we found that eoms are not necessary for initial outgrowth and guidance of ocular motor axons from the brainstem to the orbit but are required for their terminal branching and survival .
these data suggest that intermediate targets in the mesenchyme provide cues necessary for appropriate targeting of ocular motor axons to the orbit , while eom cues are responsible for terminal branching and motor neuron survival . | Methods
Generation of Mice Lacking Extraocular Muscles
Whole Mount Preparation and Imaging
Orbital Dissections and Imaging
Oculomotor Nerve Measurements
Motor Neuron Cell Counts
Results
EOMs Are Absent in
EOMs Are Not Necessary for Initial Pathfinding of Ocular Motor Axons to the Orbit
Axons of Ocular Motor Nerves Follow a Stereotypical Spatial and Temporal Trajectory in the Orbit in Both Wild-type and
Ocular Motor Nerves Fail to Develop Terminal Branches in the Absence of EOMs
Ocular Motor Neurons Die in the Absence of EOMs
Discussion |
PMC3433129 | although cdte - based czt
( cd1-xznxte ) crystals are the leading semiconductor
compounds for radiation - detection applications , the widespread deployment of czt
detectors has been limited by the high cost ( due to a low manufacturing
yield ) of detector - grade materials .
property nonuniformity has been
the major cause for both poor performance and a low yield of usable
portions of ingots .
it has long been established
that micrometer - scale defects such as tellurium inclusions / precipitates
affect carrier transport and uniformity .
however , extensive previous efforts to reduce te
particles have not resulted in an anticipated improvement in czt property
uniformity .
one special structural feature of czt crystals is their
extreme softness and their propensity to always develop a high density
of dislocations during synthesis .
experiments have shown
that when these dislocations are organized into a network of subgrains ,
they can directly affect charge carriers to cause electric
field perturbations .
clearly ,
eliminating or at least controlling dislocation subgrain structures
holds great potential to further improve czt crystals .
many
types of dislocations may be present in czt crystals , including
shuffle dislocations , glide dislocations , and dislocations ,
partial and perfect dislocations , edge , screw , mixed dislocations ,
and misfit dislocations . the understanding of the behavior of the
various combinations of these dislocations in conjunction with developing
methods for improving the corresponding dislocation network structures
are therefore challenging . the use of predictive , high - fidelity atomistic
simulations can help screen out these structure - limiting dislocation
types . here
we use a recently developed czt bond order potential ( bop ) in lammps ( large - scale atomic / molecular massively parallel simulator ) to perform selected molecular dynamic ( md ) simulations to determine
configurations , energies , and mobilities of 29 different types of
dislocations in cdte crystals .
[ the czt ternary bop is an extension of the cdte binary bop , so they are equivalent if used only for cdte .
] an efficient
method to derive activation free energy and activation volume of thermally
activated dislocation motion will be explored .
our focus is to identify
and validate important dislocations and dislocation phenomena in the
material , which can reduce future experimental efforts to improve
the dislocation network structures .
the key to high - fidelity
md simulations of dislocations is an interatomic
potential that is transferrable to the dislocated regions where the
local atomic environment is significantly different from that of the
perfect bulk crystal .
details of the czt bond order potential applied
here are omitted as they are very complex and have been fully described
previously .
it is demonstrated that the czt bop captures well
the property trends of a variety of elemental and compound structures / phases
( with local environment varying from coordination 2 to 12 ) , simulates correctly the crystalline growth of stoichiometric compounds
during both vapor deposition ( under nonstoichiometric vapor fluxes ) and melt - growth processes , and predicts precisely the configuration and density
of misfit dislocations in vapor deposited multilayers .
note that the correct simulation of growth , especially
under a nonstoichiometric environment , is a very strong validation
of the transferability of a potential as the process involves chemical
reactions and samples a variety of configurations and chemistries
statistically formed on a growth surface .
for this reason , the czt
bop is expected to produce reasonable results regarding dislocations .
different
types of dislocations can be constructed on various planes
in a zinc - blende crystal .
for instance , mobile dislocations usually
lie on the { 111 } slip planes , and misfit dislocations in multilayered
films are often seen to lie on interfacial planes , which are not necessarily parallel to { 111}. here we only
study dislocations that are on the { 111 } slip planes , with burgers
vector b being either partial < 112>a/6
or perfect < 110 > a/2 .
( a )
front projection of
( 112 ) planes , ( b ) top projection of two ( 111 ) planes a and
b showing glide perfect dislocations , ( c ) top projection of two ( 111 )
planes a and b showing glide partial dislocations , and ( d ) top projection
of two ( 111 ) planes b and c showing shuffle dislocations .
( a ) front projection
of
( 110 ) planes , ( b ) top projection of two ( 111 ) planes a and
b showing glide perfect dislocations , ( c ) top projection of two ( 111 )
planes a and b showing glide partial dislocations , and ( d ) top projection
of two ( 111 ) planes b and c showing shuffle dislocations . assuming that the dislocation line direction aligns with the z direction
and the slip occurs
on the x z plane , our definition
of different types of dislocations is shown in figures 1 and 2 , where dark and light circles
distinguish metal ( cd or zn ) and te atoms , respectively .
dislocations
defined in figure 1 are based upon an edge
dislocation coordinate system shown in figure 1a where the dislocation line is
perpendicular to one of the burgers vectors < 110>a/2 ( x axis ) .
dislocations defined in figure 2 are
based upon a screw dislocation coordinate system shown in figure 2a where is parallel
to one of the perfect burgers vectors < 110>a/2 ( z axis ) .
figures 1a and 2a indicate that a dislocation slip can occur at two different locations ,
one between two closely separated planes a and b ( where the interplane
bonds are not perpendicular to the planes ) , and the other between
the more widely separated planes b and c ( where the interplane bonds
are perpendicular to the planes ) . according to convention
, dislocations
between a and b are called glide dislocations , and dislocations between
planes b and c are called shuffle dislocations .
burgers vectors
of glide dislocations can be examined from a top
projection of two isolated planes a and b. figures 1b and 2b use such a projection to explore
perfect dislocation burgers vectors ( < 110>a/2 ) .
it can be seen
from figure 1b that in the edge coordinate
system , there are three nonequivalent perfect burgers vectors that
can be represented by the angle between b and , i.e. , = 30 ,
90 , and 150. there are other perfect burgers vectors ,
but they are equivalent to the ones listed due to crystal symmetry .
for instance , the = 330 dislocation is equivalent to
the = 30 dislocation .
similarly , it can be seen from
figure 2b that in the screw coordinate system
there are three more nonequivalent perfect burgers vectors
= 0 , 60 , and 300. figures 1c and 2c
use the same top projection of two isolated planes a and b to explore
partial dislocation burgers vectors ( < 112>a/6 ) .
it can be seen
from figure 1c that in the edge coordinate
system , there are two nonequivalent partial burgers vectors
= 0 and 120. similarly , it can be seen from figure 2c that in the screw coordinate system there are
two more nonequivalent partial burgers vectors = 90
and 330. burgers vectors of shuffle dislocations can be
examined from the
top projection of two isolated planes b and c. figures 1d and 2d use such a projection to explore
shuffle dislocation burgers vectors . here the light and dark atoms
in the b and c planes are aligned in the projection ( i.e. , only light
atoms are seen ) , and hence , there are no partial dislocation burgers
vectors for shuffle dislocations .
as indicated in figure 1d for the edge coordinate system , we explore three shuffle
dislocation burgers vectors = 30 , 90 , and 150 ,
which are the same as those in the perfect glide dislocation case
shown in figure 1b .
as indicated in figure 2d for the screw coordinate system , we explore three
more shuffle dislocation burgers vectors = 0 , 60 ,
and 300 , which are the same as those in the perfect glide dislocation
case shown in figure 2b .
dislocations
shown in figures 1 and 2 always occur between a metal ( cd or zn ) and te
{ 111 } planes .
if a dislocation has an edge component , then the dislocation
core can have extra metal atoms or extra te atoms . according to convention ,
a dislocation with more metal atoms at its core
is called an
dislocation , and a dislocation with more te atoms at its core is called
a dislocation . according to the discussion above
, we
will consider a combination
of glide and shuffle dislocations , partial and perfect dislocations ,
various burgers vectors , and and dislocations .
a cdte crystal
system with 336 ( 112 ) planes in the x direction ,
60 ( 111 ) planes in the y direction ,
and 28 ( 110 ) planes in the z direction is
used to calculate the ( 111 ) stacking fault energy sf .
the stacking fault is created by shifting the upper part of a perfect
crystal by a partial dislocation vector with respect to the lower
part ( at a glide dislocation slip plane between planes a and b , see
figure 1 or 2 ) .
molecular
statics ( ms ) energy minimization simulations are used to calculate
relaxed energies of both the perfect and the stacking faulted crystals
under the periodic boundary conditions in both x and z directions
and a free boundary condition in y direction . the stacking fault energy
is then calculated as sf = ( esf e0)/(lxlz ) , where esf and e0 are respectively the relaxed energies for stacking faulted
and perfect crystals and lx and lz are respectively
the system dimensions in x and z directions .
note that the surface energy in the y direction does
not affect the calculation as it exists for both stacking faulted
and perfect crystals and therefore cancels out .
cdte
is known to have a low stacking fault energy around 10 mj / m .
this in turn allows dislocation energies , i.e. , the
energy difference between a dislocated crystal and a perfect cdte
crystal containing the same number of atoms , to be calculated as a
function of dislocation core radius .
the key for the calculations
is to use the initial dislocation configurations that can be fully
relaxed during md and ms energy minimization simulations .
we find that
an initial dislocation created by simply positioning atoms according
to the displacement field of the linear elastic continuum solution
does not always fully relax at the core . instead
, a dislocation created
by shearing the relevant portions of the crystals using md simulations
is found to normally relax to lower energies than the other methods .
in particular , as will be described below , an md scheme illustrated
in figure 3 is effective in creating partial ,
undissociated perfect , and shuffle dislocations , and an md scheme
illustrated in figure 4 is effective in creating
dissociated perfect dislocations .
these schemes enable atom positions
to be strictly determined from atom interactions and also best reflects
realistic scenario that dislocations are created under local shear
stresses . line energy model for partial , undissociated perfect , and shuffle
dislocations .
( a ) md simulations to create dislocations and ( b ) md
and ms simulations to relax dislocations .
the computational system used is periodic in the x and z axes with free surfaces along the y axis . for the undissociated dislocations ( including partial
and shuffle dislocations ) shown in figure 3 ,
the system is divided into different regions as indicated by blue
( middle upper ) , orange ( middle lower ) , black ( left and right ) , and
white ( the rest ) colors . with the black regions held fixed and the
white regions free to relax , an md simulation is first performed to
uniformly move the blue region by half of the burgers vector 0.5b and the orange region by negative half of the
burgers vector 0.5b. for partial
dislocations , the movement of the blue and orange regions is completed
directly over a total simulated time of 0.1 ns . for perfect dislocations ,
the movement is through two stages over a total simulated time of
0.2 ns to follow the lowest energy path of two partials , i.e. , 0.5(b1 + b2 ) ( = 0.5b ) for the blue region and 0.5(b1 + b2 ) ( = 0.5b ) for the orange region .
in addition , the system temperature is uniformly decreased from 300
to 10 k during simulation .
this md procedure creates two dislocations
with opposite burgers vectors ( also note that if the dislocations
have an edge component , then one is an and the other one is
a dislocation ) as shown in figure 3a . for the dissociated perfect dislocations shown in figure 4 , the system is divided into different regions as
indicated by light and dark blue ( middle upper ) , light and dark orange
( middle lower ) , black ( left and right ) , and white ( the rest ) colors .
with the black regions held fixed and the white regions free to relax
,
an md simulation is first performed to uniformly move the light and
dark blue region by half of a shockley partial burgers vector 0.5b1 and the light and dark orange region
by negative half of the same shockley partial burgers vector 0.5b1 over a total simulated time of 0.1
ns . a second md simulation
is then performed to move the dark blue
region by half of the other shockley partial burgers vector 0.5b2 and the dark orange region by negative
half of the same shockley partial burgers vector 0.5b2 over another total simulated time
of 0.1 ns .
in addition , the system temperature is uniformly decreased
from 300 to 10 k during these two simulations .
this md procedure creates
the same two perfect dislocation burgers vectors as shown in figure 3a except that each perfect dislocation is dissociated
into two shockley partials bounding a stacking fault band as shown
in figure 4a .
( a ) md
simulations to create dislocations and ( b ) md and ms simulations to
relax dislocations .
once dislocations are created in the md simulations ,
the blue and
orange regions are released . a narrow region in the middle of the
system
is allocated as shown by the black color in figures 3b and 4b . for perfect dislocations ,
this narrow region remains a perfect crystal because the blue region
is moved by a perfect burgers vector with respect to the orange region .
for partial dislocations ,
the narrow middle region contains a stacking
fault . with left , right , and
middle black regions fixed , an ms simulation
is then conducted to relax the dislocations . in this work ,
we
use an edge dislocation geometry containing 97
( 110 ) planes in the x direction , 60 ( 111 )
planes in the y direction , and 24 ( 112 ) planes
in the z direction and a screw dislocation geometry
containing 168 ( 112 ) planes in the x direction ,
60 ( 111 ) planes in the y direction , and 14 ( 110 )
planes in the z direction to create various perfect
and dislocated computational crystals .
relaxed energies of both dislocated
and perfect crystals are calculated . to separate and
dislocation effects , the total energy is split to the left and right
parts respectively by summing up the atomic energies of the left and
right halves of the systems .
the line energies of the left and the
right dislocation , i , are then
calculated as i = ( ed , i
wilzsf)/lz , where ed and e0 are the
relaxed energies of the dislocated and the perfect crystals , respectively , lz is the dislocation length
(= system dimension in the z axis ) , sf is the stacking fault energy , w is the stacking
fault bandwidth , and the subscript i (= l or r ) indicates
the left and the right dislocations .
we find that of all the simulations using
the initial perfect dislocations , the maximum splitting distance is
only 25 after energy minimization .
table 1 indicates that the screw ( = 0 ) dislocations
have lower energies than other dislocations , being 0.38 , 0.69 , 0.79 ,
and 0.81 ev / for partial , dissociated perfect , undissociated
perfect , and shuffle dislocations , respectively .
interestingly , the
acute angle of the second lowest energy ( shuffle , glide partial , glide
perfect ) dislocations all equal 30 ( e.g. , = 30 ,
150 , and 330 ) . in addition , the energies of dissociated
perfect dislocations are about 8092% of the energies of undissociated
dislocations , suggesting that the dissociated dislocations are more
stable .
however , the observation that the perfect dislocations remain
approximately undissociated during md and energy minimization simulations
suggests an energy barrier of dissociation .
the configuration of perfect
dislocations will be examined further below in md simulations of dislocation
motion .
( a ) glide
partial dislocations , ( b ) glide perfect dislocations , and ( c ) shuffle
dislocations .
the dislocation line energy is not a constant material
property ,
but rather increases with the material volume within a radius r from the dislocation core .
the dislocation line energy
at a given radius r can be calculated using the same
approach described above except that the energies of the perfect and
dislocated crystals are taken as the sum of the atomic energies for
all the atoms within the radius .
the dislocation line energies as
a function of r thus obtained are shown in figure 5a
figure 5a c indicates that dislocation
line energies calculated with the atomistic models linearly increase
with dislocation radius r when r is larger than 40 .
this is consistent with the boundary conditions
used in figures 3 and 4 where the black regions are held fixed during simulations .
the dislocation
core energies at small radii , say r < 30 ,
come from the relaxed dislocation core structures . in the present
work , the system sizes have been chosen to be sufficiently large so
that the predicted dislocation core structures are independent of
the boundary conditions . as a result
, our simulations can give accurate
dislocation core energies at r < 30 that
can not be predicted from linear elastic theories .
it can be seen from
figure 5a c that dislocation energies
sharply decrease when the radius is decreased .
in addition ,
and dislocations have similar energy vs r curves for partial
and shuffle dislocations . while and dislocation energies
are also similar for the perfect dislocations at large radii , they
become different at the core .
in particular , the perfect dislocation
core energies are lower than the perfect dislocation core
energies .
one effective approach to simultaneously study the motion of multiple
dislocations is to examine the evolution of a dislocation loop under
a shear stress .
the computational system consists of a cdte crystal
with 120 ( 112 ) planes in the x direction ,
18 ( 111 ) planes in the y direction , and 68 ( 110 )
planes in the z direction .
periodic boundary conditions
are used in the x and z directions
and a free boundary condition is used in y direction
during the simulation . to create a dislocation loop ,
the system is
divided into different regions as shown in figure 6(a ) : the black ( top and bottom ) regions , the blue ( middle
upper ) hexagonal region , the orange ( middle lower ) hexagonal region ,
and the white ( remaining ) region . as plastic deformation usually proceeds
through a glide of dislocations
hence ,
we assume that the boundary between the upper and lower hexagons is
between planes a and b as defined in figures 1a and 2a . to create a realistic dislocation
loop with a burgers vector of b ,
an md simulation
is performed to uniformly move the top black and blue regions by 0.5b and the lower black and orange regions by 0.5b over a simulated time of 0.1 ns while allowing
the white region to relax according to newton s equation of
motion and a constant temperature of 10 k. as mentioned above , for
a partial dislocation loop , the md simulation is completed directly ,
and for a perfect dislocation loop , the md simulation is completed
in two stages to follow the low energy path of two partial dislocations .
this md simulation procedure creates a dislocation loop at a shear
strain of approximately = b / t , where b is the magnitude of the dislocation burgers
vector and t is the thickness of the sample . the
system is then redivided into three regions as shown in figure 6b : the black ( top and bottom ) regions and the white
( remaining ) region .
the evolution of dislocation loops is examined
in subsequent md simulations at a high temperature of 900 k where
the top and bottom surface black layers are further moved in positive
and negative burgers vector directions to increase the shear strain .
( a ) md simulation to create dislocation
loop and ( b ) md simulation to evolve dislocation loop . a partial
dislocation burgers vector of [ 112]a/6 is used to create an
initial dislocation loop using the procedure shown in figure 6(a ) .
further shear strain is applied
by moving the top and bottom black regions in the + x and x directions by 1.394 , respectively ,
using the procedure shown in figure 6b .
the equilibrium dislocation loop configurations obtained
at 900 k temperature are examined in figure 7 by showing the top view of four consecutive planes above and below
the slip plane , where blue and gray circles indicate cd and te atoms
respectively , and the yellow hexagon indicates the initial partial
dislocation loop at xy = 0.039 .
panels a and b in figure 7 are obtained using
exactly the same conditions except that the cd and te atoms are switched
between the two figures , so that in figure 7a , the right side of the loop is an dislocation and the left
side of the loop is a dislocation , whereas in figure 7b , the left side is and the right side is
. because the movement of a partial dislocation leaves
behind a stacking fault , the relaxed partial dislocation loop can
be identified by the boundary of the two stacking patterns demonstrated
in the figure .
it can be seen from figure 7a that the hexagonal dislocation loop becomes asymmetric under the
shear and in particular , the dislocation moves by a far longer
distance than the dislocation .
this is further verified in
figure 7b where the asymmetry of the dislocation
is flipped as a consequence of the flip of and dislocations .
this simulation strongly indicates that dislocations are much
more mobile than dislocations ( despite their similar line
energies in table 1 ) , in good agreement with
experiments conducted for many semiconductor compounds .
( a ) configurations
at strains xy = 0.039 and xy = 0.078 and ( b ) the same as in panel a
except that cd and te atoms are switched
t = 0.0025 ( a ) , 0.0100 ( b ) , 0.0250 ( c ) , and 0.0350 ns ( d ) .
one interesting observation in figure 7 is
that to accommodate the motion of the segment ( = [ 110 ] ) of the dislocation loop , two segments ( = [ 112 ] ) are created .
this means that the motion
of the mobile partial dislocations can cause elongated screw
partials . as a result
, cdte crystals may have a high density of such
type of screw partials , in agreement with its low energy shown in
table 1 . on the other hand ,
this corresponds
well to the high energy of the edge partial in table 1 .
a perfect
dislocation loop with a burgers vector of [ 110]a/2 is created
using the procedure described in figure 6a .
this procedure produces an initial shear strain of yz,0 = 0.068 . by moving the top and bottom black regions
in the + z and z directions
at a constant speed of 1.2 nm / ns during an md simulation at 900 k ,
as described in figure 6b , the system is further
deformed at a strain rate of dyz / dt = 0.338/ns .
the configuration of the dislocation
loop during the further strain is shown in figures 8a d as a function of time , where the color scheme represents
the magnitude of the slip vector developed previously to show dislocations .
figure 8a shows that
shortly after further strain at t = 0.0025 ns ( yz = 0.069 ) , the dislocation loop remains
hexagonal but some portion of the dislocation ( the lower part
of the [ 101 ] section ) bows out .
figure 8b shows that at t = 0.0100 ns ( yz = 0.071 ) , the dislocation bows out more . in figure 8c at 0.0250 ns ( yz = 0.076 )
, the dislocation is seen to have moved a significant
distance that causes it to pass through the low boundary and enter
into the image from the top boundary under the periodic boundary condition .
clearly , perfect dislocations are also much more mobile than
perfect dislocations ( despite their similar line energies
shown in table 1 ) , in good agreement with experiments . interestingly
, figure 8c indicates that , although screw dislocations also have higher mobility
than edge dislocations , they are much less mobile than
edge dislocations . when the dislocation meets the
dislocation at the top of the hexagon , they are annihilated , and the
dislocation loop becomes two vertical screw dislocations running across
the periodic dimension in the z direction , as can
be seen in figure 8d at t =
0.035 ns ( yz = 0.080 ) .
further
strain will cause the two dislocations to continuously move in the
x directions until they completely sweep the
entire area and are annihilated .
interestingly , we find that the dislocation
motion is thermally activated and does not occur when the simulation
is performed at a low temperature , say , 10 k. in addition , we find
that the dislocations do not move as straight lines , but rather through
the formation and rapid motion of kinks along the z direction as shown in figure 8c .
this is
consistent with the traditional kink model of thermally activated
motion of dislocations .
the observation
that screw dislocations are much less mobile than
edge dislocations indicates that when the sector of
a dislocation loop moves , the screw dislocation sectors ( = [ 110 ] ) will elongate . as a result
this is consistent with the low
energies of screw dislocations shown in table 1 .
in addition , no dislocation dissociation is observed in figure 8 , indicating that the perfect dislocations do not
necessarily dissociate under nonequilibrium conditions .
the details
of dislocation mobility are important to understand
because they directly control dislocation network structures . for
thermally activated motion of dislocations , the dislocation velocity
can be expressed as1where q and are respectively
the activation energy and activation volume of the dislocation motion ,
is the shear stress , k is boltzmann s
constant , t is the temperature , and v0 is a constant representing saturation dislocation velocity
for barrierless ( very high temperature or shear stress ) dislocation
motion . here q and are the primary parameters
quantifying dislocation mobility .
the discussion presented above
already indicates several distinctive
dislocation types that may determine dislocation structures .
first ,
and partial and perfect dislocations have a strong
edge component and can contribute to the climb motion responsible
for the formation of small angle grain boundaries .
dislocations
are the most mobile , resulting in special structural features of dislocation
networks . screw partial and perfect dislocations can also be important .
these screw dislocations are expected to have a high density as they
have low energies and can be created by the motion of dislocations .
the objective of the present work is not to exhaustively tabulate
the mobilities of a variety of dislocations .
hence , we focus on calculating
the activation energy barrier q and activation volume for
and edge type of perfect dislocations . such studies
will allow us to fully explore the method for calculating q and and to validate the results using known experimental
relative mobilities of and dislocations .
in addition ,
edge dislocations play an important role in dislocation network formation
due to their climb motion .
the extension of the method to other dislocations
that can provide complete inputs for simulations of dislocation network
structure evolution using larger scale ( e.g. , dislocation dynamics ) models will be performed in future work .
the activation energy barriers of dislocation motion are often
directly calculated from energy profiles using the nudged elastic
band method .
however , the stop - action observation of the dislocation migration
case shown in figure 8 indicates that the thermally
activated motion of dislocations proceeds through a variety of different
paths . without requiring an explicit treatment of migration paths ,
we perform md simulations of dislocation motion at a variety of temperatures
and shear stresses to fit an apparent activation energy barrier and
activation volume directly from ( 1 ) .
the activation energy determined
this way pertains to a free energy barrier that incorporates the multiple
path ( entropy ) effects .
in addition , this approach provides a direct
verification of ( 1 ) and , hence , the thermally activated dislocation
mechanism .
the initial crystal contains 144
( 110 ) planes in the x direction , 36 ( 111 )
planes in the y direction , and 24 ( 112 ) planes
in the z direction .
the system employs periodic boundary
conditions in x and z directions
and a free boundary condition in y direction .
first ,
the initial crystal is divided into different regions as shown in
figure 9a : the black ( left and right ) regions ,
the blue ( middle upper ) region , the orange ( middle lower ) region ,
and the white ( remaining ) region .
since we only explore glide dislocations ,
we assume that the boundary between the blue and orange regions is
between a and b planes as defined in figures 1a and 2a . to create dislocations with a perfect
burgers vector of b = b1 + b2 , an md simulation
is performed where the black regions are fixed , the white regions
are relaxed , and the blue and orange regions are first uniformly moved
by 0.5b1 and 0.5b1 , respectively , over a simulated time
of 0.001 ns and then moved by 0.5b2 and 0.5b2 , respectively ,
over another simulated time of 0.001 ns .
the crystal is then cut in
half in the x direction as shown in figure 9b with a corresponding adjustment of the periodic
length so that the two halves remain periodic in the x direction .
in particular , we use b = [ 110]a/2 , b1 = [ 121]a/6 , and b2 = [ 211]a/6 to create two crystals , one containing
an perfect dislocation , and the other one containing a
perfect dislocation . edge dislocation mobility model . ( a )
md simulation to
create
and dislocations , ( b ) isolate out and dislocations ,
and ( c ) md simulation to evolve dislocation under shear stress xy . for dislocation motion simulations , the ( half )
system is divided
into three regions : the black ( top and bottom ) regions and the white
( remaining ) region . by adding corresponding forces to the atoms in
the top and bottom regions in + x and x directions , respectively ,
molecular dynamics simulations
under an npt ( constant atom number , pressure , and temperature ) condition
are then performed to study migration of dislocations at a variety
of stresses and temperatures .
we find that in all of these simulations
the dislocation splitting is very small , further verifying the discussions
presented above that perfect dislocations do not necessarily dissociate .
assuming that the slip vector reaches
a maximum at the dislocation core , the dislocation position is calculated
as a function of time .
results obtained for and dislocations
at selected stresses and temperatures are shown in figure 10 .
it can be seen from figure 10 that all the dislocation vs time curves approximately fall
on straight lines .
this shows that the data can provide reliable information
about the steady - state dislocation velocity .
the slope of these straight
lines changes with stress and temperature , indicating that the simulated
conditions capture the thermally activated mechanisms of dislocation
motion .
figure 10a shows the effect of
temperature
on dislocation motion at a constant shear stress of 0.5 gpa .
increasing
the temperature is seen to increase the dynamics , consistent with
the thermal activation mechanism . at the same temperature ,
the
dislocation is seen to move faster than the dislocation , further
verifying the results seen in figures 7 and 8 .
figure 10b shows the effect
of shear stress at a constant temperature of 600 k. here increasing
the stress also correctly increases the dislocation velocity , and
the dislocation is again validated to move faster than the
dislocation .
( a ) effect
of temperature
at a given stress and ( b ) effect of stress at a given temperature .
the steady state velocity can be easily calculated
from the slope
of the curves shown in figure 10 .
the results
obtained at a constant shear stress of xy = 0.5 gpa are plotted as ln(v ) vs ( kt ) curves in figure 11 . here
the unfilled and filled circles represent md data for
and dislocations respectively , and the lines are fitted
to the data using equation ( 1 ) .
figure 11 indicates
that the md data falls fairly well on straight ln(v ) vs ( kt ) lines , validating
that the method indeed captures well the thermally activated dislocation
migration . through the fitting , we find that the activation energy
and activation volume are q = 0.14 ev and
= 17 for the dislocation and q = 0.27 ev and = 38 for the dislocation .
this again verifies that in the thermally activated regime ( e.g. ,
xy 1 gpa ) , the
dislocation is more mobile than the dislocation .
the
thermally activated plastic deformation of cdte has been experimentally
analyzed below room temperature . the
activation energy and activation volume
are assumed to decrease with
increasing stress in the experiments . under this assumption ,
the fitted
experimental activation energy and activation volume are approximately
0.8 ev and 5500 , respectively , at a small stress
of 0.005 gpa , and fall to < 0.5 ev and < 2250 , respectively , at a stress of 0.010 gpa . here
we assume that q and are constant and are fitted using md data
at high stresses ( e.g. , 0.5 gpa ) .
in addition , simulations target
only the peierls potential barrier mechanism of specific (
and perfect edge ) dislocations , whereas , experiments may include
point defect obstacle mechanisms and the contributions from other
dislocations . as a result
, it is expected
that the simulated activation energy and activation volume are smaller
than their experimental counterparts .
our bop - based md simulations
of dislocations in cdte crystals lead
to the following conclusions:(1) dislocations move much faster
than dislocations , in good agreement with well - known experiments
for semiconductor compounds .
this , along with previous demonstrations
that our bop captures properties of many phases and predicts crystalline
growth during md simulations of chemical vapor deposition and melt - growth , provides strong validation
that the bop correctly captures the physics of dislocation motion
in cdte .
the bop - based md simulations , therefore , can provide high - fidelity
data to construct large scale ( e.g. , dislocation dynamics ) models that can simulate the evolution of dislocation
cell structures.(2)for
partial , perfect , and shuffle
sets , screw dislocations always have the lowest energies . dissociated
glide dislocations always have lower energies than perfect glide dislocations .
however , perfect glide dislocations do not necessarily dissociate
due to the energy barrier of dissociation .
in particular , the splitting
distance of perfect glide dislocations is seen to remain very small
in our dynamic md simulations.(3)the acute angle of the second lowest
energy ( shuffle , glide partial , glide perfect ) dislocations all equal
30 ( e.g. , = 30 , 150 , 330).(4)screw dislocations are
much less mobile
than edge dislocations . as a result ,
the screw dislocation
sectors perpendicular to the sector are elongated when an
sector of a dislocation loop moves .
this suggests that screw
dislocations are likely to have a higher density than edge dislocations ,
in good agreement with the low screw dislocation energies.(5)without calculating the
energy profile
explicitly , we show that md simulations of dislocation motion at a
variety of stresses and temperatures can be used to effectively deduce
the activation free energy barrier and activation volume of thermally
activated glide of dislocations .
in particular , we found activation
free energy barriers of 0.14 and 0.27 ev and activation volumes of
17 and 36 , respectively , for and
edge types of glide dislocations .
dislocations move much faster
than dislocations , in good agreement with well - known experiments
for semiconductor compounds .
this , along with previous demonstrations
that our bop captures properties of many phases and predicts crystalline
growth during md simulations of chemical vapor deposition and melt - growth , provides strong validation
that the bop correctly captures the physics of dislocation motion
in cdte .
the bop - based md simulations , therefore , can provide high - fidelity
data to construct large scale ( e.g. , dislocation dynamics ) models that can simulate the evolution of dislocation
cell structures . for
partial , perfect , and shuffle
sets , screw dislocations always have the lowest energies . dissociated
glide dislocations always have lower energies than perfect glide dislocations .
however , perfect glide dislocations do not necessarily dissociate
due to the energy barrier of dissociation .
in particular , the splitting
distance of perfect glide dislocations is seen to remain very small
in our dynamic md simulations .
the acute angle of the second lowest
energy ( shuffle , glide partial , glide perfect ) dislocations all equal
30 ( e.g. , = 30 , 150 , 330 ) .
screw dislocations are
much less mobile
than edge dislocations . as a result ,
the screw dislocation
sectors perpendicular to the sector are elongated when an
sector of a dislocation loop moves .
this suggests that screw
dislocations are likely to have a higher density than edge dislocations ,
in good agreement with the low screw dislocation energies . without calculating the
energy profile
explicitly ,
we show that md simulations of dislocation motion at a
variety of stresses and temperatures can be used to effectively deduce
the activation free energy barrier and activation volume of thermally
activated glide of dislocations .
in particular , we found activation
free energy barriers of 0.14 and 0.27 ev and activation volumes of
17 and 36 , respectively , for and
edge types of glide dislocations . | cd1-xznxte
( czt ) crystals are the leading semiconductors for radiation
detection , but their application is limited by the high cost of detector - grade
materials .
high crystal costs primarily result from property nonuniformity
that causes low manufacturing yield .
although tremendous efforts have
been made in the past to reduce te inclusions / precipitates in czt ,
this has not resulted in an anticipated improvement in material property
uniformity .
moreover , it is recognized that in addition to te particles ,
dislocation cells can also cause electric field perturbations and
the associated property nonuniformities .
further improvement of the
material , therefore , requires that dislocations in czt crystals be
understood and controlled .
here , we use a recently developed czt bond
order potential to perform representative molecular dynamics simulations
to study configurations , energies , and mobilities of 29 different
types of possible dislocations in cdte ( i.e. , x =
1 ) crystals . an efficient method to derive activation free energies
and activation volumes of thermally activated dislocation motion will
be explored .
our focus gives insight into understanding important
dislocations in the material and gives guidance toward experimental
efforts for improving dislocation network structures in czt crystals . | Introduction
Interatomic Potential
Definition of Dislocation Types
Stacking Fault Energy
Dislocation Line Energies
Dislocation Loop Evolution under Shear
Dislocation Mobilities
Conclusion |
PMC3139143 | mental disorders , including dementia , can impair competence , but a diagnosis of dementia does not imply a complete loss of competence .
there is a wide consensus on considering competence as the capacity of a person to make a specific decision .
voting is a decision of particular interest since a consensus does not exist on which abilities the patient with dementia should retain to express a reliable choice .
thus , identifying patients who , despite the presence of dementia , maintain the capacity to vote and increasing their chance to take part in a ballot ( e.g. , allowing their caregivers to have a role in facilitating this ) would be of crucial importance .
participation in the electoral process by citizens with dementia has become especially important in recent years , both for the growing number of individuals suffering from alzheimer disease ( ad ) or other progressive cognitive disorders , and in light of the fact that in at least two cases ( 2000 us presidential elections and 2006 italian elections for the prime minister designation ) , a small number of votes had a decisive effect on the results .
it is especially in long - term facilities that inappropriate assumptions about the absence of voting capacity may deprive still capable and willing residents of the right to vote [ 4 , 5 ] .
recently , a novel test to assess the capacity to vote has been proposed : the competence assessment tool for voting ( cat - v ) , which evaluates an individual 's performance on four decision - making abilities : understanding the nature and effect of voting , appreciating the reality of voting situation , making a choice , and reasoning about voting choices . in this paper , we report the results of a study that applied a modified version of the cat - v to individuals with mild - moderate ad who were temporarily residents in a long - term care facility before 2006 italian elections for designating the prime minister .
our primary hypothesis was that although voting capacity would be inversely associated with dementia severity , the single decision - making abilities evaluated by the cat - v would be affected unequally by the dementing process .
the subjects included in the present study ( n = 38 ) represent all the patients with mild - moderate dementia ( mini - mental state examination ( mmse ) 11 ) and a clinical diagnosis of probable ad ( according to the national institute of neurological and communicative disorders and stroke ( nincds ) and the alzheimer disease and related disorders association ( adrda ) criteria ) who were admitted into the alzheimer centre of the ospedale gazzaniga ( bergamo , italy ) from sixty to thirty days before 2006 italian general elections .
although , in some respects , our centre has several characteristics of a long - term care facility , no patient is a permanent resident .
the primary requirement for a patient 's admission into our centre is the presence of behavioural abnormalities or psychopathologic symptoms in the context of a dementing syndrome but , once these features are significantly relieved , the patient is discharged .
the instrument we used to evaluate the capacity to vote was a modified version of the cat - v , an instrument that measures a person 's ability to understand the nature and effect of voting , make a choice , appreciate , and reason through a voting decision .
these criteria were operationalized into five questions preceded by an introduction reminding each person that soon he / she would have the opportunity to take part in a ballot for the election of the prime minister .
thus , as opposed to the original us version of the cat - v , in which subjects are asked to imagine that two candidates are running for governor and that the day of the interview is the election day , the scenario we proposed was real rather than hypothetical .
furthermore , in order to shorten the time of interview , unlike the original , our version of the cat - v did not include a question evaluating subjects ' appreciation of the significance of voting .
for each cat - v item , the scores assigned to each person ranged from 2 ( correct response reflecting adequate performance ) to 0 ( inadequate performance ) .
every participant was enrolled after an initial contact with his / her principal caregiver . once informed about the characteristics of the study and made sure that its results would be used exclusively for research purposes , each participant ( or his / her caregiver ) provided a written informed consent .
all of the 38 participants were interviewed and rated by one investigator ( m. sala ) , who was blinded to their mmse score .
thirty of them were also interviewed and rated by another investigator ( e. chit ) . weighted kappa and kendall tau - b
twenty - nine subjects were again interviewed by m. sala two weeks later , to evaluate the test - retest reliability .
the scores included in the main data analysis are those assigned to all participants at baseline by m. sala the spearman correlation coefficient was used to examine the association of the capacity to vote ( as expressed by the score on each of the cat - v items ) with severity of both cognitive impairment ( as expressed by the mmse score ) and behavioural and psychopathological symptoms ( as expressed by the neuropsychiatric inventory ( npi ) score ) .
each participant was administered the cat - v , the mmse , and the npi during the same session .
all the subjects who were asked to participate in the project ( n = 38 ) did complete the interview .
behavioural and psychopathological symptoms were moderately severe at baseline but were significantly relieved prior to discharge .
subjects ' performance on cat - v is shown in table 2 . over a half of
the subjects appeared to fully understand the nature of the vote , but only approximately a third was entirely able to understand its effect .
however , the great majority of participants ( ~90% ) was deemed to be completely able to make a choice .
conversely , subjects ' ability to reason about voting by comparing the choices at disposal and , above all , by evaluating the possible consequences of the preference for a candidate on their life was considerably more impaired .
in fact , only about 16% of the participants had a completely adequate performance on the latter measure . as shown in table 3 , which relates subjects ' combined performance on understanding and choice to their performance on reasoning , only three of the 38 participants ( 8% ) scored the maximum on all items .
as emerges from table 4 , there were better test - retest and interrater reliabilities for scores on understanding and choice than for scores on reasoning .
there was no relation of cat - v scores to severity of behavioural and psychopathological symptoms ( r = 0.14 , p = 0.41 ) .
conversely , as expected , lower cat - v scores were associated with lower mmse scores ( figures 1 , 2 , and 3 ) . however ,
a great variability in subjects ' performance was noted at any stage of disease . on questions evaluating understanding and choice ( figure 2 ) ,
for example , only 58% of subjects with mild ad ( mmse 20 ) obtained the maximum score but , remarkably , over one - third of those who scored the maximum was beyond mild - stage disease ( mmse < 20 ) .
lower cat - v scores were also associated with fewer years of education but , as opposed to disease severity , poor education had an impact exclusively on measures of understanding and choice ( r = 0.32 , p = 0.049 ) .
no relationship was found between education and mmse scores ( r = 0.05 , p = 0.76 ) .
among persons with mild to moderate ad , global measures of cognitive functioning , such as the mmse , can not adequately substitute for an assessment of voting capacity .
the present study has examined the capacity to vote in a sample of 38 mild - moderate ad patients using a modified version of the cat - v .
originally designed in the us , this instrument consists of a brief questionnaire , which has been translated and adapted to the italian context and by which we have explored the following functional abilities : understanding the nature and effect of voting , expressing a choice , reasoning about the choices at disposal , and reasoning about the consequences of voting .
we have also calculated the reliability of the test and its relation to dementia severity . on the basis of our results ,
a full capacity to vote ( as expressed by integrity of all above mentioned decision - making abilities ) appears to be retained by a small minority of ad subjects ( 3/38 ) , and exclusively at mild - stage disease ( mmse 20 ) . however , when we applied less restrictive criteria for determining voting competence , as those identified in the doe standard ( a legal standard based on a 2001 federal district court decision in maine ( us ) , which solely requires an intact ability to understand and make a choice ) , subjects ' voting capacity was not completely predicted by mmse scores . on understanding and choice measures , in fact , only 58% of our less deteriorated subjects ( mmse 20 ) obtained the maximum score but , remarkably , over one - third of those who scored the maximum were beyond mild - stage disease ( mmse 1319 ) . as a result , the relation of these measures to severity of cognitive impairment was only moderate ( r = 0.61 ) .
of note , over two - thirds of our ad patients , although still able to express a choice , did not appear to be entirely able to understand the nature and , especially , the effect of the vote , thereby failing to meet the doe standard . a similar pattern ( choice considerably less impaired than understanding ) has previously been reported by us investigators . in their study , however , the percentage of ad patients who failed to fulfil the doe standard was lower than that seen in our study ( 55% versus 68% ) , and there was a much more substantial link between declining voting capacity and increasing dementia severity ( r = 0.87 versus 0.61 ) .
there are several possible reasons for the discrepancy between our results and those previously reported by the us investigators .
for example , compared to these authors , we examined a sample characterized by more advanced age ( 81.1 versus 77.7 years ) , greater female preponderance ( 76% versus 52% ) , less severe impairment ( mean mmse 18.2 versus 16.4 ) , and considerably lower education ( 5.6 versus 14 years ) .
furthermore , since we excluded patients with severe ad from analyses , the range of cognitive impairment was more compressed in our sample ( mmse 1127 ) than in the us sample ( mmse 228 ) .
the exclusion of patients with severe ad may also explain the only low - moderate test - retest reliability values of our study ( understanding and choice , k = 0.42 ; reasoning , k = 0.22 ) , as well as the less satisfying agreement between our raters than between the raters of the us study ( understanding and choice , k = 0.65 versus 0.91 ; reasoning , k = 0.41 versus 0.74 ) .
subjects with severe ad have in fact a greater likelihood than those with mild - moderate disease to invariably provide completely inadequate performances , so as to be assigned the minimum score unambiguously and consistently over time .
not requiring a particular expertise , the cat - v is easily administrable , since no more than five minutes are needed for its administration .
this time might further be shortened if one decides to skip the reasoning questions because , at least in our experience , these questions were too demanding even for mildly deteriorated patients and generated performances characterized by insufficient test - retest and inter - rater reliabilities .
interpreting subjects ' performance remains , however , problematic even when analyses are restricted to the questions inherent in the doe standard ( understanding and choice ) .
clearly , performances at the extremes of the spectrum are not controversial , so that a performance generating the minimum score unequivocally indicates absence of voting capacity and , by contrast , a performance generating the maximum score indicates a full compliance with the doe standard . however , intermediate scores need a judgement to be made , the basis of which is not obvious and deserves further comments .
for example , in both the us and our study , almost all of the participants with intermediate scores appeared to be invariably capable to make a choice , while what varied was their ability to understand the nature and effect of voting .
consequently , if we had applied more liberal criteria than those identified in the doe standard for determining voting competence and , for example , we had deemed a patient to retain the capacity to vote if he / she was able to express a choice regardless of understanding , the great majority of ad subjects ( 90% in our study and 88% in the us study ) would have been categorized as such .
the limitations of this study relate to lack of data from nondemented persons , whose availability would have been extremely helpful in interpreting intermediate scores by the identification of appropriate cutoffs and of more detailed neuropsychological information than that provided by the mmse .
other issues are the relatively small sample size , the relatively narrow range of cognitive impairment , and the presence of significant behavioural and psychopathological symptoms ( although their severity did not appear to influence the performance of our patients on the cat - v ) .
since this study was restricted to persons with mild - moderate disease , and most of them had significant behavioural disturbances , its results may be not entirely representative of all patients with ad . despite these flaws ,
there was an inverse relationship between voting capacity and dementia severity but , somewhat unexpectedly , the strength of this association was not substantial .
however , as predicted , the single decision - making abilities evaluated by the cat - v were unequally impaired by the dementing process ( reasoning > understanding > choice ) . on the basis of these data ,
the use of a structured interview , such as the cat - v , may offer advantages over unstructured or clinical assessments , especially in light of the fact that global measures of cognitive functioning , such as the mmse , do not appear to be strong predictors of the capacity to vote .
further studies are needed to refine the clinicians ' approaches to identifying demented people who are still capable to vote from those who are no longer capable .
nevertheless , a tool like the cat - v can adequately assist in this distinction ( table 5 ) .
if you do not understand something of what i am saying or asking , please let me know and i will repeat it
. some of the questions might seem very simple to you , but do not worry about that .
two candidates are running for prime minister ( make the patient name the candidates or , if he / she does not remember , remind him / her of their names ) .
two candidates are running for prime minister ( make the patient name the candidates or , if he / she does not remember , remind him / her of their names ) .
understanding the nature of votingwhat will you do to pick the prime minister on election day?(if patient gives an indirect answer , describing how he / she or people in general would choose between the candidates , for example watching tv , listening to their campaign issues , ask:well , that is how you might decide who you like to be the prime minister . but
how would actually express your choice?)score of 2 : entirely correct response , for example , i will go to the polls and vote or
i will cast my vote for one or the other , and so forth.score of 1 : ambiguous or partially correct response , for example , that is why we have election day , and so forth.score of 0 : incorrect response , for example , there is nothing you can do ; the tv guys decide , and so forth .
( if patient gives an indirect answer , describing how he / she or people in general would choose between the candidates , for example watching tv , listening to their campaign issues , ask :
well , that is how you might decide who you like to be the prime minister . but how would actually express your choice ? ) score of 2 : entirely correct response , for example , i will go to the polls and vote or
i will cast my vote for one or the other , and so forth .
score of 1 : ambiguous or partially correct response , for example , that is why we have election day , and so forth .
score of 0 : incorrect response , for example , there is nothing you can do ; the tv guys decide , and so forth .
understanding the effect of votingonce the election for prime minister is over , how is it going to be decided who is the winner?score of 2 : entirely correct response , for example , the votes will be counted and the candidate with more votes will be the winner.score of 1 : ambiguous or partially correct response , for example , the better between the two candidates will be the winner
once the election for prime minister is over , how is it going to be decided who is the winner ? score of 2 : entirely correct response , for example , the votes will be counted and the candidate with more votes will be the winner .
score of 1 : ambiguous or partially correct response , for example , the better between the two candidates will be the winner
choicehand patient a card with the information in the following paragraph in large print ; allow to retain and consult this card for the remainder of the interview.for the sake of simplicity , the first candidate ( of the right party ) is willing to lower taxes by decreasing the burden of bureaucracy and public administration , in order to make people spend more as a result of higher income .
the second candidate ( of the left party ) is willing to either raise taxes or , by fighting tax elusion , keep them unchanged getting every citizen to pay , so that the rights to education and welfare remain protected .
based on either what i have just told you or what you already knew about the candidates , do you think you are able to choose between the two ?
mind that i do not want to know from you which candidate you would vote for , but only if you have made your choice?score of 2 : the patient clearly indicates the choice , including a reasoned choice not to vote or a manifestation of indetermination ( i still do not know which candidate to vote for).score of 0 : no choice is stated because the patient is unable to choose , does not understand what is asked , and so forth .
hand patient a card with the information in the following paragraph in large print ; allow to retain and consult this card for the remainder of the interview . for the sake of simplicity , the first candidate ( of the right party ) is willing to lower taxes by decreasing the burden of bureaucracy and public administration , in order to make people spend more as a result of higher income .
the second candidate ( of the left party ) is willing to either raise taxes or , by fighting tax elusion , keep them unchanged getting every citizen to pay , so that the rights to education and welfare remain protected .
based on either what i have just told you or what you already knew about the candidates , do you think you are able to choose between the two ?
mind that i do not want to know from you which candidate you would vote for , but only if you have made your choice ?
score of 2 : the patient clearly indicates the choice , including a reasoned choice not to vote or a manifestation of indetermination ( i still do not know which candidate to vote for ) . score of 0 : no choice is stated because the patient is unable to choose , does not understand what is asked , and so forth .
reasoning
comparative reasoningif the patient identifies a choice , ask : why do you think that it is worth voting for either of the candidates ? or why do you think neither of the candidates deserves to be voted for?score of 2 : entirely correct response , for example , because it is right to maintain the welfare state
, because it is right that everybody pays taxes , because the state should not empty the citizen 's pockets with too high taxes ,
because , despite different views , neither of the candidates will fulfill the promises made before election day , and so forth.score of 1 : ambiguous or partially correct response , for example , healthcare , it is better to spend more than spend less , and so forth.score of 0 : the patient fails to mention a comparative attribute of the respective candidates .
reasoning on consequencesif the patient is able to make his / her choice for either of the candidates or even in the case he / she wants to abstain from voting , ask : in your opinion , should the first candidate , that one who wants to lower taxes by decreasing the burden of bureaucracy , or second candidate , that one who wants to maintain the welfare state by fighting against tax elusion , be elected , how could that affect your life?score of 2 : entirely correct response , for example , i will have more money to spend ,
, i can not predict what will happen , because they wo n't do what they promised to do , and so forth.score of 1 : ambiguous or partially correct response ,
for example , health will improve , and so forth.score of 0 : the patient does not give a consequence for his / her life or a reason for saying that there are no personally relevant consequences .
comparative reasoningif the patient identifies a choice , ask : why do you think that it is worth voting for either of the candidates ? or why do you think neither of the candidates deserves to be voted for?score of 2 : entirely correct response , for example , because it is right to maintain the welfare state
, because it is right that everybody pays taxes , because the state should not empty the citizen 's pockets with too high taxes ,
because , despite different views , neither of the candidates will fulfill the promises made before election day , and so forth.score of 1 : ambiguous or partially correct response , for example , healthcare , it is better to spend more than spend less , and so forth.score of 0 : the patient fails to mention a comparative attribute of the respective candidates .
reasoning on consequencesif the patient is able to make his / her choice for either of the candidates or even in the case he / she wants to abstain from voting , ask : in your opinion , should the first candidate , that one who wants to lower taxes by decreasing the burden of bureaucracy , or second candidate , that one who wants to maintain the welfare state by fighting against tax elusion , be elected , how could that affect your life?score of 2 : entirely correct response , for example , i will have more money to spend ,
, i can not predict what will happen , because they wo n't do what they promised to do
, and so forth.score of 1 : ambiguous or partially correct response , for example , health will improve , and so forth.score of 0 : the patient does not give a consequence for his / her life or a reason for saying that there are no personally relevant consequences .
if the patient identifies a choice , ask :
why do you think that it is worth voting for either of the candidates ? or why do you think neither of the candidates deserves to be voted for ?
score of 2 : entirely correct response , for example , because it is right to maintain the welfare state , because it is right that everybody pays taxes , because the state should not empty the citizen 's pockets with too high taxes , because , despite different views , neither of the candidates will fulfill the promises made before election day , and so forth . score of 1 : ambiguous or partially correct response , for example , healthcare , it is better to spend more than spend less , and so forth .
score of 0 : the patient fails to mention a comparative attribute of the respective candidates . if the patient is able to make his / her choice for either of the candidates or even in the case he / she wants to abstain from voting , ask : in your opinion , should the first candidate , that one who wants to lower taxes by decreasing the burden of bureaucracy , or second candidate , that one who wants to maintain the welfare state by fighting against tax elusion , be elected , how could that affect your life ?
score of 2 : entirely correct response , for example , i will have more money to spend ,
, i can not predict what will happen , because they wo n't do what they promised to do , and so forth .
score of 1 : ambiguous or partially correct response , for example , health will improve , and so forth .
score of 0 : the patient does not give a consequence for his / her life or a reason for saying that there are no personally relevant consequences . | voting by persons with dementia raises questions about their decision - making capacity .
methods specifically addressing voting capacity of demented people have been proposed in the us , but never tested elsewhere .
we translated and adapted the us competence assessment tool for voting ( cat - v ) to the italian context , using it before 2006 elections for prime minister .
consisting of a brief questionnaire , this tool evaluates the following decision - making abilities : understanding nature and effect of voting , expressing a choice , and reasoning about voting choices .
subjects ' performance was examined in relation to dementia severity . of 38 subjects with alzheimer 's disease ( ad ) enrolled in the study ,
only three scored the maximum on all cat - v items .
mmse and cat - v scores correlated only moderately ( r = 0.59 ; p <
0.0001 ) with one another , reflecting the variability of subjects ' performance at any disease stage . most participants ( 90% ) , although performing poorly on understanding and reasoning items , scored the maximum on the choice measure .
our results imply that voting capacity in ad is only roughly predicted by mmse scores and may more accurately be measured by a structured questionnaire , such as the cat - v . among the decision - making abilities evaluated by the cat - v , expressing a choice was by far the least affected by the dementing process . | 1. Introduction
2. Methods
3. Results
4. Discussion
Italian Version of the Competency Assessment Tool for Voting |
PMC4413401 | bangladesh is divided into 64 administrative districts and has a population of 149.7 million , area of 147,570 km , and population density of approximately 1015 people / km2 .
the country has a total fertility rate of 2.3/capita gdp of $ 772 , life expectancy of 69.5 years .
the government 's expenditure on health is the third largest in the country , after education and defense .
the paradox of bangladesh lies in its gdp and its achievements in the health , education , and socio - economic sectors .
the latest international diabetes federation ( idf ) atlas estimated the incidence of type 1 dm ( t1 dm ) in bangladesh at 4.2 new cases of t1dm/100,000 children ( 014 years)/year , in 2013 .
the highest incidence of t1 dm ( children 014 years ) is estimated to be in europe and north america , with south - east asia closely following the trend at third position .
a series of studies have reported a constant global increase in the incidence of t1 dm , and multifactorial process might be involved . at the bangladesh institute of research and rehabilitation in diabetes , endocrine and metabolic disorders ( birdem ) , there has been an upward trend in the number of newly diagnosed children , from 112 cases in 2008 to 319 cases in 2013 , as documented by the changing diabetes in children ( cdic ) program at birdem .
the question , however , remains whether this upward trend is because of a real increase in the incidence of childhood dm or increased awareness and a decrease in prevalence of communicable diseases , brought about by hygienic practices , immunization against common infectious diseases , with a concomitant rise in non - communicable diseases ( ncd ) .
recent studies show that it is likely that multifactorial reasons are responsible for the increasing incidence e.g. , hygienic practices , feeding regimens ( autoimmunity to cow 's milk ) .
badas has contributed massively to educating the public about diabetes , such that the hitherto unaccepted notion that children could get and die from dm , has been translated into a relatively low threshold for testing for dm in children with polyuria , loss of weight , unconsciousness etc .
they are often considered a burden on the family , especially girls ; they have little prospect of getting married or being employed .
dm is likely to be hidden from teacher , prospective spouse and employer , often with far - reaching consequences .
lack of motivation , inability to manage common complications e.g. , hypoglycaemia , sick day management , drop out from the clinic ( which may be due to lack of motivation or extra cost involved in travel ) , psychological issues , are other common problems .
the diabetic association of bangladesh ( badas ) , which was established in 1956 , is the largest of its kind in the world , with 68 affiliated associations in 64 districts of the country .
it has more than 400,000 diabetics registered at its tertiary centre , birdem in dhaka .
prior to badas , there was no care available for patients with dm needing insulin under the government health services , so poor patients faced certain death !
episodic care was the only kind available , and patients with chronic diseases , faced an uncertain and grim future .
the motto was that no diabetic shall die untreated , unfed or unemployed , even if poor. starting of as an opd , badas has grown into a network of 64 affiliated associations , one in as many districts , and a chain of small hospitals and opds , as well as medical and nursing schools .
all patients , irrespective of socio - economic status , are entitled to free consultation and certain tests .
treatment , including insulin , is available to patients free of cost if entitled , or according to income .
badas operates on a cross - financing model , such that services for the poor are subsidized from the income earned from the well - to - do , who come to seek care or be investigated .
badas is a member of the idf and was a who collaborative centre until recently .
children with diabetes are still managed by adult physicians or occasionally by adult diabetologists , except in institutions like birdem , where paediatricians took over the care of children , and adolescents with diabetes in 1997 , as well as in dhaka shishu hospital .
children and adolescents have special needs at different stages e.g. , nutrition , schooling , growth , puberty etc .
initially , when the department of paediatrics started , the majority of patients were classed as mrdm , and classical t1 dm were a minority . however , the situation is reversed now , with an increase across all socio - economic groups of t1 dm . the burden of providing for the care of diabetic patients places a huge demand on the stretched resources of badas . t1
two programs need special mention , the changing diabetes in children ( cdic ) program , and the life for a child ( lfac ) program , the former sponsored by novo nordisk and the latter by the international diabetes federation ( idf ) .
the central one is located in dhaka and the peripheral ones are located in 2 district headquarters ; chittagong and faridpur .
the number of patients enrolled in dhaka is 1477 , in faridpur 216 and 200 in chittagong .
age of the children range from 0 - 18yrs . under the lfac program , there are 2200 registered ( 1yr-23yrs ) .
there is a slight preponderance of females over males . with the launch of these 2 programs ,
the intensity of motivation and education increased and as a result , glycaemic control has improved over time from a hba1c of more than 9% to a gradual fall to < 7.5% amongst the cdic patients .
both the programs are providing support by providing free consultation , free insulin , education , machines for measurement of hba1c , microalbumin on spot urine sample , and retinal camera .
some blood tests , annual check - up and screening for complications , growth and pubertal monitoring are done free of cost .
cdic has constructed the clinic within the premises of the women 's and children hospital ( birdem2 ) and has been providing funds for running the clinic including doctors and supporting staff salary . under its umbrella , cdic supports some health care staff , including a psychologist . and runs an enviable education programme .
the education program was set up with the help of cdic , and employs diabetes educators , a cadre specially trained in a structured diabetology course , training patients and caregivers in injection and testing techniques , and advising patients about any issues related to diabetes .
however , now diabetes educators are being paid for by patients using the cross financing model .
also certain components of the chec -up are available on payment by the well - to - do .
the lfac program is carrying out a multi - centred epidemiological study of t1 dm , which will give us an insight into the risk factors , trends etc .
it also brought to the world 's notice that dm is increasing alarmingly and every nation has a responsibility to prevent the onward march of this threat , which culminates in 14 november being declared the un world diabetes day .
diabetes in children and adolescents is increasing in bangladesh , and comprehensive diabetes care is essential to achieve good glycaemic control .
diabetes education combined with appropriate motivation of the patients and caregivers is the cornerstone of dm management .
sustainability of the programs will ensure that the child with diabetes can lead a normal life . | diabetes mellitus ( dm ) is a common endocrine disorder among children and adolescents in bangladesh .
the latest international diabetes federation atlas estimated the incidence of type 1 dm ( t1 dm ) in bangladesh as 4.2 new cases of t1dm/100,000 children ( 014 years)/year , in 2013 .
diabetes , being a lifelong disease , places a huge burden on the economy of the most densely populated , and resource - poor country of the world .
the diabetic association of bangladesh ( badas ) , the largest of its kind in the world , provides comprehensive care to the biggest number of diabetics at any one centre and is engaged in advocacy .
although sounding grandiose , it 's aims that no diabetic shall die untreated , unfed or unemployed , even if poor is pursued with a passion .
recently badas has been supported in its endeavor for children and adolescents by two programmes ; viz the changing diabetes in children program ( a joint initiative of badas , the world diabetes foundation and novo nordisk ) , and the life for a child programme ( lfac ) supported by the idf .
numerous studies from the prosperous countries have demonstrated the incidence of t1 dm is increasing .
data from the cdic clinic at birdem shows a rising trend in patients presenting with classical t1 dm .
in addition , the pattern of dm is changing . | I
S |
PMC2925389 |
dna polymerases are organized into seven families : a , b , c , d , x , y , and reverse transcriptase [ 1 , 2 ] . among these families ,
dna polymerases are involved in dna replication , dna repair , dna lesion bypass , antibody generation , and sister chromatid cohesion . despite these diverse roles , dna polymerases catalyze the nucleotidyl transfer reaction using a two divalent metal ion mechanism with at least one positively charged residue that functions as a general acid at their active site , follow a similar minimal kinetic pathway , and share a similar structural architecture consisting of the fingers , palm , and thumb subdomains [ 8 , 9 ] .
surprisingly , the polymerization fidelity of eukaryotic dna polymerases spans a wide range : one error per one to one billion nucleotide incorporations ( 10 to10 ) .
the y - family dna polymerases are known for catalyzing nucleotide incorporation with low fidelity and poor processivity .
these enzymes are specialized for translesion dna synthesis which involves nucleotide incorporation opposite and downstream of a damaged dna site .
lesion bypass can be either error - free or error - prone depending on the dna polymerase and dna lesion combination . to accommodate a distorted dna substrate , y - family dna polymerases utilize several features : a solvent - accessible and conformationally flexible active site , smaller fingers and thumb subdomains , an additional subdomain known as the little finger , the little finger and polymerase core domains move in opposite directions during a catalytic cycle , and a lack of 3 5 exonuclease activity .
unfortunately , these features , which facilitate lesion bypass , may also contribute to the low fidelity of a y - family dna polymerase during replication of a damaged or undamaged dna template .
thus , it is important to understand the mechanism and fidelity of the y - family dna polymerases .
saccharomyces cerevisiae dna polymerase ( ypol ) , a y - family dna polymerase , is critical for the error - free bypass of uv - induced dna damage such as a cis - syn thymine - thymine dimer [ 1519 ] . to date , pol remains the only y - family dna polymerase with a confirmed biological function .
ypol is organized into a polymerase domain , ubiquitin - binding zinc finger ( ubz ) domain , and proliferating cell nuclear antigen- ( pcna ) interacting peptide ( pip ) motif ( figure 1 ) .
x - ray crystal structures of ypol 's catalytic core have been solved alone as well as in complex with a cisplatin - dna adduct and an incoming nucleotide . due to a lack of structures for full - length ypol
, it is unclear if the c - terminal residues 514632 interact with dna and contribute to the polymerase function of ypol. using pre - steady - state kinetic techniques , we have measured the base - substitution fidelity of full - length and truncated ypol ( figure 1 ) catalyzing nucleotide incorporation into undamaged dna .
in addition , we have determined the dna binding affinity of both full - length and truncated ypol. our results show that the c - terminus of ypol has a minor effect on the dna binding affinity and the base substitution fidelity of this lesion bypass dna polymerase .
materials were purchased from the following companies : [ -p ] atp , mp biomedicals ( solon , oh ) ; biospin columns , bio - rad laboratories ( herclues , ca ) ; dntps , ge healthcare ( piscataway , nj ) ; oligodeoxyribonucleotides , integrated dna technologies , inc .
( coralville , ia ) ; and optikinase , usb ( cleveland , oh ) .
the primer strand 21-mer or blunt - end 16-mer was 5-radiolabeled with [ -p]atp and optikinase . then ,
the 21-mer was annealed to the appropriate 41 mer template ( table 1 ) and the palindromic blunt - end substrates were annealed as described previously .
the catalytic core of ypol ( 1513 ) containing an n - terminal mgssh6ssglvprgsh tag was purified as described previously .
all experiments were performed in reaction buffer a which contained 40 mm tris - hcl ph 7.5 at 23c , 5 mm mgcl2 , 1 mm dtt , 10 g / ml bsa , and 10% glycerol .
a rapid chemical - quench flow apparatus ( kintek , pa , usa ) was used for fast reactions . for burst assays , a preincubated solution of ypol ( 320 nm ) and
5-[p]-labeled d-1 dna ( 480 nm ) was mixed with dttpmg ( 100 m ) . to measure the dissociation rate of the ypoldna binary complex
, a preincubated solution of ypol ( 50 nm ) and 5-[p]-labeled d-1 dna ( 100 nm ) was mixed with a molar excess of unlabeled d-1 dna ( 2.5 m ) for various time intervals prior to initiating the polymerization reaction with dttpmg ( 150 and 400 m for truncated and full - length ypol , resp . ) for 15 s. for single - turnover kinetic assays , a preincubated solution of ypol ( 150 nm ) and 5-[p]-labeled dna ( 30 nm ) was mixed with an incoming dntpmg ( 0.4800 m ) .
reaction products were analyzed by sequencing gel electrophoresis ( 17% acrylamide , 8 m urea , 1 tbe running buffer ) , visualized using a typhoon trio ( ge healthcare ) , and quantitated with imagequant software ( molecular dynamics ) .
the equilibrium dissociation constant ( kd ) of the ypoldna binary complex was determined using two techniques .
first , an electrophoretic mobility shift assay ( emsa ) was employed by adding increasing concentrations of ypol ( 10450 nm ) into a fixed concentration of 5-[p]-labeled d-1 dna ( 10 nm ) in buffer a. the solution established equilibrium during a 20-minute incubation period .
then , the binary complex was separated from unbound dna using a 4.5% native polyacrylamide gel and running buffer as previously described except the final concentration of tris was adjusted to 40 mm .
increasing concentrations of ypol ( 2300 nm ) were titrated into a fixed concentration of f-8 dna ( 25 nm ) in buffer a ( devoid of bsa ) .
the f-8 dna substrate ( table 1 ) was excited at a wavelength of 312 nm with emission and excitation slit widths of 5 nm .
the emission spectra were collected at 1 nm intervals from 320 to 500 nm using a fluoromax-4 ( jobin jvon horiba ) .
emission background from the buffer and intrinsic protein fluorescence were subtracted from each spectrum . for the pre - steady - state burst assay ,
the product concentration was graphed as a function of time ( t ) and the data were fit to the burst equation ( 1 ) using the nonlinear regression program , kaleidagraph ( synergy software ) :
( 1)[product]=a[1exp ( k1t)+k2 t ] .
a represents the fraction of active enzyme , k1 represents the observed burst rate constant , and k2 represents the observed steady - state rate constant .
data for the emsa were graphed by plotting the concentration of the binary complex as a function of enzyme concentration ( e0 ) and fitting it to a quadratic equation ( 2 ) :
( 2)[edna]=0.5(kddna+e0+d0 ) 0.5[(kddna+e0+d0)24e0d0]1/2 .
d
0 is the dna concentration . for the fluorescence titration experiments , a modified quadratic equation ( 3 )
was applied to a plot of the fluorescence intensity ( f ) measured at 370 nm versus enzyme concentration :
( 3)[f]=fmax + [ ( fmin fmax ) 2d0 ] {(kddna+e0+d0 ) [(kddna+e0+d0)24e0d0]1/2}.fmax and fmin represent the maximum and minimum fluorescence intensity , respectively . for the rate of dna dissociation from the binary complex , a single - exponential equation ( 4 )
was applied to a plot of product concentration versus time :
( 4)[product]=a[exp ( kofft)]+c .
a represents the reaction amplitude , koff is the observed rate constant of dna dissociation , and c is the concentration of the radiolabeled dna product in the presence of a dna trap for unlimited time . for the single - turnover kinetic assays , a plot of product concentration versus time was fit to a single - exponential equation ( 5 ) to extract the observed rate constant of nucleotide incorporation ( kobs ) :
( 5)[product]=a[1exp ( kobst ) ] .
to measure the maximum rate constant of incorporation ( kp ) and the apparent equilibrium dissociation constant ( kd ) of an incoming nucleotide , the extracted kobs values were plotted as a function of nucleotide concentration and fit to a hyperbolic equation ( 6 ) :
( 6)[kobs]=kp[dntp](kd+[dntp ] ) .
the free energy change ( g ) for a correct and incorrect nucleotide substrate dissociating from the ednadntp complex
, r is the universal gas constant and t is the reaction temperature in kelvin .
previously , transient state kinetic techniques have been used to characterize full - length ypol at 30c .
therefore , we first performed a burst assay ( see section 2 ) to ensure that our purified proteins , truncated and full - length ypol ( figure 1 ) , behaved in a similar manner at 23c . compared to wild - type ypol , the truncated construct contains only the polymerase domain ( figure 1 ) .
a preincubated solution of ypol ( 320 nm ) and 5-[p]-labeled 21/41 mer d-1 dna ( 480 nm ) was mixed with dttpmg ( 100 m ) and quenched with edta at various times .
product concentration was plotted as a function of time and was fit to ( 1 ) , since there were two distinct kinetic phases : a rapid , exponential phase and a slow , linear phase ( data not shown ) .
biphasic kinetics of nucleotide incorporation indicated that the first turnover rate was the rate of nucleotide incorporation occurring at the enzyme 's active site while subsequent turnovers ( i.e. , linear phase ) were likely limited by the dna product release step as demonstrated by full - length ypol at 30c and other dna polymerases [ 23 , 29 , 30 ] .
the equilibrium dissociation constant for the binary complex of ypoldna ( kd ) was measured to determine if the c - terminus of ypol affects dna binding affinity ( scheme 1 ) .
for example , varying concentrations of full - length ypol ( 10450 nm ) were incubated with a fixed concentration of 5-[p]-labeled d-1 dna ( 10 nm ) before separating the binary complex from the unbound dna on a native gel ( figure 2(a ) ) . then
, a quadratic equation ( 2 ) was applied to a plot of the binary complex concentration versus ypol concentration which resolved a kd of 16 1 nm ( figure 2(b ) and table 2 ) . under similar reaction conditions ,
the kd of truncated ypol was estimated to be 34 3 nm , a binding affinity ( 1/kd ) value that is 2-fold weaker than that of full - length ypol ( table 2 ) . to corroborate these estimated kd values , we measured the true kd for the ypoldna complex using a fluorescence titration assay .
an analog of da , 2-aminopurine , was embedded into the 41 mer template of f-8 dna which is identical to 21/41 mer d-8 dna except that 2-aminopurine flanks the 5 end of the templating dc base ( table 1 ) .
the f-8 dna substrate ( 25 nm ) was excited at 312 nm , and the emission spectrum was collected from 320 to 500 nm .
after serial additions of full - length or truncated ypol in independent titrations , a decrease in the fluorescence intensity of f-8 was observed .
these changes in fluorescence intensity at 370 nm were plotted as a function of the ypol concentration and were fit to ( 3 ) to extract a kd equal to 7 4 nm for full - length ypol ( figure 2(c ) ) and 13 5 nm for truncated ypol ( table 2 ) .
these kd measurements were tighter than those determined using emsa , since the fluorescence titration assay allows ypol to associate and dissociate during data collection .
in contrast , emsa does not maintain a constant equilibrium because dissociated ypol can not reassociate with dna during electrophoresis separation .
nonetheless , there was a confirmed ~2-fold difference in the dna binding affinity between full - length and the catalytic core of ypol which indicates that the c - terminal 119 amino acid residues of ypol slightly enhance the binding of the enzyme to dna .
next , we directly measured the rate of dna dissociation from the ypoldna complex ( see section 2 ) .
a preincubated solution of ypol ( 50 nm ) and 5-radiolabeled d-1 dna ( 100 nm ) was combined with a 50-fold molar excess of unlabeled d-1 dna for various time intervals before dttp was added for 15 s to allow ample extension of the labeled d-1 dna that remained in complex with ypol. a plot of product concentration versus the incubation time with the unlabeled dna trap ( data not shown ) was fit to ( 4 ) which yielded dna dissociation rates ( koff ) of 0.008 0.001 s and 0.0041 0.0008 s for truncated and full - length ypol , respectively ( table 2 and scheme 1 ) .
interestingly , the rate of dna dissociation from full - length ypol is 2-fold slower than that from truncated ypol , which indicated that the c - terminus of ypol may slightly contribute to this polymerase 's dna binding affinity .
based on the measured kd from figure 2(c ) and koff values , the apparent second - order association rate constant ( kon = koff / kd ) of the binary complex ypoldna was calculated to be 0.62 and 0.59 m s for truncated and full - length ypol , respectively ( table 2 ) .
these similar kon values indicate that the slightly stronger dna binding affinity of full - length ypol is mainly due to a slightly slower rate of dna dissociation ( koff ) .
taken together , the data in table 2 suggest that the c - terminal 119 amino acid residues of ypol slightly hinder the dissociation of dna from the binary complex ypoldna .
this hindrance is through either direct physical interactions between the c - terminus of ypol and dna , modulation of the conformation of the polymerase domain by the c - terminus of ypol , or both . since
a pre - steady - state burst was observed for truncated ypol , we continued to investigate the nucleotide incorporation efficiency ( kp / kd ) by measuring the maximum rate of nucleotide incorporation ( kp ) and the apparent equilibrium dissociation constant ( kd ) of an incoming nucleotide under single - turnover conditions . by performing these experiments with ypol in molar excess over dna , the conversion of d - dnan to d - dnan+1 ( scheme 1 )
a preincubated solution of truncated ypol ( 150 nm ) and 5-[p]-labeled d-7 dna ( 30 nm ) was mixed with varying concentrations of datpmg ( 0.480 m ) and quenched with edta at various times ( see section 2 ) . a plot of product concentration versus time was fit to ( 5 ) to extract the observed rate constant ( kobs ) for datp incorporation ( figure 3(a ) ) .
then , the kobs values were plotted as a function of datp concentration and fit to a hyperbolic equation ( 6 ) which resolved a kp of 6.9 0.4 s and an apparent kd of 17 3 m ( figure 3(b ) ) .
the pre - steady - state kinetic parameters for the remaining 15 possible dntp : dn base pair combinations were determined under single - turnover conditions and were used to calculate the substrate specificity constant ( kp / kd ) , discrimination factor ( ( kp / kd)correct/(kp / kd)incorrect ) , and fidelity ( ( kp / kd)incorrect/[(kp / kd)correct + ( kp / kd)incorrect ] ) of truncated ypol ( table 3 ) . overall , the base substitution fidelity of truncated ypol was in the range of 10 to 10 which translates into 1 misincorporation per 100 to 10,000 nucleotide incorporations ( table 3 ) .
depending on the mispair , truncated ypol catalyzed a misincorporation with 30- to 2,700-fold ( 640-fold on average ) lower efficiency than the corresponding correct base pair . to better understand the mechanistic basis of truncated ypol 's fidelity , the equation for polymerase fidelity
can be simplified as follows :
( 8)fidelity=(kp / kd)incorrect[(kp / kd)correct+(kp / kd)incorrect](kp / kd)incorrect(kp / kd)correct=[(kp)incorrect(kp)correct]1[(kd)correct(kd)incorrect]1=(rate difference)1(binding affinity difference)1 .
thus , fidelity is inversely proportional to the rate difference and apparent binding affinity difference between correct and incorrect nucleotide incorporation . in general , the mechanistic basis of ypol 's discrimination was due to a 3- to 68-fold ( 18-fold on average ) weaker apparent binding affinity ( 1/kd ) and 5- to 220-fold ( 50-fold on average ) slower rate constant of incorporation for a mismatched dntp .
although all four correct dntps were bound with similarly high affinity ( table 3 ) , mismatched purine deoxyribonucleotides have 2- to 6-fold lower apparent kd values than mismatched pyrimidine deoxyribonucleotides .
because 5-protruding purines have been found to have stronger stacking interactions with a terminal dna base pair than 5-protruding pyrimidines , the difference in apparent kd values suggests that base - stacking interactions between an incorrect dntp and the terminal primer / template base pair da : dt ( table 1 ) play a role on the binding of dntp by truncated ypol. interestingly , we have previously demonstrated that the preferred nucleotide for template - independent nucleotide incorporation catalyzed by dpo4 , another y - family dna polymerase , is datp mainly due to its strong intrahelical base - stacking ability . to further evaluate the role of base stacking
, we first examined if truncated ypol can catalyze template - independent nucleotide incorporation of datp or dptp ( figure 4 ) onto four palindromic , blunt - end dna substrates ( be1 , be2 , be3 , and be4 in table 1 ) .
the base of dptp , a dntp analog , has four conjugated benzene rings but possesses no hydrogen - bonding abilities .
the dna substrates possess all four possible terminal base pairs and each molecule of them can be bound by a single polymerase molecule .
our radioactive experiments showed that truncated ypol was able to incorporate datp and dptp ( data not shown ) .
then , we individually measured the kinetic parameters for datp and dptp incorporation under single - turnover reaction conditions ( table 4 ) .
interestingly , the apparent kd values of datp were 3- to 5-fold smaller with a purine than those with a pyrimidine on the primer 's 3-base , indicating that base stacking is also important for the binding of datp to the binary complex of ypolblunt - end dna .
this base - stacking effect is more dramatic for dptp incorporation onto blunt - end dna because the apparent kd values of dptp are 10- to 80-fold tighter than datp incorporation onto the same blunt - end dna substrate ( table 4 ) .
thus , the binding free energy difference between datp and dptp is 1.4 to 2.6 kcal / mol .
previously , we have obtained a comparable binding free energy difference of 2.3 kcal / mol for similar blunt - end datp and dptp incorporation at 37c catalyzed by dpo4 .
although neither datp nor dptp forms any hydrogen bonds with a template base when bound by ypolblunt - end dna , the bases of these two nucleotides should have different base - stacking interactions with a terminal base pair of a blunt - end dna substrate considering that a dangling pyrene base ( 1.7 kcal / mol ) has previously been found to possess a higher base - stacking free energy than a dangling adenosine ( 1.0 kcal / mol ) .
however , the base - stacking free energy difference ( 0.7 kcal / mol ) between pyrene and adenosine is smaller than the aforementioned binding free energy difference ( 1.42.6 kcal / mol ) between dptp and datp .
one possible source is favorable van der waals interactions between pyrene and active site residues of truncated ypol. in addition , the base - stacking effect and van der waals interactions may stabilize the ternary complex of ypolblunt - end dnanucleotide and facilitate catalysis , leading to much higher kp values with dptp than those with datp ( table 4 ) . due to the differences in kp and apparent kd ,
the substrate specificity values of dptp are 100- to 1,000-fold higher than those of datp with blunt - end dna ( table 4 ) and 10- to 100-fold higher than mismatched datp with regular dna ( table 3 ) .
the base substitution fidelities of full - length and truncated ypol may differ because the c - terminal , nonenzymatic regions may alter the polymerization fidelity .
for example , the proline - rich domain of human dna polymerase has been shown to upregulate the polymerase fidelity up to 100-fold . to determine if the c - terminus of ypol influences polymerization fidelity , we measured the pre - steady - state kinetic parameters for dntp incorporation into d-1 dna ( template da ) catalyzed by full - length ypol ( table 5 ) .
the fidelity was calculated to be in the range of ( 1.4 to 2.6 ) 10 for full - length ypol ( table 5 ) .
relative to the fidelity of truncated ypol with d-1 ( table 3 ) , full - length ypol has a 3-fold higher fidelity .
therefore , the c - terminus of ypol slightly affects the base substitution fidelity . moreover , truncated ypol discriminated between a correct and incorrect dntp by ~30-fold on average based on the kp difference while the discrimination for full - length ypol was ~170-fold on average for incorporation into d-1 dna ( tables 3 and 5 ) .
the incorporation rate constant for correct dttp was ~4 s for both ypol enzymes , but the misincorporation rate was 3- to 23-fold faster for truncated ypol. this rate enhancement for truncated ypol is partially offset by a greater discrimination at the apparent ground - state binding level so that the fidelity of truncated ypol was only 3-folder lower than that of full - length ypol. our above studies demonstrated that the c - terminus of ypol enhances this enzyme 's dna binding affinity and base substitution fidelity by 2- and 3-fold , respectively .
these results suggest that the nonenzymatic , c - terminal region of ypol ( figure 1 ) has a mild impact on the n - terminal polymerase domain and its activity .
this conclusion is inconsistent with previous studies which have qualitatively demonstrated that mutations or deletions in the ubz domain or pip motif do not affect polymerase activity [ 3537 ] .
however , these reported qualitative assays are not sufficiently sensitive to detect the small perturbation on polymerase activity as described in this paper .
the presence of the c - terminal 119 residues of ypol may either interact with dna , slightly alter the conformation of the polymerase domain , or both ( see above discussion ) , thereby enhancing its dna binding affinity and polymerase fidelity .
the fidelity of several y - family dna polymerases synthesizing undamaged dna has been determined by employing steady - state [ 3848 ] , pre - steady - state [ 28 , 30 , 4953 ] , or m13-based mutation assays [ 39 , 41 , 42 , 45 , 54 , 55 ] . from these studies , the fidelity ranges from 10 to 10 . under steady - state reaction conditions , the base substitution fidelity of ypol and
human pol has been measured to be in the range from 10 to 10and 10 to 10 , respectively [ 38 , 40 ] , which is similar to our pre - steady - state kinetic results .
consistently , pol displays the highest substrate specificity for the dctp : dg base pair under both steady - state and pre - steady - state reaction conditions ( table 3 and unpublished data , brown and suo ) [ 38 , 40 ] .
this may seem surprising , since pol participates in the efficient bypass of uv - induced dna damage such as a cis - syn thymine - thymine dimer ( i.e. , a datp : dt base pair ) [ 1520 , 56 , 57 ] .
however , pol has also been shown to be efficient at bypassing guanine - specific damage such as 8-oxo-7,8-dihydro - dg [ 58 , 59 ] , 1,2-cis - diammineplatinum(ii)-d(gpg ) intrastrand cross - links [ 6063 ] , and various n2-dg lesions [ 64 , 65 ] . among the four eukaryotic y - family dna polymerases ( i.e. , pol , dna polymerase , dna polymerase ( pol ) , and rev1 ) , rev1 exhibits low fidelity on undamaged dna due to its strong preference for inserting dctp [ 46 , 52 ] while pol has an unusual preference for dgtp : dt mispairs over datp : dt due to hoogsteen base pair formation [ 51 , 69 ] .
interestingly , the lowest fidelity base pair for truncated ypol was dgtp : dt ( table 3 ) .
the two hydrogen bonds established in the wobble base pair may enhance the catalytic efficiency of ypol since hydrogen bonding is important for the efficiency and accuracy of ypol .
also noteworthy , the truncated versions of eukaryotic y - family dna polymerases have been used for many biochemical studies in literature . based on our quantitative kinetic analysis of ypol , these results suggested the nonenzymatic regions of y - family dna polymerases do not alter the polymerase activity significantly . as a y - family dna polymerase , ypol displays low fidelity on undamaged dna ( tables 3 and 5 ) .
in contrast , replicative dna polymerases in the a- and b - families have a polymerization fidelity that is 13 orders of magnitude greater than the y - family dna polymerases ( table 6 ) . dna polymerases with higher fidelity
are more proficient at using the ground - state binding affinity to discriminate between a correct and incorrect dntp .
the y - family dna polymerases provide little to no discrimination based on the kd difference while replicative dna polymerases discriminate up to almost three orders of magnitude
. this lack of selection in the ground state by the y - family dna polymerases may be due to the relatively loose and solvent - accessible active site which has minimal contacts with the nascent base pair [ 11 , 21 , 71 ] .
moreover , nucleotide selection by the y - family dna polymerases in the ground state may be mainly governed by watson - crick base pairing , since the calculated g values ( 0.951.7 kcal / mol ) are similar to the free energy differences between correct and incorrect base pairs ( 0.31.0 kcal / mol at 37c ) at the primer terminus based on dna melting studies ( table 6 ) .
however , with g values 3.0 kcal / mol , the replicative dna polymerases harness the additional 2.0 kcal / mol of energy from other sources such as a tight active site or close contacts with the nascent base pair . one common fidelity checkpoint among dna polymerases
these large differences may correspond to different rate - limiting steps ( e.g. , protein conformational change , or phosphodiester bond formation ) during nucleotide incorporation [ 9 , 30 , 71 ] .
for ypol , kinetic data suggest that correct and incorrect dntps are limited by a conformational step preceding chemistry , although , additional studies are needed to confirm these results .
this work presents the mechanistic basis of the base substitution fidelity of ypol on undamaged dna , which examined all possible dntp : dn base pair combinations for the first time .
ypol discriminates against incorrect nucleotides at both the ground - state nucleotide binding and incorporation steps .
finally , the 119 residues at the c - terminus have a mild impact on the kinetic mechanism of ypol. | understanding polymerase fidelity is an important objective towards ascertaining the overall stability of an organism 's genome .
saccharomyces cerevisiae dna polymerase ( ypol ) , a y - family dna polymerase , is known to efficiently bypass dna lesions ( e.g. , pyrimidine dimers ) in vivo . using pre - steady - state kinetic methods , we examined both full - length and a truncated version of ypol which contains only the polymerase domain . in the absence of ypol 's c - terminal residues 514632 ,
the dna binding affinity was weakened by 2-fold and the base substitution fidelity dropped by 3-fold .
thus , the c - terminus of ypol may interact with dna and slightly alter the conformation of the polymerase domain during catalysis .
in general , ypol discriminated between a correct and incorrect nucleotide more during the incorporation step ( 50-fold on average ) than the ground - state binding step ( 18-fold on average ) .
blunt - end additions of datp or pyrene nucleotide 5-triphosphate revealed the importance of base stacking during the binding of incorrect incoming nucleotides . | 1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions |
PMC4060061 |
activated lymphocytes undergo a rapid burst in cellular proliferative , biosynthetic , and secretory activities and must obtain metabolic substrates to attempt this dramatic increase in metabolism .
their insignificant intracellular store of nutrients obligates lymphocytes to markedly increase the uptake of metabolic substrates from their microenvironment .
although lymphocytes are able to use glucose , glutamine , ketone bodies , and fatty acids ( fa ) , it was determined that glucose and glutamine are quantitatively the most important fuel for activated lymphocytes . regarding the new metabolic demands of activated lymphocytes , glucose is initially retained in the cell by phosphorylation into glucose 6-phosphate by hexokinases ( hks ) .
from there , glucose 6-phosphate can be used as a substrate by aerobic glycolysis or by the pentose - phosphate pathway ( ppp ) . in the ppp ,
glucose 6-phosphate serves to generate ribose ( for the synthesis of rna and dna ) and nadph ( for fa synthesis ) [ 1 , 2 ] . for glucose 6-phosphate that enters aerobic glycolysis ,
the molecule is converted to pyruvate , after which it can be converted to lactate or acetyl - coa or be fully oxidized .
the majority is converted to lactate ( approximately 91% ) [ 47 ] , while most of the remaining pyruvate is converted to acetyl - coa , which has a central role in membrane biogenesis , serving as a precursor to phospholipids , cholesterol , and triacylglycerol [ 1 , 3 ] .
thus , only a small percentage of glucose 6-phosphate is fully oxidized in lymphocytes . in this scenario ,
the removal of citrate ( pyruvate converted to acetyl - coa plus oxaloacetate ) from the tricarboxylic acid ( tca ) cycle for biosynthetic reactions imposes the need to continue replenishing intermediates to maintain this cycle 's function [ 2 , 9 ] .
thus , beyond glucose , activated lymphocytes also increase their update of glutamine and convert it to glutamate , which is in turn converted to -ketoglutarate via glutamate dehydrogenase .
in addition to replenishing intermediates to maintain the tca cycle using glutamate , lymphocytes also convert glutamine to aspartate and ammonia , providing biosynthetic precursors , purines , and pyrimidines , for the synthesis of dna and rna .
finally , a limited percentage of glutamine can be converted to lactate or be fully oxidized . in fact , although oxidative phosphorylation still occurs in effector t lymphocytes , it seems that , of the glutamine ( and glucose ) utilized by these cells , only approximately 1.5% is oxidized . in accordance with the importance of glucose and glutamine in activated lymphocytes , early studies of these cells demonstrated that
, to meet the new bioenergetic and biosynthetic demands imposed by activation , lymphocytes also increase the maximal activity levels of enzymes , such as hk , glucose-6-phosphate dehydrogenase ( g6pdh ) and phosphate - dependent glutaminase ( glutase ) , which are key enzymes in the glycolysis , pentose - phosphate , and glutaminolysis pathways , respectively .
the mitochondrial enzyme citrate synthase ( cs ) , an important enzyme in the tca cycle , is also affected [ 6 , 7 ] . comparatively , b cell metabolism has been less well investigated than t cell metabolism ; however , the metabolic characteristics of both lymphocyte types might be similar [ 2 , 10 ] .
an outstanding feature of lymphocytes is that these cells utilize glucose and glutamine at high rates in a strictly function - dependent manner .
furthermore , more recently , studies of direct modifications to t lymphocyte metabolic pathways demonstrated that metabolic reprogramming and lymphocyte activation are intricately linked , as cellular metabolism was found to be directly controlled by the signaling pathways that drive cell survival and activity .
notably , pearce and colleagues stated that the reasons why t cells would adopt specific metabolic programs and the impacts of such programs on cell function and immunological responses were unclear . in this sense ,
t lymphocytes adopt a metabolic program that reflects their energetic and biosynthetic needs in specific states , ranging from resting to memory cell conversion . for resting lymphocytes , it is worth mentioning that , despite the name
, these cells continuously migrate through secondary lymphoid tissues to maintain immune surveillance prior to activation ; to accomplish this activity , these cells rely on the oxidative metabolism of glucose , amino acids , and lipids .
however , as previously discussed , once activated , lymphocytes grow , proliferate , differentiate , and adapt to stress , and the mixed oxidative metabolism associated with a nave resting state , which preferentially generates atp , does not support these new functions .
thus , the prioritization of the synthesis of macromolecules , rather than atp production , explains the already - mentioned dependence of activated lymphocytes on aerobic glycolysis .
in other words , the cell replaces its previous efficient atp production ( resting state ) with efficient and rapid macromolecule biosynthesis ( activated state ) .
additional support for the finding that cell metabolism is a key regulator of lymphocyte function and differentiation has been provided by the patterns of fuel usage and transcriptional and posttranscriptional factors that control metabolism in the various activated t cell lineages .
the helper t cell lineages th1 , th2 , and th17 all exhibited increased aerobic glycolysis ( as previously mentioned for activated lymphocytes ) , the posttranscriptional regulator mtorc1 was found to control glucose metabolism in th1 and th17 t cells , whereas mtorc2 controlled glucose in th2 cells .
t regulatory ( treg ) cells exhibit lipid oxidation as a primary metabolic phenotype , which is controlled by ampk .
similarly , memory t cells also oxidize lipids , although in these cells , this metabolic phenotype is controlled by the posttranscriptional regulators traf6 and ampk .
the signals and stimuli that normally control the immune system ( is ) can be affected by conditions such as obesity and type 2 diabetes ( t2d ) . in this sense
, it was proposed that the direct control of lymphocyte metabolism mediated by survival and activity - related signaling pathways could introduce the potential for metabolic changes to promote diseases .
more specifically , the inability of cell metabolism to meet the energetic and biosynthetic demands of lymphocytes could disrupt immune functionality , a process that has been observed in several immunological diseases .
for example , the inhibition of glycolytic metabolism can suppress cell proliferation and cytokine production and also compromise effector t cell differentiation .
in contrast , mitogen - induced t cell activation can reflect the glycemic statuses and insulin levels of type 1 diabetes and t2d patients .
furthermore , hyperglycemia and ketoacidosis were found to increase the levels of proinflammatory cytokines and the numbers of activated t lymphocytes in diabetic patients .
lymphocyte metabolic and/or functional dysregulation has been observed in a diet - induced obesity ( dio ) model .
these phenomena were reported to promote reductions in the treg and th2 cell populations and increases in the resident inflammatory lymphocyte population .
similarly , our laboratory recently reported that dendritic cells cultivated under leptin - free conditions exhibited a different phenotype from that of wild type cells ; this phenotype was characterized by a reduced ability to induce cd4 cell proliferation , while inducing increased treg and th17 cell differentiation .
these results proved useful for increasing our understanding of whether leptin can induce beneficial ( increased treg cell numbers ) or detrimental ( increased th17 numbers ) clinical outcomes . in lymphocytes from graves ' disease patients , the maximal activity levels of hk , g6pdh , cs , and glutase
were all reduced . in vitro , thyroid hormones were found to increase glucose and glutamine metabolism in the lymphocytes from these patients .
additionally , it was reported that concomitant acute and chronic infections in patients with several diseases , such as cancer or asthma , are associated with an imbalance of t1 ( t helper type 1 and t cytotoxic type 1 ) and t2 ( t helper type 2 and t cytotoxic type 2 ) immune functions [ 18 , 19 ] . in graft - versus - host disease ( gvhd ) ,
one of the few studies that investigated t lymphocyte metabolism in vivo demonstrated that cells activated under gvhd conditions became highly dependent on lipid metabolism , rather than exhibiting the expected increase in glycolytic metabolism . in accordance with the results of that study
, the fa metabolism dependence of allogeneic t cells distinguishes these cells metabolically from other activated t cell subsets , thus providing targets for therapeutic intervention ( e.g. , blockade of fa transport , inhibition of fa oxidation , and limitation of fuel sources ) .
cancer is another condition associated with inflammation . in fact , the concept that an inflammatory tissue injury could induce neoplasia and the existence of a close relationship between carcinogenesis and inflammation were initially postulated by the greek physician galenus approximately 2000 years ago .
current estimations suggest that approximately 25% of cancers require a chronic inflammatory microenvironment for development .
additionally , obesity , which is associated with chronic low - grade inflammation , increases the risk of developing certain types of cancers .
therefore , in accordance with the hypothesis suggested by caro - maldonato and coworkers , lymphocytes from animals and humans with cancer are expected to exhibit metabolic dysregulation or mismatches .
in fact , cells from primary effusion lymphoma ( pel ) , a subtype of b cell non - hodgkin 's lymphoma with a median patient survival duration of 6 months , provide support for this hypothesis . in vitro , these cells exhibit an increased dependence on aerobic glycolysis through the pi3 k / akt / mtor pathways that control glycolysis via glut1 .
in fact , these cells , as well as those from other b cell non - hodgkin 's lymphoma subtypes , are dependent on fa synthase to such an extent that bhatt and coworkers suggested the possibility of using this enzyme as a unique molecular treatment target in these cancers .
three possible outcomes of the occurrence of a mismatch or insufficient fuel usage in t lymphocytes were described .
in accordance with these results , the above - described outcomes for t and b lymphocytes have been observed in several immunological diseases and have also provided opportunities to selectively modulate specific immune functions by targeting glucose , lipid , and amino acid metabolism .
the studies mentioned in this section were designed in accordance with two main research approaches that have been proposed to establish a link between exercise and the is : a metabolic approach , which considers plasma glutamine concentrations / metabolism , and a neuroendocrine approach , which considers changes in the levels of immunomodulatory hormones and neurotransmitters .
interestingly , in vitro studies demonstrated that the stress hormones adrenaline and cortisol are able to modulate lymphocyte metabolism .
the organism exerts tight control over the internal environment , and any subtle disruption of the regulated limits triggers physiological feedback mechanisms to reestablish the internal milieu . among these mechanisms
is integration of the nervous and endocrine systems ( or the neuroendocrine system , nes ) with is , which is systematically controlled by the nes . of note ,
the integrated communication between the nes and the is possibly consists of the sharing of common signaling proteins and their corresponding receptors [ 28 , 29 ] . in the face of any stimuli able to disrupt homeostasis , the response of the nes is invariably ( regardless of the nature of the stress ) the same : activation of the sympathoadrenal ( sa ) system and consequent release of catecholamines ( i.e. , adrenaline and noradrenaline ) and activation of the hypothalamic - pituitary - adrenal ( hpa ) axis , which , in humans , results in cortisol production and release [ 30 , 31 ]
. the stereotypic response of the nes to stress aids in understanding of the effects of exercise upon the is because among the several molecules ( e.g. , hormones , cytokines ) able to affect immune cell function , catecholamines , and cortisol appear to be particularly involved in exercise - induced immune responses .
specifically , activation of the sa system occurs several seconds after exercise initiation , whereas the hpa response and the secretion of cortisol often need 2030 minutes before beginning .
additionally , catecholamines appear to be responsible for the initial effects of acute exercise on the is ( e.g. , the migration of lymphocyte subpopulations ) , whereas cortisol appears to exert its effects within a period of at least 2 hours
's work exercise presents a unique stress on the homeostatic conditions , and this stress is specific to the nature and configurations of the protocol and associated elements ( i.e. , the environmental conditions or nutritional status ) .
that is , the magnitude of nes activation in response to exercise stress is determined by the intensity and duration of such exercise .
consequently , the exercise protocols that most affect the is are those in which the intensity and duration ( acute variables ) and frequency ( chronic variable ) are higher .
not surprisingly , aerobic exercise protocols with longer durations ( > 1.5 hours ) and greater intensities ( 55/6075% of vo2max ) induce greater release of catecholamines and cortisol in comparison with aerobic exercise at lower intensity .
discussing the effects of different intensities , durations , and modes of exercise on the response of the nes is obviously beyond the scope of this review .
however , briefly , noradrenaline presents a curvilinear increase in response to acute exercise as workload augments , while adrenaline increases at workloads over 60% vo2max .
regarding cortisol , mild - intensity , moderate - duration aerobic exercise does not appear to alter its levels .
however , exercises with intensity above 85% vo2max [ 40 , 41 ] or with a duration greater than 60 minutes typically lead to increases in cortisol secretion .
rosa and coworkers previously demonstrated the ability of adrenaline to increase the proliferative index of mesenteric lymphocytes and to concomitantly augment the maximal activities of hk , glutase , and cs , as well as glucose and glutamine consumption .
however , the excess of systemic catecholamines induced by high - intensity , exhaustive exercise could have an immunosuppressive effect , such as a reduction in the plasma levels of interferon- ( ifn ) and an antiviral cytokine .
in addition to its antiviral property , ifn has antiapoptotic and antiproliferative effects on activated lymphocytes .
regarding the effects of this cytokine on lymphocyte metabolism , we demonstrated that the ability of ifn to limit t and b cell proliferation could be explained by the suppression of glucose and glutamine metabolism and reduced maximal g6pdh , cs , and glutase activities .
the immunosuppressive effect of glucocorticoids on mesenteric lymphocytes is associated with a 40% reduction in pyruvate utilization due to inhibition of pyruvate dehydrogenase 's maximal activity .
additionally , as mentioned , cortisol increases are typically observed during high - intensity exercises .
we recently demonstrated that basketball players participating in an official game ( stressful exercise ) presented increases in salivary cortisol and a reduction in levels of interleukin ( il)-21 , a cytokine that stimulates immunoglobulin a - secreting cells .
additionally , in accordance with the immunosuppressive ability of cortisol , it was observed that the numbers and proliferative ability of circulating lymphocytes are affected by a single bout of intense exercise .
for example , a 3050% decrease in the lymphocyte count occurs at 30 minutes after exercise .
regarding the proliferative index of lymphocytes , our results demonstrated that participation in very intense exercise protocols , an olympic triathlon ( swimming for 1.5 km , cycling for 40 km , and running for 10 km ) [ 49 , 50 ] or a simulated cycling competition ( 6 sets of 20 minutes at 90% of the individual 's anaerobic threshold ) reduced the proliferative indices of t and b lymphocytes and plasma glutamine levels .
these changes were associated with reduced cytokine production ( e.g. , il-1 , il-2 , il-4 , tumor necrosis factor ( tnf) , and interferon ( ifn) ) by mononuclear peripheral cells in response to mitogens .
it was proposed that the ifn/il-4 ratio in the culture supernatants of stimulated t cells could act as an objective indicator for monitoring the th1/th2 balance ( cell - mediated / humoral immunity ) . as such
, these studies suggested that an intense bout of acute exercise could affect this balance [ 19 , 4951 ] . to maintain homeostasis in the face of all organic changes induced by physical exercise ( e.g. , increased body temperature , dehydration , ion imbalances , hypoxia , and blood pressure changes ) ,
other hormones ( e.g. , insulin , growth hormone , aldosterone , glucagon , and thyroxin ) are necessary in addition to cortisol and catecholamines during exercise and recovery .
although the involvement of these hormones in exercise 's ability to modulate the is needs further investigation , certain evidence reinforces possible involvement .
for example , insulin can control the metabolism and functionality of lymphocytes , and its infusion into critically ill patients is used to achieve tight hyperglycemic control and to fight systemic inflammation .
thyroid hormones in turn stimulate aerobic glycolysis , glutamine consumption , and aerobic metabolism in human lymphocytes .
as previously discussed regarding the effect of an excess of catecholamines on plasma tnf , the plasma levels of other cytokines are affected by exercise , and it has been suggested that the impact of exercise on cytokine production could partially explain how this stressor modulates the is . regarding this concept , the cytokine that is most responsive to exercise is il-6 , whose levels increase up to 100 fold as the duration of exercise progresses .
the increase in il-6 levels is followed by an increase in the levels of an il-1 receptor antagonist that is an inhibitor of the inflammatory cytokines il-1 and il-10 , important anti - inflammatory cytokines .
in addition to the neuroendocrine and immune responses to exercise mentioned above , exercise may modulate immunity in alternative ways .
one of these ways is exercise - induced muscle damage , which results in the secretion of inflammatory cytokines by innate immune cells .
thus , the reduction in the levels of this amino acid after strenuous exercise could be related to immunosuppression , whereas the increase in glutamine levels induced by chronic moderate - intensity exercise would induce a positive effect upon immune function .
finally , it is worth mentioning that , in many respects , the responses and adaptations to chronic exercise are the result of the cumulative influence of repeated acute exercise bouts .
thus , to understand how exercise training can modulate lymphocyte function as well as metabolism , it is important to know the acute effects of exercise .
regarding the chronic effects of physical exercise , the protection that it offers against all - cause mortality is known to occur primarily due to the ability of exercise to protect against atherosclerosis , t2d , colon cancer , and breast cancer . the fact that the above - mentioned diseases seem to associate with low - grade chronic inflammation and the finding that acute exercise increases the systemic levels of several anti - inflammatory cytokines ( especially exercise that is not high intensity or exhaustive ) suggest the possibility that regular exercise can protect against the chronic conditions associated with low - grade inflammation via an anti - inflammatory effect . in accordance with certain authors ,
the potential underlying mechanisms of the anti - inflammatory effects of regular moderate exercise primarily include a reduction in visceral fat mass , increased production , and release of myokines and reduced expression of toll - like receptors on monocytes and macrophages ; however , these mechanisms likely also include inhibition of monocyte and macrophage infiltration into adipose tissues , a reduction in the number of circulating proinflammatory monocytes and an increase in the number of circulating treg cells .
the modulation of lymphocyte metabolism by exercise , however , is not included on this list . in accordance with the proposal that regular moderate - intensity exercise has beneficial anti - inflammatory ( and immunomodulatory ) effects
, it seems that when chronic , this exercise intensity is able to reverse the age - associated reduction in th1 cell numbers or th1-cell derived cytokine levels that are normally observed in older adults .
moreover , other studies have reported that moderate exercise training increased the production of th1 response - associated cytokines in both humans and rats [ 5963 ] .
still , experimental evidence suggests that chronic moderate exercise could normalize il-4 concentrations and increase il-2 concentrations in a heart failure model in which the t2-type response had been initially elevated .
it was demonstrated that when chronic , this level of exercise stimulates a type 2 t cell phenotype in trained individuals and that this tendency might be associated with the high incidence of upper respiratory infection episodes among athletes .
in fact , two recent studies have suggested that lifelong participation in high volumes of intense exercise could compromise immune functionality [ 65 , 66 ] .
together , these studies reported that young athletes had fewer cd4 t lymphocytes and that these cells exhibited reduced functionality and a higher degree of differentiation in comparison with those from young nonathletes .
regarding cd8 t lymphocytes , it was observed that despite , the higher number of these cells , there was a lower frequency of thymic emigration .
however , this altered lymphocyte functionality observed in young athletes was not found in elderly athletes ; however , in the latter group , natural killer cells exhibited increased activation and degranulation
. therefore , it is possible that , in elderly individuals , the is can adapt itself to the detrimental effects of lifelong exhaustive exercise .
thus , considering the strict relationship between function and metabolism in these cells , it is important to understand how glucose and glutamine consumption in lymphocytes is modulated by regular moderate- and high - intensity exercise .
evidence suggests that these effects of exercise are at least partly due to the ability of exercise to modulate cell nutrient metabolism and particularly glucose and glutamine metabolism .
for example , navarro and colleagues demonstrated the ability of chronic moderate - intensity exercise ( eight weeks of treadmill running ) to modulate the activation , proliferation , cytokine production , and glucose and glutamine metabolism of t and b lymphocytes . in support of the statement that lymphocytes utilize glucose and glutamine at high rates according to their specific immune functions ,
the exercise - induced metabolic changes observed by navarro and colleagues were accompanied by concomitant alterations in functionality .
for example , increased expression of il-2 and its receptor ( il-2r ) and decreased expression of il-4 and its receptor ( il-4r ) were observed in t cells relative to b lymphocytes .
these data suggest that chronic moderate exercise in healthy animals primarily enhances the th1 response phenotype . as several immunological disorders
have been associated with a dysregulated th1/th2 balance , this is an important finding . regarding humoral immunity ,
chronic moderate exercise was shown to increase igg production in lymphocytes from trained rats compared with lymphocytes from sedentary animals , thus indicating an improvement in humoral immunity . in support of the finding that both cellular and humoral immune functions improved in response to exercise , increases in the expression and modulation of cd8 , cd54 , and cd30 were observed , potentially indicating improvements in both types of immunity .
it was observed that the changes in lymphocyte function were accompanied by a differential effect of moderate exercise on t and b lymphocyte metabolism .
specifically , t lymphocytes increased glutamine utilization by shifting the metabolism of this amino acid to an aerobic pathway ( as previously mentioned herein , only a minor percentage of glutamine is oxidized in lymphocytes ) .
concomitantly , these cells reduced their glucose consumption and lactate production levels ( lymphocytes typically convert most of their glucose to lactate ) .
in contrast , b lymphocytes exhibited increases in both glucose and glutamine consumption , although only glutamine aerobic metabolism was increased .
all of these lymphocytic changes were possible because key glucose and glutamine metabolic enzymes were targets of the modulatory effect of chronic exercise .
therefore , in accordance with enhanced aerobic glutamine metabolism , the maximal activities of glutase and cs increased in t lymphocytes ' response to exercise .
in addition to these 2 enzymes , the maximal activities of hk and g6pdh were also augmented in b lymphocytes in response to chronic exercise .
the effects of chronic moderate - intensity exercise were also investigated in animal models of chronic diseases .
recently , activated t lymphocytes were proposed as a model to understand carcinogenesis . through changes in transporter expression and isozyme switching , both activated lymphocytes and cancer cells
become highly glycolytic and glutamine dependent to promote growth , proliferation , and differentiation . therefore ,
an understanding of the metabolism of activated t cells could facilitate the identification of new therapeutic strategies that would selectively target tumor metabolism or inflammatory immune responses .
thus , the effects of exercise were investigated in lymphocytes obtained from walker-256 tumor - bearing rats . in response to the tumor , the metabolism and function of these cells
were compromised ; t and b cells from the tumor - bearing rats exhibited lower proliferative indices relative to those of cells from sedentary animals and increased glucose consumption and lactate production in comparison with cells from control animals .
eight weeks of moderate - intensity treadmill running suppressed tumor growth and reversed the repressive effects of the walker-256 tumors on the lymphocytes ' proliferative indices .
additionally , exercise training reversed the effect of the tumor by reducing glucose consumption and lactate production while counterbalancing the effects of the disease on the maximal activities of g6pdh , hk , and cs .
therefore , the immunomodulatory effects of exercise were characterized by a reversion of the tumor - induced changes .
finally , the exercise - induced effects on lymphocyte function and metabolism were accompanied by altered plasma hormone levels ( e.g. , growth hormone , testosterone , and corticosterone ) and beneficial changes in cytokine levels ( e.g. , il-1 , il-2 ) . rheumatoid arthritis ( ra ) is an autoimmune disease that causes several disturbances in immunological functioning [ 72 , 73 ] .
previously , it had been speculated that because certain immune functions are exacerbated in ra , improved is functioning with exercise could theoretically be detrimental to ra patients .
however , the opposite was found to be true , as studies performed in the 1990s regarding exercise and inflammatory disease demonstrated that nearly any type of exercise was superior to a sedentary lifestyle for ra patients [ 74 , 75 ] .
additionally , it was observed that exercise appeared to be beneficial for individuals with ra because of its anti - inflammatory effects [ 22 , 74 ] .
therefore , we decided to verify whether alterations in glucose and glutamine metabolism were present in a model of experimental arthritis ( collagen - induced arthritis , cia ) and whether a chronic swim training regimen could counterbalance the deleterious effects of ra by modulating the metabolism of these nutrients .
initially , we observed that lymphocytes from cia animals consumed more glucose , despite exhibiting reduced lactate production relative to lymphocytes from healthy animals , thus indicating that cia induced defective lymphocyte activation .
additionally , cia reduced glutamine consumption and glutamate / aspartate production , and these metabolic changes were associated with an elevated proliferative index in the cells from cia animals .
however , an eight - week moderate - intensity swim training regimen reduced the proliferative index and glucose consumption of lymphocytes from the cia animals and increased their glutamine metabolism ( figure 1 ) . to obtain a more complete understanding of the immunomodulatory effects of exercise
trained cia rats exhibited lower levels of the proinflammatory hormone prolactin and higher levels of the immunosuppressive hormones progesterone and corticosterone .
interestingly , chronic exercise also increased the plasma levels of il-2 , a cytokine that can both initiate and terminate inflammation under different conditions , as well as increased plasma levels of glutamine . taken together
, the data from the trained cia animals suggested that the ability of swim training to counterbalance several effects of cia resulted from the modulation of lymphocyte metabolism and the balance between proinflammatory and anti - inflammatory hormones and cytokines . despite the common belief that high - intensity exercise is immunosuppressive
, it is important to note that , in this case , high intensity means chronic exercise of high intensity and volume , such as those physical training regimens used by athletes .
that is , it is possible to speculate that high - intensity exercise at a low / moderate volume would induce beneficial effects on the is because this type of exercise would allow the organism to become adapted to it . in support of this speculation ,
an eight - week anaerobic jumping training ( a high - intensity exercise ) regimen was reported to increase the expression of the proapoptotic protein bax and reduce the expression of the antiapoptotic protein bcl-2 ; these findings were associated with reduced walker-256 tumor growth consequent to apoptosis .
subsequently , our group observed that walker-256 tumor - bearing rats subjected to high - intensity running training for an 8-week period ( 85% vo2max thirty minutes per day for five days ) exhibited a 40% reduction in tumor growth and a 35.5% increase in lifespan relative to sedentary , tumor - bearing rats .
these changes were accompanied by reduced lactate production in the walker-256 tumor cells , suggesting that the tumor cells had become less glycolytic . moreover , in the trained animals , the tumor cells exhibited increased glutamine consumption and glutamate and aspartate production . despite these findings , glutamine consumption due to aerobic metabolism was reduced . regarding lymphocyte function and metabolism , the same study demonstrated that the exercise training protocol counterbalanced the effects of the walker-256 tumors on lymphocyte metabolism .
lymphocytes from trained animals exhibited an increased proliferative index , reduced glucose consumption ( aerobic and anaerobic ) , and reduced lactate production in comparison with immune cells from sedentary , tumor - bearing rats .
similarly , the high - intensity exercise program reversed the tumor - induced effects on glutamine metabolism .
notably , the modulatory effect of exercise was accompanied by increased cytokine levels ( e.g. , il-1 , il-2 , and tnf ) and changes in plasma hormone levels ( e.g. , increased corticosterone levels , reduced growth hormone levels ) .
therefore , the immunomodulatory effects of exercise occurred in response to a complex interaction of hormones , cytokines , and metabolic changes .
acute exercise and chronic exercise affect lymphocyte function in a manner associated with the modulation of glucose and glutamine metabolism .
although further studies are necessary , the primary experimental evidence suggests that the well - known anti - inflammatory and immunomodulatory effects of exercise are at least partly characterized by the ability of chronic exercise to adjust the energetic and biosynthetic demands of lymphocytes in response to physiological and pathological conditions . | glucose and glutamine are important energetic and biosynthetic nutrients for t and b lymphocytes .
these cells consume both nutrients at high rates in a function - dependent manner .
in other words , the pathways that control lymphocyte function and survival directly control the glucose and glutamine metabolic pathways .
therefore , lymphocytes in different functional states reprogram their glucose and glutamine metabolism to balance their requirement for atp and macromolecule production . the tight association between metabolism and function in these cells was suggested to introduce the possibility of several pathologies resulting from the inability of lymphocytes to meet their nutrient demands under a given condition .
in fact , disruptions in lymphocyte metabolism and function have been observed in different inflammatory , metabolic , and autoimmune pathologies .
regular physical exercise and physical activity offer protection against several chronic pathologies , and this benefit has been associated with the anti - inflammatory and immunomodulatory effects of exercise / physical activity .
chronic exercise induces changes in lymphocyte functionality and substrate metabolism . in the present review
, we discuss whether the beneficial effects of exercise on lymphocyte function in health and disease are associated with modulation of the glucose and glutamine metabolic pathways . | 1. Glucose and Glutamine Metabolism and Lymphocyte Function
2. Lymphocyte Metabolic Dysregulation and Disease
3. Mediators of Exercise-Induced Immunomodulation
4. Anti-Inflammatory and Immunomodulatory Effects of Chronic Exercise
5. Chronic Exercise-Mediated Modulation of Lymphocyte Nutrient Metabolism
6. Conclusion |
PMC4211185 | our understanding on genetic factors affecting nerve injury and regeneration is primarily derived from numerous mouse models .
alterations of nerve regeneration are then observed in these mice ( osterloh et al . , 2012 ; wilhelm et al . , 2012
) . although critical information has been learnt from these models , it is often difficult to know how these findings can be translated into humans
. the mechanical force during each injury can not be the same , which makes any controlled study formidable among humans with nerve injures .
are there any individuals or families with specific genetic mutations that would render neurological deficits when mechanical stress on these individuals is no more than ordinary physical activities ? these mechanical stresses result in no symptoms in normal subjects , but are sufficient to cause dysfunction of the nerves with the mutation .
hereditary neuropathy with liability to pressure palsies ( hnpp ) says yes to this question .
as its name denotes , this autosomal dominant inherited disorder typically presents with focal sensory loss and/or muscle weakness when the related peripheral nerves are challenged by mechanical stress .
for instance , a patient with hnpp sits with one leg crossed on the other leg , which imposes mechanical pressure on the peroneal nerve at the fibular head .
a half hour of this benign pressure is often sufficient to induce a foot drop on the crossed leg that may last hours to months in patients with hnpp ( earl et al . , 1964 ; li et al . ,
strenuous physical activities in hnpp patients , such as running 10 miles with a 50 lb backpack , may lead to severe arm paralysis and protracted recovery ( horowitz et al . , 2004 ) .
gene mapping has revealed that patients with hnpp are associated with a heterozygous deletion of chromosome 17p12 ( c17p12 ) ( chance et al . , 1993 ) .
humans with a heterozygous truncation mutation of pmp22 manifest an hnpp phenotype identical to that in patients with the heterozygous deletion of c17p12 , supporting a causal role of loss of pmp22 function but not other genes in c17p12 ( nicholson et al .
mice with heterozygous knockout of pmp22 gene recapitulate the pathology of humans with hnpp ( adlkofer et al . , 1995 ) .
application of mechanical compression on pmp22 mouse nerves induced conduction block ( failure of action potential propagation ) more rapidly than that in pmp22 mouse nerves .
this finding is well in line with the focal sensory loss and muscle weakness in hnpp patients when their nerves are exposed to mild mechanical stress ( li et al .
utilizing the pmp22 mouse model , molecular mechanism underlying the impaired action potential propagation in hnpp has been investigated lately ( guo et al . , 2014 ) .
it is primarily localized in adult myelinating schwann cells , while its expression is diffuse in the developing nervous system ( parmantier et al . , 1995 , 1997 ;
interestingly , demyelination is not found until the late stage of the disease ( bai et al . , 2010 ) .
although demyelination is widely regarded as one of the most important mechanisms which alter nerve conduction , effective nerve conduction is also thought to require a proper myelin seal through myelin junctions ( such as tight junctions , adherens junctions ) .
these junctions seal the spaces between adjacent myelin lamellae as well as spaces between the myelin and axolemma ( hartline and colman , 2007 ) .
we found that deficiency of pmp22 dislocates junction protein complexes in myelin ( figure 1 ) .
this change yields excessively permeable myelin that allows an entry of dextran molecules up to a size of 70 kda ( guo et al . , 2014 )
. myelin junction disruption ( modified from figure 6h i in guo et al . , annals of neurology 2014 ) .
upper panel : myelin junctions in a pmp22 nerve fiber are depicted in paranodes and mesaxons .
schmidt - lanterman incisures in inter - nodes are omitted since junctions in the incisures have changes similar to those in paranodes and mesaxons .
lower panel : a pmp22 nerve fiber shows disruption or loss of junction protein complexes ( tight junctions , ad - herens junctions ) in paranodes and/or mesaxons . these junction pro - teins may be found in aberrant locations , including pernuclear regions of myelinating schwann cells .
abnormal assembly of these junctions ( including jam - c transmembrane adhesion ) also loosens adhesion between paranodal myelin lamellae ( arrow on the right ) .
these changes increase myelin permeability that shunts current out of nerve fiber in the absence of demyelination , called functional demyelination .
the severity of abnormally increased myelin permeability was found to vary in different nerve fibers ( guo et al . , 2014 ) , and would produce two different populations of myelinated nerve fibers .
those in the first group have severely leaky myelin that would shunt current out of nerve fibers , leading to failure of action potential propagation in the absence of demyelination .
those in the second group have a mildly increased permeability of myelin , which still allows action potential to propagate , but would compromise the safety factor of action potential propagation .
the nodes of ranvier in myelinated nerve fibers typically generate depolarizing currents five times higher than the minimum required for the induction of an action potential .
this surplus is called the safety factor ( kaji et al . , 2000 ) .
this partially compromised safety factor would put the pmp22-deficient nerve fiber at risk to conduction failure if the fiber is challenged by additional external factors , such as mechanical stress .
taken together , studies in hnpp reveal a new concept that specific human genetic factor , such as pmp22 , may critically affect human nerve susceptibility to injures and recovery after the trauma . one mechanism to achieve this biological effect is through pmp22 's regulation of myelin junction formation and stability .
there are three types of junctions in myelin , including tight junctions , adherens junctions , and septate junctions . the first two are often autotypic junctions between myelin laminae of the same cell .
the third one is situated between the most inner lamina of schwann cells and axolemma ( hartline and colman , 2007 ) .
desmosomes were initially reported in myelin , but were later proven to be adherens junction ( fannon et al . , 1995 ) . under freeze - fracture electron microscopy , tight junctions appear as micro - strands extruding out of the membrane ( tetzlaff , 1978 ) . in the peripheral nerve myelin , these junctions are localized in non - compact myelin such as paranodal loops , schmidt - lanterman incisures , and inner / outer mesaxons ( poliak et al . , 2002 ) .
the strands are formed by polymerization of claudins , a family of tetraspan membrane proteins .
c - terminals of claudins interact with a group of cytoplasmic proteins containing pdz - domains such as zo1 or zo2 ( itoh et al . , 1999 ) . on the other hand
, these pdz - containing proteins also interact with actins and link the tight junction strands to the cytoskeleton for junction stabilization ( hartsock and nelson , 2008 ) .
e - cadherins have a large glycosylated extracellular domain , a single transmembrane domain and a cytoplasmic tail at the c - terminal that interacts with catenins ( -catenin , -catenin and p120 catenin ) .
there is another family of proteins , called jam ( jam - a , jam - b and jam - c ) that expresses nearby junctions .
jam contains a large extracellular ig - domain , a transmembrane domain , and a cytoplasmic c - terminal .
interactions between the ig - domain of jam from two opposing membranes may form homotypic dimers .
2000a ) , sealing the opposing membranes juxtaposed to tight / adherens junctions , and further strengthening the seal of myelin inter - membrane space ( bazzoni et al . , 2000b ; ebnet et al . , 2000 ) .
ablation of jam - c in mice results in hnpp - like pathology and alters nerve conduction ( scheiermann et al . , 2007 ) . finally , septate junctions are localized between paranodal myelin loops and axolemma .
the protein constituents of septate junction include neurofascin-155 ( nf155 ) located at the tips of paranodal myelin loops and the nf155-interacting partners ( caspr and contactin ) on the axolemma . under freeze fracture
em , septate junctions ( also called transverse bands ) are ridges spiraling along the paranodal axolemma which bridge the tips of paranodal myelin loops and axolemma ( rosenbluth , 2009 ) .
removal of any septate junction protein detaches paranodal myelin loops from the axolemma and impairs action potential propagation ( bhat et al .
, 2001 ; boyle et al . , 2001 ; sherman et al . , 2005 )
our observation in pmp22 mice shows abnormal formation and maintenance of these tight / adherence junctions in pmp22 deficiency .
this finding not only offers a novel mechanism to explain nerve conduction defect in the disease ( guo et al . , 2014 ) , but also has additional physiological implications .
after the developmental stage , when the myelinated nerve fiber has matured , its diameter , internodal length and myelin thickness remain stable .
our study provides an alternative mechanism that may fine - tune conduction by tightening or loosening the myelin junctions .
acquired or sporadic diseases can be studied by utilizing genetic models . a typical example is those studies in amyotrophic lateral sclerosis ( als ) .
a variety of rodent genetic models , such as the sod1 and tdp43 transgenic mice , have been used to study the pathogenic mechanisms of the disease . while als patients with mutations in sod1 or tdp43 are rare , investigations using these transgenic mice have made remarkable contributions to our understanding in the pathogenesis of als in general ( swarup and julien , 2010 ) .
we believe that a similar advance in nerve injures could be achieved through the use of genetic models .
. this strategy should motivate investigators to seek more families that might reveal additional genetic factors relevant to nerve injures .
sh3tc2 ( src homology 3 domain and tetratricopeptide repeats ) may become the next candidate gene .
autosomal recessive mutations in sh3tc2 have been associated with an inherited peripheral nerve disease , called charcot - marie - tooth disease type-4c ( cmt4c ) .
patients with cmt4c usually present with an early onset neuropathy with severe axonal loss and dysmyelination ( kessali et al . , 1997 ) .
however , humans with heterozygous mutations in sh3tc2 may present with carpal tunnel syndrome only ( lupski et al . ,
it is tethered to cellular membrane via its myristic acid anchor and is involved in regulation of endosome recycling through its interaction with rab11 ( stendel et al . , 2010 ) .
how these molecular functions relate to nerve resistance to mechanical stress is still unknown . finally , nerve entrapments ( focal compression ) are common neurological conditions in humans ; including median nerve entrapment at the wrist ( carpal tunnel syndrome ) , ulnar nerve across the elbow , and peroneal nerve across the fibular head . | genetic factors may be learnt from families with gene mutations that render nerve - injury susceptibility even to ordinary physical activities .
a typical example is hereditary neuropathy with liability to pressure palsies ( hnpp ) .
hnpp is caused by a heterozygous deletion of pmp22 gene .
pmp22 deficiency disrupts myelin junctions ( such as tight junction and adherens junctions ) , leading to abnormally increased myelin permeability that explains the nerve susceptibility to injury .
this finding should motivate investigators to identify additional genetic factors contributing to nerve vulnerability of injury . | Introduction
Nerves in humans with PMP22 deficiency exhibit susceptibility to mechanical pressure
Junction disruption and abnormally increased permeability in PMP22-deficient myelin
Lessons may be learnt from additional genetic mutations |
PMC3465969 | alzheimer 's disease ( ad ) is a progressive neurodegenerative disease characterized by accruing cognitive and noncognitive deficits . among the latter , aberrant motor behavior with either overall slowing or hyperactivity , and depression are common symptoms [ 15 ] . in patients with ad , presence of apathy
as indicated by decreased motor activity and decreased agitation / hyperactivity as indicated by increased motor activity is associated with faster functional and cognitive decline [ 57 ] .
levels of dopamine , noradrenaline , and serotonin are decreased in patients with ad [ 811 ] . in animal models of ad , dopamine , noradrenaline , and serotonin all
bupropion is a monoamine reuptake inhibitor selective for dopaminergic and to a lesser extent noradrenergic neurotransmission but has no effect on serotonergic neurotransmission .
bupropion improved visual memory in patients with major depressive disorder . in rodent animal models ,
bupropion significantly decreased the duration of immobility on the forced swim test and increased locomotor activity in freely moving animals .
citalopram is a potent selective inhibitor of serotonin reuptake and increases extracellular serotonin concentrations in the hippocampus of rats but has no effect on the uptake of noradrenaline and dopamine .
citalopram was recently reported to improve spatial memory in rats and to decrease immobility time in the forced swim test .
a standard paradigm for the assessment of cognition in animal research is the investigation of spatial learning in water and dry - land mazes [ 2325 ] . while early work on spatial orientation in rodents was performed in complex dry - land mazes , since the mid 1980s complex spatial tasks predominantly are assessed in water mazes .
hence , water maze learning likely not only assesses cognitive processes of spatial memory but is confounded by noncognitive components such as anxiety .
it was the goal of the present study to assess whether common substances used for treatment of depression in humans affect spatial learning in a transgenic animal model of ad .
three groups of 4.5-month - old male app23 mice ( charles river ) and one group of same aged male wild - type mice were used in this study .
three or four animals were housed in a cage and were maintained on a 12 h light / dark cycle in a temperature ( 22 2c ) and humidity ( 55 5% ) controlled room similar to previous protocols [ 23 , 27 , 28 ] .
injection of bupropion ( 20 mg / kg body weight ) , another with citalopram ( 10 mg / kg body weight ) , respectively , starting fourteen days prior to onset of experiments .
one group of app23 mice and wild - type mice were nacl - sham treated with the same protocol . to assess spatial memory
, we used a complex maze that has been described previously ( figure 1 ) .
starting on the date of the last treatment , animals were trained four times a day for three consecutive days and two times on the fourth day .
they had a maximum time of 300 s to find the exit , where they were rewarded with a food pellet .
different parameters , for example , duration , moving and resting time were recorded by a tracking system ( multitrack , accuscan , usa ) .
all statistical analyses were carried out using the statistics program spss ( spss 17.0 for windows , spss inc .
both wild - type and app23 animals improved their performance during repeated exposure to the dry - land maze as shown by a significant effect of trial on total time ( two - way anova ; f
13,154 = 6.751 , p < 0.001 ) , resting time ( two - way anova ; f
13,154 = 5.042 , p < 0.001 ) , and moving time ( two - way anova ; f
13,154 = 9.157 , p < 0.001 ) .
both groups increased their running speed over all trials ( two - way anova ; f
13,154 = 11.577 , p < 0.001 ) with wild - type animals more so than app23 animals ( two - way anova ; f
1,154 = 28.676 , p < 0.001 ) ( figure 2(a ) ) . to catch the efficacy of spatial learning ,
both wild - type and app23 animals improved their performance during repeated exposure to the dry - land maze as shown by a significant effect of trial on distance ( two - way anova ; f
13,154 = 4.942 , p < 0.001 ) ( figure 2(b ) ) .
path length was shorter for wild - type animals than for app23 animals ( two - way anova ; f
1,154 = 37,767 , p < 0.001 ) . to assess the effect of treating app23 animals
, we performed a two - way anova with treatment groups sham treatment , bupropion treatment , and citalopram - treatment .
there was a significant effect of treatment group on total time ( f
2,224 = 12.794 , p < 0.001 ) with fisher lsd multiple comparison testing indicating significant differences ( table 1 , figure 3 ) .
likewise , a significant effect of treatment group was found on resting time ( f
2,224 = 18.103 , p < 0.001 ) with fisher lsd multiple comparison testing indicating significant differences for treatment with bupropion but not for citalopram treatment ( table 1 , figure 3 ) .
contrary , no overall effect of treatment was found on moving time ( f
2,224 = 2.224 , p = 0.111 ; table 1 , figure 3 ) .
analysis of running speed featured significant treatment differences ( f
2,224 = 17.507 , p < 0.001 ) with fisher lsd multiple comparison testing showing that bupropion - treated animals run faster while citalopram - treated animals run with less speed than sham - treated animals ( table 2 , figure 4(a ) ) .
further there was no significant effect of treatment group on distance ( f
2,224 = 0.124 , p = 0.883 ) ( table 2 , figure 4(b ) ) .
maze studies are an established means to investigate spatial learning in experimental animals , both in water mazes and dry - land mazes . compared to wild - type animals , the total , moving , and resting time to find the goal zone
were decreased in app23 transgenic animals and the running speed was increased . however , path length was also shorter in wild - type than app23 animals .
we conclude that the difference between wild - type and app23 animals reflects both , better spatial learning and higher psychomotor activity .
this is in good harmony with previous studies showing diminished learning in middle - aged app23 animals [ 29 , 30 ] .
reduced running speed in app23 is in good harmony with a previous study with reduced psychomotor activity in another transgenic mouse model of ad , the app / ps1 transgenic model .
citalopram has been used in dosages from 0.01 mg / kg to 8 mg / kg .
the effects on spatial cognition remain ambiguous and depend on dosage , paradigm , species , and the interaction thereof . at low dosages
, citalopram did not affect spatial learning or even improved it but had negative effects in dosages higher than 4 mg / kg in rats . at moderate dosages of about 5 mg / kg , citalopram decreased immobility time in the forced swim test . to our knowledge
, the present study is the first to address the effects of citalopram on spatial learning in a transgenic mouse model of ad . in this model ,
high dosages of citalopram did not change path length , total , moving , or resting time , while running speed was slightly decreased .
we interpret this such as to indicate that spatial learning per se is unaffected but that psychomotor activity is slightly decreased .
this is in good harmony with previous reports showing either a slight decrease of psychomotor activity on administration of comparable dosages of citalopram or unchanged locomotor activity . at least in clinical studies ,
no benefit on cognition was found on treating ad patients with citalopram [ 35 , 36 ] . on treatment of app23 animals with bupropion ,
resting time decreased and running speed increased while length of path and moving time remained unchanged .
we interpret this finding such as to indicate that bupropion does not improve spatial learning per se but that it increases psychomotor activity .
this is in good harmony with a previous study in mice showing an increased locomotor activity in dosages from 1 mg / kg to 20 mg / kg .
altogether , bupropion and citalopram differentially affect psychomotor activity in the app23 transgenic mouse model of ad while spatial learning per se is unaffected . |
background . incipient alzheimer 's disease
is often disguised as depressive disorder . over the course of ad , depressive symptoms are even more frequent .
hence , treatment with antidepressants is common in ad .
it was the goal of the present study to assess whether two common antidepressants with different mechanisms of action affect spatial learning in a transgenic animal model of alzheimer 's disease .
methods .
we assessed spatial memory of male wild - type and b6c3-tg(appswe , psen1de9)85dbo ( app23 ) transgenic animals in a complex dry - land maze .
animals were treated with citalopram ( 10 mg / kg ) and bupropion ( 20 mg / kg ) .
results . moving and resting time until finding the goal zone decreased in 4.5-month - old sham - treated wild - type animals and , to a lesser extent , in app23 animals . compared with sham - treated app23 animals , treatment with bupropion reduced resting time and increased speed . on treatment with citalopram , moving and resting time were unchanged but speed decreased .
length of the path to the goal zone did not change on either bupropion or citalopram .
conclusion .
bupropion increases psychomotor activity in app23 transgenic animals , while citalopram slightly reduces psychomotor activity .
spatial learning per se is unaffected by treatment with either bupropion or citalopram . | 1. Introduction
2. Methods
3. Results
4. Discussion |
PMC3579363 | betagene participants are mexican - american adults ( both parents and three or more grandparents are mexican or of mexican descent ) who are 1 ) women who had gestational diabetes mellitus ( gdm ) within the previous 5 years , 2 ) siblings or cousins of those with a history of gdm , or 3 ) women with normal glucose levels during pregnancy in the past 5 years .
women documented with and without previous gdm were identified from the los angeles county / university of southern california medical center , kaiser permanente southern california s delivery population , and obstetrical / gynecological clinics at local southern california hospitals .
women without previous gdm were frequency - matched to gdm cases by age , bmi , and parity . details regarding recruitment have been previously described ( 14 ) .
all protocols for betagene were approved by the institutional review boards of participating institutions , and all participants provided written informed consent before participation .
research data were collected in two separate visits to the general clinical research center at the university of southern california . the first visit consisted of a physical examination , dietary and pa questionnaires , a 2-h , 75-g oral glucose tolerance test ( ogtt ) , and fasting blood for lipid measurements ( 15 ) .
participants with fasting glucose < 7.0 mmol / l were invited for a second visit , which consisted of a dual - energy x - ray absorptiometry scan for body composition and an insulin - modified intravenous glucose tolerance test ( ivgtt ) for measurement of insulin sensitivity and -cell function ( 16 ) .
plasma glucose was measured on an autoanalyzer using the glucose oxidase method ( ysi model 2300 ; yellow springs instruments , yellow springs , oh ) .
insulin was measured by a two - site immunoenzymometric assay ( tosoh biosciences , san francisco , ca ) that has <
0.1% cross - reactivity with proinsulin and intermediate split products . trained bilingual ( english / spanish ) interviewers administered both pa and dietary questionnaires .
the amount and intensity of pa was assessed by questionnaires developed in the hawaii - los angeles multiethnic cohort study ( 17,18 ) . this questionnaire is comprised of a list of structured questions describing various types of activity ( sitting , strenuous sports , vigorous work , and moderate activities including sports and work ) during the past year .
responses were then used to estimate the total minutes of moderate and vigorous activity per week .
the questionnaire was validated against doubly labeled water and showed a correlation coefficient of 0.3 between hours of total activity and total energy expenditure ( unpublished data ) . the u.s .
department of health and human services ( dhhs ) recommends at least 75 min / week of vigorous or 150 min / week of moderate activity for americans ( 19 ) .
individuals were categorized into three mutually exclusive groups according to how their reported pa matched these dhhs guidelines : low , < 75 min / week of vigorous and
< 150 min / week of moderate activity ; moderate , either 75 min / week of vigorous or 150 min / week of moderate activity ; high , reported both 75 min / week of vigorous and 150 min / week of moderate activity ( 20 ) .
dietary intake was assessed using the 126-item semiquantitative harvard food - frequency questionnaire ( 21 ) . the food - frequency questionnaire consisted of a list of foods with a standardized serving size and a selection of nine frequency categories ranging from never or less than one serving per month to more than six servings per day , during the past year .
an open - ended free text section was used to capture food items that did not appear on the standard list and included information on usual serving size and number of servings consumed per week for incorporation in the dietary intake calculation for each subject .
insulin response to oral glucose ( 30-min insulin ) was computed as the 30-min ogtt insulin concentration minus the fasting insulin concentration .
total and incremental ( above basal ) areas under ogtt glucose and insulin curves were calculated by trapezoid method .
insulin sensitivity ( si ) , glucose effectiveness ( sg ) , and the incremental insulin response to glucose ( airg ) during the first 10 min of the ivgtt were determined using the millenium version of the bergman minimal model ( 22 ) .
the disposition index ( di ) , a measure of -cell function , was computed as the product of airg and si .
characteristics of the study cohort were presented by means , medians , and interquartile ranges .
fasting insulin , postchallenge insulin , insulin response , si , di , sg , and total calories were log transformed to approximate normal distribution prior to analysis .
geometric means were presented for these variables , and the associated standard errors were calculated by the delta method ( 23 ) .
the relationship between diabetes - related metabolic measures and pa was assessed by testing trend association between the metabolic measures and levels of pa in categories ( low , moderate , and high ) using generalized estimating equations to account for correlations among related individuals within families .
the impact of sex on the associations was assessed by including sex as a covariate and testing for significant interaction between pa group and sex in the model .
if no significant interaction was detected , analyses were conducted using the entire sample with adjustment for age and sex .
the impact of body composition on the association was assessed through covariate adjustment for bmi or percent body fat and/or waist - to - hip ratio .
betagene participants are mexican - american adults ( both parents and three or more grandparents are mexican or of mexican descent ) who are 1 ) women who had gestational diabetes mellitus ( gdm ) within the previous 5 years , 2 ) siblings or cousins of those with a history of gdm , or 3 ) women with normal glucose levels during pregnancy in the past 5 years .
women documented with and without previous gdm were identified from the los angeles county / university of southern california medical center , kaiser permanente southern california s delivery population , and obstetrical / gynecological clinics at local southern california hospitals .
women without previous gdm were frequency - matched to gdm cases by age , bmi , and parity . details regarding recruitment have been previously described ( 14 ) .
all protocols for betagene were approved by the institutional review boards of participating institutions , and all participants provided written informed consent before participation .
research data were collected in two separate visits to the general clinical research center at the university of southern california . the first visit consisted of a physical examination , dietary and pa questionnaires , a 2-h , 75-g oral glucose tolerance test ( ogtt ) , and fasting blood for lipid measurements ( 15 ) .
mmol / l were invited for a second visit , which consisted of a dual - energy x - ray absorptiometry scan for body composition and an insulin - modified intravenous glucose tolerance test ( ivgtt ) for measurement of insulin sensitivity and -cell function ( 16 ) .
plasma glucose was measured on an autoanalyzer using the glucose oxidase method ( ysi model 2300 ; yellow springs instruments , yellow springs , oh ) .
insulin was measured by a two - site immunoenzymometric assay ( tosoh biosciences , san francisco , ca ) that has <
the amount and intensity of pa was assessed by questionnaires developed in the hawaii - los angeles multiethnic cohort study ( 17,18 ) . this questionnaire is comprised of a list of structured questions describing various types of activity ( sitting , strenuous sports , vigorous work , and moderate activities including sports and work ) during the past year .
responses were then used to estimate the total minutes of moderate and vigorous activity per week .
the questionnaire was validated against doubly labeled water and showed a correlation coefficient of 0.3 between hours of total activity and total energy expenditure ( unpublished data ) . the u.s .
department of health and human services ( dhhs ) recommends at least 75 min / week of vigorous or 150 min / week of moderate activity for americans ( 19 ) .
individuals were categorized into three mutually exclusive groups according to how their reported pa matched these dhhs guidelines : low , < 75 min / week of vigorous and
< 150 min / week of moderate activity ; moderate , either 75 min / week of vigorous or 150 min / week of moderate activity ; high , reported both 75 min / week of vigorous and 150 min / week of moderate activity ( 20 ) .
dietary intake was assessed using the 126-item semiquantitative harvard food - frequency questionnaire ( 21 ) .
the food - frequency questionnaire consisted of a list of foods with a standardized serving size and a selection of nine frequency categories ranging from never or less than one serving per month to more than six servings per day , during the past year .
an open - ended free text section was used to capture food items that did not appear on the standard list and included information on usual serving size and number of servings consumed per week for incorporation in the dietary intake calculation for each subject .
insulin response to oral glucose ( 30-min insulin ) was computed as the 30-min ogtt insulin concentration minus the fasting insulin concentration .
total and incremental ( above basal ) areas under ogtt glucose and insulin curves were calculated by trapezoid method . insulin sensitivity ( si ) , glucose effectiveness ( sg ) , and the incremental insulin response to glucose ( airg ) during the first 10 min of the ivgtt
the disposition index ( di ) , a measure of -cell function , was computed as the product of airg and si .
characteristics of the study cohort were presented by means , medians , and interquartile ranges .
fasting insulin , postchallenge insulin , insulin response , si , di , sg , and total calories were log transformed to approximate normal distribution prior to analysis .
geometric means were presented for these variables , and the associated standard errors were calculated by the delta method ( 23 ) .
the relationship between diabetes - related metabolic measures and pa was assessed by testing trend association between the metabolic measures and levels of pa in categories ( low , moderate , and high ) using generalized estimating equations to account for correlations among related individuals within families .
the impact of sex on the associations was assessed by including sex as a covariate and testing for significant interaction between pa group and sex in the model .
if no significant interaction was detected , analyses were conducted using the entire sample with adjustment for age and sex .
the impact of body composition on the association was assessed through covariate adjustment for bmi or percent body fat and/or waist - to - hip ratio .
a total of 1,250 participants were recruited into the betagene study with completed ivgtts ; of these , 1,152 completed ogtts and pa questionnaires .
the mean age was 34.7 years ( range , 17.965.6 years ) , mean bmi was 29.6 kg / m , mean percent body fat was 34.7% , and 72.3% of the cohort were female .
normal glucose tolerance was present in 707 ( 61.4% ) , impaired glucose tolerance in 361 ( 31.3% ) , and diabetes by 2-h glucose 11.1
mmol / l ( 24 ) in 84 ( 7.3% ) participants , respectively . among females ,
the median durations for vigorous and moderate activity were both 45 min / week , considerably below the dhhs recommendation of 150 min / week of moderate activity or 75 min / week of vigorous activity for chronic disease prevention ( 19 ) .
cohort characteristics ( n = 1,152 ) of the study participants , 501 ( 43% ) were classified as low in pa , 448 ( 39% ) as moderate , and 203 ( 18% ) as high .
the higher activity groups were significantly younger ( mean age = 35.3 , 34.2 , 34.0 years in the low , moderate , and high groups , respectively ; p = 0.012 for trend ) .
the distribution of females versus males in the three pa groups was significantly different ( p < 0.001 ) and showed a pattern that indicated lower pa among females : low = 52 vs. 22% for females vs. males , moderate = 33 vs. 55% , and high = 16 vs. 23% , respectively .
table 2 presents the comparison of the age - adjusted means for the metabolic measures among the three pa groups .
although no significant association was observed between pa and bmi ( p = 0.28 ) , increasing pa was significantly associated with decreasing percent body fat ( p < 0.0001 ) , 2-h glucose ( p = 0.001 ) , fasting insulin ( p = 0.0003 ) , and 2-h insulin ( p = 0.0001 ) and increasing -cell function ( p = 0.004 ) .
an increasing level of pa was marginally associated with an increasing level of airg ( p = 0.09 ) .
the associations between pa and diabetes - related traits appeared to be similar between males and females ( interaction test p > 0.28 for each trait after including sex in the model , details by sex analyses ) ( supplementary tables 1 and 2 ) except ogtt fasting glucose ( p = 0.037 ) .
age - adjusted fasting glucose decreased with increasing pa in women ( mean sem for low = 5.1 0.04 mmol / l , moderate = 5.0 0.04
mmol / l , and high = 4.9 0.06 mmol / l ; p = 0.013 ) but not in men ( low = 5.2 0.08 mmol / l , moderate = 5.1 0.05
mmol / l , and high = 5.2 0.06 mmol / l ; p = 0.35 ) . after further adjustment for sex ,
the association between pa and percent body fat was no longer significant ( p = 0.38 ) ( table 2 ) .
the significant associations for 2-h glucose , fasting , and 2-h insulin and di observed in the age - adjusted analyses remained after further adjustment for sex ( table 2 ) .
an increasing level of pa was significantly associated with a lower waist - to - hip ratio ( p = 0.012 ) and marginally associated with decreasing fasting glucose ( p = 0.10 ) and increasing si ( p = 0.076 ) after adjustment for age and sex .
comparison of metabolic traits across the three pa groups * we assessed the relative contribution of body fat to the observed associations between pa and diabetes - related traits by additionally adjusting for percent body fat , bmi , or waist - to - hip ratio .
age- , sex- , and percent body fat adjusted means for ogtt fasting and 2-h glucose and insulin are shown in fig .
analogously , adjusted means for si , airg , and di are shown in fig .
2 . associations between pa and 2-h glucose , fasting insulin , 2-h insulin , and di remained significant after further adjusting for percent body fat , and the regression coefficients were only slightly reduced ( < 15% change ) . by contrast , adjustment for body fat reduced the association between pa and si by 26% and increased the association with airg by 27% , although these associations remained insignificant ( fig .
results were similar when models were adjusted for bmi or waist - to - hip ratio instead of percent body fat ( data not shown ) . including waist - to - hip ratio in addition to percent body fat or bmi in the model adjustment did not change the conclusion ( data not shown ) .
age- , sex- , and percent body fat adjusted means and 95% cis for ogtt fasting and 2-h glucose and insulin by the three pa groups : low , moderate , and high .
p values were from the trend association between pa groups and each of the metabolic traits .
age- , sex- , and percent body fat adjusted means and 95% cis for insulin sensitivity ( si ) , acute insulin secretion ( airg ) , and -cell compensation for insulin resistance ( di ) by the three pa groups : low , moderate , and high .
p values were from the trend association between pa groups and each of the metabolic traits . because higher pa level was typically accompanied by higher caloric intake , we further adjusted for caloric intake .
caloric intake did not significantly alter the age- and sex - adjusted associations between pa and any diabetes - related traits .
pa was not significantly associated with bmi ( p = 0.66 ) or percent body fat ( p = 0.54 ) after further adjustment for caloric intake .
further adjustment for caloric intake had little impact on the age- , sex- , and percent body fat adjusted associations between pa and fasting glucose , 2-h glucose , fasting insulin , 2-h insulin , airg , si , and di ; the additional adjustment resulted in < 10% change in the corresponding regression coefficients , with adjusted p values of 0.098 , 0.017 , 0.0003 , 0.016 , 0.21 , 0.16 , and 0.019 , respectively .
in this study , we observed that an increasing level of self - reported pa was significantly associated with decreasing levels of fasting , 2-h insulin , and 2-h glucose and an increasing level of -cell function assessed as the di .
these associations were not modified significantly by adjustment for percent body fat , bmi , waist - to - hip ratio , or caloric intake .
these findings suggest that more pa , even in a free - living environment , is beneficial to -cell function and glucose regulation . the impact of exercise on glucose and insulin metabolism has been evaluated by several studies .
animal studies have demonstrated that exercise increases glucose uptake by stimulating glut4 translocation in muscle cells and increasing glucose uptake by the liver ( 5 ) . additionally , an exercise training study among type 2 diabetic patients revealed that exercise enhances the whole - body glucose disposal ( 6 ) . although the association between pa and insulin sensitivity did not make the statistical significance based on a p value < 0.05 cut point in this cohort , there was a trend that subjects with higher pa levels had better insulin sensitivity ( p = 0.076 after adjustment for age and sex ) .
this attenuated relationship was consistent with the lack of association between pa and bmi or percent body fat and could be due to the fact that no exercise training and interventions were applied in this cohort .
thus , our results are consistent with the findings from exercise trainings and support the concept that more pa may contribute to lower ogtt glucose and insulin levels and , to a lesser significant extent , better insulin sensitivity .
the most novel finding in this study was the significant association between pa and -cell function .
of note , > 75% of the participants in this cohort were overweight or obese ( 25th percentile of bmi was 25.5 kg / m ) . two previous studies evaluated short - term exercise training and changes in -cell function before and after training .
one of the studies included 12 subjects > 60 years of age ( 11 ) .
the other study included overweight adults and demonstrated that both moderate and vigorous exercise training improved insulin sensitivity and -cell function , although the improvement of -cell function was not statistically significant for vigorous activity ( 12 ) .
although the biological mechanisms of the impact of pa on -cell function have not been clarified , there has been evidence that exercises expanded -cell mass by stimulating its proliferations and preventing its apoptosis ( 25,26 ) . in these rat studies
, exercises were shown to enhance the expression of insulin receptor substrate-2 , which is crucial for -cell growth and survival .
the beneficial effect of exercises on -cell function may also be the improvement of adipose tissue biology such as increasing adiponectin and reducing inflammation ( 27 ) .
recently , we showed that declining adiponectin was significantly associated with -cell function deterioration in a longitudinal study independent of weight gain ( 28 ) .
the mechanism for this association may promote -cell function and survival by increasing ceramidase activity , decreasing intracellular ceramide levels , and increasing antiapoptotic metabolite sphingosine-1 phosphate levels ( 29,30 ) . taken together , the results from this report and previous reports suggest that greater amounts of daily living activity might protect -cell function , even in overweight and obese individuals . a cross - sectional study in
mexican children suggested that the impact of pa on -cell function could be mediated by body fat ( 31 ) .
they found that the higher cardiorespiratory fitness , which reflects chronic pa behavior , was significantly correlated with -cell function as well as insulin resistance .
we did not observe a significant association between percent body fat or bmi and self - reported pa groups after adjustment for age and sex .
in addition , the adjustment for percent body fat and bmi did not significantly reduce the association between pa and -cell function .
the lack of association between pa and bmi or percent body fat in this adult cohort may be due to the fact that our cohort is primarily composed of women with a history of gdm and their family members , the majority of whom are overweight or obese and are presumably at higher than normal risk for diabetes .
we did not measure fitness levels , and pa was self - reported and included work - related activities such as moving heavy furniture , loading trucks , and gardening , as well as sports activities such as aerobics .
our finding was consistent with the results from several large studies with long - term follow - up , which showed that self - reported pa was associated with a lower incidence of diabetes independent of bmi ( 32 ) .
in other previous studies , energy intake was shown to confound the effect of exercise on body weight or insulin resistance ( 33,34 ) . however , in our analysis , the associations between -cell function , glucose , insulin profiles , and pa were not significantly changed by the adjustment for caloric intake . therefore , our results indicated a more direct contribution of pa in preserving -cell function . moreover , our findings are consistent with the results of a recent study in rats , which demonstrated that voluntary exercise was beneficial for sustaining -cell compensation without preventing dyslipidemia or obesity ( 35 )
. one possible mechanism for such an effect would be mitigation of the adverse metabolic effects of obesity .
since pancreatic biopsies can not be performed , a surrogate measure would have been the computed axial tomography scan estimate of peritoneal fat or an ultrasound assessment of hepatic fat content .
although the questionnaire has been used in previous studies in mexican americans ( 18 ) , it is well known that self - reported pa tends to be overreported on questionnaires ( 36,37 ) .
dhhs recommendation for pa instead of using the minutes of pa as a continuous variable to reduce the impact of measurement errors . as evidence of potential overreporting in this cohort ,
57% of participants reported meeting or exceeding the dhhs pa guideline , as compared with 36% for mexican americans in a national report of participation in aerobic activity ( 38 ) .
however , we also note that our questionnaire included work - related pas , which might explain the higher than expected percentage .
second , we did not measure physical fitness , which is more objective and a better predictor than self - reported pa for many health outcomes , such as diabetes and cardiovascular diseases ( 39,40 ) .
third , the cross - sectional and observational design of our study precludes us from examining the dynamic impact of pa on the change in metabolic traits .
the strength of the current study is the unique sample , which includes a large cohort of mexican americans with detailed ogtt- and ivgtt - based measures of glucose tolerance , insulin sensitivity , and -cell function . unlike other studies that mostly examined the short - term effect of exercise training / intervention on insulin secretion and insulin resistance with small sample sizes , we described the association with pa in a free - living environment to provide a more realistic model than the impact of a specific , short - term exercise intervention .
we showed that increasing pa is associated with better -cell function in a population without overt diabetes .
our results suggest that an effect on diabetes prevention by lifestyle change ( 1,2 ) may be mediated by the improvement of -cell function .
our findings have implications for a real - world approach to the delay , prevention , and/or early treatment of type 2 diabetes . in conclusion
, our study indicates that a greater level of pa might play a role in improving glucose tolerance and protecting -cell function in mexican americans who are at high risk of developing diabetes .
the beneficial impact of pa on -cell function does not depend on bmi , percent body fat , and energy intakes .
our findings have important public health implications to prevent / slow down the deterioration of -cell function that leads to type 2 diabetes . | objectiveto examine the association between self - reported physical activity ( pa ) and diabetes - related quantitative traits.research design and methodsthe observational cohort was 1,152 mexican american adults with dual - energy x - ray absorptiometry , oral and intravenous glucose tolerance tests , and self - reported dietary and pa questionnaires .
pa was categorized into three mutually exclusive groups according to the u.s .
department of health and human services pa guidelines for americans : low ( vigorous < 75 min / week and moderate < 150 min / week ) , moderate ( vigorous 75 min / week or moderate 150 min / week ) , and high ( vigorous 75 min / week and moderate 150 min / week ) . trends in pa groups were tested for association with metabolic traits in a cross - sectional analysis.resultsthe participants mean age was 35 years ( range , 1866 years ) , mean bmi was 29.6 kg / m2 , and 73% were female . among them , 501 ( 43% ) , 448 ( 39% ) , and 203 ( 18% ) were classified as having low , moderate , and high pa , respectively .
after adjustment for age , a higher pa was significantly associated with lower 2-h glucose , fasting insulin , and 2-h insulin and greater -cell function ( p = 0.001 , 0.0003 , 0.0001 , and 0.004 , respectively ) .
the association did not differ significantly by sex .
results were similar after further adjustment for age , sex , bmi , or percent body fat.conclusionsan increasing level of pa is associated with a better glucose and insulin profile and enhanced -cell function that is not explained by differences in bmi or percent body fat .
our results suggest that pa can be beneficial to -cell function and glucose regulation independent of obesity . | RESEARCH DESIGN AND METHODS
Study participants
Testing procedures and assays
PA and dietary assessment
Data analysis
RESULTS
CONCLUSIONS
Supplementary Material |
PMC4897246 | variations in root canal morphology , especially in multirooted teeth , are a constant challenge for diagnosis and successful endodontic therapy .
root canal treatment can be highly guaranteed when all root canals are identified , thoroughly cleaned and shaped , and obturated with an inert filling material .
the clinician should be able to mentally visualize the pulp spaces from the coronal aspect to the apical foramen and should always be aware of the common internal root morphology and the possible variations which might be encountered .
otherwise , these anatomical variations may complicate endodontic treatment and compromise therapy outcomes [ 2 , 3 ] .
mandibular first molars are the first permanent posterior teeth to erupt and are most prone to suffer from caries , leading to requirement of endodontic treatment .
it is generally accepted that mandibular first molars have two roots , a mesial root and a distal root and three or four canals , two in the mesial root and one or two in the distal root .
the most relevant variable related to the number of roots is the presence of a third distolingual root .
this macrostructure , first mentioned by carabelli , was called radix entomolaris , which in general is smaller than mesial and distobuccal roots and can be separate from or partially fused with these other roots .
when located at the mesiobuccal side , this additional root is called the radix paramolaris .
anatomical variations in the number and location of canals have been reported by several studies .
these include the presence of a third canal in the mesial root , three canals in the distal root , and mandibular first molar with five , six , or even seven canals [ 10 , 11 ] .
various techniques have been adopted to evaluate root canal morphology , and it has been reported that tooth demineralization and canal staining of extracted teeth are a very accurate method to detect root morphology .
other techniques include access cavity preparation and radiographs with files placed in root canals , in vivo root canal treatment with radiographic evaluation , and macroscopic sectioning .
however , these methods had serious limitations as the relationship between the external structure and the pulp might get lost during preparation of study samples .
advancements in the field of radiology have drawn attention to the use of computed tomography ( ct ) for imaging teeth .
technological advances have witnessed the invention of cone beam computerized tomography ( cbct ) which was introduced in the field of endodontics in 1990 by tachibana and matsumoto .
cbct scans were recently shown to be a valuable tool in various stages of endodontic treatment as they deliver immediate and accurate three - dimensional radiographic images .
preoperatively , these images offer superior diagnostic performance over conventional radiographic images and are used to improve the assessment of root canal systems as they provide information about the internal and external tooth anatomy including number and location of roots and canals , root and canal curvatures , size of the pulp chamber , and the degree of calcification .
investigated the use of cbct scanning in identifying root canal systems and compared it with images obtained by using digital radiography .
they concluded that cbct images always resulted in the identification of greater number of root canal systems than digital images .
additionally , cbct technology aids in the diagnosis of endodontic and nonendodontic pathosis , assessing vertical root fractures , analysis of root resorption defects , and presurgical assessment of apicoectomy procedures [ 19 , 20 ] .
postoperatively , cbct scans can help in assessment of the technical quality of the root canal filling as well as healing of periapical lesions .
many studies of root canal morphology in mandibular molars have been conducted because these teeth present a complex morphology that often complicates endodontic treatment .
a review of the literature showed a lack of studies on root canal morphology of mandibular permanent first molar teeth in a palestinian population .
the purpose of this study was to investigate canal number and internal morphologies of permanent mandibular first molar using a sample of cbct images obtained from extracted teeth of a palestinian population .
a sample of 320 extracted double - rooted mandibular permanent first molars from palestinian population was collected from the tooth bank at the department of oral surgery .
teeth included in this study were selected according to the following criteria : permanent mandibular first molars with intact roots and fully formed apices .
teeth with broken roots , serious defects , and root canal fillings , posts , and crown restorations and teeth with signs of root resorption were excluded from the study .
an ethical approval was obtained from the ethics committee of the university , and patients that were undergoing extraction signed an informed consent in regard to providing his / her tooth after being extracted for use in this study .
teeth were stored in 10% formalin for disinfection and were cleaned of calculus and remaining soft tissue by using an ultrasonic scaler .
was carried out on extracted teeth , gender and age of the patients were not recorded . to integrate the samples positions ,
the teeth were fixed in some molds made of silicone impression molding materials ( zetaplus , zhermack , rovigo , italy ) in 6-tooth groups .
the mounted teeth were scanned with a cbct scanner ( mct-1 [ ex-2 f ] , morita manufacturing corp , kyoto , japan ) with image capture parameters set at 80 kv and 5.0 ma and an exposure time of 17.5 s. tomographic sections of 1 mm in 3 planes ( axial , coronal , and sagittal ) were created .
the cbct images were analyzed using xorancat software version 3.1.62 ( xoran technologies , ann arbor , usa ) on a dell precision t5400 workstation ( dell , round rock , tx ) , with a 32-inch dell lcd in a darkroom .
the contrast and brightness of the images were adjusted using the image processing tool in the software to ensure optimal visualization .
all mandibular first molars were thoroughly examined in the three planes ( axial , sagittal , and coronal ) at 1.0 mm intervals by continuously moving the toolbar from the floor of the pulp chamber to the apex .
the following observations were registered : number of root canals per root and canal configuration in each root based on vertucci 's classification ( figure 1 ) .
two endodontists evaluated the radiographic images simultaneously to reach a consensus . in cases where a consensus was not reached , a third professional oral radiologist with endodontic experience
the number and type of root canals of mandibular first molars are summarized in table 1 .
all selected teeth had two separate roots . in total , 174 ( 54.4% ) of the mandibular first molars had three canals ( mesiobuccal , mesiolingual , and distal ) , 132 teeth ( 41.3% ) had four canals ( mesiobuccal , mesiolingual , distobuccal , and distolingual ) , and only four teeth had two canals ( 1.2% ) .
analysis of the mesial root revealed one canal in 1.3% , two canals in 95.6% , and three canals in 3.1% . in the distal root , one canal was found in 57.5% and two canals were found in 42.5% of the examined roots ( table 2 ) .
examples of canal distribution of examined teeth taken in an axial section are shown in figure 2 .
types of canal configuration for each root are summarized in table 3 . of the examined teeth ,
the most common canal configuration in the mesial roots was vertucci type iv ( 53.8% ) followed by type ii ( 38.8% ) ( figure 3 ) . in the distal roots ,
the most prevalent canal configuration was vertucci type i ( 57.5% ) followed by type ii ( 22.5% ) and type iii ( 10.6% ) ( figure 4 ) .
morphologic variation in the anatomy of the root canal system should always be considered at the beginning of root canal treatment .
each case , independent of which tooth is to be treated , should be examined clinically and radiographically in a thorough manner to detect possible anatomic variations .
it is generally accepted that a major cause of root canal treatment failure is an inability to locate and adequately treat all canals of the root canal system . in a statistical analysis of retreatment cases ,
allen et al . analyzed a total of 1300 endodontic subjects for factors that may have contributed to the failure of the original treatment and reported that untreated canals were responsible for failure in 114 cases , with 8.8% prevalence .
in another investigation , hoen and pink found that missed canals were the main cause of endodontic retreatment in 42% of cases studied . in our study , all evaluated teeth were two rooted .
unfortunately , the tooth bank at our dental school from which the examined teeth have been selected did not contain any three - rooted mandibular first molars .
no studies have been conducted on the incidence of radix entomolaris and radix paramolaris in palestine .
radiographic examination is an essential part of endodontic management , from initial diagnosis to monitoring treatment results .
a periapical radiograph is the most common method used to assess the configuration of root canal systems during root canal treatments .
it provides much needed information about root canal morphology especially if taken from different horizontal angels
. however , periapical radiographs produce a 2-dimensional image of 3-dimensional objects which prevents complete understanding of root morphology and canal anatomy .
tooth clearing and canal staining had been considered for a long time the gold standard for studying root canal morphology where fine internal anatomic details can be visualized . in this study , we used cbct scanning to evaluate the number of canals and canal configuration of mandibular first molars .
compared several methods used to evaluate root canal morphology and reported that cbct was as accurate in identifying root canal systems as the modified canal staining and tooth clearing technique . in fields of dentistry where 3-dimensional imaging is necessary ,
cbct is considered by some to be the standard of care with many advantages .
cbct is an office - based imaging technique so can be conveniently used in vivo when required for diagnosis and preoperative assessment .
additionally , cbct scanning has been shown to be more accurate than digital radiographs in determining root canal systems . when compared with 2-dimensional digital radiographs ,
cbct reduces or even eliminates the superimposition of the surrounding structures that normally overlap and enables clinicians to identify more canals in multicanal teeth that can then be instrumented and obturated , thereby increasing the likelihood of a successful outcome .
moreover , cbct technology uses an extra - oral imaging scanner at a considerably lower radiation dose and higher resolution than conventional ct [ 18 , 30 ] .
the number of root canals and canal configuration of mandibular first permanent molars reported in the literature vary on the basis of the different ethnic populations and different methods used ( table 4 ) .
our study showed that 54.4% of mandibular first molars had three canals and 41.3% had four .
these results are in agreement with those of zhang et al . who reported an incidence of a fourth canal in 43% of a chinese population .
chen et al . reported an incidence of 45.9% in a taiwanese population . in their study on mandibular molars in a jordanian population using clearing technique , al - qudah and awawdeh reported a fourth canal in 46% of the studied sample .
the incidence of two canals in the mesial roots was found to be 95.6% . in their systematic review , de pablo et al
. found a similar incidence ( 94.4% ) in the 17 studies included accounting for 4535 mesial roots investigated . in the present study , ten mesial roots ( 3.1% )
the clinical significance of an extra canal means that failing to locate and properly treat this canal could lead to treatment failure .
the presence of the middle mesial canal was reported up to 14.8% incidence . in a study of 760 mandibular molars
, fabra - campos found that 20 ( 2.6% ) teeth had three canals in the mesial root . in 13 of
these ( 65% ) the third canal joined the mesiobuccal canal in the apical third of the root and in 6 ( 30% ) they converged with the mesiolingual canal , also in the apical third ; the third canal ended as an independent canal in only 1 case . an overall same incidence ( 2.6% )
in the mesial root , type iv configuration was most prevalent ( 53.8% ) followed by type ii ( 38.8% ) canal configuration .
these results are consistent with the findings of most of the earlier studies [ 5 , 12 , 15 , 17 ] in their systematic review , de pablo et al . reported a prevalence in mesial roots of 52.3% for type iv and 35% with type ii .
an exception was reported by al - nazhan with type ii being the most prevalent followed by type iv . in the present study
, ten mesial roots showed an additional configuration type ( 3 - 2 ) as described by gulabivala et al . and
type ( 3 - 2 - 1 ) as described by al - qudah and awawdeh .
identification , preparation , and obturation of type iv and type ii are relatively straightforward .
however , the presence of additional types needs extra efforts , because failure to debride and disinfect this complex anatomy might have a direct effect on the treatment outcome .
the most prevalent canal configuration in the distal roots was type i ( 57.5% ) followed by type ii ( 22.5% ) , type iii ( 10.6% ) , and type iv ( 8.1% ) .
the high prevalence of the vertucci type i canal configuration in the distal canals is consistent with the previous observations [ 12 , 17 , 27 , 32 ] . in terms of type iv configuration ,
the incidence ( 8.1% ) was lower than reported by previous studies [ 5 , 12 , 14 , 17 , 27 ] . in a korean population , kim et al
. reported a higher incidence ( 76.9% ) of mesial roots with type iv and ( 66.6% ) of distal roots with type i configuration .
these higher incidences were also reported by zhang et al . in a chinese population : 81% of mesial roots with type vi and 84% of distal roots with type i configuration .
these differences may be related to study design ( in vivo versus in vitro ) , study technique , sample size , and sample population .
the present study indicates that cbct is helpful for the investigation of root canal morphology .
when root canal assessment is possible from traditional periapical images or clinical procedures , the use of cbct may not be necessary .
when there are abnormal findings on traditional periapical films or variations detected clinically , it may be impossible to evaluate the root canal system effectively .
in such situations , it is necessary to adopt cbct for further diagnosis , whilst at the same time ensuring that the patients ' exposure to radiation is kept as low as reasonably possible .
cbct scans allow the identification of anatomic features and variations of the root canal system . within the limitations of this study
, it can be concluded that , in a palestinian population , cbct scans show that mandibular first permanent molars commonly have four canals with a lower incidence ( 41.3% ) compared to previously reported data .
therefore , to treat mandibular first permanent molars , dentists need to be aware of the possible existence of a separate distolingual root canal before they initiate endodontic treatment . |
aim .
the purpose of this study was to investigate the number of canals and variations in root canal configuration in the mandibular permanent first molar teeth of a palestinian population using cone - beam computed tomography ( cbct ) .
methods . a sample of 320 extracted double - rooted mandibular permanent first molars from palestinian population was collected for this study and scanned with cbct scanner .
the following observations were made : number of root canals per root and canal configuration in each root based on vertucci 's classification .
results . of the 320 mandibular first molars analyzed , 174 ( 54.4% ) had three canals , 132 teeth ( 41.3% ) had four canals , and only four teeth had two canals .
the most common canal configuration in the mesial roots was vertucci type iv ( 53.8% ) followed by type ii ( 38.8% ) . in the distal roots , the most prevalent canal configuration was vertucci type i ( 57.5% ) followed by type ii ( 22.5% ) and
type iii ( 10.6% ) . conclusion .
our results showed that the number of canals and canal configuration in palestinian population were consistent with previously reported data .
the present study also indicates that cbct is helpful as a diagnostic tool for the investigation of root canal morphology . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion |
PMC4939228 | preliminary estimates show that 11% of the global burden of disease is manageable by surgery ( 1 ) .
even with conservative estimates , each year 7 million people suffer from complications caused by surgery , while probably half of them are preventable .
studies have also shown that the rate of major complications caused by surgery for hospitalized patients in developed countries is 3 - 6% , accounting for 0.4 - 0.8% of total mortality , as opposed to 5 - 10% in developing countries ( 2 ) .
what is more , the performance of organizations providing surgical care in low- and middle - income countries ( lmics ) is not satisfactory ( 3 ) .
only 3.5% of major surgical procedures are performed in low - income countries , while they account for 1.3% of the world s population ( 4 ) .
in addition to the quality of surgical procedures , the cost - effectiveness of surgeries is a very important issue which is different between countries .
available evidence suggests that the cost - effectiveness of essential surgical care in lmics is not appropriate ( 1 ) .
it seems that although surgery is an integral component of health care , it is generally neglected in these countries ( 5 , 6 ) . to tackle the above problems and reduce inequity in the quality and quantity of surgical care in the world , especially in lmics , in december 2005 , the world health organization ( who ) launched a global initiative on emergency and essential surgical care .
this initiative includes educational materials , designing a tool for data collection regarding the quantity and quality of surgical care and emergency care ( standard tool for situational analysis ) , and preparing and completing a global atlas for essential surgical care ( 5 ) .
the first level comprises primary health care , the second level delivers both inpatient and outpatient specialized health services by hospitals and health centers , and the third level provides subspecialty services as inpatient and outpatient services .
the medical universities based on their educational , research , and medical facilities , and also on the indictors of provincial health are divided into three types .
type i universities have more facilities and better health indicators than the other two types ( 7 ) .
based on this grouping , 31% of hospitals are managed by type i universities , 52% by type ii , and 17% by type iii universities .
this study , originally presented as a phd thesis , aims to address the current dearth of information on the status of surgical care in iran and also to introduce and implement strategies suggested by the who for the universal and effective management of essential surgical care in iran .
this research was a descriptive and cross - sectional study performed at 42 first - referral district hospitals in iran in 2013 selected via the randomized sampling method . in june 2012 ,
the study protocol was approved by the ethics committee of iran university of medical sciences ( project # 344 ) .
general , governmental and non - educational hospitals which had active operating rooms were included in this study .
general private or charity hospitals as well as those in provincial capitals were excluded from the study .
first , the hospitals that met the inclusion criteria were determined . as is shown in table 1 ,
there were 68 hospitals in type i universities , 113 in type ii , and 37 in type iii .
then , due to the large number of the qualified hospitals , according to the proportion of each type of university hospitals , it was decided to choose 3 hospitals for each type i university , 2 hospitals for each type ii university , and one hospital for each type iii university .
consequently , via random sampling , the present study included 73 hospitals , 42 of which responded with completely answered questionnaires and they were eligible to be included in the final data analysis .
the study tool was a questionnaire developed by the who in 2003 for the situational analysis of emergency and essential surgical care ( 8 , 9 ) .
this tool is used to assess surgical needs in many developing countries and has been translated into farsi and validated and employed in iran by mouseli ( 2009 ) ( 10 - 12 ) . however , the researcher improved and completed the translation of the tool before it was utilized .
the questionnaire consisted of 138 questions in four parts , encompassing general information about the hospital , surgical and anesthetic infrastructure , human resources for surgery , surgical interventions and services , and equipment for surgery and resuscitation .
the reliability of the questionnaire was evaluated by calculating cronbach s for 7 filled questionnaires , which yielded 77.7% internal co - efficiency .
thereafter , the questionnaire was filled after obtaining the consent of the directors of the studied hospitals and ensuring the confidentiality of the information .
the data were analyzed via descriptive statistics ( i.e. frequency , percentage , and ratio ) using the spss software ( version 16 ) .
among the studied hospitals , there were 16 ( 38.1% ) type i , 19 ( 45.2% ) type ii , and 7 ( 16.7% ) type iii university hospitals .
the total population covered by these 42 hospitals was 5637688 , and the mean of the population covered by each hospital was 134230.67 67496.22 .
the average number of the active hospital beds was 94.55 44.74 , and the mean number of the operating rooms was 3.36 1.62 .
the mean distance to the next health center in the next level was 120.57 88.66 km .
as the data presented in table 2 show , the highest numbers of approved beds and active beds were 200 and 220 , respectively .
the highest number of the operating rooms was 8 , with a mean number of 3.36 . additionally , the mean number of the admitted patients was 15186.10 , the mean number of the patients that underwent surgery was 3249.73 , the mean number of the operated children younger than 15 years old was 279 , and the mean number of the patients that were referred to more equipped centers for surgery was 233.89 .
regarding infrastructure , our results showed that all the hospitals had oxygen cylinders , running water , electricity , functioning anesthesia machines , emergency departments , archives of medical records , and functioning x - ray machines .
the least available facilities were central oxygen ( 27 [ 64.3% ] hospitals ) and blood banks ( 28 [ 66.71% ] hospitals ) . in all the hospitals ,
regardless of their types , specialists in surgery , anesthesia , and obstetrics and gynecology were available full time . to complete expert staff ,
in addition , none of the general practitioners or nurses that were qualified or had performed surgery or anesthesia was reported as hospital staff .
number of the paramedics and nurses is related to all the wards of the hospital and not just surgery .
as is shown in table 5 , cricothyroidotomy was offered only in 85.7% of the hospitals and foreign body removal in 85.7% . among urgent procedures ,
neonatal surgeries were conducted in 14.3% of the hospitals , open treatment of fractures in 71.4% , and amputation in 66.7% . from non - urgent procedures ,
acute burn management was conducted in 38.1% of the hospitals , urethral stricture dilatation in 64.3% , cleft - lip repair in 31% , and release of contracture tissue in 52.4% .
the present study showed that the most common shortage was in arm and leg splints , reported by 47.6% of the hospitals .
cricothyroidotomy sets and artery forceps were available in 57.1% and 78.5% of the hospitals , correspondingly .
the other required supplies were always available in most of the hospitals ( over 85% ) .
among the studied hospitals , there were 16 ( 38.1% ) type i , 19 ( 45.2% ) type ii , and 7 ( 16.7% ) type iii university hospitals .
the total population covered by these 42 hospitals was 5637688 , and the mean of the population covered by each hospital was 134230.67 67496.22 .
the average number of the active hospital beds was 94.55 44.74 , and the mean number of the operating rooms was 3.36 1.62 .
the mean distance to the next health center in the next level was 120.57 88.66 km .
as the data presented in table 2 show , the highest numbers of approved beds and active beds were 200 and 220 , respectively .
the highest number of the operating rooms was 8 , with a mean number of 3.36 .
additionally , the mean number of the admitted patients was 15186.10 , the mean number of the patients that underwent surgery was 3249.73 , the mean number of the operated children younger than 15 years old was 279 , and the mean number of the patients that were referred to more equipped centers for surgery was 233.89 .
regarding infrastructure , our results showed that all the hospitals had oxygen cylinders , running water , electricity , functioning anesthesia machines , emergency departments , archives of medical records , and functioning x - ray machines .
the least available facilities were central oxygen ( 27 [ 64.3% ] hospitals ) and blood banks ( 28 [ 66.71% ] hospitals ) .
in all the hospitals , regardless of their types , specialists in surgery , anesthesia , and obstetrics and gynecology were available full time . to complete expert staff ,
in addition , none of the general practitioners or nurses that were qualified or had performed surgery or anesthesia was reported as hospital staff .
number of the paramedics and nurses is related to all the wards of the hospital and not just surgery .
as is shown in table 5 , cricothyroidotomy was offered only in 85.7% of the hospitals and foreign body removal in 85.7% . among urgent procedures ,
neonatal surgeries were conducted in 14.3% of the hospitals , open treatment of fractures in 71.4% , and amputation in 66.7% . from non - urgent procedures ,
acute burn management was conducted in 38.1% of the hospitals , urethral stricture dilatation in 64.3% , cleft - lip repair in 31% , and release of contracture tissue in 52.4% .
the present study showed that the most common shortage was in arm and leg splints , reported by 47.6% of the hospitals .
cricothyroidotomy sets and artery forceps were available in 57.1% and 78.5% of the hospitals , correspondingly .
the other required supplies were always available in most of the hospitals ( over 85% ) .
the present study provides a general view of the current status of surgical and anesthesia services in first - level hospitals in the referral system in iran , based on the who tool .
our findings identified the gaps in the infrastructure , human resources , surgical interventions , and essential equipment and indicated that suitable facilities and equipment , human resources , and infrastructure are available in the district hospitals in iran at a standard higher than that in many of the lmics which have evaluated and published the status of their own essential surgical care ( 11 - 17 ) .
our results also showed a significant improvement in status by comparison with the situation 5 years ago in iran .
this is the second study in iran to have evaluated surgical care in the district hospitals using the who tool .
the results of the present study , conducted 5 years after the previous one , showed that iran has made significant progress in the different aspects of surgical care over the recent years ( 10 ) . in the infrastructure domain , despite its wide geographical expanse , iran has endeavored to provide its population with access to essential surgical care .
our results revealed that the mean number of the hospital beds in the 42 evaluated hospitals was 97.24 , which shows a good increase compared with the figure ( 69 beds ) reported in the previous study .
more than 92% of the studied hospitals had management guidelines for surgery , anesthesia , pain relief , and emergency conditions .
the reasons for the availability of these guidelines were the implementation of clinical governance and safe surgery services in the hospitals , as well as the requirement for accreditation programs for the hospitals ( 18 ) . in comparison with 5 years ago ,
the surgical services have made headway in all aspects of the infrastructure domain ( 10 ) .
this indicates the good status of iran s general hospitals in terms of infrastructure , which also suggests that more surgical services can be defined for the hospitals in this level .
our findings also demonstrated that the status of surgical care in iran compared with that in many developing and even some developed countries is good . in regard to infrastructure , the situation of iran compared with that of other developing countries , which were studied using the similar assessment tool , was significantly better , such that it places iran in the range of developed countries . in mongolia , only 45% of the hospitals had power generators and 23% had blood banks ( 13 ) . in afghanistan , 41% of the hospitals had anesthesia machines and 40% had access to running water ( 14 ) . in gambia ,
oxygen sources , running water , and electricity were available in 77.8% , 50% , and 44.4% of the hospitals , correspondingly .
furthermore , most of the studied hospitals in these countries did not have management guidelines ( 15 ) .
in terms of human resources , the iranian ministry of health has made great strides in promoting surgical care by training more surgeons . presently , 52 medical universities and medical faculties are active in iran , which yearly accept 150 residents in general surgery in 27 universities , 181 anesthesia residents in 26 universities , and 224 obstetrics and gynecology residents in 27 universities ( 19 ) .
there is no shortage of human resources in iran , but their distribution is far from equitable ( 20 , 21 ) insofar as the expert human resources are less likely to work full time in disadvantaged provinces .
it is notable that surgical services were provided only by surgeons , anesthesiologists , and obstetrics in the studied hospitals .
performing surgery and anesthesia by general physicians is illegal ; therefore , none of the surveyed hospitals permitted general practitioners or other health care personnel to perform any surgery or anesthesia .
considering human resources , iran also has a very good condition compared to many developing countries .
for example in afghanistan , a study reported that only 64.7% of the hospitals had surgeons and 29.4% had anesthesiologists .
in addition , 30% of afghanistan s hospitals did not have gynecologists , which was one of the main reasons for the referral of patients to the next health level ( 14 ) . in mongolia ,
the presence of surgeons and anesthesiologists was not reported even as part time and only in limited hospitals did general physicians have the permission to perform anesthesia and surgery ( 13 ) . in some studies , for example in sierra leone ,
likewise , in the east region of africa , there were 400 surgeons for more than 200 million people ( 17 , 22 ) . in the surgical intervention domain ,
life - saving services such as resuscitation and chest tube insertion were offered by 100% of the hospitals .
cricothyroidotomy and foreign body removal were offered in 85.7% of the hospitals , and the other hospitals referred their patients to a higher level .
for emergency surgeries like neonatal operations , most of the hospitals ( 85.7% ) also referred their patients to a higher level .
services for acute burn , dilatation of urethral strictures , and cleft lip were provided in 38.1% , 64.3% , and 31% of the hospitals under study , respectively .
the main reason for not offering these services as stated by the other hospitals was a lack of specialists , followed by a paucity of equipment .
mouseli in his study reported neonatal surgery and cleft - lip repair in 40% and acute burn treatment and dilatation of urethral stricture in 45% of the hospitals ; these figures are not very different from our results ( 10 ) . in the present study ,
all the studied hospitals provided anesthesia services , and only 26.2% of them did not offer anesthesia with ketamine .
the principal reason for this shortcoming was a lack of experts and equipment . in this domain , iran s status is far more desirable than that in many developing countries . in tanzania , equipment shortages precluded the provision of many life - saving procedures such as oxygen tubing , pulse oximetry , and pediatric intubation ( 11 ) . in gambia due to the inadequacy of human resources , cesarean section and appendectomy were performed only in 58.8% of the hospitals ( 15 ) .
with respect to equipment , the iranian ministry of health has tried to improve surgical care by equipping hospitals and paying special attention to operating rooms .
equipment and supplies , renewable items , and supplementary equipment were always available in all the hospitals in the current study . only items like arm and leg splints ( 47.6% ) and cricothyroidotomy sets ( 57.1% ) were not always available in the studied hospitals .
the availability of equipment showed that there has been a good improvement in this area since the last study in iran ( 10 ) . in this regard , iran s situation was superior to that in many other developing countries . in mongolia ,
the availability of arm and leg splints and cricothyroidotomy sets was reported in 14% and 18% of the hospitals , correspondingly .
in addition , oxygen sources , suction pumps , and resuscitator bag valves and masks ( for adults ) were only supplied in 9% of the hospitals in mongolia ( 13 ) .
in contrast , our findings showed that the same pieces of equipment were , respectively , available in 100% , 97.6% , and 95.2% of the hospitals evaluated .
the limited number of the studied hospitals compared with the total district hospitals ( 42 vs. 218 ) may limit the generalizability of our results to all surgical care in all districts and is , as such , one of the limitations of the present study .
another drawback of note is that the quality of the provided surgical services was not assessed in this study .
furthermore , it is possible that because of the weaknesses in the hospital information system ( his ) in some hospitals , the accurate number of the patients referred to next levels was not reported . considering the defined level of services for these hospitals on the basis of the findings of the current study
, it is advisable that a revision be made to these services with a view to expanding the spectrum of the provision of care . by encouraging health care personnel , especially surgeons and anesthesiologists , to render their services in small towns
, it will be possible to provide better quality services at higher levels and reduce referral rates .
moreover , the attainment of this goal requires a more equitable distribution of specialists to reflect the acceptable status of equipment distribution in iran .
it is deserving of note that very few studies have hitherto been conducted on the performance of district hospitals , not least in the field of surgical services .
the present study , thus , sought to present a general view of the current status of surgical services at iranian district hospitals . | background : surgery is an essential component of health care , yet it has usually been overlooked in public health across the world.objectives:this study aimed to perform a situational analysis of essential surgical care management at district hospitals in iran.materials and methods : this research was a descriptive and cross - sectional study performed at 42 first - referral district hospitals of iran in 2013 .
the world health organization ( who ) tool for the situational analysis of emergency and essential care was used for data collection in four domains of facilities and equipment , human resources , surgical interventions , and infrastructure .
data analysis was conducted using simple descriptive statistical methods.results:in this study , 100% of the studied hospitals had oxygen cylinders , running water , electricity , anesthesia machines , emergency departments , archives of medical records , and x - ray machines
. in 100% of the surveyed hospitals , specialists in surgery , anesthesia , and obstetrics and gynecology were available as full - time staff .
life - saving procedures were performed in the majority of the hospitals . among urgent procedures ,
neonatal surgeries were conducted in 14.3% of the hospitals . regarding non - urgent procedures ,
acute burn management was conducted in 38.1% of the hospitals .
also , a few other procedures such as cricothyrotomy and foreign body removal were performed in 85.7% of the hospitals.conclusions:the results indicated that suitable facilities and equipment , human resources , and infrastructure were available in the district hospitals in iran .
these findings showed that there is potential for the district hospitals to provide care in a wider spectrum . | 1. Background
2. Objectives
3. Materials and Methods
4. Results
4.1. The Characteristics of Centers
4.2. Basic Infrastructure
4.3. Human Resources
4.4. Surgical Interventions
4.5. Equipment and Supplies
5. Discussion |
PMC5259925 | fundamental to this , however , is a reliable , quick , inexpensive , and accurate diagnosis .
surgical histopathology remains the gold standard for diagnosis , but is generally unavailable in most developing countries and comes with challenges of equipment , expertise , and cost to facilities and patients at large .
it involves obtaining tissue diagnosis of subcutaneous and other tumors with the aid of a special needle that with imaging techniques may allow its use in a wider scope .
the procedure is regarded as inexpensive , requiring simple equipment , less time consuming , and can be applied to a wide range of diseases .
detailed explanation of the procedure and how it can be applied throughout the body has been documented and has been adopted in clinical practices , especially for quick bedside diagnosis of various malignancies .
the dangers of false positives and the inevitability of false negatives can not be overemphasized because a negative cytology report is not definitive , especially when there is a mass .
a good cytopathology practice also requires access to surgical pathology to clarify cases for which cytopathology gives uncertain or no diagnosis and for validation and quality control purposes .
fine needle aspiration cytology ( fnac ) clinics and cytology practices have been running in the pathology department of our hospital since 2010 . after brief closure of the department in 2004 ,
much assistance in re - establishing the department to include a cytology unit came from the breast health global initiative .
there was also collaboration between our hospital and a teaching hospital in europe to train pathologists and technologists in 2005 . in this article
, we present a detailed retrospect of experience in running cytopathology services in kumasi , ghana after its establishment .
this will provide a clear picture on the performance with respect to this service as offered by the department .
this was a retrospective study conducted using reports on all cytology procedures performed in our department from january 2010 to december 2011 .
this retrospective study was approved and supervised by the research and development unit ( ethics committee ) of our institution with approval from the department .
participant informed consent was not obtained ; hence , patient records / information was anonymized and de - identified prior to analysis . in routine cytology practice in our centre , pathologists performed fine - needle aspirations with the assistance of biomedical scientists .
the method applies to various body sites and its interpretation was documented by a previous documented review of the methodology .
fluid specimens ( urine and pleural effusions ) are centrifuged to obtain the sediment , which is then used to prepare direct smears .
wet mounted smears are stained with papanicolaou stain , giemsa , and sometimes hematoxylin and eosin .
neelsen ( zn ) staining was performed on smears when tuberculosis was clinically suspected or if granulomatous inflammation was noted on the routine stains .
no gynecological smears were , however , received in the department during the period under review .
this was due in part to the unavailability of cyto - screeners to assist in the anticipated workload at the time .
data from the reports were entered into an excel spreadsheet and analyzed using the statistical package for social scientists , version 16.0 ( spss , il ) .
this was a retrospective study conducted using reports on all cytology procedures performed in our department from january 2010 to december 2011 .
this retrospective study was approved and supervised by the research and development unit ( ethics committee ) of our institution with approval from the department .
participant informed consent was not obtained ; hence , patient records / information was anonymized and de - identified prior to analysis .
in routine cytology practice in our centre , pathologists performed fine - needle aspirations with the assistance of biomedical scientists .
the method applies to various body sites and its interpretation was documented by a previous documented review of the methodology .
fluid specimens ( urine and pleural effusions ) are centrifuged to obtain the sediment , which is then used to prepare direct smears .
wet mounted smears are stained with papanicolaou stain , giemsa , and sometimes hematoxylin and eosin .
neelsen ( zn ) staining was performed on smears when tuberculosis was clinically suspected or if granulomatous inflammation was noted on the routine stains .
no gynecological smears were , however , received in the department during the period under review .
this was due in part to the unavailability of cyto - screeners to assist in the anticipated workload at the time .
data from the reports were entered into an excel spreadsheet and analyzed using the statistical package for social scientists , version 16.0 ( spss , il ) .
a total of 836 cases were reviewed , an average of approximately 418 cases were performed a year .
this accounted for approximately 5.7% of the total workload of the department including surgical pathology , cytopathology , and autopsy .
approximately one - fifth ( 19.4% ) of the cases reviewed , however , had no age specified .
more than half ( 58.0% ) of the cases received had no clinical diagnosis indicated on the forms .
although a number of cases had no indicated gender , this formed only 4.3% of the total cases [ table 1 ] .
characteristics of cytological cases received at the department relative to gender the most aspirated sites in decreasing order was the breast ( 20.2% ) , lymph nodes ( 20.0% ) , thyroid ( 12.0% ) , soft tissue ( 9.6% ) , salivary glands ( 4.7% ) , ascitic fluid ( 4.1% ) , pleura ( 5.1% ) , csf ( 0.7% ) , liver ( 0.6% ) , and other miscellaneous sites , which accounted for 23.0% of the total .
more females had samples aspirated from the breast ( 27.9% ) and thyroid ( 13.4% ) than males ( 5.6% and 8.8% , respectively ) .
there were more benign than malignant cases with respect to all sites aspirated except the breast ( 18.3% ) , lymph nodes ( 35.0% ) , and soft tissues ( 11.7% ) where the reverse occurred [ table 2 ] .
site distribution of cases according to age , sex , and nature of diagnosis in table 3 , the age and sex distribution of cases received relative to cytological diagnosis is shown .
there was an equal distribution ( 40.5% ) of reported benign and malignant cases among those aged 5 years and below .
there was an almost equal distribution of malignant ( 38.0% ) and benign ( 35.0% ) cases among males .
diagnosis , however , could not be ascertained in 28 ( 3.3% ) of the cases .
reasons for this included inadequate smear , inadequate cellular material , and no palpated lumps for aspiration .
cytopathology is a relatively inexpensive , quick , and reliable method of diagnosis in pathology settings , especially in developing countries .
, we sought to evaluate the frequency of cases , efficacy of diagnosis , its impact , and relevance to referring clinicians .
most of the fnacs performed were aspirates from the breast ( 20.2% ) , followed by that from the lymph nodes ( 20.0% ) .
our findings show that the use of cytopathology for the diagnosis of the abovementioned conditions is very important , in the triage of such patients and the provision of a quick diagnosis for prompt attention by referring clinicians .
in contrast to our findings , a study conducted in cameroon showed that lymphadenopathy was the most common indication for fnac followed by breast . in most of the cases under review , cytopathology was very useful in distinguishing benign and malignant cases .
benign cases were , however , more than malignant cases , a reassurance that with the right treatment patients are likely to have a very good prognosis .
this is contrary to a finding by spillane et al . , who found a high percentage of malignant lesions being diagnosed by fine - needle aspiration biopsy ( fnab ) .
however , this was of benefit by reducing the number of corresponding open biopsy rates .
the percentage of the total workload accounted for by cytopathology was relatively small . because pathology practice in ghana is still in its infancy with high rate of autopsy practice , cytopathology is usually not considered by clinicians , especially those in peripheral hospitals .
laboratory request forms , which provide detailed information about the patient and clinical history , were not properly filled or completed by referring clinicians .
approximately one - fifth of all the forms received did not specify sex , and more than half ( 58.0% ) of the cases did not have any clinical diagnosis indicated by referring clinicians .
this could lead to a possible delay in diagnosis as well as reliability of the diagnosis .
a similar finding was also reported in a study conducted in ghana on the evaluation of request forms received in a hematology laboratory , and seems to be the trend in most hospitals in ghana .
seventy - seven percent of the time , a working diagnosis was achieved with a definite diagnosis in 73.4% of cases
. however , there is a limitation in determining the specificity and sensitivity of diagnosis .
poor requests from referring clinicians , varied sampling techniques by pathologists and biomedical scientists may account for the high rate of nondiagnostic cases .
going forward , histology and cytopathology correlation may be necessary to reduce the high rate of non - diagnostic cases and to ascertain unclear cases .
it has been suggested that having a pathologist on site for fnac improves sampling and enables assessment of clinical signs and symptoms to enhance diagnostic accuracy .
this was a major advantage in this setting , as seen in the percentage of diagnosis obtained
. more work , however , needs to be done to improve efficiency and diagnosis rate .
cytopathology practice in our centre has improved and is promising . improving and expanding on the current practice will ensure that pathologists in practice sustain and improve diagnostic cytopathology , as well as provide materials for training young pathologists . as the number of pathologists on site increases , sub - specialization in cytopathology may be necessary to enhance the diagnostic capability of the department . | aim : surgical pathology service is generally unavailable in most developing countries and comes with challenges .
cytopathology is a reliable , inexpensive adjunct to surgical histopathology .
we present a retrospective review of the various cytopathology cases received at the department.materials and methods : a retrospective review of 836 cytopathology cases from january 2010 to december 2011 at the department of pathology of our hospital was conducted .
all cytopathology reports and records from the department were retrieved and analyzed using the statistical package for the social sciences version 16 for windows.results:a total of 836 ( mean age 38.18 22.18 ) cases were reviewed , at an average of approximately 418 cases performed a year ( 5.7% of the total workload ) .
more than half ( 58.0% ) of the cases received had no clinical diagnosis indicated on request forms .
seventy - seven percent ( 77% ) of the cases were diagnosed as either definite or nondefinite .
the breast was the most aspirated specimen site ( 20.2% ) .
benign cases formed 45.0% of all the cases and 29.0% were malignant .
there were more benign than malignant cases with respect to all sites aspirated except the breast ( 18.3% ) , lymph nodes ( 35.0% ) , and soft tissues ( 11.7% ) where the reverse occurred.conclusion:patronage of cytopathology in kumasi is increasing and serves as a quick , cheap , and effective alternate means for diagnosis . improving and expanding on the current practice will ensure that pathologists in practice sustain and improve diagnostic cytopathology and provide material for training young pathologists . | Introduction
Materials and Methods
Design/Site
Ethical consent
Sample collection and reporting
Data handling
Results
Discussion
Conclusion
Financial support and sponsorship
Conflicts of interest |
PMC4433968 | gait initiation ( gi ) is the transition from an upright stance to a steady - state gait .
it is
a movement that can be completed in the absence of plantar surface feedback1 and is presumably preprogrammed2 . in quiet stance , the inhibition of the
soleus ( sol ) and the activation of the tibialis anterior ( ta ) result in a dorsiflexion
torque , forward propulsive force , and a backward movement of the center of pressure
( cop)3,4,5 . the cop and center of mass ( com ) become
decoupled , and a fall forward is initiated .
in addition , the stepping - limb hip abductors
create movement of the cop toward that limb6 . thus , muscle activity at the ankle and hip propels the com forward
and toward the intended stance limb to allow stepping - limb toe - off ( to ) and the first
step7,8,9 .
of interest has been the
modulation of ground reaction forces ( grfs ) and muscle activity before stepping - limb to with
changes in intended velocity . as would be expected ,
the posterior displacement of the cop
and the propulsive force have been shown to increase , and the time to this peak force
decrease , with an increase in the intended velocity of gi4 , 10,11,12,13 .
the duration of both stepping and stance ta activities increase
with the velocity of gi4 , 5 , 14 .
significant
correlations between kinetic events , time to stepping - limb to , amplitude and duration of ta ,
and intended velocity of gi have been reported5
. similar patterns of modulation seem to occur when stepping to a new
height15 or over an obstacle7 , 16,17,18,19,20,21 .
previous studies have described the velocity - dependent characteristics of gi . however , no
single study has reported electromyogram ( emg ) and grf data of both the stance and stepping
limbs and their relation to the intended velocity of gi .
one reason for this has been the
use of a single force plate12 , 13 , 22,23,24,25,26,27 and the other is that emg activity was not measured1 , 28,29,30,31,32,33,34,35,36,37 in many studies .
information on both the stepping and stance limbs is valuable in a rehabilitation setting
where accurate patient education and documentation of change is required .
this information
has uses when an asymmetrical lower - limb function is caused by injury or disease , and the
patient not only has to generate the required forces , but also strategize as to which limb
should be the stepping limb .
for example , time to stepping - limb to has been shown to be
slower in stroke patients when the hemiplegic limb is the stance limb28 , yet others have reported the initiation velocity to be
greater if the hemiplegic limb is the stance limb30 ,
35 .
tokuno and eng35 reported decreased anteroposterior ( a - p ) forces from the
hemiplegic limb , regardless of whether it was leading or trailing , and questioned whether
the hemiplegic limb should be the stepping or trailing stance limb .
those with a lower - limb
amputation tend to initiate gait with the prosthetic limb .
the force generated by this limb
is low , and a rescaling of the force of the sound limb is necessary for an adequate first
step length36 .
information on the interaction of the stance and stepping limbs during gi could help guide
rehabilitation decisions .
moreover , how this interaction changes with the velocity of gi is
also important , given that an increase in gait velocity is a positive indicator of
recovery38,39,40,41 . if the velocity and strategy employed to initiate gait is
important in rehabilitation , the collection of more comprehensive data from healthy subjects
is required .
the purpose of this study was , therefore , to investigate the effects of
accuracy constraints ( targets ) placed on the stepping - limb heel - strike ( hs ) on muscle
activities and grf during gi .
we hypothesized that the velocity of gi and stepping over an
obstacle would decrease when accuracy constraints were placed on the stepping - limb hs , and
our results show this decrease was the result of the modulation of grfs and muscle
activities of both the stance and stepping limbs before stepping - limb to .
the study sample consisted of twenty healthy participants ( ten males and ten females ; mean
age , 29.2 2.9 years ; range , 2434 years ) with no known neurological or orthopedic
deficits .
the study was approved by the university institutional review board , and all
participants signed an informed consent form . the study participants characteristics are
summarized in table 1table 1.subject characteristicsgendernumberage ( years)height ( cm)weight ( kg)male1030.2 ( 2.78)174.8 ( 5)71.2 ( 3.79)female1028 ( 2.69)164.6 ( 5.24)53.4 ( 3.09)total2029.2 ( 2.9)169.9 ( 7.3)62.8 ( 9.72 ) .
surface electrodes were applied to the center of the muscle bellies of the ta and sol of
the stance and stepping limbs . each recording electrode consisted of two silver - silver
chloride 1-cm - diameter electrodes , embedded in an epoxy - mounted preamplifier system ( 35 ) ,
and their centers were spaced 2 cm apart .
the emg signals were high - pass filtered ( 20 hz4 khz ) using a zero - lag
filter ( therapeutics unlimited , iowa city , ia , usa ) to remove movement artifacts and
full - wave rectified online .
the final amplification was 10 k. the collection and processing
of emg data followed seniams recommendations42 . two force platforms ( advanced mechanical technology , inc . ,
newton , ma , usa ) , embedded in a
level walkway ( 5 m length , 1.22 m width ) , measured the grfs of the stance and stepping
limbs .
the grfs ( kinetic data ) were collected at 1,000 hz and later filtered with a 4th
order , butterworth , zero lag filter , with a cutoff frequency of 50 hz .
foot switches ( b
& l engineering , los angeles , ca , usa ) were placed in the shoes to measure the hs of the
stepping limb .
the emg and foot switch signals were synchronized with the kinetic data to
simultaneously start both the emg and foot switch data capture .
both the emg and the force
platform data were recorded simultaneously on a personal computer at a sampling rate of
1,000 hz for 4 s ( biopac systems , goleta , ca , usa ) .
the test conditions included the use of
an obstacle ( 10 cm height , 10 cm depth , and 140 cm width ) made of wood .
for each trial , participants stood in a predetermined position with each foot on a force
platform .
participants were asked to begin walking or stepping over the obstacle , at a fast
speed , with the limb they naturally preferred to initiate gait with , after receiving the
verbal cue go , and to continue to walk a minimum of three steps . before the experimental
trials , the average position of the stepping hs was determined for each subject using a
video analysis . for half of the experimental trials ,
a 3-cm - diameter target was placed on
the ground to dictate the position and accuracy of the stepping - limb hs .
all participants
performed two practice trials to familiarize themselves with the experimental procedure .
participants completed 10 trials under each of the following four conditions : gi , gi to the
target , stepping over the obstacle , and stepping over the obstacle to the target .
all participants were required to wear flat - soled shoes normally used for everyday
walking or sports activities .
two - way analysis of variance ( initiation condition accuracy ) for repeated measures was
performed to determine the main and interaction effects of a - p grfs , muscle response , and
temporal events .
post hoc analysis using turkey s hsd was used to determine the
between - group mean differences if the analysis of variance found a significant main effect
or interaction of initiation condition accuracy
the independent variables were initiation condition ( gi and stepping over an obstacle ) and
accuracy ( target , no target ) .
the dependent measures included emg amplitude , slopes and peak
of the propulsive grf , and timing events of gi .
the amplitude of stance- and stepping - limb
ta emg was determined between the onset of movement and time to peak a - p grf of the stepping
limb ( taemg1 ) , and between time to stepping - limb peak a - p grf and stepping - limb to ( taemg2 ) .
the amplitude and duration of stepping - limb sol activity were also calculated . the onset and
offset of sol activity
time to stepping - limb to ,
stepping - limb hs , stance - limb heel - off , and stance limb to were also determined .
timing data
were referenced from the onset of movement , which was defined as the first detectable onset
of force platform activity .
an investigator blinded to the experimental trials performed all
determinations of muscle activity timing and change in grf .
the temporal events are shown in table
2table 2.means ( sd ) of temporal events ( ms)dependent variablesgait initiationsteppingno targettargetno targettargetstepping limb peak fx340 ( 49)423 ( 42)329 ( 47)368 ( 73)stepping limb toe - off511 ( 76)613 ( 81)475 ( 69)548 ( 91)stepping limb heel - strike890 ( 74)1008 ( 86)968 ( 71)1071 ( 108)stance limb toe - off1024 ( 76)1219 ( 156)1093 ( 85)1257 ( 153)values represent mean standard deviation ( sd ) . * significant main effect of target
( p<0.01 or p<0.05 )
. significant main effect of initiation condition
( p<0.01 or p<0.05 ) . significant interaction ( p < 0.05 ) .
significant difference between conditions ( p<0.01 or
p<0.05 ) .. although there was an interaction ( f(1 , 8) = 6.47 , p <
0.05 ) of the time to stepping - limb peak a - p grf ( the target condition for stepping over an
obstacle was greater than that for gi ) , the mean times to peak a - p grf in the target
conditions were significantly greater than in the no - target conditions .
the time to
stepping - limb to showed significant main effects of both the initiation and target
conditions ( f(1 , 8) = 10.87 , p
< 0.05 and f(1 , 8) =
19.29 , p < 0.01 , respectively ) .
time to stepping - limb to was longer in stepping to the
target but shorter in the obstacle condition .
the times to stepping - limb hs and stance - limb
to showed main effects of the initiation and target conditions ( f(1,7 ) =
7.83 to 18.24 , p < 0.01 to p < 0.05 ) . the times increased in both the target and
stepping conditions .
values represent mean standard deviation ( sd ) . * significant main effect of target
( p<0.01 or p<0.05 ) .
significant main effect of initiation condition
( p<0.01 or p<0.05 ) . significant interaction ( p < 0.05 ) .
the amplitude of the ta emg was determined from both the onset of force platform activity
to stepping - limb peak a - p grf ( taemg1 ) , and from stepping - limb peak a - p grf to to of the
stepping limb ( taemg2 ) .
there was a significant main effect of target on the stepping - limb
taemg1 ( f(1 , 8) = 7.75 , p < 0.05 ) which showed decreased amplitudes in
the target conditions ( table 3table 3.means ( se ) of emg dependent variables ( ms.v)dependent variablesgait initiationsteppingno targettargetno targettargetstance limb taemg1340 ( 49)423 ( 42)329 ( 47)368 ( 73)stance limb taemg2511 ( 76)613 ( 81)475 ( 69)548 ( 91)stepping limb taemg1890 ( 74)1008 ( 86)968 ( 71)1071 ( 108)stepping limb taemg21024 ( 76)1219 ( 156)1093 ( 85 ) 1257 ( 153)values represent mean standard deviation ( sd ) .
however , there was no significant effect on the taemg2 of the stepping
limb .
taemg : tibialis anterior
electromyogram . * significant main effect of target ( p<0.01 or p<0.05 ) .
significant interaction ( p<0.05 ) .
stance taemg1 showed a significant interaction ( f(1 , 8) = 12.12 , p <
0.05 ) ( table 3 ) .
stance taemg1 was greater in gi
than in the stepping over the obstacle with no target , but showed no difference between the
target conditions .
stance taemg2 was significantly greater in the no - target conditions than
in the target conditions ( f(1,8 ) = 13.31 , p < 0.01 ) ( table 3 ) .
the mean data show that except for taemg2 of the
stepping limb , the ta amplitude was clearly greater in the no - target conditions .
the
stepping - limb sol emg amplitude was similar in all the conditions ; however , its duration was
24 ms greater in the target conditions ( f(1 , 8) = 8.05 , p < 0.05 ) .
there was a significant main effect of target on both the stepping - limb peak a - p grf
( f(1 , 8) = 30.88 , p < 0.001 ) and the slope to stepping - limb peak a - p
grf ( f(l , 8) = 18.2 , p < 0.01 ) .
values were greater in the no - target
conditions ( table 4table 4.means ( sd ) of peak ( % bw ) and slope ( % bw / s ) force plate dependent
variablesdependent variablegait initiationsteppingno targettarget no targettargetstepping limb peak fx39.6 ( 6)26 ( 7.8)40.8 ( 9.6)25.7 ( 7.2)stepping limb slope fx125.2 ( 42)62 ( 22.5)120.6 ( 26.4)75.6 ( 32.3)stance limb peak fx81.8 ( 13)52.6 ( 12)66.3 ( 15)47.5 ( 13)stance limb slope fx318.2
( 63)208.2 ( 53)313.1 ( 87)192.5 ( 54)values represent mean standard deviation ( sd ) . fx : anteroposterior ground reaction
force ; % bw : percent body weight ; % bw / s : percent body weight per second .
there was no significant difference between the gi and stepping over the
obstacle conditions for either dependent variable .
although there was a significant
interaction of the stance - limb peak a - p grf ( f(1 , 8) = 13.64 , p < 0.01 ) ,
there were also main effects of the stepping ( f(1 , 8) = 30.07 , p < 0.01 )
and target ( f(1 , 8) = 24.80 , p < 0.01 ) conditions .
stance - limb peak a - p
grf was greater in the gi and no - target conditions ( table 4 ) .
the slope to peak a - p grf showed a main effect of target only and the
slope was greater in the no - target conditions ( f(1 , 8) = 22.27 , p <
fx : anteroposterior ground reaction
force ; % bw : percent body weight ; % bw / s : percent body weight per second . *
significant interaction
( p<0.05 ) . significant difference between conditions ( p<0.01 or
p<0.05 ) .
a unique relation exists between force profiles and muscle activity in different movement
strategies .
an increase in the degree of accuracy of a task causes a decrease in the
velocity of movement43,44,45 .
therefore ,
there must be a change in strategy with an accuracy constraint , but only if the accuracy
demands are sufficiently sensitive to dictate movement velocity46 .
furthermore , on the basis of previous studies of gi and
stepping7 , 8 , it could be concluded that by manipulating the limb trajectory , for
example , stepping over an obstacle as opposed to gi , forces would remain invariant . by
investigating
the component of gi thought to dictate the velocity of movement , better data
on how forces are modulated during voluntary movement from a quiet stance could be
provided .
the current study investigated both gi and stepping over an obstacle and the interaction of
the stance limb with the stepping limb , when target constraints were placed on the
stepping - limb hs .
the results show that the initiation velocity increase in no - target
conditions was due to the modulation of the stance- and stepping - limb grfs and the
corresponding increase of the ta activity in both limbs before stepping - limb to .
this was
achieved by significantly increasing the stepping- and stance - limb taemg1 in the no - target
conditions .
it seems , therefore , that taemg1 and the slope to stepping - limb peak a - p grf
contributed to the intended velocity of initiation ( gi or stepping over an obstacle ) . in the present study ,
stepping - limb to marks the first significant event , and in the no - target condition it
occurred approximately 90 ms earlier than in the target conditions .
this decrease in time
was , in part , due to a 15% ( 61 ms ) decrease in time to stepping - limb peak a - p grf of the
no - target conditions .
this decrease in time to peak a - p grf was due to a 78% increase in
slope to peak a - p grf that resulted in a 55% increase in peak a - p grf of the stepping limb .
given the relative changes in time and slope , it seems that subjects modulated the rate of
increase of force and kept the time to peak force relatively constant to achieve the
increase in the peak acceleration force of the stepping limb . as the ta controls the
backward movement of the cop3 , 4 , 10 , 47
, there was a significant increase in stepping - limb taemg1
in the no - target conditions .
the relation between the backward movement of the cop10 , 48
or ta activity and gait velocity has previously been recognized . of interest ,
the time from
stepping - limb to to stepping - limb hs remained invariant for both gi and stepping over the
obstacle , a finding supported by the earlier data of brenire and colleagues3 , 10 , 47 . in the present study , the mean difference
between the accuracy conditions was only 23 ms .
furthermore , there was no main effect of the
amplitude of stepping - limb taemg2 or sol , and it has been previously recognized that sol
creates heel rise in preparation for the first step but does not contribute toward gait
velocity48 .
the contribution of the
stepping limb to the velocity of initiation ( gi or stepping ) seems , therefore , to be
determined by taemg1 and the slope to stepping - limb peak a - p grf48 . for the stance limb , there was a significant target effect on both taemg1 and taemg2 . in
both stepping over the obstacle and gi ,
the amplitude of taemgi was far greater than that of
taemg2 , and only minimal acceleration force was generated until the slope to stance - limb a - p
grf appeared .
the onset of this slope coincided with the peak a - p grf of the stepping limb ,
our selected time division between taemg1 and taemg2 . during this phase of gi and stepping
over an obstacle ,
this transition
from unloading to loading occurs slightly before peak a - p grf of the stepping limb . given
the greater amplitude of taemg1 of the stance limb but smaller peak force
, we consider that
the smaller a - p grf is probably related to the unloading of the stance limb , and slope to
stance - limb peak a - p grf to the loading of that limb before stepping - limb to .
the slope to
stance - limb peak a - p grf was the same for gi and stepping over the obstacle , but peak a - p
grf was less when stepping over the obstacle .
peak a - p grf coincides with stepping - limb to ,
and the earlier to for stepping ( and therefore smaller peak a - p grf ) is thought to be
related to the trajectory of the stepping limb7 . because of the greater trajectory , the time to stepping - limb hs
from to was longer for stepping over the obstacle .
the slope to stance - limb peak a - p grf increased in the no - target condition .
there was an
increase of approximately 50% in the no - target condition , with only a 14% difference in time
to peak a - p grf .
the increase in the slope to peak a - p grf of the stepping limb was 78% . for
the stance limb
, these data differ from a previous report8 that used both a large and a small target .
however , it should be
noted that the target size used in the present study was 50% smaller than the small target
used in the previous study8 . in an
isometric plantar flexion task , the slopes to peak forces
were also found to be the same
once the target size had reached a critical level .
the fact
that an explicit instruction to increase the velocity of gi ( as opposed to accuracy
constraints ) resulted in an increase in the slope to stance - limb peak a - p grf supports this
interpretation7 .
because the experiment was
only conducted on a relatively small sample of healthy young adults ( gi in a
non - pathological setting ) , our findings can not be generalized to the population with a
pathological gi .
moreover , much of the pathological gi condition is asymmetrical , yet data
is only presented for right foot gait initiation .
furthermore , data was only collected at a
fast speed , and we used experimental manipulations such as a target and obstacle - crossing
instead of asking the participants to simply initiate their gait at slow , preferred , and
fast speeds . in conclusion , this study investigated changes in emg and grf responses during gi at
different intended velocities of initiation .
the velocity of gi and stepping over an
obstacle increased when no accuracy constraints were placed on the stepping - limb hs .
this
increase was due to the modulation of both the stance- and stepping - limb grfs before
stepping - limb to .
the time to these peak forces remained relatively constant ( 100-ms less in
the no - target conditions ) .
that is , the slope to the stepping- and stance - limb peak a - p grf
was modulated , whereas the time to peak a - p grf remained relatively constant .
this notion is
supported by the strong correlations between the slope to stepping - limb a - p grf and time to
stepping hs ( r = 0.84 ) , and the more modest correlation ( r
= 0.64 ) of the slope to stance - limb peak a - p grf .
furthermore , there were no differences in
the slopes to either stepping- or stance - limb peak a - p grf between gi and stepping over the
obstacle .
this concurs with upper - extremity studies showing that the distance moved does not
affect the rate of increase of force . on the basis of these data , gi and/or stepping over an
obstacle
this would be preferable in
upper - extremity experiments , for which the subject sits and the extremity is stabilized .
voluntary movement from an upright stance may be useful for assessing changes in performance
after rehabilitation .
hemiparetic patients show an asymmetric gait pattern and prefer initiating gait with the
affected leg to utilize the higher stability of the supporting limb .
however , hemiparetic
patients are encouraged to initiate gait with the intact limb to increase the weight - bearing
ability of the affected leg .
similarly , unilateral amputees in rehabilitation training are
also encouraged to lead with the intact limb when stepping up , and with their prosthetic
limb when stepping down49 .
however , a
recent study30 questions whether
initiating the gait of hemiplegic patients with the intact limb is related to a higher risk
of fall . the movement pattern of the cop and com is altered in patients with hemiparetic
stroke when initiating gait with the unaffected leg
. this difference may be attributable to
the higher incidence of falls when initiating gait with the unaffected leg due to the
altered movement pattern of the cop and com .
the present study provides insight into the
working mechanisms of the stepping and stance limbs and shows the clear need to further
investigate whether the intact or affected limb should be used to initiate gait during
rehabilitation and prosthetic training . | [ purpose ] this study investigated the effects of accuracy constraints ( targets ) placed on
the stepping - limb heel - strike ( hs ) on the electromyogram ( emg ) and ground reaction forces
( grfs ) during gait initiation . [ subjects and methods ] twenty healthy subjects ( 29.2
2.9 years ) were asked to begin walking or stepping over a 10-cm - high obstacle at a fast
speed .
a 3-cm - diameter target was placed on the ground to dictate the position and
accuracy of the stepping - limb hs .
[ results ] the results showed that the initiation
velocity increase in the no - target conditions was due to modulation of the stance- and
stepping - limb grfs and a corresponding increase in the tibialis anterior ( ta ) activities
of both limbs before stepping - limb toe - off .
this was achieved by significantly increasing
the stepping- and stance - limb taemg1 ( determined between the onset of movement and time to
peak anteroposterior ( a - p ) grf of the stepping- and stance- limb ) for the no - target
conditions .
it seems , therefore , that taemg1 and the slope to stepping - limb peak a - p grf
contributed to the intended velocity of initiation .
[ conclusion ] these data indicate that
gait initiation and/or stepping over an obstacle may prove to be tasks by which motor
control can be measured .
the present study provides insight into the working mechanisms of
the stepping and stance limbs and shows a clear need to further investigate whether the
intact or affected limb should be used to initiate gait during rehabilitation and
prosthetic training . | INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION |
PMC3335422 | bovine colostrum is an opaque white liquid which is a natural source of nutrition . five main components in bovine colostrum are water , fats , proteins ( e.g. , caseins , albumins , and globulins ) , sugars ( essentially lactose ) , and mineral salts . since bovine colostrum spoils easily , it is sometimes processed into dairy products .
the most durable form of bovine colostrum is bovine colostrum powder , which is produced by the dehydration of raw bovine colostrum . in developing countries ,
casein is the major protein in cow 's milk , and comprises about 80% of the total protein content of which the rest , 20% , are the whey or serum protein [ 1 , 2 ] .
whey proteins contain alphalactalbumin ( -la ) , betalactoglobulin ( -lg ) , immunoglobulin , lactoferrin , and bovine serum albumin .
alphalactalbumin ( -la ) and betalactoglobulin ( -lg ) are two kinds of whey protein in bovine colostrum and specifically produced in the mammary epithelial cells only during lactation . as a monomer , alphalactalbumin ( -la ) and betalactoglobulin ( -lg ) strongly bind calcium and zinc ions and may possess bactericidal or antitumor activity . high - effect capillary electrophoresis ( hpce ) ,
this technology already has applied to protein and aminofacid broadly for many years . due to the appealing advantages in separation efficiency and resolution , small amounts of solvent and sample , ce
have rapidly been applied for the assay in the protein separation field . for a long time , the capabilities of matrix - assisted laser desorption / ionization ( maldi ) mass spectrometry for the rapid evaluation of proteins have been proved .
furthermore , it has also been tested to be a valid tool in the dairy industry .
although the technique does not determine the exact concentrations of bovine colostrum proteins , the relative proportions of several main bovine colostrum proteins can be identified . in this experiment ,
another portion was separated with cpe method and detectable differences in their maldi spectra were found .
the present work describes alphalactalbumin ( -la ) and betalactoglobulin ( -lg ) in the rehydration of bovine colostrum powder were successfully separated by cloud point extraction using a nonionic surfactant triton x-114 .
the separation was mainly based on their contrasting hydrophobicities , and our main goal was to achieve the highest concentration of alphalactalbumin ( -la ) and betalactoglobulin ( -lg ) in the surfactant - poor phase obtaining high efficiency of cpe method .
since separation of these proteins using cpe depends on several variables , such as type and concentration of surfactant , ph , net charge , size and sample volume [ 6 , 7 ] , the optimization design was concluded in .
the matrix - assisted laser desorption ionization - time of flight mass spectrometry ( maldi - tof ms ) was used to confirm the separation of alphalactalbumin ( -la ) and betalactoglobulin ( -lg ) from the rehydration of bovine colostrum powder .
bovine colostrum powder ( sunlife bovine colostrum powder ) was purchased in a local chinese market . all the water used in
the experiment was milli - q deionized water ( millipore , billerica , ma , usa . ) .
acetonitrile ( hplc grade ) was obtained from sk chemicals ( ulsan , korea ) .
the -cyano-4-hydroxycinnamic acid ( chca ) was obtained from applied biosystems ( foster city , ca , usa ) .
trifluoroacetic acid ( tfa ) was obtained from sigma - aldrich ( steinheim , germany ) .
nacl , isooctylphenyl ether ( triton x-114 ) , and sucrose were obtained from sigma - aldrich ( steinheim , germany ) .
the factors were triton x-114 concentration , sample volume , nacl and sucrose concentration , and ph . the surfactant solution was prepared with 10 mmol / l tris - hcl ( ph 7.4 ) , 150 mmol / l nacl , and 6% ( w / v ) sucrose .
200 l bovine colostrum sample was added to the surfactant solution , and the solution was homogenized again .
the ce was performed using a p / ace mdq capillary electrophoresis system ( beckman coulter , fullerton , ca , usa ) under a normal polarity separation mode . a capillary ( beckman coulter , fullerton , ca , usa ) with 50 cm effective length ( 60 cm total ) and
an inner diameter ( i.d . ) of 75 m was used with an applied voltage of 25 kv at 20c .
prior to use the capillary for the first time , a rinse for 1 min ( 30 psi ) with 0.1 mol / l hcl was performed .
a rinse for 10 min ( 30 psi ) with the running buffer was performed then equilibrate the capillary for 10 min ( 25 kv ) .
finally a rinse for 10 min ( 30 psi ) was performed the temperature of the separation capillary column was thermostated at 20 c. among each runs , the capillary was rinsed with 0.1 mol / l hcl for 0.5 min and then with the electrophoresis buffer for 1.5 min .
the separation buffer was 50 mm citrate buffer ( ph 3.0 ) , and the applied voltage is 25 kv . the electrophoresis buffers were filtered through 0.22 m filter before use .
the samples were dissolved in 1 ml of buffer and filtered [ 0.22 m ( millipore ) ] for ce analysis .
samples were injected into the capillary using pressure at 0.5 psi for 10 s. the absorbance was measured at a wavelength of 214 nm for the detection of protein . to prepare a saturated solution of chca matrix : 10 mg of the dry chca matrix
was added to a tube containing 1 ml solution of water / acetonitrile/0.1% tfa ( 4/5/1 , v / v / v ) .
0.5 l of the sample / matrix ( 1/5 , v / v ) mixture was spotted per well on a stainless steel maldi sample plate .
all the maldi measurements were performed on a 4700 proteomics analyzer ( applied biosystems , foster city , ca , usa ) equipped with a nd : yag laser ( 355 nm ) operating at 200 hz .
ions formed by a pulsed laser beam were accelerated to 20 kilo volts in positive ion linear mode with a 740 ns delay times .
the acquisition method was set as follows : 60 subspectra were acquired on one spot and 25 shots were averaged per subspectrum , so one maldi spectrum was the accumulated spectra of a total of 1500 shots per spot . for each sample ,
five spots were equally spotted and examined , and the acquisition method ran three times per spot .
the final data were the trimmed means of the results of these 15 parallel tests as a whole .
this instrument operating parameters were optimized for ion peaks corresponding to apomyoglbin ( horse , m / z 16952 ) , thioredoxin ( e. coli , m / z 11674 ) , and insulin ( bovine , m / z 5734 ) .
to highlight the extraction of whey proteins from bovine colostrum and their separation from casein proteins by cloud point extraction , ce was used to identify -lactalbumin and -lactoglobulin extracted from the rehydration of bovine colostrum powder .
the electropherograms of standard and cloud point extraction sample from the rehydration of bovine colostrum powder were shown in figures 1(a ) and 1(b ) and figure 1(c ) , respectively .
the retention time of -lactalbumin and -lactoglobulin was about 10 and 15 min under the optimal separation condition . in figure 1(c ) , there were only two whey protein peaks ( -lactalbumin and -lactoglobulin ) .
it was proved right with maldi - tof ms in the next experiment . from figure 1(c ) , it is also proved the efficiency of the extraction procedure is successful . to highlight the extraction of whey proteins from bovine colostrum and their separation from casein proteins by cpe , maldi - tof ms to
intact samples of the two protein fractions was applied . a simple sample treatment that only involved dilution of precipitated proteins was performed . figure
2(a ) displays the mass spectra obtained , and it summarizes different proteins identified in the surfactant poor and water phases .
the theoretical molar masses are in agreement with those previously described for whole cow bovine colostrum protein analyses by maldi - tof ms .
the mass spectra of figure 2(a ) show ions of ca.m / z11958 and 23919 corresponding to the -casein and -casein , respectively .
those ions in figure 2(b ) of higher m / z values correspond to -lactalbumin ( m / z14152 ) and -lactoglobulin ( m / z18318 ) .
finally , the ion of ca.m / z 14152 in figure 2(b ) probably corresponds to species originating from lactose addition to -lactalbumin .
the main proteins identified in the surfactant poor phase ( figure 2(b ) ) were -lactalbumin and -lactoglobulin , which are fractions of the whey proteins . besides such proteins , -casein and -casein were also detected in the water phase .
in fact , both last proteins also present amphiphilic character [ 4 , 5 ] , corroborating their presence in the surfactant poor phase .
cpe was efficiently employed to cow bovine colostrum samples being able to extract and separate whey from casein proteins using only 200 l of sample volume , sample volume without any sample pretreatment .
( 1% , w / v ) , 150 mmol / l nacl , and sucrose ( 6% , w / v ) were used at ph 7.0 for protein separations . at such conditions ,
good partition coefficient was obtained , allowing the separation of -lactalbumin and -lactoglobulin ( present in the surfactant poor phase ) from casein proteins ( present in the water phase ) in only 15 min and with minimum costs , indicating that the proposed factorial design was successfully applied in the experimental domain employed and the main objective of this work attained .
although the partition coefficient was obtained , it is important to comment that some hydrophobic proteins were achieved in the surfactant poor phase and vice versa , as demonstrated through maldi - tof ms analysis .
after cloud point extraction , the capillary electrophoresis was used to check the efficiency of the extraction procedure .
the results had been effectively confirmed by the characterization with matrix - assisted laser desorption ionization time of flight mass spectrometry ( maldi - tof ms ) . finally , the adopted strategy could also be used for separating those low - abundant proteins in bovine colostrum after properly optimizing the cpe method for such task . | alphalactalbumin ( -la ) and betalactoglobulin ( -lg ) in the rehydration of bovine colostrum powder were successfully separated by cloud point extraction using a nonionic surfactant triton x-114 .
the effects of different factors , including the surfactant concentration , sample volume , electrolyte , and ph were discussed . the optimized conditions for cloud point extraction of alphalactalbumin ( -la ) and betalactoglobulin ( -lg )
can be concluded that the best surfactant is 1% ( w / v ) triton x-114 , 200 l of sample volume , 150 mmol / l nacl , and 6% ( w / v ) sucrose .
after cloud point extraction , the capillary electrophoresis is used to check the efficiency of the extraction procedure .
the results had been effectively confirmed by the characterization with matrix - assisted laser desorption ionization time of flight mass spectrometry ( maldi - tof ms ) . | 1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion |
PMC3894757 | it is recognized that teeth with furcation involvement may undergo a more extensive and
rapid clinical probing attachment loss and are lost with greater frequency than
single - rooted teeth .
although
regeneration of periodontal tissue is the ultimate goal of periodontal therapy ,
regeneration is not a predictable healing outcome after conventional treatment . with the advent of regenerative techniques ,
a new concept of periodontal treatment based
on the principle of cellular selection and guided tissue regeneration , permits the treatment of teeth with
furcation lesions by conservative surgical methods .
various studies reported gains in
horizontal and vertical clinical attachment , with complete closure of the furcation in
up to 67% of the cases using different types of mechanical barriers . despite positive results based on reports of substantial attachment
gain and bone fill of furcations , guided - tissue regeneration ( gtr ) is technique
sensitive and could be associated with increased postoperative problems . a major
drawback to expanded polytetrafluoroethylene
( eptfe ) and other non - resorbable membranes
is the need for their removal after 4 to 6 weeks , often with a surgical procedure that
adds to patient morbidity and may disturb the healing tissue .
bone replacement grafts have achieved similar results to gtr , demonstrating a 55%
overall improvement , with either complete or partial furcation fill compared to 52% for
gtr
. the use of bioactive glass
( bg ) when compared to an eptfe barrier may produce similar clinical results .
when used in a variety of periodontal
and oral surgical applications , bg has shown positive clinical and histological
outcomes . in the past decade ,
several cytokines , or growth factors , have received attention
because they can regulate migration , attachment , proliferation and/or differentiation of
periodontal ligament cells into hard tissue cells and have been shown to enhance
regeneration with in vivo experiments .
a convenient
technique to obtain transforming growth factor
( tgf ) and platelet - derived growth factor ( pdgf ) is by preparing
autologous platelet - rich plasma ( prp ) .
pdgf has shown to exert a favorable effect on periodontal
regeneration as measured by gain in clinical attachment and defect fill in
humans .
considering that the most favorable outcome of periodontal regenerative procedures in
furcations lesions has been achieved with a combination of graft material and
gtr , the goal of the present study was to investigate , histologically
and histometrically , the potential adjunctive benefits of prp when used with gtr and bg
in the treatment of class ii furcation lesions .
nine adult female mongrel dogs were included in the experiment ( mean weight=15 kg ) .
the
surgical procedures were performed under general anesthesia with intravenous injection
of a 3% sodium pentobarbital solution ( 0.5 ml / kg ) .
the maxillary and mandibular second
and fourth premolars had been bilaterally extracted previously and the extraction sites
had been allowed to heal for 2 months .
bilateral class ii furcation lesions were
surgically created at the buccal aspect of mandibular third premolars . in order to
create the defects
osteotomy was done in the furcation area with high - speed diamond burs with
constant irrigation and with nos . 1 and 2 oschenbein chisels
. the furcations lesions
were standardized with a millimeter probe and measured 5.0 mm in the apico - occlusal
direction and 2.0 mm in the buccolingual direction ( figure 1 ) , following a technique previously described .
clinical aspect of class ii furcation defect ( 5x 2 mm ) the defects were filled with gutta - percha to induce an inflammatory response due to
plaque accumulation for a period of one month ( figure
2 ) .
after this period , scaling and root planing was performed and a regimen of
daily brushing plus topical application of 0.1% chlorhexidine gluconate was instituted
for 7 days prior to the surgical procedures
. clinical aspect of class ii furcation defect filled with gutta - percha in the same day of the treatment of the mandibular defects , similar defects were created
at the buccal aspect of maxillary third premolars and were exposed to plaque
accumulation for 1 month .
forty - five days after this surgical intervention , the
maxillary defects were assigned to receive the same treatments .
three tubes
( 5 ml ) , containing 0.5 ml of 3.2% sodium citrate solution as an anticoagulant , were
drawn from each dog .
the blood was thus separated into 3 basic parts : red blood cells ( at the
bottom of the tube ) , platelet enriched plasma ( a discrete grey line in the middle of
the tube ) and platelet poor plasma ( at the top of the tube ) .
the portion
corresponding to the platelet poor plasma was discarded from each tube and the
remaining content was centrifuged again at 1,200 rpm for 15 min .
four hundred
microliter of the middle portion , corresponding to the platelet enriched plasma , were
pipetted from each tube . in order to obtain the gel ,
30 l of 10% calcium chloride
was added to prp and heated in a water bath at 37c .
mucoperiosteal flaps were carefully reflected on the buccal aspect of the
experimental sites , the granulation tissue was removed by curettage , and the exposed
root surface was instrumented . on the root surface ,
the base of the defect was marked
with curettes to establish a landmark for the histomorphometric analysis .
each
contralateral defect in each animal was randomly assigned to one of the following
treatments : gtr+bg ( perioglas , us biomaterials , alachua , fl , usa ) - control group : bg particles
were applied filling the defect and absorbable membrane ( resolut xt
gore - tex , gore associates , flagstaff , az , usa ) was adapted to the defect ; gtr+bg+prp - test group : bg was incorporated to the prp ( 1:1 ) and this assemblage was
immediately taken to a water bath at 37c what enabled the formation of a mixture of
the gel and the bg graft .
this mixture was applied filling the defect and an
absorbable membrane ( resolut xt gore - tex , gore associates ) was adapted to
the defect .
primary , tension - free wound closure was accomplished following defect treatment , with
the gingival flaps positioned and sutured with interrupted sutures ( vicryl , ethicon
inc , so jos dos campos , sp , brazil ) in order to cover the membranes . immediately
after the surgeries , an intramuscular injection of penicillin ( 1.5 ml - 150,000 iu )
was given to the animal .
forty five days after the mandibular treatment , the maxillary defects ( 2 defects/
animal ) were randomly assigned to receive the same treatments ( group a and group
b ) .
postoperative plaque control was performed by irrigation with a solution of 1%
chlorhexidine gluconate ( every other day ) .
dogs were sacrificed 90 days after the
first treatment , under general anesthesia , with a perfusion of 10% neutral formalin
solution .
they were decalcified in a solution of equal parts of 50%
formic acid and 20% sodium citrate for 5 months .
the decalcified specimens were
washed in running water , dehydrated and embedded in paraffin .
mesiodistal 7-m - thick
sections were obtained from the tissue blocks longitudinally , in a buccolingual
direction , in a way to obtain a panoramic view of the furcation area .
the sections
were stained with hematoxylin and eosin . for histometric and descriptive histological analyses , 5 sections
per site representing the middle portion of the defect ( 30 m
apart )
were used to obtain the mean for each parameter in each dog , measured with an
image analysis system ( image pro plus version 3.0 , media cybernetics , silver spring ,
md , usa ) .
the following linear measurements ( mm ) were obtained in the mesial and distal roots
under the furcation :
defect extension ( de ) : linear extension of the total root surface
between the two notches ;
new cementum ( nc ) : linear extension of the root surface covered by
new cementum ;
connective tissue adaptation ( ca ) : extension of the root surface
covered by connective tissue without cementum .
new bone ( nb ) : measured from a horizontal line connecting both
notches ( apical limit of the defect ) to the most coronal extent of new bone .
the following
parameters ( mm ) were obtained by the point counting technique :
mineralized bone area ( mba ) : portion of the defect area filled with
mineralized bone tissue ;
residual bg particle area ( rbg ) : portion of the defect area filled
with bg remaining particles ;
non - mineralized bone area ( nmba ) : portion of the defect area filled
with non - mineralized tissue ; the measurements were performed using a microscope ( zeiss axioskop 2 , carl zeiss
instruments , gottingen , lower saxony , germany ) with a 1.25/.035 objective associated
with a video camera / computer / software ( image pro plus version 3.0 , media
cybernetics ) .
the mean
values for all groups were determined using the individual means from the 9 dogs .
after testing the assumptions confirming normal distribution , two - way anova was
carried out to check any significance among the different treatment groups , any
significance among the different time periods and the interaction between them .
three tubes
( 5 ml ) , containing 0.5 ml of 3.2% sodium citrate solution as an anticoagulant , were
drawn from each dog .
the blood was thus separated into 3 basic parts : red blood cells ( at the
bottom of the tube ) , platelet enriched plasma ( a discrete grey line in the middle of
the tube ) and platelet poor plasma ( at the top of the tube ) .
the portion
corresponding to the platelet poor plasma was discarded from each tube and the
remaining content was centrifuged again at 1,200 rpm for 15 min .
four hundred
microliter of the middle portion , corresponding to the platelet enriched plasma , were
pipetted from each tube . in order to obtain the gel ,
30 l of 10% calcium chloride
was added to prp and heated in a water bath at 37c .
mucoperiosteal flaps were carefully reflected on the buccal aspect of the
experimental sites , the granulation tissue was removed by curettage , and the exposed
root surface was instrumented . on the root surface , the base of the defect was marked
with curettes to establish a landmark for the histomorphometric analysis . each
contralateral defect in each animal was randomly assigned to one of the following
treatments : gtr+bg ( perioglas , us biomaterials , alachua , fl , usa ) - control group : bg particles
were applied filling the defect and absorbable membrane ( resolut xt
gore - tex , gore associates , flagstaff , az , usa ) was adapted to the defect ; gtr+bg+prp - test group : bg was incorporated to the prp ( 1:1 ) and this assemblage was
immediately taken to a water bath at 37c what enabled the formation of a mixture of
the gel and the bg graft .
this mixture was applied filling the defect and an
absorbable membrane ( resolut xt gore - tex , gore associates ) was adapted to
the defect .
primary , tension - free wound closure was accomplished following defect treatment , with
the gingival flaps positioned and sutured with interrupted sutures ( vicryl , ethicon
inc , so jos dos campos , sp , brazil ) in order to cover the membranes . immediately
after the surgeries ,
an intramuscular injection of penicillin ( 1.5 ml - 150,000 iu )
was given to the animal .
forty five days after the mandibular treatment , the maxillary defects ( 2 defects/
animal ) were randomly assigned to receive the same treatments ( group a and group
b ) .
postoperative plaque control was performed by irrigation with a solution of 1%
chlorhexidine gluconate ( every other day ) .
dogs were sacrificed 90 days after the
first treatment , under general anesthesia , with a perfusion of 10% neutral formalin
solution .
they were decalcified in a solution of equal parts of 50%
formic acid and 20% sodium citrate for 5 months .
the decalcified specimens were
washed in running water , dehydrated and embedded in paraffin .
mesiodistal 7-m - thick
sections were obtained from the tissue blocks longitudinally , in a buccolingual
direction , in a way to obtain a panoramic view of the furcation area .
the sections
were stained with hematoxylin and eosin . for histometric and descriptive histological analyses , 5 sections
per site representing the middle portion of the defect ( 30 m
apart )
were used to obtain the mean for each parameter in each dog , measured with an
image analysis system ( image pro plus version 3.0 , media cybernetics , silver spring ,
md , usa ) .
the following linear measurements ( mm ) were obtained in the mesial and distal roots
under the furcation :
defect extension ( de ) : linear extension of the total root surface
between the two notches ;
new cementum ( nc ) : linear extension of the root surface covered by
new cementum ;
connective tissue adaptation ( ca ) : extension of the root surface
covered by connective tissue without cementum .
new bone ( nb ) : measured from a horizontal line connecting both
notches ( apical limit of the defect ) to the most coronal extent of new bone .
the following
parameters ( mm ) were obtained by the point counting technique :
mineralized bone area ( mba ) : portion of the defect area filled with
mineralized bone tissue ;
residual bg particle area ( rbg ) : portion of the defect area filled
with bg remaining particles ;
non - mineralized bone area ( nmba ) : portion of the defect area filled
with non - mineralized tissue ; the measurements were performed using a microscope ( zeiss axioskop 2 , carl zeiss
instruments , gottingen , lower saxony , germany ) with a 1.25/.035 objective associated
with a video camera / computer / software ( image pro plus version 3.0 , media
cybernetics ) .
the mean
values for all groups were determined using the individual means from the 9 dogs .
after testing the assumptions confirming normal distribution ,
two - way anova was
carried out to check any significance among the different treatment groups , any
significance among the different time periods and the interaction between them .
no suppuration or
abscess formation was observed during the 90 days and no exposure of membranes was
observed in both groups during the healing period .
a clinical examination performed
immediately before sacrifice , disclosed that the gingival margin of all sites was
clinically normal and positioned coronal to the fornix of the buccal furcation .
there were no cases of epithelial downgrowth , ankylosis or extensive root resorption
observed in all sites .
a similar healing pattern was observed in the two groups , in both
periods , regarding the filling of the defect ( figure
3 ) .
mesiodistal histologic section of class ii furcation lesion stained with
hematoxilyn and eosin ( h&e ) , ( original magnification x20 ) ; a ) site of the
control group guided - tissue regeneration + bioactive glass ( gtr+bg ) after 45
days ; b ) site of the test group guided - tissue regeneration + bioactive glass +
platelet - rich plasma ( gtr+bg+prp ) after 45 days ; c ) site of the control group
after 90 days ; d ) site of the test group after 90 days .
both treatments promote
defect filling in both periods however a greater mineralized bone area was
observed for test group in both periods when compare with the control group a continuous layer of new cementum had formed in the specimens of both groups
extending coronally from the apical notches to a varying degree .
however , in the
group b , a more evident layer of new cementum was observed in the fornix region
( coronal portion of the defect ) . in both groups ,
some small areas of the defects
presented a very dense connective tissue surrounding the remaining bg particles .
however , connective tissue adaptation with collagen fibers running parallel to the
root surface seemed to be more frequent in group a. various defects in all groups presented bone surrounding bg particles and bone in
direct contact with these graft particles ( figure
4 ) .
however , in group b the defects were filled by a higher density bone
with a more evident mineralized bone area with islands of highly cellular bone
marrow . in group a , the bone presented larger marrow spaces what could indicate a
more immature bone when compared to group b. a ) photomicrograph of a mesiodistal histologic section of class ii furcation
lesion platelet - rich plasma + bioactive glass + guided - tissue regeneration
( prp+bg+gtr ) 90 days after treatment defect ( masson trichrome staining ,
original magnification x40 ) .
remaining bg particles were observed surrounded by new bone ( nb ) ,
connective tissue ( ct ) and areas suggesting the presence of immature bone ( ib )
in contact with bg ; b ) histologic section of the prp+bg+gtr defect , 90 days
after staining with hematoxilyn and eosin ( h&e ) , ( original magnification
x100 ) .
some
areas indicated the new bone formation occurring around bg particles
( arrows ) the histometric results are shown in tables 1
and 2 .
data analysis showed a superior length
of new cementum for group b in both periods ( 45 days and 90 days ) ( p<0.05 ) .
no
statistically significant differences were observed between periods for the linear
parameters for the two groups .
the addition of prp provided a greater mineralized
bone area in both periods ( p<0.05 ) .
the non - mineralized bone area was greater in
the control group ( p<0.05 ) in both periods while no statistically significant
difference was observed in the area occupied by the remaining bg particles between
groups .
significant differences between periods were observed in the mineralized bone
area and non - mineralized bone area ( p<0.05 ) .
mean and standard deviation ( sd ) for the linear parameters evaluated after all
the treatments ( bioactive glass+guided - tissue regeneration and bioactive
glass+platelet - rich plasma+guided - tissue regeneration ) in both periods .
capital letters compare treatments within each time and non - capital letter
compare times within each treatment .
means followed by different letters are
statistically different mean and standard deviation ( sd ) for the area parameters evaluated after all
the treatments ( bioactive glass+guidedtissue regeneration and bioactive
glass+platelet - rich plasma+guided - tissue regeneration ) in both periods capital letters compare treatments within each time and non - capital letter
compare times within each treatment .
no suppuration or
abscess formation was observed during the 90 days and no exposure of membranes was
observed in both groups during the healing period .
a clinical examination performed
immediately before sacrifice , disclosed that the gingival margin of all sites was
clinically normal and positioned coronal to the fornix of the buccal furcation .
there were no cases of epithelial downgrowth , ankylosis or extensive root resorption
observed in all sites . no inflammation and foreign body reactions indicating toxicity
were observed .
a similar healing pattern was observed in the two groups , in both
periods , regarding the filling of the defect ( figure
3 ) .
mesiodistal histologic section of class ii furcation lesion stained with
hematoxilyn and eosin ( h&e ) , ( original magnification x20 ) ; a ) site of the
control group guided - tissue regeneration + bioactive glass ( gtr+bg ) after 45
days ; b ) site of the test group guided - tissue regeneration + bioactive glass +
platelet - rich plasma ( gtr+bg+prp ) after 45 days ; c ) site of the control group
after 90 days ; d ) site of the test group after 90 days .
both treatments promote
defect filling in both periods however a greater mineralized bone area was
observed for test group in both periods when compare with the control group a continuous layer of new cementum had formed in the specimens of both groups
extending coronally from the apical notches to a varying degree .
however , in the
group b , a more evident layer of new cementum was observed in the fornix region
( coronal portion of the defect ) . in both groups ,
some small areas of the defects
presented a very dense connective tissue surrounding the remaining bg particles .
however , connective tissue adaptation with collagen fibers running parallel to the
root surface seemed to be more frequent in group a. various defects in all groups presented bone surrounding bg particles and bone in
direct contact with these graft particles ( figure
4 ) .
however , in group b the defects were filled by a higher density bone
with a more evident mineralized bone area with islands of highly cellular bone
marrow . in group a , the bone presented larger marrow spaces what could indicate a
more immature bone when compared to group b. a ) photomicrograph of a mesiodistal histologic section of class ii furcation
lesion platelet - rich plasma + bioactive glass + guided - tissue regeneration
( prp+bg+gtr ) 90 days after treatment defect ( masson trichrome staining ,
original magnification x40 ) .
remaining bg particles were observed surrounded by new bone ( nb ) ,
connective tissue ( ct ) and areas suggesting the presence of immature bone ( ib )
in contact with bg ; b ) histologic section of the prp+bg+gtr defect , 90 days
after staining with hematoxilyn and eosin ( h&e ) , ( original magnification
x100 ) .
some
areas indicated the new bone formation occurring around bg particles
( arrows )
data analysis showed a superior length
of new cementum for group b in both periods ( 45 days and 90 days ) ( p<0.05 ) .
no
statistically significant differences were observed between periods for the linear
parameters for the two groups .
the addition of prp provided a greater mineralized
bone area in both periods ( p<0.05 ) .
the non - mineralized bone area was greater in
the control group ( p<0.05 ) in both periods while no statistically significant
difference was observed in the area occupied by the remaining bg particles between
groups .
significant differences between periods were observed in the mineralized bone
area and non - mineralized bone area ( p<0.05 ) .
mean and standard deviation ( sd ) for the linear parameters evaluated after all
the treatments ( bioactive glass+guided - tissue regeneration and bioactive
glass+platelet - rich plasma+guided - tissue regeneration ) in both periods .
capital letters compare treatments within each time and non - capital letter
compare times within each treatment .
means followed by different letters are
statistically different mean and standard deviation ( sd ) for the area parameters evaluated after all
the treatments ( bioactive glass+guidedtissue regeneration and bioactive
glass+platelet - rich plasma+guided - tissue regeneration ) in both periods capital letters compare treatments within each time and non - capital letter
compare times within each treatment .
the present study evaluated if the application of prp on class ii furcation lesions
would enhance periodontal regeneration when associated with gtr and bioactive glass
( bg ) .
clinical reports have demonstrated that the association of gtr , bone substitutes
and prp could provide good results in the treatment of intrabony defects and class ii
furcation lesions , as revealed by gain in clinical attachment , defect fill at reentry ,
reducing probing depth , vertical defect fill and horizontal defect fill .
however , to the best of our knowledge , this is the first study that
evaluated histologically the effect of the association of prp with gtr and bg on
periodontal regeneration of class ii furcation lesions in dogs .
it has been demonstrated that when a bone graft or substitute is combined with the gtr
procedure in furcation lesions , a greater amount of defect fill is observed .
this advantage can be explained by the
osteoconductive and/or osteoinductive properties of the graft materials .
one of the bone
substitutes that have been investigated is the bg , a material that has been suggested as
an osteoconductive graft .
the use of bg in the present study was not associated with any
foreign body reaction .
it was observed that some remaining bg granules showed
dissolution of their core with the formation of new bone , as described by vogel , et
al .
the sites treated with grt+bg ( group a ) showed new cementum formation and new bone
regeneration in both periods .
these results are in accordance with previous reports ,
which demonstrated a beneficial effect of the association of gtr and bone
substitutes .
however , clinical studies do not provide information
about the nature of the healing process after these procedures .
in contrast with previous studies , in the present investigation , there were no instances of epithelial
downgrowth in all sites .
( 2007 ) observed a correlation between the exposure of the
membrane and the epithelial migration .
the presence of epithelial tissue was observed in
all histological sections of the sites that shown a membrane exposure and was ascribed
to the degradation of the exposed portions of this material .
the use of prp for periodontal regeneration has been based on the idea that this
preparation contains polypeptide growth factors ( pgfs ) .
some specific pgfs , like pdgf
and tgf , could promote the growth and differentiation of the periodontal ligament and
alveolar bone cells and could be responsible for the clinical improvement observed in
experimental sites
. other interesting feature of prp is its sticky
consistency due to its high fibrin content .
the fibrin component of prp may work as a
hemostatic agent aiding in stabilizing the graft material and the blood clot .
blood clot immobilization has been
suggested as an important event in the early phases of wound healing of periodontal
regenerative procedures .
it has been previously shown that prp may stimulate pdl cells and fibroblastic cell
proliferation but may suppress epithelial cell division in
vitro .
consequently , by
ordering these cellular responses into a series of related events , prp may facilitate
wound - healing and tissue regeneration . in the present study ,
the sites in the group b
showed a superior cementum formation , in both observation periods , when compared with
the sites of group a ( p<0.05 ) . to the best of our knowledge ,
no study provided direct
evidence that the prp could stimulate the differentiation of cells from the periodontal
ligament to cementoblasts , inducing cementogenesis .
however , previous investigations
have suggested that the treatment with recombinant growth factors , such as pdgf and igf
may enhance the formation of cementum on denuded dentin surfaces .
in addition to it , a similar result was
observed when the prp was associated with a subepithelial connective tissue graft for
the treatment of gingival recessions created in dogs .
the positive effect of prp in the bone response was demonstrated by previous
studiesand could be evidenced in the present study by the
largest area of mineralized bone observed in group b. sites treated with prp showed a
superior mineralized bone area with a concomitant inferior non - mineralized bone area , in
both periods , when compared with group a ( p<0.05 ) . however , the difference between
treatments is more evident in the first period ( 45 days ) when the area of mineralized
bone in group b was almost twice the one observed in group a. this observation is in
agreement with an in vitro study that suggested that pdgf acts upon osteoblastic cell
proliferation , exerting most of its effects during the early phase of wound healing .
although the mineralized bone formation was superior in group b of the present study , it
should be noticed that recent animals studies could not demonstrate that prp increases
bone regeneration when used in peri - implant bone defects . to date
, there is insufficient information about growth factor interactions and how they
influence the activations of gene expression and protein production .
also , the ideal
platelet and growth factor concentrations to promote periodontal regeneration has not
been established .
( 2003 ) reported that growth factors may act at specific times and at appropriate
concentrations and this may be other explanation for the different results in studies
with prp . in the present study ,
the achieved percentage mean of platelet concentration
was 227.02 % ( mean of 463,015 platelets/l ) in relation to blood platelet count .
the technique to produce prp used in this study was previously described by suaid , et
al.(2008 ) .
this technique
enables to produce a platelet concentration generally four times greater than the whole
blood .
( 2003 )
performed a study that measured the concentrations of bfgf , vegf , pdgf - bb and tgf - b1
released from platelet concentrate and whole blood , before and after the addition of
calcium alone , thrombin alone and various concentrations of calcium and thrombin .
they
observed that calcium chloride , regardless of the use of thrombin , released platelet
growth factors . in this study ,
the concentration of growth factors that was released was
sufficient to promote endothelial cell proliferation in vitro .
since the use of thrombin in this type
of investigation is not allowed in our country , instead of thrombin , 10% calcium
chloride was added to prp and heated in a water bath at 37c .
within the limits of this animal study , it was concluded that prp in association with
gtr and bg may enhance the amount of new cementum and provide a more mineralized bone
( mature ) in a shorter period of time when applied to the treatment of class ii furcation
lesions in dogs .
future studies are required to clarify the mechanism of action of prp
in the periodontal regeneration process and the ideal platelet and growth factor
concentrations to adequately promote periodontal regeneration .
the authors thank the financial support of fapesp ( the state of so paulo research
foundation , # 04/12428 - 6 ) and cnpq ( national counsel of technological and scientific
development # 300817/2007 - 0 ) . | objective this study was designed to evaluate the potential adjunctive benefits of
platelet - rich plasma ( prp ) when used with guided - tissue regeneration ( gtr ) and
bioactive glass ( bg ) in the treatment of class ii furcation lesions .
material and methods bilateral class ii furcation lesions were surgically created and allowed to
become chronic in the mandibular third premolars of 9 dogs .
the defects were
randomly assigned to : a ) gtr+bg and b ) gtr+bg+prp .
similar defects were created in
the maxillary third premolars and received the same treatments after 45 days .
dogs
were sacrificed 90 days after the first treatment .
the histometric parameters
evaluated were : connective tissue adaptation , new cementum , new bone , mineralized
bone area , non - mineralized bone area , and residual bg particle area .
results data analysis showed a superior length of new cementum and a greater mineralized
bone area for group b in both periods ( p<0.05 ) .
the non - mineralized bone area
was greater in the control group ( p<0.05 ) in both periods .
conclusion within the limits of this study , it can be concluded that the use of prp in the
treatment of class ii furcation defects may enhance the amount of new cementum and
provide a more mineralized bone in a shorter period of time . | INTRODUCTION
MATERIAL AND METHODS
PRP Preparation
Defect treatment
Linear measurements
Area measurements
Statistical analysis
RESULTS
Clinical observations
Histological observations
Histometric measurements
DISCUSSION
CONCLUSIONS
ACKNOWLEDGEMENTS |
PMC3376778 | feet provide the foundation for static support for body and dynamic support during walking or running .
the deformity includes four components : metatarsus adductus , cavus , hindfoot varus and equinus .
the cause of clubfoot has long been debated by the medical community . according to the journal of children s orthopaedics ,
some scientific investigators concluded that the condition was caused by malformed bones , abnormalities of muscle , joint or vascular lesions and/or abnormal ligaments and tendons .
another opinion is that congenital clubfoot results when external forces put the foot or the feet in a faulty position while the fetus is developing .
nowadays , there are two main hypotheses that say that congenital clubfoot is caused by neurogenic disorders in neuromuscle balance or gene variations .
any feet deformation should be corrected as early as possible to provide physiological function of proprioceptors that gives good balance and helps learning to walk .
treatment for clubfoot has evolved from a minimal surgery to a casting technique and then to extensive surgery . within the years , the principles of surgical technique in treatment for congenital clubfoot were changed and modified many times .
the most popular types of surgical procedures that are used in treatment for congenital clubfoot are listed below.medial side incision ( evans , dega , turco)two skin incisions medial and lateral side ( carroll , sotirow , uglov)circumferential ( cincinnati ) ( mckay , crawford)semi - cincinnati medial side incision ( evans , dega , turco ) two skin incisions medial and lateral side ( carroll , sotirow , uglov ) circumferential ( cincinnati ) ( mckay , crawford ) ponseti described his method in late 1950s .
his method is an innovative , conservative treatment for clubfoot involving a gentle manipulation of the child s foot and the application of toe - to - groin plaster casts that is followed by bracing and tenotomy .
the procedure consists of manual redresions , started as early as possible , which corrects a longitudinal arc of the foot and an abduction of a forefoot . with this treatment ,
a plaster cast is applied after each weekly session to retain the degree of correction obtained and to soften the ligaments .
after 46 weeks of the treatment , when adduction and supination of the calcaneum bone is corrected , the tight achilles tendon is cut in a minor procedure ( tenotomy ) to perform correction of the equinal deformation .
the corrected foot is put in a holding cast for 3 weeks to allow the tendon to regenerate .
then , when the final cast is removed , a foot abduction brace a.k.a .
this device consists of a pair of shoes attached to an adjustable bar at a specific width and angle .
the main objective of this paper is to present the early results of treatment congenital clubfoot by ponseti method .
the paper is based on data for 35 children25 boys and 10 girls with 47 clubfeet treated during the period of 20072011 .
we monitored the deformation before each redresion and casting , and before and after the tenotomy . to evaluate the effects of treatment
, we used pirani s scale with its six main features concerning external edge , medial crease and covering of the head of the talus bone in the midfoot region and posterior crease , incorrective equinal deformation and empty heel in hindfoot region .
according to pirani s scale , a total score of 6 points represents a severe clubfoot with a score of 0 points representing a normal foot .
all 35 patients were newborns with 7 of them suffering from bilateral deformation . according to bensachel
dimeglio s classification , 40 feet represented stift type of deformation and most of the patients had a maximal or medium grade of equinal , varus and adduction deformation ( table 1).table 1angular deformation before treatment most of them were medium or maximal ( 4090)result after tenotomysagital planepassive dorsal flexionmaximal719025median417017minimalbelow 405 angular deformation before treatment most of them were medium or maximal ( 4090 ) we started treating the patients as early as possible . for 25 patients , redresions and
clubfeet were corrected with manipulations every 57 days followed by plaster - cast applications .
then , we measured the residual deformation for varus and adduction through passive correction ( table 2 ) . for all treated patients , we accomplished satisfactory results.table 2angular deformation for varus and adduction after redresions and casting treatment during passive correction
we reached 05 in all treated patientsdeformationgrade of varus and adduction deformationnumber of feetspontaneous correction152045passive correction0545full correction02 angular deformation for varus and adduction after redresions and casting treatment during passive correction
we reached 05 in all treated patients when the correction of adduction and supination of the calcaneum bone was completed , the tenotomy of achilles tendon was performed .
this minor surgical procedure , conducted under a light sedation , was applied to 34 children out of the total number of 35 treated children . for one child
, excellent results were achieved after redresions followed by bracing so that no surgery was required .
the remaining patients underwent the tenotomy followed by a 3-week - long holding cast period that allowed the tendon to regenerate longer . in 85% of treated feet , we achieved sufficient correction in sagittal plane and ability of passive dorsal flexion ( table 3 ) .
for the final correction in all treated cases , we used tibax or clubax device , which is a simple instrument to perform corrections in all three planes .
such orthosis helped to preserve the good results after surgery ( fig . 1 ) .
however , those four children did not use that device strictly according to our recommendation.table 3correction in sagittal plane and ability of passive dorsal flexion after tenotomy we reached good results in 85% of treated feetresult after tenotomysagital planepassive dorsal flexionnumber of feet ( % ) good051039 feet ( 85%)satisfactory103008 feet ( 15%)fig .
1tibax and clubax orthosis correction in sagittal plane and ability of passive dorsal flexion after tenotomy
we reached good results in 85% of treated feet tibax and clubax orthosis during the analysis of our final results , we presented data after follow - up of all patients ( median 21 months ) , together with a subanalysis of patients according to pirani s scale .
after the treatment , we accomplished a score of 0.51.5 points in all 46 treated feet ( table 4 ) .
we evaluated the results of treatment as good or very good for 75% of patients . for the remaining patients ,
the results were evaluated as satisfactory.table 4pirani scale before and after treatment we accomplished 0.51.5 point in all 33 treated feetbefore treatment by ponseti method842 months after achilles tenotomypointsnumber of feetpointsnumber of feet6101.5955.5281164.590.520 pirani scale before and after treatment we accomplished 0.51.5 point in all 33 treated feet
the main objective of treatment for congenital clubfoot is to obtain pain - free , plantigrade foot , with good mobility and without calluses . in general ,
children who suffer from such deformation undergo some type of surgery to complete the correction .
this can range from a percutaneous heelcord lengthening to a wide release of medial , posterior and lateral structures , with or without transfer of the anterior tibial tendon [ 7 , 8 ] .
this leads ponseti to develop his method in the 1950s , repopularized in the beginning of 2000 in the usa and europe .
currently , manipulation , redresions and casting according to ponseti method are the gold standard , and this method is endorsed by the american association of orthopedic surgeons .
napiontek in 2004 introduced and widely popularized ponseti method in poland , and since 2007 , we have been using it in our clinic .
treatment should be initiated as soon as possible , preferably within the first week of life .
the majority of clubfeet can be corrected in infancy in about 68 weeks with the proper gentle manipulations and plaster casts followed by tenotomy .
however , the technique requires a lot of training , experience and practice . in our study ,
nonoperative procedures took usually 58 weeks which is a similar period to ponseti standards and that what other author report [ 11 , 12 ] .
for the treatment performed at our clinic , tenotomy of the achilles tendom was crucial for the completion of a successful correction of the foot .
all of them consist of a bar with shoes that are attached at the ends of the bar in external rotation .
the device is worn 23 h a day for 34 months and then at nighttime for 24 years . in our clinic , we used tibax or clubax device which was similar to orthosis recommended by posna ( pediatric orthopaedic society of north america ) as : the dobbs dynamic abduction brace , the markell or mitchell abduction brace .
severity of the deformity at birth is not a reliable indicator of the odds for a relapse ; therefore , almost all clubfoot patients are held to the same bracing protocols in order to provide them with the best protection against regression [ 13 , 14 ] . in early results
, we noticed that all children who did not use the orthosis as it was prescribed experienced high recurrence rate .
we have to add that in some severe cases , more invasive surgery treatment is needed to correct the position of the clubfoot despite using ponseti method .
most often , a more invasive surgery is this is needed in cases when a child has other developmental problems as , e.g. , arthrogryposis .
we also want to point out that the time when the child begins treatment by ponseti method is an important factor .
most authors consider that a congenital clubfeet treatment has to start no more than a few months after birth .
however , some resent researches showed that the ponseti method is effective for children as old as ten , even in cases of a failed surgery [ 16 , 17 ] .
we agree with ponseti s thesis that the basic of deformity in the congenital clubfoot is fibrosis of the soft tissue .
therefore , soft tissue in infants is more responsive to redresions , casting and finally bracing .
in essence , our study supports the principles of ponseti s method , because in cases when these principles were strictly obeyed , all patients accomplished satisfactory results and there have been no recurrences that are known to us [ 5 , 18 ] .
therefore , we strongly recommend using this method to avoid more invasive surgery in the future .
this method is of the particular value for the initial treatment of congenital clubfoot deformity . | the purpose of this study was to evaluate the early results of the ponseti method in reducing extensive corrective surgery rates for congenital idiopathic clubfoot in patients treated in children s orthopaedic clinic and rehabilitation department medical university of lublin between the years 20072011 .
thirty - five patients with 47 idiopathic clubfeet were followed prospectively while being managed with the ponseti method .
clubfoot severity was graded with use of the dimeglio system .
the initial correction was achieved , and early results were measured by using pirani scoring method . | Introduction
Aim of the paper
Materials and methods
Results
Discussion |
PMC4047242 | nordic walking is a physical activity consisting of walking with poles similar to ski
poles .
the poles are designed for
the purpose of activating the upper body during walking .
they are equipped with rubber or
spike tips , and the walking itself resembles nordic - style skiing .
beginners are asked to
walk in an upright position , not leaning forward or backward .
the head should be up and
facing forward , and the poles are held close to the body .
when the leading foot is moving
forward , the opposite arm swings forward to waist height .
the opposite pole strikes the
ground level with the heel of the leading foot .
the poles remain pointing diagonally backward and are pushed as far
back as possible .
the arm straightens and the hand opens , coming off the grip by the end of
the arm swing .
nordic walking is not expensive and can be performed throughout the year . but
handling of the poles is difficult , the technique required to handle them can not be easily
acquired by community - dwelling elderly individuals .
an intriguing phenomenon is the extent to which the human motor system can transfer
relevant strategies learned in one context to other similar situations .
experimental
paradigms using upper limb tasks show the ability of the nervous system to generalize
learned motor skills to similar movements , contexts , or workspaces and even to the opposite
limb1,2,3,4 .
such studies have yielded valuable insights into how the nervous system codes and
generalizes motor skills .
walking is a motor task that is highly flexible in its adaptation
to different situations .
extensive work has shown the ability
of the locomotor system to adapt to changes in sensory input from the limbs5,6,7 .
in addition , the walking pattern is easily
modified to accommodate different external constraints , for example , navigating over or
around obstacles8 or different slopes of
the walking surface9 , 10 .
therefore , it is
important for elderly individuals to train with movements in the backward direction to
prevent falling forward .
nordic backward walking ( nbw ) is a very difficult form of walking
for beginners , and we do not currently know the effect of repetition of nbw on movement in
the backward direction in the elderly .
the aims of this study were : 1 ) to confirm the motor learning process of nbw in older adult
community - dwelling volunteers and 2 ) to check the change in psychological condition
resulting from nbw .
we evaluated 19 older adult
community - dwelling volunteers ( 11 female and 8 male ; mean age 644 ) ( table 1table 1 .
general characteristics of participants ( n=19)indexesmeansdage ( years)644gender ( male / femal)11/11functional reach test ( cm)28.17.5bone density ( g / cm)2.40.2mini - mental state examination ( point)291 ) . the volunteers ( n=19 ) were included based on the following criteria : 1 )
self - report of good health and 2 ) absence of a history of psychiatric or neurological
disorder .
all participants gave informed consent , and the study was approved by the ethics
committee of kansai university of welfare sciences .
we measured nbw consecutively
five times and then measured it a sixth time one hour later .
no participants reported fatigue ,
likely because of the short walking distance and limited number of trials .
the primary variables of interest were walking
speed , stride length , and visual analogue scale ( vas ) .
the vas was used as an index of sense
of fear for walking backward .
a paired t - test was used to compare the mean front and back scores . the alpha level for
statistical significance
all data were analyzed using the
statistical package for the social sciences ( spss ) version 19 .
our results demonstrated that following repeated sessions of backward walking , the walking
speed during nbw in the first , second , third , fourth , and fifth sessions were 73 m / min , 80
m / min , 86 m / min , 88 m / min , and 91 m / min , respectively .
significant differences were only
found between the first and second sessions and between the second and third sessions .
the
walking speed in the sixth session ( 86 m / min ) , measured one hour later , was decreased in
comparison with that in the fifth session .
the stride lengths during nbw in the first ,
second , third , fourth , and fifth sessions were 0.52 m , 0.56 m , 0.58 m , 0.59 m , and 0.59 m ,
respectively .
significant differences were only found between the first and second sessions
and between the second and third sessions .
the stride length in the sixth session ( 0.56 m ) ,
measured one hour later , was decreased in comparison with that in the fifth session .
the vas
scores after nbw for the first , second , third , fourth , and fifth sessions were 4.03 cm ,
3.46 cm , 2.95 cm , 2.47 cm , and 2.00 cm , respectively .
in addition , the vas score for the sixth
session ( 2.00 cm ) , measured one hour later , was increased compared with that for the fifth
session ( table 2table 2 .
motor learning during nordic backward walking ( n=19)1st2nd3rd4th5th6thbackward walking speed ( m / min)732180*2086*21882491288623stride length ( m)0.520.100.56*0.120.58*0.110.590.130.590.120.560.11visual analogue scale ( cm)4.032.393.46*2.352.95*2.252.47*2.392.00*2.291.662.04values are presented as the averagesd .
this study investigated the learning process in beginners learning how to perform nbw using
nordic poles .
our results demonstrated that following repeated sessions of backward walking ,
the walking speed during nbw showed significant differences only between the first and
second sessions and between the second and third sessions .
the walking speed in the sixth
session , measured one hour later , was decreased in comparison with that in the fifth
session .
unlike the subjects studied by lake and cavanagh11 , our subjects demonstrated a clear increase in efficiency , likely
as a result of increased efficiency of motor unit recruitment due to practice .
the differing
results can most likely be explained by considering the differences in the type of exercise
used .
backward walking is truly a novel task , whereas even individuals who do not
specifically train with forward running do engage in forward walking .
therefore , the motor
pathways used for any forward locomotion are at least somewhat trained , whereas those used
for backward locomotion are untrained .
significant differences in stride length during nbw were only found between the first and
second sessions and between the second and third sessions .
the stride length in the sixth
session , measured one hour later , was decreased in comparison with that in the fifth
session . specifically , the increase was significant between the fourth and sixth week of
training .
schwane et al.12 suggested that
a novel activity may require increasingly greater motor unit recruitment in order to
complete a task .
although we did not specifically examine muscle activity , backward walking
requires greater sustained emg activity of the quadriceps than forward walking13 , 14 .
thus , we believe our findings are the first to demonstrate that an
individual may perform backward walking more efficiently after repeated sessions of backward
walking training .
significant differences in vas score after nbw were found for each session . in addition ,
the stride length in the sixth session , measured one hour later , was decreased in comparison
with that in the fifth session .
if the energy cost decreases as a person learns a new motor
skill , as suggested by schwane et al.12 ,
the prescribed exercise workload , per the regression equations reported by myatt et al.15 and clarkson et al.16 , may not have the intended training effect .
our findings
are consistent with the hypothesis that efficiency increases when a task is no longer
novel .
therefore , it is important for elderly individuals to train with backward movements to
prevent falling forward .
the skill of the subjects in performing nbw rose with every
repetition , but they learned at a slow pace .
also , their level of skill tended to decrease
when they took a break between sessions .
however , their sense of fear did not worsen even if
they took a break .
this finding suggests that nbw is indeed a novel task and that motor
learning occurs as a result of practice , leading to a more efficient recruitment of motor
units .
therefore , nbw was a form of training that matched with the preferences of the
elderly and was thought to be useful as a backward walking exercise for the elderly .
nbw can
be performed anywhere and anytime , and therefore , widespread clinical application of nbw is
possible . | [ purpose ] the aims of this study were : 1 ) to confirm the motion learning process of
nordic backward walking ( nbw ) in older adult community - dwelling volunteers and , 2 ) to
check the change in psychological condition resulting from nbw .
this study examined
whether the learning process and psychological condition become more efficient after
repeated sessions of nbw .
[ methods ] the subjects were 19 community - dwelling elderly
individuals between the ages of 64 and 78 years .
[ results ] significant differences in
walking speed during nbw were only found between the first and second sessions and between
the second and third sessions .
the walking speed in the sixth session , measured one hour
after the fifth session , was decreased in comparison with that in the fifth session .
significant differences in stride length during nbw were only found between the first and
second sessions and between the second and third sessions .
the stride length in the sixth
session , measured one hour after the fifth session , was decreased in comparison with that
in the fifth session .
significant differences in vas score ( sense of fear ) after nbw were
found for each session .
vas score for the sixth session , measured one hour after the fifth
session , was decreased in comparison with that for the fifth session .
[ conclusions ] the
findings in the present study suggest that nbw is indeed a novel task and that motor
learning occurs as a result of practice , leading to a more efficient recruitment of motor
units . | INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION |
PMC3038693 | alzheimer 's disease ( ad ) is a progressive neurological disorder resulting from the deposition of alzheimer -amyloid peptide ( a ) as senile plaques , the appearance of neurofibrillary tangles , and selective neuronal loss .
the most abundant forms of a are 40 and 42 amino acid residues long and referred to as a40 and a42 , respectively .
the endocytic pathway has been implicated in the extracellular secretion of a40 and a42 [ 2 , 3 ] .
these peptides are derived from the endoproteolytic cleavage of the amyloid precursor protein ( app ) with -secretase , followed by -secretase .
the endosomal contents can be either secreted from the cell [ 79 ] or transferred to the lysosome .
the endosome has been found to be quite acidic ( ph 6 ) with the recycled endosome slightly less acidic ( ph 6.5 ) [ 11 , 12 ] .
exposure of a to endosomal ph conditions has been found to induce various conformational and oligomeric states [ 1315 ] .
many oligomeric forms of a have been proposed and characterized as intermediates in the pathway to forming the amyloid fibre .
some of these structures include trimers , pentamers , high molecular weight a-derived diffusible ligands ( addls ) , protofibrils , and fibrils [ 1621 ] .
here we present a method for generating a mixed population of a conformations using model endocytic conditions . using this method ,
we demonstrate that when a is exposed to endosomal conditions for an extended period of time , the ability of the peptide to bind and internalize into living rat adrenal pheochromocytoma ( pc12 ) cells increases with time until it reaches a maximum beyond which its ability to interact with pc12 cells diminishes drastically .
a40 and a42 were synthesized and purified as described previously . before cleavage from the resin , the fluorophore , n-(9-fluorenylmethoxycarbonyl)-n-tetramethylrhodamine-(5-carbonyl)-l - lysine ( molecular probes , eugene , or ) ( abbreviated tmr ) ,
the crude peptides were purified by hplc using a superdex tricorn 10/300 gl peptide column ( amersham biosciences , piscataway , nj ) with 30 mm nh4oh running buffer . to maintain stock peptide solutions free from fibril seeds ,
solutions were stored at ph 10 and 4c immediately after chromatographic separation of monomeric peptides .
a preparations were never lyophilized , as this process may allow for seeds to form .
these solution conditions have been previously shown to maintain the monomeric state [ 16 , 23 , 24 ] .
peptide purity and identity was confirmed using both maldi mass spectrometry and amino acid analysis .
concentrations of stock peptide solutions were determined using amino acid analysis and confirmed by either tyrosine absorbance ( 275 nm , = 1390 cm m ) or tmr absorbance for labelled peptides ( 550 nm , = 92000 cm m ) .
at least three separate synthesized lots of a were used in this study and each displayed identical cell association rates when compared for quality assurance .
oligomeric samples were prepared by diluting stock a samples to 30 m with 30 mm nh4oh and reducing to ph 6 with 0.2 m hcl and incubating for zero ( a ) , 3 ( a ) , 24 ( a ) , or 90 hours ( a ) in the dark at 20c .
hydrodynamic radius ( rh ) measurements were made at 20c with a dynapro dls instrument ( protein solutions inc . ,
peptide samples ( 30 m ) were reduced to ph 6 using 0.2 mm hcl , centrifuged at 12000 g for 3 minutes and then rapidly added to a 1 cm path length cuvette and left in the instrument .
dls data was collected at various time points over 90 hours . particle translational diffusion coefficients were calculated from decay curves of autocorrelation of light scattering data and converted to hydrodynamic radius ( rh ) with the stokes - einstein equation .
histograms of intensity versus rh were calculated using dynamics data analysis software ( protein solutions inc . , piscataway , nj ) .
a samples ( 30 m ) were either not filtered or spin - filtered for 30 minutes at 14,000 g using 10 , 30 , 100 kda ( amicon ultra cellulose kmwo ) , or 0.1 m ( amicon pvdf ) spin filters .
absorbance at 550 nm was collected on a molecular devices spectramax m5 ( molecular devices corp . ,
sunnyvale , ca ) and graphs were created normalizing the absorbance signal from each filtered sample to the corresponding unfiltered sample .
fluorescence measurements were obtained using 200 l of 30 m a samples , within a 96-well plate , after addition of 5-fold molar excess of thioflavin - t and incubation at room temperature for 30 minutes .
emission at 485 nm was collected using 440 nm excitation on a molecular devices spectramax m5 ( molecular devices corp . ,
pc12 cells were maintained in dmem / f12 containing 10% fetal bovine serum ( hyclone , logan , ut ) with 100 units / ml penicillin and 100 g / ml of streptomycin . to induce differentiation of pc12 cells and for cell imaging , they were plated at 2.2 10 cells / cm in lab - tech chambered cover glass chambers and suspended in phenol red free dmem / f12 containing n2 supplement and 10 ng / ml ngf .
cells were differentiated for 72 hours before media was replaced and peptide treatments ( final concentration 1.5 m ) were performed .
tmr - labelled a samples ( 6 m ) were prepared in phenol red free dmem / f12 media containing n2 supplement and 10 ng / ml ngf .
either fresh media or cultured supernatants , obtained from cell culture after 72 hours , were used . to avoid interference from cell culture components , molecular weights of tmr - labelled a were obtained by selective monitoring of tmr absorbance at 550 nm .
sedimentation experiments were performed at 20c on a beckman xli analytical ultracentrifuge using an an50-ti rotor .
molecular weights were calculated using beckman xli data analysis software in which absorbance versus radial position data were fitted to the sedimentation equilibrium equation using nonlinear least - squares fitting .
three - dimensional stacks of fluorescence micrographs were taken at 20c with a confocal laser - scanning system consisting of an lsm 510 zeiss meta nlo confocal microscope with a c - apo 40x water immersion objective ( numerical aperture 1.2 ) and hene laser with a 543 nm laser line .
the displayed images were captured using zeiss lsm image version 4 and prepared using imagej version 1.37v and represent a single cross - section through the cells .
differentiated pc12 cells were treated with 50 m monodansylcadaverine ( mdc ) for 20 minutes at 37c with 5% carbon dioxide . after inhibitor treatment , a ( final concentration 1.5 m ) was added to the media containing mdc and imaged after 4 hours at 37c .
a ( final concentration 1.5 m ) was also added to differentiated pc12 cells media for 4 hours at either 37c or 4c prior to imaging .
differentiated pc12 cells were first treated with a ( final concentration 1.5 m ) for 6 hours at 37c followed by treatment with 40 nm mitotracker deep red and 50 nm lysotracker green dnd-26 for 20 minutes .
after treatment , the media was exchanged with fresh phenol red free dmem / f12 media containing n2 supplement and 10 ng / ml ngf with 1.5 m dapi for nuclear staining .
cells were imaged using argon laser with 488 nm laser line for lysotracker , hene laser with 633 nm laser line for mitotracker , hene laser with 543 nm laser for tmr - labelled a , and a tunable chameleon laser at 730 nm for two - photon excitation of dapi .
2d histograms and correlation coefficients were determined using image j version 1.42q with colocalisation threshold plugin .
differentiated pc12 cell media was replaced with media containing 0.6 to 20 m a , a , a , or 10 m melitin as a positive control for 48 hours .
cell survival was quantified using the sulforhodamine b assay , and absorbance was measured at 560 nm using molecular devices spectramax m5 ( molecular devices corp . , sunnyvale , ca ) .
lc50 values were determined as the concentration of a required to kill fifty percent of the cells from an absorbance versus a concentration plot .
to study the interaction between a and live cells , we synthesized and fluorescently labelled a40/42 .
the synthesized a was maintained in solution from purification to storage and was never lyophilized , as these solution conditions are known to significantly reduce the formation of a aggregation seeds [ 16 , 23 , 24 ] .
we covalently attached tetramethylrhodamine ( tmr ) to the n - terminus of a via a flexible glycine linker to generate tmr - a. the n - terminus of a is highly accessible even in the fibril state [ 21 , 27 , 28 ] and attaching a fluorescent label to this site has been shown to neither alter its amyloidogenic properties [ 16 , 29 , 30 ] , nor its solubility behaviour [ 31 , 32 ] .
we have previously shown that treating cultured cells with 1.5 m monomeric tmr - a42 leads to the formation of visible aggregates on the surface of pc12 cells within one hour of treatment .
we initiated our current study by investigating whether a aggregation could occur in cell culture media alone . using analytical ultracentrifugation , we measured the molecular weights of a40 and a42 present in phenol red free cell culture medium , both freshly prepared and conditioned media taken from differentiated pc12 cells 3 days postdifferentiation .
following the addition of 6 m a to each medium and subsequent 24-hour incubation at room temperature , both media preparations were centrifuged to equilibrium at room temperature in an analytical ultracentrifuge in order to determine the molecular weight of a conformations .
the molecular weights of a40 and a42 from both media preparations were measured to be approximately 4103 da and 4425 da , respectively .
these values both correspond to the expected monomeric molecular weights of a , falling within the 95% confidence intervals of 36504570 da for a40 and 36504770 da for a42 .
thus , the aggregation of a seen by confocal microscopy apparently occurs only after interaction with the differentiated pc12 cells and not with cultured supernatants .
it should be noted that the concentration previously used to treat cells ( 1.5 m ) and the concentration used for ultracentrifugation ( 6 m ) are considerably lower than the reported 20 to 50 m range required for in vitro aggregation [ 3335 ] .
since a40 and a42 are generated through endoproteolytic cleavage [ 79 ] , and the ph of the endosome and recycled cellular vesicles is equivalent to ph 6 , we characterized the a conformations formed under endosome conditions .
a ( 30 m ) was reduced to ph 6 , and hydrodynamic radius calculations were collected after zero ( a ) , 3 ( a ) , 24 ( a ) , and 90 ( a ) hours using dynamic light scattering ( figure 1 ) .
the average hydrodynamic radius of a40 was found to increase from 2.0 nm at time zero ( a40 ) to 216 nm after 90 hours at ph 6 ( a40 ) .
a more striking increase was found with a42 , beginning with 1.7 nm at time zero ( a42 ) to 451 nm after 90 hours at ph 6 ( a42 ) .
for each of these samples , the development of increasingly higher ordered aggregates was observed over time and the samples that were treated for 90 hours contained particles over 1000 nm in radii . to further investigate the relative levels of peptide aggregation at endocytic ph , we filtered the a40 and a42 samples through various molecular weight cutoff ( mwco ) spin filters ( figure 2 ) .
approximately 67% of a40 , 60% of a40 , and 55% of a40 were recovered through the 10 kda mwco spin filter and approximately 80% were recovered through the 100 kda mwco filter , except for a40 with only 60% recovered . in contrast , only 55% of a42 , a42 , and a42 were recovered through the 10 kda filter and approximately 60% were recovered through the 100 nm filter .
these results indicate that the majority of peptide conformations present under these conditions were able to pass through a 10 kda molecular weight filter , but that just over 40% could not be recovered through the 100 nm filter for the a40 , a42 , a42 , and a42 samples .
we also used thioflavin - t to assess the time - dependence of the extent of amyloid fibril formation at endocytic ph .
thioflavin - t is a dye known to shift its fluorescence from 430 nm to 490 nm upon binding specifically to the cross--structure of amyloids but not to monomeric or small oligomeric complexes [ 36 , 37 ] .
we observed enhanced thioflavin - t fluorescence at all time points ( figure 3 ) ; however , thioflavin - t bound most strongly to a40 , a42 , and a42 .
the high thioflavin - t binding to a samples suggests that these late stage a conformations are the most aggregated . using confocal microscopy
we have previously shown that monomeric a42 associates with cells more rapidly than a40 , with significant staining observable after six hours of treatment . to determine whether the aggregation state of a affects cell association , we exposed each of the a40 and a42 samples to differentiated pc12 cells and monitored the kinetics of association by confocal microscopy . upon treating cells with a40 , the cell surface association
moreover , a40 was observed to significantly internalize into these cells after only 6 hours , whereas significant internalization of a40 was only visualized around 24 hours ( figure 4 ) .
similarly , a42 internalized into differentiated pc12 cells after only one hour of treatment , whereas a42 treatment only became observable at 24 hours ( figure 4 ) .
interestingly , when differentiated pc12 cells were treated with late - stage a40 or a42 , very few cells underwent internalization or even exhibited cell surface interaction with these aggregated peptide forms ( figure 4 )
. to illustrate the contrast between these treatments , we collected the three - dimensional image slices through a cell from the base to the apex for the 6-hour treatment with a42 , a42 , and a42 ( figure 5 ) .
a42 was only observed around the periphery of the cell , whereas some a42 was located inside the cell . by contrast
, a42 treatment seemed to localize to extracellular regions and did not produce the punctate pattern as observed with the a42 sample . to quantify the frequency of cells that exhibited peptide internalization , we randomly selected five fields of view from at least three separate 6-hour treatment experiments and plotted the percentage of cells having internalized a ( figure 6 ) .
was found to have a large number of aggregates with hydrodynamic radius over 1000 nm ( figure 1 ) and were found to significantly bind thioflavin - t ( figure 3 ) , then these aggregates may favour self - association over cell association . to determine if the internalization of endocytic a is mediated through cellular processes , such as receptor - mediated endocytosis or through direct peptide - mediated processes like membrane pore formation , we monitored the effects of temperature and monodansylcadaverine ( mdc ) on internalization .
mdc is a known inhibitor of receptor - mediated clathrin - dependent endocytosis [ 38 , 39 ] . at physiological temperature ( 37c ) , after a 4-hour treatment of cells with a40 and a42 , internalization was observed ( figure 7 ) . at 4c membrane vesicle formation
when a40 or a42 association with differentiated pc12 cells was monitored at 4c , none of the cells were found to have internalized these peptides ( figure 7 ) .
similarly , when differentiated pc12 cells were treated with mdc , neither a40 nor a42 were observed within the cells .
these observations indicated that a was internalized through a cell - directed import mechanism , rather than an independent penetration route through the cell membrane .
the location of deposited intracellular a40/42 was examined using intracellular organelle markers in differentiated pc12 cells .
cells treated with a40 and a42 for 6 hours were visualized using lysotraker green dnd-26 for the lysosome , mitotracker deep red for the mitochondria , and dapi for nucleus staining ( figure 8) .
the staining pattern of each cellular organelle marker in the same image plane as the a40 and a42 treatment was determined ( figure 8) and quantified ( figure 9 ) . only the signal from the lysosome marker costained with both internalized a40 and a42 . the red signal from the peptides costained well with green signal from the lysosomes , resulting in the yellow signal in the merged image . when quantified , 71% of the lysosome signal intensity colocalised with the a40 channel and 80% with the a42 channel ( figure 9 ) .
whereas only 9% of the mitrochondria signal intensity colocalised with a40 channel and less than 1% with the a42 channel ( figure 9 ) .
the intensity of both a40 and a42 fluorescence signals was found to increase in the lysosomes over time , which may reflect the accumulation of a40/42 in the lysosome . to determine the relative toxicity of these a conformations ,
a cytotoxicity assay was performed where differentiated pc12 cells were treated with various peptide concentrations for 48 hours ( figure 10 ) .
we calculated the lethal concentration at which 50% of cells were killed ( lc50 ) , for each of the a conformations .
we found that a40 was moderately more toxic than a40 , with a lower lc50 value ( figure 10 ) .
surprisingly , a40 was also found to have a lower lc50 value compared to a40 .
a42 was also found to be moderately more toxic than a42 , whereas a42 had a much higher lc50 value compared to a42 ( figure 10 ) .
we have developed a method to produce a collection of a conformations that differ in their extent of aggregation and investigated the interaction between these states of a40 and a42 and differentiated pc12 cells .
others have described the methods to isolate individual soluble oligomeric forms of a , using various chemical reagents and protocols [ 4144 ] .
in addition , purified oligomeric a molecules from cell culture [ 45 , 46 ] or transgenic mice have also been monitored .
these purified sources of oligomeric a offer great potential in understanding the progression of a aggregation ; however , they can not be directly visualized over the time course of their effects on cells during maturation from earlier to later conformations .
since it is well established that a monomers can oligomerize under the physiological ph of the endosome [ 1315 ] , and a is formed through endoproteolytic cleavage , we have utilized this condition to determine which conformations are present and how these mixed conformations interact with differentiated pc12 cells .
it is known that the extent of oligomerization / fibrillation is very dependent on experimental conditions .
have indicated that a can be induced to form fibrils via dilution from 100 mm naoh to neutral ph in the presence of 10 mm hepes/100 mm nacl buffer , while dilution into phosphate buffered saline results in oligomer formation . in our experiments , the peptide was diluted from 30 mm ammonium hydroxide ( ph 10 ) to ph 6 with final condition of 1 mm ammonium chloride as the only additional chemical .
we do note that the normal human physiological concentration of ammonium chloride in blood and cerebrospinal fluid is approximately 20 to 50 m [ 49 , 50 ] , and that hyperammonemia has been linking to alzheimer type ii astrocytosis [ 51 , 52 ] .
when extracellular monomeric a associates to the surface of cells , we speculate there are three possible outcomes : ( 1 ) it can act as a stable template to allow further a aggregation ; ( 2 ) the peptide can penetrate through the cell membrane depositing in the cytoplasm ; or ( 3 ) the peptide may be internalized into the cell within endocytic vesicles , which would result in a reduction in the surrounding ph . in the third case
, the endocytic vesicles containing a can theoretically be recycled back to the cell surface or directed to the lysosome where a further reduction in ph would occur .
we have previously visualized oligomeric a on the surface of neuronal cell lines , and here sought to determine first whether this aggregation occurred prior to cell surface deposition in the cell culture mediaor are aggregation and cell surface binding concomitant and linked processes.using ultracentrifugation analysis of a preparations either in freshly prepared cell culture media or in conditioned media removed from cultured cells after 3 days , we did not observe any conformation larger than the monomer .
these results indicate that components in our cell media or secreted factors from cultured cells are not responsible for the observed a aggregation present on the surface of neuronal cells . after adding monomeric a to cells
, we noted a maturation time for the visual appearance of a on the cell surface and in the cell interior ( figure 4 ; ) .
we postulate that the peptides on the cell surface might undergo a series of ph reductions through endocytic recycling , before becoming visible punctae on and inside the cell . since ph 6 is approximately endocytic ph , we characterized the kinetic effects of this condition on peptide conformations . exposing a40 for up to 24 hours at ph 6
, we observed an increase in the average hydrodynamic radius from 2.0 nm ( a40 ) to 183 nm ( a40 ) ( figure 1 ) .
in addition , approximately 60% of these peptides were recovered through a 10 kda mwco filter and 80% were recovered through a 100 kda mwco filter ( figure 2 ) , and only minor thioflavin - t binding was found ( figure 3 ) . following exposure of up to 24 hours at ph 6 , a42 had an increase in the average hydrodynamic radius from 1.7 nm ( a42 ) to 246 nm ( a42 ) ( figure 1 ) , approximately 55% were recovered through 10 kda mwco filter and only 60% were recovered through 100 nm filter ( figure 2 ) and more significant thioflavin - t binding was measured ( figure 3 ) .
both a40 and a42 treated for 90 hours at ph 6 were observed to have large average hydrodynamic radius particles , with 40% of these samples being withheld by a 100 nm filter and both displayed significant thioflavin - t staining . also , these early - and late - stage a samples were observed to have an ensemble of a conformations ( figure 1 ) .
the interactions of these early - and late - stage a conformations were monitored with differentiated pc12 cells . whereas late stage a40 and a42 exhibited very few interactions with cells ( figures 4 , 5 ) and internalized at a very low frequency ( figure 6 ) , early stage a40 and a42 interacted with cells rapidly ( figure 4 ) and internalized into the majority of the cells present ( figure 6 ) .
interestingly , a40 showed a more rapid cellular association and internalization compared to a40 ( figure 4 ) .
similarly , a42 was found to internalize more rapidly than a42 , indicating that a specific conformation may play a role in cell binding and internalization .
furthermore , our model of endocytic a toxicity ( figure 10 ) is in agreement with previous findings utilizing chemically - produced oligomers of a which showed that a fibrils were less toxic than soluble oligomers [ 41 , 53 ] .
our results with a are also in agreement with studies that have indicated that regions with large amyloid plaques do not correlate directly with regions of significant neuronal loss [ 54 , 55 ] .
for example , the peptide sequences corresponding to tat ( 4860 ) , penetratin , and oligoarginine are known to internalize into live cells .
cellular import of tat ( 4860 ) and penetratin was shown to be temperature dependent , indicating the possible role of endocytosis for internalization [ 56 , 57 ] .
in contrast , oligoarginine containing c - terminal tryptophan was shown to follow a nonendocytic pathway , independent of energy requirements and temperature .
another type of internalization process has been studied in the bacterium clostridium septicum , whose alpha toxin contains functional domains responsible for oligomerization and cellular pore formation . using specific domains that bind cell surface receptors ,
alpha toxin monomers oligomerize to form pores in human cells and thus impose direct entry . by exposing a to endosomal conditions
, we were able to observe internalization of a40/42 , which was inhibited at 4c or with the endocytosis inhibitor mdc ( figure 7 ) . throughout the course of our confocal experiments
, we did not observe any disruption of the cell membrane , which would have been expected if membrane channels or pores were being formed by a. thus , the structural conformation of a that was observed to internalize into cells seems to follow a cell - mediated import mechanism .
our results with the various a conformations correlate directly with previous reports that indicate the deposition of a in the lysosome [ 15 , 59 , 60 ] and with the previous study that internalized a can persist undegraded for days . from our findings taken together with previous studies , we present the following model for the interaction of a with neuronal cells ( figure 11 ) .
as monomeric a binds the cell surface , this event could lead to a conformational change allowing for the further catalytic aggregation of a. recently , it has been shown that oligomeric a binds to gm1 ganglioside and alters physical properties of the plasma membrane which stimulates the amyloidogenic processing pathway of app [ 62 , 63 ] .
a that is bound to the cell surface may become internalized and recycled , allowing for acidification and further aggregation , which in turn may stimulate additional a generation .
once the surface a reach some specific structural form , they may be internalized and directed to the lysosome for potential degradation . however , the reduced ph of the lysosome may instead cause further aggregation and persistence of a , resulting in lysosomal overload and cell death
. the recently reported high turnover rate of monomeric a [ 64 , 65 ] and the formation of large aggregate pools of a may indicate that the human body uses these mechanisms to sequester and remove the toxic a oligomers from neuronal cells . with age , the turnover rate for
monomeric a may slow down , which could then result in the initiation of our proposed mechanism and may contribute to the late onset of alzheimer 's disease .
it is also possible that endocytosis of a and lysosomal targeting are mechanisms by which the cell clears a aggregates from the cell surface .
also , certain large aggregates of a may just be too large for the cell to internalize , and they may represent the precursor for extracellular amyloid formation .
our study shows that using a simple method to generate various a conformations , the rates of cellular interaction and targeting can be followed with live cell cultures . using this model
, we found that early endocytic conformations , rather than highly aggregated late forms , serve as the major contributors to rapid cell internalization .
the mechanism of internalization likely involves a cell surface receptor - mediated process instead of peptide - mediated direct entry , resulting in accumulation in the lysosome .
this method allows for conformation - specific therapeutics and conditions to be screened with live cells , circumventing the need to purify specific a conformations . | the amyloid peptides , a40 and a42 , are generated through endoproteolytic cleavage of the amyloid precursor protein .
here we have developed a model to investigate the interaction of living cells with various forms of aggregated a40/42 .
after incubation at endosomal ph 6 , we observed a variety of a conformations after 3 ( a3 ) , 24 ( a24 ) , and 90 hours ( a90 ) . both a4224 and a4024
were observed to rapidly bind and internalize into differentiated pc12 cells , leading to accumulation in the lysosome .
in contrast , a40/4290 were both found to only weakly associate with cells , but were observed as the most aggregated using dynamic light scattering and thioflavin - t .
internalization of a40/4224 was inhibited with treatment of monodansylcadaverine , an endocytosis inhibitor .
these studies indicate that the ability of a40/42 to bind and internalize into living cells increases with degree of aggregation until it reaches a maximum beyond which its ability to interact with cells diminishes drastically . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion |
PMC3484823 | it starts with bleeding gums , but can finally lead to tooth loss in case of negligence .
periodontal disease ranges from mild gingivitis to severe disease that results in the destruction of the supporting tissue and alveolar bone loss .
previous epidemiological studies in developing countries have shown that the periodontal status of the population that determined by the presence of gingival inflammation , dental plaque and dental calculus was worse compared to countries with higher developmental levels .other epidemiological studies [ 35 ] suggested that subjects in developing countries did not necessarily have a higher prevalence and severity of period on titis than subjects in industrialized nations even though they may have more dental plaque , calculus and gingival inflammation .
previous studies have reported general conclusions regarding the assessment of periodontal status , as they were based on pre - selected population samples .
miyazaki reviewed the community periodontal index of treatment needs ( cpitn ) data according to the previous classification of this index from independent surveys conducted in 79 countries and reported little difference in the prevalence of periodontal pockets between developing and industrialized countries .
very few epidemiological studies that have been carried out in african countries showed low extent and severity of periodontal pockets or attachment loss , while more studies and reports are available from eastern european and scandinavian countries .
similar investigations have not been carried out on a wide spectrum of population samples in greece during the last decades . comparisons with the 1985 findings
indicate that severe periodontal diseases may be declining in greece , whereas gingivitis may be increasing . in order to test the characterization of prevalence and severity of periodontal disease , significance of differences between full - mouth examination and partial recording protocols
for this reason , the mentioned protocols for assessment of periodontal status include several periodontal indices , such as russell s index , periodontal disease index , gingival periodontal index , gingival bone count index , extent and severity index and community periodontal index .
therefore , the purpose of the present study was to examine the periodontal condition of a sample of adults in three isolated regions in a city in greece and estimate the association of demographic , behavioural and clinical factors with periodontal disease .
in the present study , 640 individuals , 300 males and 340 females , 2069 years of age were selected .
all the selected subjects were inhabitants of three isolated villages in n. w achaia , one of the biggest municipalities in greece , who visited a private practitioner for their regular dental follow - up which was organized by the greek dental association for all the greek population annually .
the whole population of the mentioned villages was estimated as 1,023 inhabitants according to the local authorities .
the majority of the inhabitants worked in the capital of achaia , patra and attended the local university .
inco - operation between the greek dental association and the local authorities , they emphasized the necessity and importance for an annual dental follow - up .
an additional motivation for participation was the distribution of printed instructions on proper oral hygiene aspects to the individuals examined .
the sample was divided into five groups based on age ; namely , group i ( 120 individuals ) , 20 to 29 years old ; group ii ( 130 individuals ) , 30 to 39 years old ; group iii ( 140 individuals ) , 40 to 49 years old ; group iv ( 130 individuals ) , 50 to 59 years old ; group v ( 120 individuals ) , 60 to 69 years old .
the participants of the study underwent an oral clinical and physical examination and filled a similar self - administered questionnaire that included several epidemiological variables . the oral health study presented here took place between may and october 2011 at the mentioned private practice .
the present study was not an experimental one . in greece , only experimental studies must be reviewed and approved by authorized committees ( such as the greek dental association , ministry of health ) .
all participants were informed about the evaluation to which they would be submitted and gave their informed consent for participation .
if an index tooth was absent , the closest distal tooth was used as a substitute . in the absence of a tooth distal to the index tooth ,
molar or premolar index teeth were never replaced by the examination of an anterior tooth . in individuals with extensive destruction of the cemen to - enamel junction due to dental caries , erosion ,
abrasion , presence of prosthetic restoration or dental calculus , the location of the junction was estimated according to the location of adjacent teeth .
individuals who had undergone a previous periodontal treatment , conservative or surgical , within the previous six months were excluded from the study sample .
edentulous adults ( n= 0 ) were excluded from the study due to the impossibility of evaluating the periodontal conditions in this population .
two independent physical examinations were performed , one blinded and one non - blinded to the medical history . just before the physical examination , participants filled in a similar self - administered questionnaire that included several epidemiological variables such as age , gender , educational level ( primary , secondary , college , university ) , smoking status ( current regular smoker , occasional smoker and non - smoker ) , dental visits ( frequency and reasons for the last dental visit ) , oral hygiene habits ( tooth brush frequency , use of dental floss / mouthwash ) and data regarding their variables of their general health .
socioeconomic status of the sample was excluded from the study because of the current economic crisis in greece , as it could be a negative motivation for the individuals in order to participate . on the other hand
, the study sample consisted of individuals who were permanent inhabitants in isolated areas and no significant differences regarding their socieconomic status would be expected .
one well - trained and calibrated dentist who was also registered as an active member . in the hellenic society of periodontology ( hsp ) and european federation of periodontology ( efp ) performed the examinations of the participants and the clinical measurements .
the clinical measurements concerned the following teeth ( according to ramfjord index ) : 16 , 21 , 24 , 36 , 41 and 44 .
the reason that ramfjord index was used is that it represents a simple , easy , reproducible and representative as possible periodontal index , especially in isolated societies with a low - level of oral hygiene and it is one of the most appropriate indices for partial - mouth recording protocols .
the variables that were measured clinically were the following :
assessment of dental plaque and dental calculus ( 0= no calculus , 1= supra - gingival calculus , 2= subgingival calculus ) .
presence or absence of supra - gingival plaque was recorded after disclosing soft deposits using erythromycin solution ( 3% ) for a period of 30 seconds ; the teeth and gingival were dried with compressed air while dental unit light was used as the light source for the inspections .
dental plaque and calculus indices were recorded from the mesio - buccal site of each index tooth .
the plaque index ( pli ) , which was used was a modification of the one determined by le .scores 2 and 3 were difficult to be distinguished from each other and therefore were combined into a single category ( i.e. , 0= no dental plaque , 1= plaque detected after running the probe across the cervical area of the index tooth , 2= plaque that could be detected visually).probing depth ( pd ) assessmentclinical attachment level ( cal)location of the free gingival margin ( fgm ) relative to the cemento - enamel junction(cej ) , fgm - cejbleeding on probing ( bop ) assessment of dental plaque and dental calculus ( 0= no calculus , 1= supra - gingival calculus , 2= subgingival calculus ) .
presence or absence of supra - gingival plaque was recorded after disclosing soft deposits using erythromycin solution ( 3% ) for a period of 30 seconds ; the teeth and gingival were dried with compressed air while dental unit light was used as the light source for the inspections .
dental plaque and calculus indices were recorded from the mesio - buccal site of each index tooth . the plaque index
( pli ) , which was used was a modification of the one determined by le .
scores 2 and 3 were difficult to be distinguished from each other and therefore were combined into a single category ( i.e. , 0= no dental plaque , 1= plaque detected after running the probe across the cervical area of the index tooth , 2= plaque that could be detected visually ) .
probing depth ( pd ) assessment clinical attachment level ( cal ) location of the free gingival margin ( fgm ) relative to the cemento - enamel junction(cej ) , fgm - cej bleeding on probing ( bop ) the above mentioned indices ( pd ) , ( cal ) , ( fgm - cej ) and ( bop ) were recorded at six sites on each index tooth . a william s probe ( pcp10-se , hu - friedy mfg .
, chicago , il , usa ) color coded at 1 , 2 , 3 , 5 , 7 , 8 , 9 , 10 mm was used for periodontal examinations and pd and cal were measured to the nearest millimeter .
mean cal ( averaged across sites in each individual)was used to describe the cumulative amount of periodontal destruction .
prevalence of periodontal destruction was determined as the percent of individuals who showed a 4.06.0 mm or higher than 7.0 mm cal at their most severely affected sites . the extent of mild , moderate and severe periodontal destruction was estimated by the percent of sites per individual with a cal of 2.04.0 mm , 4.06.0 mm and higher than 7.0 mm .
the reason why the mentioned classification was not based on the american academy of periodontology , is that , as mentioned in an isolated population , the oral hygiene level is poor enough and the presence of deep pockets and severity of clinical attachment loss could be expected extensively .
a randomly chosen sample of 128(20% ) individuals was re - examined clinically by the same dentist in order to establish the intra - examiner variance .
after consideration of the code numbers of the double examined participants no differences were recorded between the 1 and 2 clinical assessments .
it is obvious that in this case the in traexaminer consistency of clinical recordings per sextant was not estimated by k index .
the individual was the statistical unit . for each participant , the mean values of the clinical parameters ;
namely , the dental plaque , dental calculus , pd , cal and fgm - cej were calculated .
multiple linear regression analysis was used in order to assess the association of the mean cal with demographic , behavioural and clinical parameters .
associations were assessed by adding each independent variable to the fully adjusted model and testing whether the explained variable was increased significantly .
tooth brushing frequency was classified into three levels ( daily , occasionally , never ) .
frequency of dental visits was dichotomized ( yearly or more frequently vs. less frequently or never ) .
gender and smoking were classified as dichotomous variables and age , mean plaque and mean calculus scores ( averaged across sites in each individual ) were used as continuous variables .
the final multivariate model contained variables that were significant after adjusting for all other variables in the model .
the variables included in the final model for mean cal were then included in regressions using mean pd and mean fgm - cej as dependent variables .
the purpose for using the above mentioned procedure was to assess whether the association of the independent variables with the mean cal was related to variations in mean pd and/or to variations in the location of the fgm relative to the cej .
the data analysis was performed using the statistical package of spss ver.17.0 ( spss inc . ,
chicago , il , usa ) . a p value less than 5% ( p<0.05 ) was considered as statistically significant .
in the present study , 640 individuals , 300 males and 340 females , 2069 years of age were selected .
all the selected subjects were inhabitants of three isolated villages in n. w achaia , one of the biggest municipalities in greece , who visited a private practitioner for their regular dental follow - up which was organized by the greek dental association for all the greek population annually .
the whole population of the mentioned villages was estimated as 1,023 inhabitants according to the local authorities .
the majority of the inhabitants worked in the capital of achaia , patra and attended the local university .
inco - operation between the greek dental association and the local authorities , they emphasized the necessity and importance for an annual dental follow - up .
an additional motivation for participation was the distribution of printed instructions on proper oral hygiene aspects to the individuals examined .
the sample was divided into five groups based on age ; namely , group i ( 120 individuals ) , 20 to 29 years old ; group ii ( 130 individuals ) , 30 to 39 years old ; group iii ( 140 individuals ) , 40 to 49 years old ; group iv ( 130 individuals ) , 50 to 59 years old ; group v ( 120 individuals ) , 60 to 69 years old .
the participants of the study underwent an oral clinical and physical examination and filled a similar self - administered questionnaire that included several epidemiological variables . the oral health study presented here took place between may and october 2011 at the mentioned private practice
, only experimental studies must be reviewed and approved by authorized committees ( such as the greek dental association , ministry of health ) .
all participants were informed about the evaluation to which they would be submitted and gave their informed consent for participation .
if an index tooth was absent , the closest distal tooth was used as a substitute . in the absence of a tooth distal to the index tooth , the next tooth in the mesial location was measured .
molar or premolar index teeth were never replaced by the examination of an anterior tooth . in individuals with extensive destruction of the cemen to - enamel junction due to dental caries , erosion ,
abrasion , presence of prosthetic restoration or dental calculus , the location of the junction was estimated according to the location of adjacent teeth .
individuals who had undergone a previous periodontal treatment , conservative or surgical , within the previous six months were excluded from the study sample .
edentulous adults ( n= 0 ) were excluded from the study due to the impossibility of evaluating the periodontal conditions in this population . similarly , wisdom teeth and individuals with systemic disorders were excluded from the study .
two independent physical examinations were performed , one blinded and one non - blinded to the medical history . just before the physical examination , participants filled in a similar self - administered questionnaire that included several epidemiological variables such as age , gender , educational level ( primary , secondary , college , university ) , smoking status ( current regular smoker , occasional smoker and non - smoker ) , dental visits ( frequency and reasons for the last dental visit ) , oral hygiene habits ( tooth brush frequency , use of dental floss / mouthwash ) and data regarding their variables of their general health .
socioeconomic status of the sample was excluded from the study because of the current economic crisis in greece , as it could be a negative motivation for the individuals in order to participate .
on the other hand , the study sample consisted of individuals who were permanent inhabitants in isolated areas and no significant differences regarding their socieconomic status would be expected .
one well - trained and calibrated dentist who was also registered as an active member . in the hellenic society of periodontology ( hsp ) and european federation of periodontology ( efp ) performed the examinations of the participants and the clinical measurements .
the clinical measurements concerned the following teeth ( according to ramfjord index ) : 16 , 21 , 24 , 36 , 41 and 44 .
the reason that ramfjord index was used is that it represents a simple , easy , reproducible and representative as possible periodontal index , especially in isolated societies with a low - level of oral hygiene and it is one of the most appropriate indices for partial - mouth recording protocols .
the variables that were measured clinically were the following :
assessment of dental plaque and dental calculus ( 0= no calculus , 1= supra - gingival calculus , 2= subgingival calculus ) .
presence or absence of supra - gingival plaque was recorded after disclosing soft deposits using erythromycin solution ( 3% ) for a period of 30 seconds ; the teeth and gingival were dried with compressed air while dental unit light was used as the light source for the inspections .
dental plaque and calculus indices were recorded from the mesio - buccal site of each index tooth .
the plaque index ( pli ) , which was used was a modification of the one determined by le .scores 2 and 3 were difficult to be distinguished from each other and therefore were combined into a single category ( i.e. , 0= no dental plaque , 1= plaque detected after running the probe across the cervical area of the index tooth , 2= plaque that could be detected visually).probing depth ( pd ) assessmentclinical attachment level ( cal)location of the free gingival margin ( fgm ) relative to the cemento - enamel junction(cej ) , fgm - cejbleeding on probing ( bop ) assessment of dental plaque and dental calculus ( 0= no calculus , 1= supra - gingival calculus , 2= subgingival calculus ) . presence or absence of supra - gingival plaque was recorded after disclosing soft deposits using erythromycin solution ( 3% ) for a period of 30 seconds ; the teeth and gingival were dried with compressed air while dental unit light was used as the light source for the inspections .
dental plaque and calculus indices were recorded from the mesio - buccal site of each index tooth .
the plaque index ( pli ) , which was used was a modification of the one determined by le .
scores 2 and 3 were difficult to be distinguished from each other and therefore were combined into a single category ( i.e. , 0= no dental plaque , 1= plaque detected after running the probe across the cervical area of the index tooth , 2= plaque that could be detected visually ) . probing depth ( pd ) assessment clinical attachment level ( cal ) location of the free gingival margin ( fgm ) relative to the cemento - enamel junction(cej ) , fgm - cej bleeding on probing ( bop ) the above mentioned indices ( pd ) , ( cal ) , ( fgm - cej ) and ( bop ) were recorded at six sites on each index tooth . a william s probe ( pcp10-se , hu - friedy mfg .
, chicago , il , usa ) color coded at 1 , 2 , 3 , 5 , 7 , 8 , 9 , 10 mm was used for periodontal examinations and pd and cal were measured to the nearest millimeter .
mean cal ( averaged across sites in each individual)was used to describe the cumulative amount of periodontal destruction .
prevalence of periodontal destruction was determined as the percent of individuals who showed a 4.06.0 mm or higher than 7.0 mm cal at their most severely affected sites . the extent of mild , moderate and severe periodontal destruction was estimated by the percent of sites per individual with a cal of 2.04.0 mm , 4.06.0 mm and higher than 7.0 mm .
the reason why the mentioned classification was not based on the american academy of periodontology , is that , as mentioned in an isolated population , the oral hygiene level is poor enough and the presence of deep pockets and severity of clinical attachment loss could be expected extensively .
a randomly chosen sample of 128(20% ) individuals was re - examined clinically by the same dentist in order to establish the intra - examiner variance .
after consideration of the code numbers of the double examined participants no differences were recorded between the 1 and 2 clinical assessments .
it is obvious that in this case the in traexaminer consistency of clinical recordings per sextant was not estimated by k index .
the individual was the statistical unit . for each participant , the mean values of the clinical parameters ;
namely , the dental plaque , dental calculus , pd , cal and fgm - cej were calculated .
multiple linear regression analysis was used in order to assess the association of the mean cal with demographic , behavioural and clinical parameters .
associations were assessed by adding each independent variable to the fully adjusted model and testing whether the explained variable was increased significantly .
tooth brushing frequency was classified into three levels ( daily , occasionally , never ) .
frequency of dental visits was dichotomized ( yearly or more frequently vs. less frequently or never ) .
gender and smoking were classified as dichotomous variables and age , mean plaque and mean calculus scores ( averaged across sites in each individual ) were used as continuous variables .
the final multivariate model contained variables that were significant after adjusting for all other variables in the model .
the variables included in the final model for mean cal were then included in regressions using mean pd and mean fgm - cej as dependent variables .
the purpose for using the above mentioned procedure was to assess whether the association of the independent variables with the mean cal was related to variations in mean pd and/or to variations in the location of the fgm relative to the cej .
the data analysis was performed using the statistical package of spss ver.17.0 ( spss inc . ,
the total number of the participants who visited the mentioned practice was 712 , of which 673 met the selection criteria and 640 of them accepted the invitation to participate in the study giving a response rate of 95.1% .
the educational level , tobacco consumption , reasons for seeking dental care and oral hygiene habits of the sample of the study are shown in figure 1 .
table 1 presents the extent ( percent of affected sites per subject by age group and gender ) of cal , periodontal pockets , gingival recession , bop , dental plaque and calculus .
the final multivariate model ( stepwise method ) showed that age ( p=0.000 ) , gender(p=0.016 ) and presence of calculus ( p=0.000 ) were associated with mean cal .
age(p=0.018 ) and gender ( p=0.000 ) were significantly associated with mean pd , while according to the same age model ( p=0.000 ) , gender ( p=0.000 ) and the presence of dental plaque ( p=0.027 ) were significantly associated with mean gingival recession ( table 3 ) .
the present study has important limitations because the sample was not randomly drawn from the population but consisted of patients that visited a private dental practice for their regular dental follow - up which was organized by the greek dental association annually . on the other side
similar studies have not been carried out in greece and data from the present study are only comparable to those reported for similar studies which were carried out in other countries .
mamai - homata et al . recorded the periodontal status of greek adults aged 3544 years in relation to socio - demographic and behavioral parameters and collected limited data comparable to those reported in the present study regarding few aspects only . according to the results of the present study , the sample presented a relatively high prevalence , but low extent of cal .
, the prevalence of a cal higher than 4 mm was 92% among 30 to 39-year - old individuals and 100% among 50 to 59-year - old individuals , whereas contrary to the present study regarding the extent of cal , the average percentage ( % ) of sites affected was 84% among 30 to 39-year - old individuals and 93% among 50 to 59-year - old individuals .
the high prevalence of the assessed cal was 72.1% ( 5 mm ) and 60.9% ( 7 mm ) .
in addition , 95% of the 35 to 44-year - old individuals and 99.2% of the 65 to 74-year - old individuals presented a cal higher than 3 mm and 100% of the older than 50 years old individuals higher than showed a higher than 5 mm cal , while the lower prevalence that was assessed was 19.7% ( > 5 mm ) .
the extent of a higher than 4 mm cal that was assessed was 11.8% in a vietnamese population and 20% in a younger adult population showing a higher level than that of the present study . according to the above observations , the mean cal and the percentage of sites / individual with cal 2 mm in the present study are comparable to those mentioned studies that had regular access to dental care and preventive dentistry , while are less severe that those reported for populations in the european and other countries . those differences could be attributed to several factors such as the heterogeneous population samples , the different interests that have been showed by the population samples regarding the value of oral hygiene and the need for a regular dental follow - up , the origin of the sample collected ( such as the dental hospital or private practice ) , the fact that most of the reviewed studies assessed the periodontal conditions using full - mouth clinical examinations with four or six sites per tooth , while others used two facialsites per tooth in half of the dentition .
in addition , the sample of the present study concerned individuals who sought dental treatment in a private dental practice and we could not consider it as random one . if it is supposed that a site with cal 46 mm presents moderate periodontitis and that rates greater than 7 mm are consistent with a diagnosis of advanced periodontitis , according to table 2 which shows the prevalence of individuals with at least one affected site , at least 68.2% of the 60 to 69-year - old individuals showed sites with moderate periodontitis and at least 27.6% of the subjects of the same age group showed advanced periodontitis .
however , the final model showed that age , gender and dental plaque were the most important factors associated with gingival recession ; whereas , age , gender and calculus were the factors that were associated with cal . in a study by corraini et al .
, multivariate analysis identified dental plaque , calculus and age as risk indicators for cal 5 mm .
. found that an older age and a high percentage of sites with calculus were significant positive predictors of a high percentage of sites with cal 4 mm while the same parameters were statistically significant predictors of a high percentage of sites with cal 7 mm .
similar results regarding age and cal were observed in a study by holtfreter et al . and also a study by wang et al . .
however , bouchard et al . found that age and gender were powerful independent predictors of cal . in the present study ,
the prevalence of pocket depth increased with age up to the 40 to 49-years - old age group for a pd of 46 mm and then decreased , while for severe pockets ( 7 mm ) the prevalence increased with age up to the 5059-years - old age group .
previous studies have recorded higher prevalence rates of probing depths such as 75% ( 5 mm ) , 69.7%( 4 mm ) , 25.3%( 6 mm ) and 11.3% ( > 5.5 mm ) , while other studies have recorded lower prevalence rates of probing depths such as 30% ( 4 mm ) , less than 5 % ( 6 mm ) and 43.3% ( 4 mm )
. similar findings with higher prevalence rates have been recorded and assessed ; 84%( 4 mm ) among 3039-year - old individuals and 93% ( pd 4 mm ) among 50 - 59-year - old individuals while in another study 76.9% ( 4 mm ) was detected among 35 - 44-year - old individuals and 87.7% ( 4 mm ) among 65 to 74-year - old individuals .
the extent of the pocket depths in the present study was lower than the pocket depths in a study of a vietnamese population and another study including young adults , while it was higher than the pocket depths in a study in the amazon rain forest .
as mentioned , gingival inflammation was determined by the percentage of sites per individual that showed bop . according to the results of the present study , gingivitis increased significantly with age except in the 60 to 69-year - old age group .
however , the above - mentioned finding was not in agreement with a previous study performed by ronderos et al . .
bop ( 57.8% ) was lower than a study in the usa , which was estimated as 82.8% , while in another study in brazil , it occurred in 97.9% of the samples .
similar observations were recorded in a study by silva - boghossian et al . in brazil , whereas in a study in greece , bop was 16.2% . according to the mentioned observations , the present study suggests that even without prior therapeutic or preventive period on talintervention , subjects with extensive dental plaques , gingivitis and calculus do not present severe loss of period on taltissue .
this finding is in agreement with a previous study which showed that destructive periodontal disease is not necessarily an inevitable consequence of long - standing gingival inflammation .
in addition , the present study does not support that periodontal disease is more extensive among poor societies and populations with minimal access to dental care and preventive dentistry , situations that are common in isolated villages / towns .
bop was not associated with cal , pd or gingival recession , while previous studies showed that bop was significantly associated with cal and increased pd , but not with gingival recession .
this finding regarding age is in agreement with observations of other studies in several age groups .
this relationship between the occurrence of gingival recession and age could be attributed to the longer period of exposure to factors that cause gingival recession and the cumulative effects of the lesion itself . regarding the role of dental plaque and gingival inflammation in the development of gingival recession
, previous studies have showed that gingival inflammation was the most frequent precipitating etiological factor of gingival recession .
they suggested that a localized inflammatory process causes the breakdown of connective tissue destruction and have also reported that gingival recession was associated with a high level of dental plaque while a study by goutoudi et al
. revealed that gingival margin recession was associated with both high inflammatory and plaque scores .
in addition , a significant association between gingival recession and periodontal disease was recorded .
a study by slutzkey and levin showed a negative correlation between dental plaque on the buccal tooth aspect and gingival recession , while loe et al . emphasized the role of poor oral hygiene , dental plaque and calculus in gingival recession .
pd was significantly associated with age and gender , but not dental plaque or calculus .
found that the prevalence of periodontal pockets greater than 6 mm was associated with gender .
the multivariate analysis indicated an association between dental plaque and gingival recession and this association was independent of calculus .
furthermore , tobacco consumption , educational level , tooth brushing frequency and reasons for dental visit were not significantly related to the mean attachment loss .
bergstrm , bokor - bratic and many investigators have shown the effects of smoking on the periodontium and periodontal health .
these studies have shown that cigarette smoking is clearly established as one of the most significant risk factors in the development and the progression of periodontal disease . regarding the role of dental hygiene habits ,
it is known that proper use of tooth brush and use of dental floss are the most effective tools for removal of dental plaques from tooth surfaces , especially in terproximal surfaces .
similarly , a regular dental follow - up can prevent the development , extent and severity of all forms of periodontal diseases .
the role of educational level regarding oral hygiene habits is also known , since more educated individuals have less gingival inflammation or other forms of periodontal diseases , which might be attributed to the fact that more educated individuals have realized the value and importance of preventive dentistry and oral hygiene procedures , applying proper habits and standards of oral hygiene , and following regular dental check - ups .
similar observations have been recorded in other studies , indicating that the level of education is the most important contributor to periodontal destruction .
recorded that low educational subjects exhibited significantly more periodontal attachment loss and had significantly fewer healthy gingival units , while zini et al . showed that a lower level of education was associated with severe chronic periodontitis .
periodontal disease of this sample was mainly associated with gingival inflammation and presence of dental plaque and calculus . however , the majority of the individuals showed cal and gingival recession rather than deep pockets . despite the extensive gingival inflammation and poor oral hygiene of the sample , it did not present destructive forms of periodontal disease .
study results showed that a strong need exists for improvement of the population s self - awareness of oral hygiene and better oral health education mainly in rural regions of greece .
in addition , it is important to focus more on periodontal aspects of dental care and effective prevention programmes and better control of periodontal disease are required . | objective : the aim of the present study was to examine the periodontal condition of an adult population in three isolated regions in greece and to determine the association of periodontal disease with several demographic , behavioral and environmental factors.materials and methods : the study population consisted of 640 individuals , aged 20 to 69 years from three isolated regions .
the following indices were assessed : pocket depth ( pd ) , clinical attachment level ( cal ) , dental plaque , calculus and bleeding on probing ( bop ) .
statistical analysis was accomplished by multiple linear regression model which was used to assess the association between the mean clinical attachment loss and clinical , demographic and behavioral parameters.results:the samples of the study showed high levels of dental plaque , dental calculus and bop .
the final multivariate model showed that age ( p=0.000 ) , gender ( p=0.016 ) and presence of calculus ( p=0.000 ) were associated with the mean clinical attachment loss .
age ( p=0.000 ) , gender ( p=0.000 ) and dental plaque ( p=0.027 ) were associated with gingival recession , while age ( p=0.018 ) and gender ( p=0.000 ) were associated with probing depth .
bleeding on probing , dental plaque , toothbrush frequency , level of education , tobacco consumption and reasons for dental visits were not associated with the mean clinical attachment loss.conclusion:periodontal disease consists of a complicated destructive condition of the periodontal tissue with a. multi - factorial etiology . oral hygiene instructions and
a regular dental follow - up could play a significant role in the prevention of periodontal disease . | INTRODUCTION
MATERIALS AND METHODS
Study population:
Ethics:
Inclusion criteria:
Exclusion criteria:
Questionnaire:
Clinical Examination:
Reproducibility:
Statistical analysis:
RESULT
DISCUSSION
CONCLUSIONS |
PMC4203578 | jellyfish ( aurelia aurita ) were obtained from the new england aquarium and maintained in 20-l aquaria at 20 c . for recording
, individual animals were placed into a glass filming vessel ( 30 10 25 cm ) filled with filtered seawater .
recordings of free - swimming animals were acquired by a high - speed digital video camera ( fastcam 1024 pci ; photron ) at 1,000 frames per second .
detailed swimming kinematics ( 2d ) were obtained using imagej v1.46 software ( national institutes of health ) to track the x and y coordinates of the apex of the jellyfish bell margin over time .
bell margin speed was calculated from the change in the position of the margin tip over time as :
u=((x2x1)2+((y2y1)2))1/2t2t1
jellyfish were illuminated with a laser sheet ( 680 nm , 2w continuous wave ; lavision ) oriented perpendicular to the camera s optical axis to provide a distinctive body outline for image analysis and to ensure the animal remained in - plane , which ensures the accuracy of 2d estimates of position and velocity .
fluid motion created by the jellyfish while swimming was quantified using 2d digital particle image velocimetry ( dpiv ) . using the setup described in gemmell et al .
the velocities of particles illuminated in the laser sheet were determined from sequential images analyzed using a cross correlation algorithm ( lavision software ) .
image pairs were analyzed with shifting , overlapping interrogation windows of a decreasing size of 64 64 pixels to 32 32 pixels . | the ability of animals to propel themselves efficiently through a fluid medium is ecologically advantageous .
flexible components that influence vortex interactions are widespread among animal propulsors . however the mechanisms by which vortices are enhanced and appropriately positioned for thrust generation are still poorly understood . here , we describe how kinematic propulsor movements of a jellyfish can enhance and reposition a vortex ring that allows the recapture of wake energy for secondary thrust generation and efficient locomotion .
we use high - speed video and digital particle image velocimetry ( dpiv ) to resolve kinematics simultaneously with fluid structures .
these results provide new insight into how animals can manipulate fluid structures to reduce metabolic energy demands of swimming muscles and may have implications in bio - inspired design . | Materials and Methods |
PMC4297096 | they are the most common cause of severe long - term pain and physical disability , and affect hundreds of millions of people s quality of life all around the world .
osteoarthritis ( oa ) , a progressive , degenerative disease of the ( articular ) joints ( hips , knees , neck , lower back , or small joints of the hands ) , represents the most common musculoskeletal disease .
the etiology of the disease is not well understood and oa is considered a complex disease to which both genetic and acquired or environmental factors contribute .
misalignment of the joint and joint instability resulting from ( untreated ) cartilage defects , previous surgery ( e.g. , partial or total meniscectomy ) or from other traumas and injuries to the joint , as well as intensive loading due to obesity or intensive sports activities , are all particular risk factors to develop the disease .
oa affects all structures within the joint , but an early hallmark is a progressive degeneration of the articular cartilage accompanied by bony remodeling ( subchondral bone sclerosis and osteophyte formation ) , occasional flares of inflammation , mild synovitis ( in some patients ) , and osteitis .
localized loss of cartilage can increase focal stress across the joint leading to further cartilage destruction .
this then eventually results in a misalignment and destabilization of the whole joint and progression and aggravation of the disease
. early diagnosis of oa - associated cartilage alterations would be very beneficial for disease prevention and control , not at the least because current treatment of established oa is symptomatic and palliative with an emphasis on control of joint pain and maintenance of function .
the lack of a cure and associated huge unmet medical need provide an opportunity for the development of novel approaches and medicines to treat oa , especially for disease - modifying therapies that can halt and/or reverse the onset and progression of the disease .
however , the development of therapeutic approaches is largely hampered by the lack of quantifiable readouts in preclinical models .
refinement and validation of the readout technology for oa biology would be tremendously valuable for the animal studies on joint degeneration , and should allow us capturing subtle differences and thus broader hallmarks of the disease , carefully map the disease progression in the different tissues and animal models and , ultimately , asses the beneficial changes of those hallmarks when testing the candidate disease - modifying treatment . currently , preclinical efficacy of novel therapies for cartilage repair in small animal models is commonly assessed by histopathology .
the modified mankin scoring system is often used for semiquantitative histological assessment of oa because of its already widespread adoption , ease of use , and its similarity to scoring systems used for oa in humans .
this method focuses , among others , on the structural changes observed , such as lesions or brillations and losses in proteoglycan staining .
histopathology has a high discriminative power but is destructive , labor intensive , costly in terms of time and resources , and , in standard settings it only provides 2-dimensional ( 2d ) information because of the loss of data by the thickness of the sections , thus spatial and quantitative 3d assessment of the cartilage structure and quality are not feasible .
additionally , the modified mankin scoring is currently not annotated automatically , with consequent interreader variability .
the said limitations prompt the current need for a 3d imaging technique that is able to quantify the cartilage properties in the murine joint and can replace histopathology with a more efcient , high throughput , longitudinal imaging method .
recent advances in 3d imaging techniques , such as high - resolution microfocus x - ray computed tomography ( microct ) imaging , have led to successful quantification of the changes in the subchondral bone structure in the mouse knee joint during the progression of oa . the use of ionic partition equilibrium contrast agents , such as anionic ( i.e. , hexabrix ; covidien , hazelwood , mo ) or cationic contrast agents , as well as lead - based contrast agents have shown to allow quantification of functional and structural information of the cartilage . however , the limited spatial image resolution of most microct devices does not allow distinguishing calcified cartilage from the subchondral bone . additionally , most studies implementing new structural and functional readouts refrain to show equivalence to standard histopathological scorings
. moreover , although several 3d cartilage characteristics have been quantified , no validation compared with the golden standard of histopathological scoring was performed yet . in a previous study
, we evaluated the potency of contrast - enhanced nanofocus x - ray computed tomography ( ce - nanoct ) to simultaneously image in 3d the subtissue architecture of cartilage at high contrast and spatial image resolution without invasive , labor - intensive sample preparation . in this study , we have shown , in an established mouse model for oa , that ce - nanoct can be used as an imaging tool for virtual 3d histopathology . by direct comparison with standard histopathology
we showed equivalence of ce - nanoct images for the scoring of the structure , quality , and pathophysiology of the chondro - osseous junction .
additionally , the quantitative nature of the 3d ce - nanoct data sets allowed the development of an empirical model based on a limited set of 3d cartilage characteristics measured by 3d image analysis , which is predictive for the cartilage quality and structure as can be evaluated by histopathological scoring .
this model thus could be a potential replacement of or complimentary tool for the modified mankin scoring when assessing preclinical efficacy of novel therapies for cartilage repair .
destabilization of the medial meniscus ( dmm ) by medial meniscotibial ligament transection leads to mild oa with various degrees of cartilage lesions . to show the potential of ce - nanoct for virtual histopathology and 3d quantification of cartilage characteristics , 4 treatment groups were assessed .
right knee joints were harvested from adult ( rj : nmri - foxn1/foxn1 ) mice that were sham operated ( sham ) , or that underwent a medial meniscus destabilization procedure ( dmm ) and were sacrificed 8 weeks later . as a third group ( dmm / ha ) , right knee joints were harvested after 8 weeks from mice that underwent a dmm procedure and were injected after 2 and 5 weeks with hyaluronic acid , which is in clinical use as viscosupplementation treatment . a final group contained age - matched nonsurgical controls ( am ) .
we have worked with the nude mouse model to assess the effect of dmm on the cartilage structure and quality , because it allows testing a cellular therapy in the mouse using human cells ( ongoing work ) .
these 4 research - relevant groups were selected to obtain a broad spectrum of cartilage defects on a per - animal basis ( i.e. , from bad to unaffected ) that one might encounter in preclinical oa research , thus to show the potency of ce - nanoct to be implemented as a standard technique in the oa field rather than to interpret the biology behind dmm - induced oa .
all knee joints were imaged using ce - nanoct , after which some were randomly sampled to allow a comparison with the histological gold standard .
animal experiments were performed in accordance with the belgian legislation under the national authorization number la1210530 and were approved by the ethical committee of the faculty of biomedical sciences of ku leuven . to enable ce - nanoct from the 4 mouse knee joint groups ,
briefly , the knee joint capsules were dislocated to expose the tibial and femoral cartilage layers .
subsequently , all joints were immersed for 30 minutes in a solution of hexabrix 320 diluted 4/5 in phosphate - buffered saline , then wrapped in parafilm , and were stably positioned in the nanoct system . as the negatively charged ioxaglate of which
hexabrix 320 is partially composed , will be locally repelled by the anionic glycosaminoglycans ( sgag ) in the cartilage , ioxaglate accumulation in the cartilage is inversely related to the sgag content .
the nanoct system applied in this study was a phoenix nanotom s ( ge measurement and control solutions , munich , germany ) .
it was equipped with a tungsten target and was operated , for all scans , at a voltage of 60 kv and a current of 140 a .
the exposure time was 500 ms and a frame averaging of 1 and image skip of 0 was applied , resulting in a scanning time of 20 minutes per sample .
as described in kerckhofs et al . , to quantify the 3d characteristics of the noncalcified and underlying calcified cartilage layers , the cartilage region was selected using ctan software ( bruker micro - ct , kontich , belgium ) by manually drawing a consistent region of interest ( roi ) in every 20th cross - section throughout the whole tibial plateau . briefly , delineating the total cartilage area ( i.e. , both noncalcified and calcified ) was performed based on ( a ) the difference in grayscale between the noncalcified cartilage and the surrounding ( i.e. , liquid ) and ( b ) the difference in structure compared with the subchondral bone and noncalcified cartilage for the calcified cartilage ( i.e. , having a porous structure ) .
after automatic otsu segmentation , the noncalcified and calcified cartilage were segmented and analyzed in 3d by calculating the volume , the thickness distribution , and the average thickness .
dataviewer ( bruker micro - ct , kontich , belgium ) was used on the ce - nanoct images to perform the modified mankin scoring for cartilage structure and quality , as well as to quantify the loss in sgag content . after ce - nanoct imaging , a set of representative knee joints were processed for histology .
they were fixed in 10% formalin , decalcified in 4% formic acid , and embedded in paraffin .
sections of 5 m were made every 60 m and were stained with safranin - o and counterstained with fast green .
the medial side of the tibial plateau in the histological sections ( n = 10 per joint ) and coronal ce - nanoct images ( n = 10 with a 60 m interspacing ) was graded using a modified mankin scoring system specifically for cartilage structure and quality . in brief , for both the histological sections and the ce - nanoct images , articular cartilage structure was graded from 0 to 11 . concerning the cartilage quality , the safranin - o staining for the histological sections and the hexabrix staining for the ce - nanoct images were graded between 0 and 8 . for both parameters , grade
a grade of 11 for the cartilage structure was indicative for clefts / loss of articular cartilage extending into the subchondral bone .
grade 8 for cartilage quality implied a severe loss in safranin - o ( or increase in hexabrix ) staining in more than half of the depth of the noncalcified cartilage thickness and involving more than half of the plateau .
grades of each sample ( n = 10 ) were averaged and summed for the 2 scoring parameters , whereby 0 represented completely unaffected hyaline cartilage and the maximum mankin score of 19 represented a highly degenerated oa knee . to quantify the sgag content in the hyaline cartilage , the intensity of the hexabrix excluded signal in the noncalcified layer of the hyaline cartilage was normalized to the signal of the growth plate cartilage ( i.e. , in the same coronal ce - nanoct cross - sections ) .
a grayscale histogram was plotted along an arbitrary 600 m line through the noncalcified cartilage on the medial side of the tibial plateau ( fig .
the normalized sgag content in the cartilage compared to the healthy growth plate cartilage was then calculated according to equation ( 1 ) .
the normalized sgag content was measured in the same coronal ce - nanoct cross - sections as used for the modified making scoring .
( a ) a typical coronal contrast - enhanced nanofocus computed tomography ( ce - nanoct ) image and ( b ) the corresponding grayscale histogram along the white line drawn in ( a ) through the noncalcified cartilage on the medial ( left ) side of the tibial plateau ( gray curve ) with the average grayscale value ( black curve ) .
( c ) a typical coronal ce - nanoct image and ( d , e ) the corresponding grayscale histograms ( d ) along the white line annotated with 1 drawn in ( c ) through the background of the image ( gray curve ) with the average grayscale value ( black curve ) and ( e ) along the white line annotated with 2 drawn in ( c ) through the growth plate cartilage ( gray curve ) with the average grayscale value ( black curve ) .
1b ) along an arbitrary line through the noncalcified cartilage on the medial side of the tibial plateau ( fig .
1d ) along a 600 m long line through the background in the same image ( i.e. , liquid , fig .
1e ) along a 400 m long line through the growth plate cartilage in the same image ( fig .
a computational model was used to determine whether a combined set of morphological cartilage characteristics could quantitatively and in 3d predict the level of cartilage damage in a comparative manner to histological cartilage pathology scoring ( using the modified mankin scoring as a measure for cartilage structure and quality ) .
a linear modeling formalism was used that was already shown to be capable of relating quantitative contributions of multiple signals to a ( single ) measured response , namely partial least square regression ( plsr ) .
plsr is able to identify the information content within the set of measured characteristics that most closely maps onto the output response ( i.e. , modified mankin score ) .
the resulting mapping of lumped signals to corresponding responses allowed to identify the most important structural cartilage characteristics
plsr uses linear algebra to reduce the dimensionality of multivariate data sets by defining principal component axes from the original data set that contain the most important information on a specific output variable . for the optimization of the number of model dimensions ,
the root mean squared error ( rmse ) between the measured level of cartilage damage and the level predicted by plsr models with increasing numbers of principal components was investigated .
each cartilage characteristic was individually withheld from the training set to construct a cross - validation model , in which an rmse of the prediction could be assessed by predicting the withheld sample . the predicted rmse was minimal in this study when using two principal components . to generate the plsr model , the software package jmp 8 ( sas institute inc .
statistical analysis was done using a statistical software add - in for microsoft excel for windows , namely , analyse - it version 2.26 excel 12 + .
to evaluate the significance level between the groups , one - way analyses of variance were used with a confidence level of 95% .
destabilization of the medial meniscus ( dmm ) by medial meniscotibial ligament transection leads to mild oa with various degrees of cartilage lesions . to show the potential of ce - nanoct for virtual histopathology and 3d quantification of cartilage characteristics , 4 treatment groups were assessed .
right knee joints were harvested from adult ( rj : nmri - foxn1/foxn1 ) mice that were sham operated ( sham ) , or that underwent a medial meniscus destabilization procedure ( dmm ) and were sacrificed 8 weeks later . as a third group ( dmm / ha ) , right knee joints were harvested after 8 weeks from mice that underwent a dmm procedure and were injected after 2 and 5 weeks with hyaluronic acid , which is in clinical use as viscosupplementation treatment . a final group contained age - matched nonsurgical controls ( am ) .
we have worked with the nude mouse model to assess the effect of dmm on the cartilage structure and quality , because it allows testing a cellular therapy in the mouse using human cells ( ongoing work ) .
these 4 research - relevant groups were selected to obtain a broad spectrum of cartilage defects on a per - animal basis ( i.e. , from bad to unaffected ) that one might encounter in preclinical oa research , thus to show the potency of ce - nanoct to be implemented as a standard technique in the oa field rather than to interpret the biology behind dmm - induced oa .
all knee joints were imaged using ce - nanoct , after which some were randomly sampled to allow a comparison with the histological gold standard .
animal experiments were performed in accordance with the belgian legislation under the national authorization number la1210530 and were approved by the ethical committee of the faculty of biomedical sciences of ku leuven .
to enable ce - nanoct from the 4 mouse knee joint groups , samples were processed as described in kerckhofs et al .
briefly , the knee joint capsules were dislocated to expose the tibial and femoral cartilage layers .
subsequently , all joints were immersed for 30 minutes in a solution of hexabrix 320 diluted 4/5 in phosphate - buffered saline , then wrapped in parafilm , and were stably positioned in the nanoct system . as the negatively charged ioxaglate of which hexabrix 320 is partially composed , will be locally repelled by the anionic glycosaminoglycans ( sgag ) in the cartilage , ioxaglate accumulation in the cartilage is inversely related to the sgag content .
the nanoct system applied in this study was a phoenix nanotom s ( ge measurement and control solutions , munich , germany ) .
it was equipped with a tungsten target and was operated , for all scans , at a voltage of 60 kv and a current of 140 a .
the exposure time was 500 ms and a frame averaging of 1 and image skip of 0 was applied , resulting in a scanning time of 20 minutes per sample .
as described in kerckhofs et al . , to quantify the 3d characteristics of the noncalcified and underlying calcified cartilage layers , the cartilage region was selected using ctan software ( bruker micro - ct , kontich , belgium ) by manually drawing a consistent region of interest ( roi ) in every 20th cross - section throughout the whole tibial plateau . briefly , delineating the total cartilage area ( i.e. , both noncalcified and calcified ) was performed based on ( a ) the difference in grayscale between the noncalcified cartilage and the surrounding ( i.e. , liquid ) and ( b ) the difference in structure compared with the subchondral bone and noncalcified cartilage for the calcified cartilage ( i.e. , having a porous structure ) .
the roi was automatically interpolated for the 19 intermediate cross - sections . after automatic otsu segmentation ,
the noncalcified and calcified cartilage were segmented and analyzed in 3d by calculating the volume , the thickness distribution , and the average thickness .
dataviewer ( bruker micro - ct , kontich , belgium ) was used on the ce - nanoct images to perform the modified mankin scoring for cartilage structure and quality , as well as to quantify the loss in sgag content .
after ce - nanoct imaging , a set of representative knee joints were processed for histology .
they were fixed in 10% formalin , decalcified in 4% formic acid , and embedded in paraffin .
sections of 5 m were made every 60 m and were stained with safranin - o and counterstained with fast green .
the medial side of the tibial plateau in the histological sections ( n = 10 per joint ) and coronal ce - nanoct images ( n = 10 with a 60 m interspacing ) was graded using a modified mankin scoring system specifically for cartilage structure and quality . in brief , for both the histological sections and the ce - nanoct images , articular cartilage structure was graded from 0 to 11 . concerning the cartilage quality , the safranin - o staining for the histological sections and the hexabrix staining for the ce - nanoct images were graded between 0 and 8 . for both parameters , grade
a grade of 11 for the cartilage structure was indicative for clefts / loss of articular cartilage extending into the subchondral bone .
grade 8 for cartilage quality implied a severe loss in safranin - o ( or increase in hexabrix ) staining in more than half of the depth of the noncalcified cartilage thickness and involving more than half of the plateau .
grades of each sample ( n = 10 ) were averaged and summed for the 2 scoring parameters , whereby 0 represented completely unaffected hyaline cartilage and the maximum mankin score of 19 represented a highly degenerated oa knee .
to quantify the sgag content in the hyaline cartilage , the intensity of the hexabrix excluded signal in the noncalcified layer of the hyaline cartilage was normalized to the signal of the growth plate cartilage ( i.e. , in the same coronal ce - nanoct cross - sections ) .
a grayscale histogram was plotted along an arbitrary 600 m line through the noncalcified cartilage on the medial side of the tibial plateau ( fig .
the normalized sgag content in the cartilage compared to the healthy growth plate cartilage was then calculated according to equation ( 1 ) .
the normalized sgag content was measured in the same coronal ce - nanoct cross - sections as used for the modified making scoring .
( a ) a typical coronal contrast - enhanced nanofocus computed tomography ( ce - nanoct ) image and ( b ) the corresponding grayscale histogram along the white line drawn in ( a ) through the noncalcified cartilage on the medial ( left ) side of the tibial plateau ( gray curve ) with the average grayscale value ( black curve ) .
( c ) a typical coronal ce - nanoct image and ( d , e ) the corresponding grayscale histograms ( d ) along the white line annotated with 1 drawn in ( c ) through the background of the image ( gray curve ) with the average grayscale value ( black curve ) and ( e ) along the white line annotated with 2 drawn in ( c ) through the growth plate cartilage ( gray curve ) with the average grayscale value ( black curve ) .
1b ) along an arbitrary line through the noncalcified cartilage on the medial side of the tibial plateau ( fig .
1d ) along a 600 m long line through the background in the same image ( i.e. , liquid , fig .
1e ) along a 400 m long line through the growth plate cartilage in the same image ( fig .
in this study , a computational model was used to determine whether a combined set of morphological cartilage characteristics could quantitatively and in 3d predict the level of cartilage damage in a comparative manner to histological cartilage pathology scoring ( using the modified mankin scoring as a measure for cartilage structure and quality ) .
a linear modeling formalism was used that was already shown to be capable of relating quantitative contributions of multiple signals to a ( single ) measured response , namely partial least square regression ( plsr ) .
plsr is able to identify the information content within the set of measured characteristics that most closely maps onto the output response ( i.e. , modified mankin score ) .
the resulting mapping of lumped signals to corresponding responses allowed to identify the most important structural cartilage characteristics
plsr uses linear algebra to reduce the dimensionality of multivariate data sets by defining principal component axes from the original data set that contain the most important information on a specific output variable . for the optimization of the number of model dimensions ,
the root mean squared error ( rmse ) between the measured level of cartilage damage and the level predicted by plsr models with increasing numbers of principal components was investigated . each cartilage characteristic was individually withheld from the training set to construct a cross - validation model , in which an rmse of the prediction could be assessed by predicting the withheld sample . the predicted rmse was minimal in this study when using two principal components . to generate the plsr model , the software package jmp 8 ( sas institute inc . , cary , nc ) was used .
statistical analysis was done using a statistical software add - in for microsoft excel for windows , namely , analyse - it version 2.26 excel 12 + . to evaluate the significance level between the groups , one - way analyses of variance were used with a confidence level of 95% .
in kerckhofs et al . , it was already shown that ce - nanoct has a similar discriminative power as histology with safranin - o staining to differentiate bone , noncalcified and calcified cartilage in mouse knee joints , and to identify and assess local alterations in cartilage sgag content ( i.e. , a decrease in sgag content vs. chondral lesions ) . to validate the potency of the ce - nanoct images to be used for the modified mankin scores , that is , the gold standard for histological assessment with safranin - o staining , these scores were determined on the histological sections and on the ce - nanoct coronal sections ( fig .
since full histological scoring was only performed on animals that were not imaged by ce - nanoct , no comparison for the same animal was possible .
however , figure 2a shows that , within one treatment group , there were no significant differences between the histology - based and the ce - nanoct - based scoring for cartilage structure .
this supports the equivalence of ce - nanoct images to safranin - o stained histological sections for modified mankin score determination .
the apparent difference between both scores was rather a result of the large variability in manifestations of the symptoms between different animals within one treatment group .
( a ) the modified mankin ( mm ) scoring for the cartilage structure , determined on histological and contrast - enhanced nanofocus computed tomography ( ce - nanoct ) sections for the different treatments groups .
the animals used for the histological scoring ( n = 3 or 4 per group ) were not the same individuals that than the ones assessed by the ce - nanoct scoring ( n = 3 or 4 per group ) .
the score for the cartilage structure only and the sum for cartilage structure and quality are shown for the different treatments groups .
( c ) correlation plot between the percentage of sgag ( anionic glycosaminoglycans ) loss normalized to the growth plate cartilage and the mm scoring for the cartilage quality ( as measured by hexabrix staining intensity ) , both determined on the individual 2-dimensional sectional ce - nanoct images throughout the different treatments groups and ( d ) the same correlation plot as ( c ) but the % sgag loss and mm score were averaged over all scored sections of each animal ( n = 10 ) .
the modified mankin subscore for the cartilage structure on the 2d ce - nanoct images , as well as the sum of the cartilage structure and quality ( fig .
2b ) , showed significant differences between the am group and the animals that had undergone a dmm procedure ( i.e. , dmm and dmm / ha ) , as well as between the sham - operated group and the group that had received a 2-fold injection of hyaluronic acid after dmm ( i.e. , dmm / ha ) .
this indicates that for the 4 different groups , having a broad spectrum of cartilage defects ( i.e. , from bad in case of the dmm group to almost unaffected for the am and sham - operated groups ) , ce - nanoct provides valid readouts over this whole range , thus showing the potency of ce - nanoct to be implemented as a standard technique for evaluation and quantification of cartilage damage in the oa field .
next , instead of visually scoring in a semiquantitative way the degree of noncalcified cartilage staining in the ce - nanoct images , as was done for the modified mankin subscore for cartilage quality , the normalized sgag loss was quantified by image analysis on the same images ( fig .
every data point in figure 2c represents one ce - nanoct image . for the dmm group , ce - nanoct images with both a low and a high modified mankin scoring ( and sgag loss )
although a strong correlation ( r = 0.79 ) was found between this analysis and the cartilage quality score , the latter sometimes varied for the same percentage of normalized sgag loss .
moreover , for the same cartilage quality score , a range in percentage of normalized sgag loss was found , thus showing the higher sensitivity of the quantification of the normalized sgag loss by ce - nanoct image analysis compared to the modified mankin scoring for cartilage quality . when plotting the averaged modified mankin score per animal ( n = 10 ) , an even higher correlation was found ( r = 0.92 ) . within the dmm group ,
some animals had a high modified mankin scoring ( and sgag loss ) , while there was one animal that had a modified mankin scoring ( and sgag loss ) comparable to animals in the sham and dmm / ha groups .
ce - nanoct scoring thus allows not only to discriminate intact versus highly damaged cartilage ( fig .
the in vivo medial meniscus destabilization , which serves as a model for oa , is characterized by cartilage lesions of various degrees of severities on the medial side ( fig .
, creating 3d ce - nanoct models of the different cartilaginous layers allowed a straightforward quantification of the defects . a representative ( a ) cross - sectional ,
( b ) coronal , and ( c ) sagittal contrast - enhanced nanofocus computed tomography ( ce - nanoct ) image of a tibial plateau of a mouse of which 8 weeks in vivo medial meniscus destabilization ( dmm ) resulted in a local chondral defect ( indicated by the crossed lines ) .
scale bars = 1 mm . while no significant differences were found between the different groups for both calcified cartilage volume ( fig .
4b ) , the overlying noncalcified hyaline cartilage showed significant differences between the dmm group and all other groups .
( a ) the volume and ( b ) average thickness of noncalcified and calcified cartilage on the medial tibial plateau of the 4 different treatments groups ( n = 3 or 4 ) ( p < 0.05 = significantly different ) , based on quantitative analysis of 3-dimensional ce - nanoct images . the data for the sham and dmm ( destabilization of the medial meniscus ) group have already been presented in kerckhofs et al .
the averaged thickness distribution plots ( n = 3 or 4 per group ) of noncalcified and calcified cartilage ( fig . 5a and b , respectively ) showed similar findings . for the calcified cartilage thickness , the distribution plots for all different treatment groups coincided , and the distribution maxima were not significantly different ( fig .
the noncalcified cartilage thickness of the dmm group displayed a wide spread distribution with a higher frequency of small thicknesses compared with the other groups ( fig .
5a ) , reflecting a higher presence of thinner cartilage ( in particular lesions ) and loss of sgags throughout the noncalcified cartilage . consequently , the maximum of the distribution
the ( a ) noncalcified and ( b ) underlying calcified cartilage thickness distribution on the medial tibial plateau of the different treatments groups ( n = 3 or 4 ) .
the data for the sham and dmm ( destabilization of the medical meniscus ) group have already been presented in kerckhofs et al .
plot averaged per animal group ( n = 3 or 4 ) , both for the noncalcified and calcified cartilage layers ( p < 0.05 = significantly different ) . to determine whether a specific combination of quantitative 3d structural and quality cartilage parameters could quantitatively predict the severity of cartilage damage as determined by the modified mankin scoring for cartilage structure and quality , a plsr model was developed .
the resulting 2-component plrs model was used to examine the correlation between the measured modified mankin score , as was assessed by multiple blinded readers on histological sections , and the predictions for each 3d cartilage parameter ( fig .
the plsr model combined the ( a ) noncalcified hyaline cartilage volume , ( b ) maximum of the noncalcified cartilage thickness distribution , and ( c ) averaged normalized sgag loss ( fig .
a high predictability of 90.26% was obtained for the model . including more measured characteristics ( e.g. , total cartilage volume [ noncalcified and calcified cartilage ] , average thickness of the total cartilage layer , and calcified cartilage volume and average thickness ) led to a lower predictability of the model due to inclusion of irrelevant information ( noise ) .
inclusion of less measurement characteristics than the 3 mentioned above also led to a reduction in predictability due to the omission of relevant information .
( a ) the measured modified mankin ( mm ) score versus the cross - validated predictions for each 3-dimensional cartilage parameter , ( b ) the residuals for the cross - validated predictions , and ( c ) the three 3-dimensional cartilage parameters that comprise the partial least square regression model , resulting in the following predictive equation : mm scoring = 3.55 + 33.70 * ( noncalcified cartilage volume in mm ) 0.0375 * ( maximum of the noncalcified cartilage thickness distribution plot in m ) + 14.41 * ( average sgag loss , nondimensional value ) .
in kerckhofs et al . , it was already shown that ce - nanoct has a similar discriminative power as histology with safranin - o staining to differentiate bone , noncalcified and calcified cartilage in mouse knee joints , and to identify and assess local alterations in cartilage sgag content ( i.e. , a decrease in sgag content vs. chondral lesions ) . to validate the potency of the ce - nanoct images to be used for the modified mankin scores , that is , the gold standard for histological assessment with safranin - o staining , these scores were determined on the histological sections and on the ce - nanoct coronal sections ( fig .
since full histological scoring was only performed on animals that were not imaged by ce - nanoct , no comparison for the same animal was possible .
however , figure 2a shows that , within one treatment group , there were no significant differences between the histology - based and the ce - nanoct - based scoring for cartilage structure .
this supports the equivalence of ce - nanoct images to safranin - o stained histological sections for modified mankin score determination .
the apparent difference between both scores was rather a result of the large variability in manifestations of the symptoms between different animals within one treatment group .
( a ) the modified mankin ( mm ) scoring for the cartilage structure , determined on histological and contrast - enhanced nanofocus computed tomography ( ce - nanoct ) sections for the different treatments groups .
the animals used for the histological scoring ( n = 3 or 4 per group ) were not the same individuals that than the ones assessed by the ce - nanoct scoring ( n = 3 or 4 per group ) .
the score for the cartilage structure only and the sum for cartilage structure and quality are shown for the different treatments groups .
( c ) correlation plot between the percentage of sgag ( anionic glycosaminoglycans ) loss normalized to the growth plate cartilage and the mm scoring for the cartilage quality ( as measured by hexabrix staining intensity ) , both determined on the individual 2-dimensional sectional ce - nanoct images throughout the different treatments groups and ( d ) the same correlation plot as ( c ) but the % sgag loss and mm score were averaged over all scored sections of each animal ( n = 10 ) .
the modified mankin subscore for the cartilage structure on the 2d ce - nanoct images , as well as the sum of the cartilage structure and quality ( fig .
2b ) , showed significant differences between the am group and the animals that had undergone a dmm procedure ( i.e. , dmm and dmm / ha ) , as well as between the sham - operated group and the group that had received a 2-fold injection of hyaluronic acid after dmm ( i.e. , dmm / ha ) .
this indicates that for the 4 different groups , having a broad spectrum of cartilage defects ( i.e. , from bad in case of the dmm group to almost unaffected for the am and sham - operated groups ) , ce - nanoct provides valid readouts over this whole range , thus showing the potency of ce - nanoct to be implemented as a standard technique for evaluation and quantification of cartilage damage in the oa field .
next , instead of visually scoring in a semiquantitative way the degree of noncalcified cartilage staining in the ce - nanoct images , as was done for the modified mankin subscore for cartilage quality , the normalized sgag loss was quantified by image analysis on the same images ( fig .
every data point in figure 2c represents one ce - nanoct image . for the dmm group , ce - nanoct images with both a low and a high modified mankin scoring ( and sgag loss )
although a strong correlation ( r = 0.79 ) was found between this analysis and the cartilage quality score , the latter sometimes varied for the same percentage of normalized sgag loss .
moreover , for the same cartilage quality score , a range in percentage of normalized sgag loss was found , thus showing the higher sensitivity of the quantification of the normalized sgag loss by ce - nanoct image analysis compared to the modified mankin scoring for cartilage quality . when plotting the averaged modified mankin score per animal ( n = 10 ) , an even higher correlation was found ( r = 0.92 ) . within the dmm group ,
some animals had a high modified mankin scoring ( and sgag loss ) , while there was one animal that had a modified mankin scoring ( and sgag loss ) comparable to animals in the sham and dmm / ha groups .
ce - nanoct scoring thus allows not only to discriminate intact versus highly damaged cartilage ( fig .
the in vivo medial meniscus destabilization , which serves as a model for oa , is characterized by cartilage lesions of various degrees of severities on the medial side ( fig .
, creating 3d ce - nanoct models of the different cartilaginous layers allowed a straightforward quantification of the defects . a representative ( a ) cross - sectional , ( b ) coronal , and ( c ) sagittal contrast - enhanced nanofocus computed tomography ( ce - nanoct ) image of a tibial plateau of a mouse of which 8 weeks in vivo medial meniscus destabilization ( dmm ) resulted in a local chondral defect ( indicated by the crossed lines ) .
scale bars = 1 mm . while no significant differences were found between the different groups for both calcified cartilage volume ( fig .
4b ) , the overlying noncalcified hyaline cartilage showed significant differences between the dmm group and all other groups .
( a ) the volume and ( b ) average thickness of noncalcified and calcified cartilage on the medial tibial plateau of the 4 different treatments groups ( n = 3 or 4 ) ( p < 0.05 = significantly different ) , based on quantitative analysis of 3-dimensional ce - nanoct images . the data for the sham and dmm ( destabilization of the medial meniscus ) group have already been presented in kerckhofs et al .
the averaged thickness distribution plots ( n = 3 or 4 per group ) of noncalcified and calcified cartilage ( fig . 5a and b , respectively ) showed similar findings .
for the calcified cartilage thickness , the distribution plots for all different treatment groups coincided , and the distribution maxima were not significantly different ( fig .
the noncalcified cartilage thickness of the dmm group displayed a wide spread distribution with a higher frequency of small thicknesses compared with the other groups ( fig .
5a ) , reflecting a higher presence of thinner cartilage ( in particular lesions ) and loss of sgags throughout the noncalcified cartilage . consequently ,
plot for the am group was significantly different compared with the other groups . the ( a ) noncalcified and ( b ) underlying calcified cartilage thickness distribution on the medial tibial plateau of the different treatments groups ( n = 3 or 4 ) .
the data for the sham and dmm ( destabilization of the medical meniscus ) group have already been presented in kerckhofs et al .
plot averaged per animal group ( n = 3 or 4 ) , both for the noncalcified and calcified cartilage layers ( p < 0.05 = significantly different ) .
to determine whether a specific combination of quantitative 3d structural and quality cartilage parameters could quantitatively predict the severity of cartilage damage as determined by the modified mankin scoring for cartilage structure and quality , a plsr model was developed .
the resulting 2-component plrs model was used to examine the correlation between the measured modified mankin score , as was assessed by multiple blinded readers on histological sections , and the predictions for each 3d cartilage parameter ( fig .
the plsr model combined the ( a ) noncalcified hyaline cartilage volume , ( b ) maximum of the noncalcified cartilage thickness distribution , and ( c ) averaged normalized sgag loss ( fig .
a high predictability of 90.26% was obtained for the model . including more measured characteristics ( e.g. , total cartilage volume [ noncalcified and calcified cartilage ] , average thickness of the total cartilage layer , and calcified cartilage volume and average thickness ) led to a lower predictability of the model due to inclusion of irrelevant information ( noise ) .
inclusion of less measurement characteristics than the 3 mentioned above also led to a reduction in predictability due to the omission of relevant information .
( a ) the measured modified mankin ( mm ) score versus the cross - validated predictions for each 3-dimensional cartilage parameter , ( b ) the residuals for the cross - validated predictions , and ( c ) the three 3-dimensional cartilage parameters that comprise the partial least square regression model , resulting in the following predictive equation : mm scoring = 3.55 + 33.70 * ( noncalcified cartilage volume in mm ) 0.0375 * ( maximum of the noncalcified cartilage thickness distribution plot in m ) + 14.41 * ( average sgag loss , nondimensional value ) .
current diagnostics for oa usually do not discover the disease until it is in advanced stages when joint damage may already have occurred and the patient presents with pain . a method for early diagnosis could open a window of opportunity for prevention , thereby reducing further damage . measuring subtle alterations in cartilage structure and quality
is equally needed to evaluate the ( pre)clinical efficacy of new treatment options for cartilage damage .
small animal models of oa are essential and thus quantitative readouts with functional relevance are key for advancing skeletal research . nowadays , preclinical efficacy of novel therapies for cartilage repair in small animal models is still commonly assessed by histopathology , and quantified by using scoring systems such as the modified mankin scoring .
such scoring systems are already in clinical use to assess the severity of the cartilage damage .
although validated for being an accurate and user - independent technique , histopathology remains a visual , subjective , and nonquantitative scoring approach and might not allow one to distinguish small differences in therapeutic response because of the restricted 2d character . as demonstrated in kerckhofs
, ce - nanoct provides equivalent images compared to safranin - o staining on histological sections , with a high contrast between noncalcified and calcified cartilage . in this study
, we have shown that this allows performing the modified mankin scoring also on ce - nanoct images of a murine knee joint , which can be assessed immediately as it avoids time - consuming and destructive histoprocessing steps , such as decalcification , embedding , or sectioning , which may take several weeks or , for larger species , even months
. moreover , ce - nanoct generates much more sections throughout the tibial plateau than histology and allows objective and quantitative scoring .
grayscale quantification of the noncalcified cartilage has shown a good correlation with the modified mankin scoring for cartilage quality , with the advantage of being user independent and quantitative .
although in this study the calculation of the normalized sgag content was still limited to manual image analysis , by implementation of more sophisticated image analysis algorithms this could evolve toward a semiautomated tool . therefore , ce - nanoct is a valuable tool for virtual histopathology .
it has to be mentioned that , when the noncalcified cartilage is fully depleted of sgags , the ce - nanoct images might not allow detection of the difference with a cartilage lesion , and histological sectioning might be required .
however , as shown by the quantitative measurements of the sgag loss based on the hexabrix staining , very low concentrations of sgags can still be discriminated from the conditions where cartilage is absent or fully depleted of sgags .
additionally , ce - nanoct does not yet allow visualization of the cartilage at the cellular level .
ce - nanoct also allows , because of its 3d character , analysis of different morphological parameters of the bone cartilage unit .
although many studies have used epic - ct ( i.e. , equilibrium partitioning of an ionic contrast agent via microct ) images to quantify 3d cartilage parameters , their correlation to validated histopathological scoring techniques has not been shown . nor has it been shown which of the measured 3d cartilage parameters should be included to correlate with validated histological scores . in this study , we have shown , using plsr modeling , that a limited set of easily quantifiable 3d morphological parameters ( volume and maximum thickness of the noncalcified cartilage and the averaged normalized sgag loss ) is predictive for the mm scoring of the cartilage structure and quality .
these 3d parameters are thus minimally required to draw conclusions on the severity of the damage of the articular cartilage in the knee joint .
the model could potentially replace the mm scoring based on visual inspection as the latter does not involve any quantification of cartilage quality or structure .
the 3d measurements give more information on size and shape ( and thus severity ) of the cartilage damage compared with 2d histopathology . in this study
however , as microct images ( and thus also ce - nanoct ) do allow for this quantification , including these analyses in the plsr model could even enhance the correlation of the ce nanoct analysis with the gold standard mm scoring above 90% . finally , this study assessed 4 treatment groups using ce - nanoct that were selected because they serve in many studies as control groups to assess the efficacy of a cell therapy for cartilage repair , and they represent a broad range of cartilage defects ( i.e. , from bad to unaffected ) .
mice with a destabilized meniscus displayed lesions with different degrees of severity and loss in sgag content .
twofold injection of hyaluronic acid after dmm ( dmm / ha ) was proven to be a valid positive control group for this model and follow - up period , since the cartilage characteristics , both determined in 2d and 3d , were similar to the ones of nonoperated or sham - operated animals .
ce - nanoct - based virtual histopathology and 3d morphological quantification of a limited set of 3d cartilage parameters allowed us to distinguish the 4 groups , and thus it is a valid tool to be implemented as a standard technique for the evaluation and quantification of cartilage damage in the oa field .
this study has shown the potential of a noninvasive 3d imaging methodology , namely ce - nanoct , for virtual 3d histopathology as it provides images equivalent to safranin - o - stained histological sections of murine knee joints .
because of its noninvasive and 3d character , and because it reveals differences in structure and composition between calcified , noncalcified cartilage , and the underlying bone in small animal models with high spatial and contrast resolution , ce - nanoct provides faster and more quantitative data on cartilage structure and quality compared with standard histopathology .
moreover , it was shown that a limited set of these quantitative 3d measures is able to reproduce the standard histopathological scoring , with the advantage of being user independent and semiautomated , thus being a potential replacement of the histopathological scoring . to conclude
, ce - nanoct has demonstrated its equivalence to standard histopathology in preclinical small animal studies addressing cartilage - related pathologies and therapeutic interventions . | objective : one of the early hallmarks of osteoarthritis ( oa ) is a progressive degeneration of the articular cartilage
. early diagnosis of oa - associated cartilage alterations would be beneficial for disease prevention and control , and for the development of disease - modifying treatments .
however , early diagnosis is still hampered by a lack of quantifiable readouts in preclinical models.design:in this study , we have shown the potency of contrast - enhanced nanofocus x - ray computed tomography ( ce - nanoct ) to be used for virtual 3-dimensional ( 3d ) histopathology in established mouse models for oa , and we compared with standard histopathology.results:we showed the equivalence of ce - nanoct images to histopathology for the modified mankin scoring of the cartilage structure and quality .
additionally , a limited set of 3d cartilage characteristics measured by ce - nanoct image analysis in a user - independent and semiautomatic manner , that is , average and maximum of the noncalcified cartilage thickness distribution and loss in glycosaminoglycans , was shown to be predictive for the cartilage quality and structure as can be evaluated by histopathological scoring through the use of an empirical model.conclusions:we have shown that ce - nanoct is a tool that allows virtual histopathology and 3d morphological quantification of multitissue systems , such as the chondro - osseous junction .
it provides faster and more quantitative data on cartilage structure and quality compared with standard histopathology while eliminating user bias .
ce - nanoct thus should allow capturing subtle differences in cartilage characteristics , carefully mapping oa progression and , ultimately , asses the beneficial changes when testing a candidate disease - modifying treatment . | Introduction
Materials and Methods
Mouse Model
Contrast-Enhanced Nanofocus Computed Tomography
Three-Dimensional Image Visualization and Analysis
Histology
Modified Mankin Scoring
Quantification of the Normalized sGAG content
Partial Least Square Regression Modeling
Statistical Analysis
Results
CE-nanoCT Images Are Equivalent to Histological Sections for the Modified Mankin Scoring
CE-nanoCT Allows 3D Quantification of the Cartilage Structure at the Subtissue Level
Three-Dimensional Cartilage Characteristics Determined by CE-nanoCT Are Predictive for the Cartilage Function and Structure
Discussion
Conclusions |
PMC4878209 | successful endodontic treatment begins with an accurate diagnosis , which is the cornerstone for a successful treatment plan wherein , the clinician should go through various diagnostic modalities before diagnosis and treatment plan .
thorough clinical examination , detailed case history , radiographs , and pulp sensibility tests together aid in the diagnosis of dental pulp health and not the outcome of only one specific test . in clinical practice ,
pulp sensibility tests ( thermal and electric pulp tests [ epts ] ) assess the integrity of the nerve fibers in the dentin - pulp complex by applying the stimulus to the outer surface of the tooth , whereas pulp vitality tests should assess the blood supply of the dental pulp .
electronic root apex locators , pulp vitality tester , and early caries lesion detectors are some of such devices .
the ability of ept to indicate pulp sensibility is based on the sensitivity of neural transmission .
ept is based on stimulation of intact a nerves in the pulp - dentine complex by applying an electric current on the tooth surface , which results in an ionic shift in the dentinal fluid within the tubules causing local depolarization and subsequent generation of an action potential from the intact nerve and relies on subjective assessments and comments from the patient .
the various factors that influence ept result for false positive or false negative response include the thickness of enamel and dentine , concentration of pulpal neural elements , direction of dentinal tubules , amount of dentinal fluid , and the distance between the electrode tip and the pulp .
ept is an important aid and when properly used , is a safe clinical test that can provide useful information regarding health and disease of pulp tissue .
dental fluorosis is a developmental disturbance of dental enamel caused by excessive exposure to high concentration of fluoride during tooth development . in routine clinical scenario
they pose many challenges in diagnosis and treatment planning to the clinician right from pulp sensibility tests , shade selection , shade mimicking , etching and bonding procedures during composite restorations .
the factors that influence ept are the thickness of enamel and dentine , morphology of teeth , concentration of pulpal neural elements , direction of dentinal tubules , and the distance between the electrode tip and the pulp .
many studies were done earlier to evaluate the optimal electrode placement site and threshold levels of ept with varied opinions .
the hypermineralized enamel in fluorosed teeth may influence the fluid concentration in the dentinal tubules , which is thought to play a vital role in the conduction of electric impulses . in fluorosed enamel
, hydroxyapatite crystals are substituted by fluorapatite crystals , which may result in ionic shift in the dentinal fluid .
to best of our knowledge until date , no study has been done to evaluate threshold response and optimal electrode placement site on fluorosed teeth .
therefore , the aim of this study was to evaluate lowest threshold response and optimal electrode placement site for ept in fluorosed anterior teeth .
this clinical study was carried out in the department of conservative dentistry and endodontics , kamineni institute of dental sciences , narketpally , nalgonda ( district ) , telangana , state , india .
nalgonda district is globally recognized for the incidence of severe systemic and dental fluorosis with fluoride concentration up to 14ppm .
ethical clearance for the study was obtained from the research and ethics committee of the kamineni institute of dental sciences .
eighty volunteers of either gender , between 20 and 40 years old with intact maxillary central incisors with thylstrup and fejerskov index ( tfi ) score 1 - 4 were selected [ figure 1 ] .
clinical examination was done independently by two different investigators , after wiping dry the teeth to assess the fluorotic severity according to the tfi .
based on the incidence of fluorosis , total of 80 volunteers were divided into two groups as :
group 1- volunteers with incidence of dental fluorosis ( tfi score was between 1 and 4)group 2- volunteers without incidence of dental fluorosis ( tfi score was 0 ) .
group 1- volunteers with incidence of dental fluorosis ( tfi score was between 1 and 4 ) group 2- volunteers without incidence of dental fluorosis ( tfi score was 0 ) . the teeth with mild flourosis and intact crown ( thylstrup and fejerskov index score-2 ) selected for the study .
to standardize the study , we selected either of the maxillary central incisors with an intact crown from each volunteer [ figure 1 ] . baseline bitewing and periapical radiographs were taken for teeth which had neither history of orthodontic treatment nor trauma .
the teeth with signs of surface loss ( erosion , abrasion , attrition , abfraction ) , teeth with surface pitting , ( tfi 5 - 9 ) , cracks , inter proximal caries , metallic restorations , malformed teeth , traumatized teeth , endodontically treated teeth and teeth with periapical pathology were excluded from the study .
subjects taking narcotics , alcohol or nonsteroidal anti - inflammatory drugs and with a history of mental and emotional instability were excluded from the study .
the participants were informed fully about the procedure being carried out and informed consent was obtained . tooth to be tested was isolated with rubber dam without the clamp .
the test was conducted with a digitest ept ( parkell , farmingdale , ny , usa ) in accordance with the manufacturer 's instructions ( a monopolar ept with anodal electrode probe tip and diameter of 2 mm was used in the study ) .
the circuit was completed when the subject rested a finger on the lip clip of the ept .
the tip of the electrode was lightly coated with toothpaste as a conducting medium , a gentle pulsed stimulus was applied to the tooth until the subject felt and noted a sensation such as tingling , stinging , warmth , or heat .
they were instructed to release the clip on the first detection of a warm , tingling or painful sensation .
the digital display readout of the pulp tester at which the subject first noted the above mentioned sensation was defined as the threshold .
four sites ( incisal edge , incisal third , middle third , and cervical third ) on the labial surface of each tooth were tested .
four readings were made on the labial surface of each site in sequence , starting from the incisal edge to cervical third . to eliminate the phenomenon of nerve accommodation , at least 1 min was allowed to elapse before the tooth was revisited .
means of variables from each location were compared using one - way anova and tukey 's post - hoc test .
a total of 80 volunteers were recruited in this study and were categorized into two groups of 40 each .
three hundred and twenty electrode placement sites on 80 teeth were studied and analyzed statistically .
the mean standard deviation threshold levels in the fluorosed group were 9.32 1.35 , whereas in nonfluorosed group , it was 5.57 1.40 .
the higher threshold values were recorded in the fluorosed group when compared to that of nonfluorosed group and were statistically significant ( p < 0.05 ) . when multiple comparisons were done among the groups ,
it was observed that there was a significant difference between incisal edge , middle and cervical thirds ( p < 0.05 ) . but
no statistically significant difference between incisal edge and incisal third was noted ( p > 0.05 ) [ table 1 ] .
many studies earlier were done to evaluate the optimal electrode placement site and threshold levels of ept with varied opinions .
concluded that placing the electrode at the incisal edge of anterior teeth evoked a response with the least amount of electrical current .
conducted a study on premolar and stated that the buccal cusp tip of both maxillary and mandibular premolars were the site with the lowest threshold to ept stimulation . in this study , maxillary central incisors with intact crowns
were selected which differ from the earlier studies in relation to the sample size , population characteristics , and study design .
nalgonda district of telangana , india consists of areas with various levels of fluoride in drinking water , starting from below - optimum to optimum and above - optimum levels ( up to14ppm ) .
the earliest manifestation of dental fluorosis is an outer hypermineralized layer with a porous hypomineralized subsurface ; the pores are occupied by enamel secretory proteins that are retained due to the effect of the excessive fluoride level on ameloblasts .
clinically , the porosity in the subsurface of enamel reflects as the opacity of the enamel , ranging from fine white opaque lines running across the tooth on all parts of the enamel to entirely chalky white teeth .
high fluoride concentration on fluorosis enamel indicates the substitution of oh by f increasing the surface roughness of enamel surface . in the present study to measure dental fluorosis ,
this index allows correlation between the clinical appearance of fluorosis and pathological changes and is normally the index of choice for evaluation of fluorotic severity . in the present study ,
maxillary central incisors were selected , as these teeth are of prime aesthetic concern and are considered to be most at risk for fluorosis .
volunteers within a narrow range of age group were selected to avoid sensitivity variation caused by secondary dentine deposition which causes a reduction in the volume of pulp , dental caries and tooth wear . in the present study ,
fluorosed teeth showed high threshold response when compared to nonfluorosed teeth , possible reasons may be hypermineralized enamel , which might influence the fluid concentration in the dentinal tubules , which is thought to play a vital role in the conduction of electric impulses . in fluorosed enamel hydroxyapatite crystals
are substituted by fluorapatite crystals which may result in ionic shift in the dentinal fluid . in the present study , in both fluorosed and nonfluorosed teeth incisal edge showed lowest threshold levels for ept when compared to incisal , middle and cervical thirds because the enamel is thinnest incisally with the highest concentration of neural elements in the pulp horns while in the cervical area the number of nerve fibers decrease .
the straight course of dentinal tubules at the incisal edges of anterior teeth offers faster flow rate to the electric current .
this explains why the threshold values were lower at the incisal edge region than in the cervical third .
these results were in accordance with bender et al . who found that the lowest threshold response was at the incisal edge .
structural and morphological variations of fluorosed anterior teeth did not show any variation in the optimal electrode placement site when compared to nonfluorosed teeth but have shown higher threshold levels to ept .
the possible reasons could be because of hypermineralized enamel , which might influence the fluid concentration in the dentinal tubules , which is thought to play a vital role in the conduction of electric impulses . in fluorosed enamel hydroxyapatite crystals
are substituted by fluorapatite crystals which may result in ionic shift in the dentinal fluid . during the study
, it was observed that teeth with mild fluorosis which mimics nonfluorosed teeth reported high threshold levels for ept which could be misread as delayed response of nonfluorosed teeth . in such cases ,
this base line study was done only on the anterior teeth with mild fluorosis with intact enamel surface .
further studies are required in posterior and severe fluorosed teeth for better understanding of the threshold response of fluorosed teeth to ept .
within the limitations of the study , it was observed that fluorosed teeth responded to high threshold levels , and the appropriate electrode placement site was the incisal edge .
the authors of this manuscript declare that they have no conflicts of interest , real or perceived , financial or non - financial in this article .
the authors of this manuscript declare that they have no conflicts of interest , real or perceived , financial or non - financial in this article . | background : accurate diagnosis is key to success . diagnosing the pulpal status in varied clinical situations poses a challenge to the clinician . electric pulp test ( ept ) is one of the valuable attempts in evaluating the sensibility of pulp tissue .
the aim of this study was to find out and compare the threshold levels and optimal electrode placement site for ept in fluorosed and nonfluorosed anterior teeth.materials and methodology : eighty volunteers recruited for this study were divided into two groups based on the incidence of dental fluorosis .
electric pulp testing was done on either of the central incisors in fluorosed and nonfluorosed group .
four sites on each crown were tested 4 times with digitest electric pulp tester , and the mean of the threshold responses was recorded .
the data were analyzed with spss , version 11 .
means of variables from each location were compared using one - way anova and tukey 's post - hoc test while the critical level of significance was set at p < 0.05.results:the mean and standard deviation of threshold levels in fluorosed teeth were greater when compared to that of nonfluorosed teeth at all sites with incisal edge showing the least mean threshold levels for both the groups ( p > 0.05).conclusion : within the limitations of this study , it can be concluded that fluorosed teeth respond to higher threshold levels than the non - fluorosed teeth , and incisal edge was the optimal electrode placement site . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION
Financial support and sponsorship
Conflicts of interest |
PMC4155217 | moreover , restrictions of physical functioning among
older adults are very important1 , 2 .
spinal pain is the most common of all
chronic pain disorders . in a previous study ,
including back and
neck pain , spinal pain negatively affects mobility and aerobic capacity of the elderly .
chronic diseases and pain in aging can also saliently affect the balance ability of older
adults4 .
physical functioning including
balance ability and aerobic capacity is important for the activities of daily living and
static and dynamic posture .
chronic diseases and aging may affect physical functioning5 , 6 .
musculoskeletal disorders may occur due to many reasons that affect sensorial and motor
control systems and may affect functional capacity negatively . with aging , musculoskeletal
capacity is reduced , which results in loss of muscle mass and strength . a decrease in
aerobic capacity and
physical activity levels can cause changes in gait and balance7 , 8 . in
addition
, chronic musculoskeletal pain negatively affects emotional status and activities of
daily living ( adl)9 , 10 .
the aim of this study was to show the impact of chronic musculoskeletal pain of the spinal
column and lower extremities on physical functioning , emotional status , and independency in
older adults .
the study sample consisted of two hundred and fifty - eight independent older adults who were
randomly selected from volunteers .
the exclusion criteria were being dependent in daily living activities , having a cognitive impairment , having a neurological diseases such as stroke , parkinson disease , multiple sclerosis or
spinal cord injuries .
the study was approved by the ethics committee for non - invasive clinical research of
pamukkale university denizli , turkey ( 2012/16 - 03 ) .
sociodemographic data including age , gender , marital status , and education level were
recorded through a face to face interview with a physiotherapist .
body mass index ( bmi ) was calculated for each older adults to define their obesity
level .
we asked the older adults who reported musculoskeletal pain to indicate the localization on
their pain on a body diagram . only the older adults who reported spinal or lower extremity
pain
the six - minute walk test ( 6mwt ) was used to evaluate aerobic capacity .
the 6mwt was
performed in a 30-m - long corridor , and the participants were instructed to walk as fast as
possible without running .
they could stop walking if they felt any discomfort , fatigue , or
shortness of breath .
the timed up and go ( tug ) test was used to evaluate dynamic balance .
the tug test as
described by podsiadlo and richardson is a simple timed test used to qualify functional
mobility11 . in the tug test ,
participants were asked to stand up from a standard chair with a seat height of between 40
and 50 cm , walk a 3-m distance at a normal pace , turn , walk back to the chair , and sit down .
go and ended when the
participant s back touched the backrest of the chair , with a shorter time indicating better
balance ability12 .
the yesavage geriatric depression scale ( gds ) was used to detect depressive symptoms in
order to describe emotional status .
this scale was developed as a self - report measure for
depression in older adults .
since this version proved both time - consuming and
difficult for some patients to complete , a 15-item version was developed .
the sensitivity
and specificity of the gds-15 have been assessed in a general elderly population13 .
the
turkish internal validity of the gds was confirmed by aktrk et al14 . for the 15-item short form gds ,
04 points is normal , 59
points indicates mild depression , and 1015 points indicates more severe depression .
the lawton - brody instrumental activities of daily living scale ( iadl ) was used to evaluate
independency in daily living activities .
the iadl scale measures complex functional
activities of ability including using a telephone , handling finances , self - administration
medication , use of transportation , shopping , housekeeping , laundry , and cooking .
one point
was awarded if a patient required little / no help in performing a task , and no points were
awarded if significant / total help was required . for the lawton - brody iadl ,
08 points is
evaluated as dependent , 916 points is evaluated as semi - dependant , and 1724 points is
evaluated as independent
the data were analyzed using the statistical package for social science ( spss , chicago ,
il , usa ) version 16.0 software for windows . to evaluate sociodemographic variables ,
descriptive statistical methods were used [ mean standard deviation ( sd ) and frequencies
( count and percentage ) ] . to compare the two groups ,
the data were analyzed using the statistical package for social science ( spss , chicago ,
il , usa ) version 16.0 software for windows . to evaluate sociodemographic variables ,
descriptive statistical methods were used [ mean standard deviation ( sd ) and frequencies
( count and percentage ) ] . to compare the two groups , the independent samples t - test was
used .
table 1table 1 . baseline characteristics of participants with spinal and lower extremity
painspinal pain(group i)lower ext .
pain(group ii)n ( % ) mean ( sd)n ( % ) mean ( sd)total 130 ( 100 ) 82 ( 100 ) gendermale 63 ( 48.4)41 ( 31.5 ) female 67 ( 51.5 ) 41 ( 31.5 ) bmi score27.45 ( 3.95 ) 28.14 ( 4.35)marital statussingle 41 ( 31.5 ) 33 ( 40.2)married 89 ( 68.4 ) 49 ( 59.7 ) chronic illnessno 16 ( 12.3 ) 6 ( 7.3 ) yes 114 ( 87.6 ) 76 ( 92.6 ) body mass index ( bmi , kilograms per square meter ) 30 , sd : standard deviation ,
* significant shows the baseline data of the groups .
spinal pain was reported by 61.3% of the participants ( neck and
lower back ; n=130 ) , and 38.7% reported pain in the lower extremities ( n=82 ) .
the mean age of
the participants was 71.98 5.86 years ; 131 ( 50.4% ) were male , and 127 ( 48.8% ) female .
the
mean vas score during activities was 5.061.89 cm for group i and 5.44 2.24 cm for group
ii .
no differences between the two groups in terms of the vas score were found ( p>0.05 )
( table 2table 2 .
pain(group ii)(n=130)(n=82)mean ( sd)mean ( sd)vas ( cm ) 5.06 ( 1.89 ) 5.44 ( 2.24)tug ( sn ) 24.37 ( 16.49 ) 24.24 ( 11.31)6mwt ( m ) 363.73 ( 278.66 ) 284.17 ( 195.78 ) * * gds - score 6.04 ( 3.63)7.12 ( 4.01)**iadl - score 9.20 ( 4.99)10.76 ( 5.68)***the independent samples t - test was used .
sd , standard deviation ; vas , visuel analog
scale ; tug : timed up and go test ; gds , geriatric depression scale ; iadl , instrumented
activities of daily living . * * significant ) . body mass index ( bmi , kilograms per square meter ) 30 , sd : standard deviation ,
* significant * the independent samples t - test was used .
sd , standard deviation ; vas , visuel analog
scale ; tug : timed up and go test ; gds , geriatric depression scale ; iadl , instrumented
activities of daily living . * * significant table 2 also shows the tug , 6mwt , gds , and iadl
results .
except for the tug scores , all outcome measurements showed significant differences
between the groups .
namely , the participants in group ii ( with lower limb pain ) had a lower
score in terms of aerobic capacity than those in group i ( p=0.027 ) .
although the two groups
had scores below the cutoff point for the gds , group ii had a higher score than group i
( p=0.045 ) .
the two groups had more or less the
same scores in terms of the iadl ( p=0.036 ) .
so , all participants in both groups were
semi - dependent according to the iadl scores .
the partial correlation coeffidents in the spinal pain ( group i ) ( after
correcting for age and gender)control variablesage and gender1 2 31 - 6mwt2- gds0.090 3-iadl0.142 0.3126mwt , 6 minute walk test ; gds , geriatric depression scale ; iadl , instrumental
activities of daily living ; * significant and 4table 4 .
the partial correlation coeffidents in the lower extremity pain ( group ii )
( after correcting for age and gender)control variablesage and gender1231 - 6mwt2-gds0.088 3-iadl0.025 0.420 6mwt , 6 minute walk test ; gds , geriatric depression scale ; iadl , instrumental
activities of daily living ; * significant show that there were significant inverse correlations between the gds and iadl
scores .
6mwt , 6 minute walk test ; gds , geriatric depression scale ; iadl , instrumental
activities of daily living ; * significant 6mwt , 6 minute walk test ; gds , geriatric depression scale ; iadl , instrumental
activities of daily living ; * significant
most of the older adults this study complained of
musculoskeletal pain in their spinal column and lower extremity .
the same complaints can also be found in the literature . moreover , helme and gibson , dawson
et al . , and robin et al .
reported that lower extremity pain including hip , knee , and foot
pain are very common15,16,17 . in our study ,
we studied 258 older adults who reported musculoskeletal pain in their spinal column and
lower extremities ( n=212 ; 82.1% ) . in this descriptive and comparative study
the results showed that lower extremity pain affected functioning ,
especially physical functioning , in older adults more than spinal pain .
in addition to this ,
the results obtained from the 6mwt also showed that the older adults with lower extremity
pain had the worst aerobic capacity compared with those with spinal pain . that is , our
results showed that chronic musculoskeletal pain in the lower extremity led to a decreased
aerobic capacity18,19,20,21,22,23 . on the other hand ,
the older adults evaluated in this study showed decreased independency
in daily living activities .
the same results can be found
in the literature24 , 25 . since depressive symptoms are common during aging , we also detected depressive symptoms in
our study .
although there
have been some finding indicating that chronic musculoskeletal pain increases depressive
symptoms in older adults , our results did not support this idea26 , 27 .
the results obtained in this study also supported the inverse relation between depressive
symptoms and independency in daily living activities .
this show that increased depressive
symptoms led to decreased independency in daily living activities .
this is not an unexpected
result , as it has been reported in previous related literature9 , 25 , 26
. the limitations of this study are as follows : ( 1 ) the sample size was too small to make a general comment .
( 2 ) we studied older adults with spinal or lower extremity pain , but we could not compare
our sample with older adults without pain . despite to the limitations mentioned above , the study has a major strength : this study is
the first one showing the effects of pain localization in older adults completed in
turkey . keeping in mind the limitations of the present study , we plan to perform further research
with a larger sample size so we will be able to make general comments with regard to this
field . in conclusion ,
the results of this study indicate that evaluation and relief of chronic
musculoskeletal pain are vital in geriatric rehabilitation programs . for this reason ,
health
providers , including physiotherapists , ergotherapists , medical doctors , and nurses , should
consider chronic musculoskeletal pain in older adults .
in particular , physiotherapists and
ergotherapists should focus on chronic lower extremity pain to improve physical functioning
and aerobic capacity of older adults to make them more mobile and improve their aerobic
capacity . | [ purpose ] the aim of this study was to show the impact of chronic musculoskeletal pain
of the spinal column and lower extremities on physical functioning , emotional status , and
independency in older adults . [ subjects ] in this cross - sectional study , 258 older adults
( mean age , 71.985.86 years , 50.8% males , 49.2% females ) living in their own residences
were evaluated .
[ methods ] pain intensity was analyzed using a visual analogue scale .
physical functioning was evaluated with the timed up and go test ( tug ) and a six - minute
walk test .
the geriatric depression scale was used to determine emotional status .
the
independency in daily living of the participants was evaluated using the lawton brody iadl
scale .
all participants were divided into two groups in accordance with the pain
localization : the ( 1 ) spinal pain and ( 2 ) lower extremity pain groups .
[ results ] when the
pain scores were compared , no significant differences between the two groups were found .
the same results were found in terms of tug scores .
the spinal pain group had higher
scores in terms of aerobic capacity than the lower extremity pain group .
[ conclusion ] the
results indicate that chronic musculoskeletal pain in the lower extremities decreased
aerobic capacity much more than spinal pain in older adults . | INTRODUCTION
SUBJECTS AND METHODS
Statistical analysis
RESULTS
DISCUSSION |
PMC4411900 | physical activity , or rather the lack of it , has become a significant risk factor for chronic diseases in modern society .
the consequences of hypokinesis are widespread across highly developed ( 1 3 ) and developing countries ( those with average and low income ) ( 4 , 5 ) .
while confronting this phenomenon , public health campaigns around the globe put forward the increase of physical activity among inactive people as their primary goal ( 6 ) ( inactive meaning those who do not undertake vigorous or moderate physical activity , also known as sedentary society ) ( 7 ) . however , the sedentary lifestyle is not the exact opposite ofthe physically active lifestyle ( 2 , 8 , 9 ) .
although both the lack of physical activity and prolonged sitting are serious risk factors for chronic diseases , their consequences are not dependent on each other ( 3 , 1012 ) .
scientists have proven that the physiological effects of sedentary behaviours are often more harmful ( 13 ) than insufficient amounts of physical activity itself ( 14 , 15 ) .
it appears that activities requiring low energy expenditure such as sitting down , watching tv , reading , working in front of a computer , computer games , lying down or napping , or driving a car ( 1.01.5 met ) ( 16 , 17 ) may have a stronger relationship with the risk of metabolic syndrome than the lack of any physical activity at all ( 14 , 18 , 19 ) .
watching tv , in particular , is linked with obesity ( 20 , 21 ) , cardiovascular disease ( 22 , 23 ) or type ii diabetes ( 20 ) . on the other hand ,
the sedentary lifestyle is not necessarily asynonym for lack of physical acti - vity ( 24 ) , as shown by individuals who meet or exceed the recommended amount of physical activity , even though they spend a long time on sitting ( 16 ) . in this case
, the consequences of a prolonged sitting might be independent of the protective effects of regular physical activity .
there are individuals who do not meet the who recommendations , even though they do not spend a long time sitting . instead
regardless of the relationships described above , prolonged sitting is akey problem facing public health ( 13 ) .
it appears that adults employed on a full - time basis spend the majority of their working hours sitting ( 25 ) .
the rapid increase in the use of a car as the main means of transport leads to congestion on the roads and , in consequence , extends travel time ( especially during the peak hours ) ( 26 ) . spending leisure time on watching
first , it requires lower energy expenditure than any other sedentary behaviour ( for example , driving a car , writing or reading ) ( 28 , 29 ) .
scientists report that in a typical american household , the tv is switched on for longer than 8hrs per day ( 27 ) , with the average above 2hrs per day ( 25 )
. the sitting / lying down time median in other investigated countries is 5h per day ( from 3h per day in portugal , brazil and columbia , up to 6h per day in taiwan , norway , hong kong and saudi arabia ) ( 4 ) .
the above data indicate that attention should be turned towards understanding the mechanisms underpinning sedentary behaviours .
further exploration of these behaviours should change the direction of health research ( 7 , 30 ) .
qualitative and quantitative data collection is extremely important in this particular case , as it would contribute to our understanding of this constantly growing problem .
the aim of the current investigation was to evaluate the sedentary behaviours among the polish population , with a particular focus on the relationship between these behaviours and the possibility of compensating their effects with various forms of leisure - time physical activities .
the relationships between sitting time and individual factors of polish citizens ( age , gender , level of education ) and environmental factors ( the accessibility of the sports classes and venues , the ratings of these classes and the ratings of the local authorities work in the realm of sport and recreation ) are analysed in the current work .
the survey was conducted between 10 october 2012 and 14 november 2012via computer - assisted telephone interviews ( cati ) .
the interviews were administered by qualified and supervised surveyors who delivered the questions in a way that was equally easy to comprehend for all respondents .
the research tools in the current investigation were the polish long version of international physical activity questionnaire ( ipaq ) and the bespoke survey on the determinants of sport activity among the polish population .
the survey included questions about the frequency , duration , place and the type of sport for all activities .
additionally , the participants were asked to rate the attractiveness of these classes and to provide ratings of the work done by local authorities within sport and recreation .
the ipaq provided the information on physical activity in specific domains ( such as occupational , commuting , domestic chores , sports and leisure ) as well as the duration of sitting time during the weekdays and weekends ( 31 ) .
the respondents were asked about the duration of sitting at work , at home , while studying and in their leisure time in the last 7 days prior to the survey .
this included the time they spent sitting by the desk , sitting when visiting friends , while reading , watching tv or lying down ( sleep was not included ) .
the time of sitting in / on the vehicles ( i.e. , in a car , on a bus or on a motorcycle ) was also specified in minutes per day .
the total and average sitting times ( during the weekdays and weekends ) were calculated according to the formulae below .
total sitting time min / week = sitting time in minutes during weekdays x 5 days + sitting time in minutes during weekend x 2 days average sitting time min / day= sitting time in minutes during weekdays x 5 days + sitting time in minutes during weekend x 2 days / 7 the total weekly duration of sitting was obtained by adding the time spent on commuting / transportation per week . for the purpose of the current study , only information on physical activity undertaken in leisure time ( in the last week prior to the start of the survey ) was used . after performing a standardised calculation of the durations and the frequencies of declared physical activities ( vigorous , moderate activities and walking ) ( 31 ) , and with the who recommendations on the amount of physical activity needed to maintain a good health taken into account , the respondents were divided into two groups : those who follow the recommendations ( n=874 ) and those who do not ( n=630 ) .
furthermore , those who declared undertaking 150minutes of moderate physical activity or 75 minutes of vigorous physical activity per week ( or the equivalent combination of moderate and vigorous physical activity ) were classified as physically active .
the investigated sample consisted of 1,505 polish citizens ( age range 1864 years old ) .
they were selected through the stratified - quota sampling method , with the demographic variables such as age , gender , education and the place of residence as strata .
the quotas were established proportionally , based on the data that were made accessible to the researchers by the central statistical office .
furthermore , in order to increase the representativeness of the sample , a data - weighting procedure was usedfor the variables listed above .
the quotas were proportionally assessed based on the data provided by the central statistical office ( data sources : national census of population and local data bank ) .
the characteristics of the polish population statistical analysis was run on ibmspss statistics version 21 .
the descriptive statistical analysis provided detailed characteristics of the dependent variables by using the mean ( x ) , median ( me ) and standard deviation ( sd ) .
the kolmogorov - smirnov test was used for assessing whether the data are distributed normally ( the sitting time during the weekdays and weekends , the sitting time in the vehicles and the total sitting time were taken into account ) .
given that the assumption of normality in the collected data was violated ( p<0.05 ) , further statistical analyses required use of chi and non - parametric tests such as u mann - whitney , kruskall wallis and wil - coxon .
the significant differences between dependent and independent variables ( individual : age , gender , education , participation in sport for all ; environmental : the number of and the state of free and paid sports facilities , the costs and attractiveness of the classes offered by paid public sports and leisure centres , local authorities input in promoting physical activity ) were examined ( p<0.05 ) .
the relationships between the time spent on sitting and the individual and environmental factors among polish citizens were investigated through log - linear analysis .
the respondents were divided into two groups : those who spent more than 2545min per day on sitting and those who spent less than 2545min per day on sitting ( the median of the duration of sitting was the cutoff point ) .
there were two analyses run separately : one taking into consideration the individual factors and one for the environmental factors .
the strength of the relationship between these variables and the time polish citizens spend on sitting was expressed through the odds ratios ( or ) ( with 95% confidence intervals ) .
the descriptive statistical analysis provided detailed characteristics of the dependent variables by using the mean ( x ) , median ( me ) and standard deviation ( sd ) .
the kolmogorov - smirnov test was used for assessing whether the data are distributed normally ( the sitting time during the weekdays and weekends , the sitting time in the vehicles and the total sitting time were taken into account ) .
given that the assumption of normality in the collected data was violated ( p<0.05 ) , further statistical analyses required use of chi and non - parametric tests such as u mann - whitney , kruskall wallis and wil - coxon . the significant differences between dependent and independent variables ( individual : age , gender , education , participation in sport for all ; environmental : the number of and the state of free and paid sports facilities , the costs and attractiveness of the classes offered by paid public sports and leisure centres , local authorities input in promoting physical activity ) were examined ( p<0.05 ) .
the relationships between the time spent on sitting and the individual and environmental factors among polish citizens were investigated through log - linear analysis .
the respondents were divided into two groups : those who spent more than 2545min per day on sitting and those who spent less than 2545min per day on sitting ( the median of the duration of sitting was the cutoff point ) .
there were two analyses run separately : one taking into consideration the individual factors and one for the environmental factors .
the strength of the relationship between these variables and the time polish citizens spend on sitting was expressed through the odds ratios ( or ) ( with 95% confidence intervals ) .
the total sitting time of polish citizens ( during the weekdays , weekends and when commuting ) averages 9.45.1h / day ( 46.926.5h / week ) .
it appears that there is no difference in the average sitting time during weekdays and weekends ( 4.02.8h / day ; 242.9169.1h / weekend 4.02.7h / day ; 240.8162.9h / week , respectively ) .
the average time spent sitting in a vehicle was 78.4111.0min / day ( table 2 ) .
the significant relationships were not found when the socio - demographic factors were taken into account , with one exception : gender .
the results showed that men ( 251.1170 min / week ) spend significantly more time on sitting during weekends ( chi=45.0 ; p=0.04 ) than women ( 230.9155 min / week ) .
average ( sd ) sitting time ( in min / day ) and the total sitting time ( in min / week ) of the polish population during weekdays , weekends , spent in mechanical vehicles ( when commuting ) depending on the socio - demographic factors engaging in any type of physical activity of a continued duration of 10 minutes in leisure time was declared by 67.8% of the polish population ( table 3 ) .
it appears that participation in sport for all depends on gender ( chi=11.7 ; p<0.001 ) , age ( ch=58.7 ; p<0.001 ) and education ( ch=28.7 ; p<0.001 ) .
men ( 48.0% ) , young adults ( aged 1824 : 61.1% ; aged 2529 : 52.5% ) and those educated to a degree level ( 56.7% ) participated in sport for all more often than the remaining groups of respondents . women ( 60.8% ) , those in the 5059 and 6064 age groups ( 34.5% and 31.5% , respectively ) and respondents with primary education ( 64.1% ) more often declared lack of engagement in physical activities .
the most popular sports are cycling ( 21.2% ) , swimming ( 13.4% ) and running ( 12.2% ) .
cycling as a main type of physical activity is declared mostly by the respondents who are in the age range of 5059 ( 44.7% ) and over 60 years old ( 42.6% ) .
younger adults ( 1824 years old ) , on the other hand , choose swimming and running more of ten ( 26.9% and 35.2% , respectively ) .
overall , both men and women tend to choose cycling as their favourite type of sport ( 31.1% and 42.9% , respectively ) .
the frequency and duration time of sport activities in which polish citizens ( n=1,505 ) participated in their leisure time despite a relatively high proportion of respondents declaring participation in sport for all , only 9.6% of polish citizens exercise on a daily basis ( table 3 ) .
as many as 13.9% of the respondents engage in a physical activity 34 times per week and 24.3% exercise sporadically ( a few times per month or year ) .
the results show that more men than women exercised 12 times ( chi=7.3 ; p<0.05 ) and 3itimes ( chi=11.0 ; p<0.05 ) per week .
the proportion of men who did sports 1 2 times per week was 22.8% , whereas this proportion for women was 17.3% .
the same pattern was shown for the group of people declaring engaging in a physical activity 34 times per week ( 16.9% of men and 11.0% of women ) .
interestingly , women declared lack of any physical activity more often than men did ( 35.2% vs. 29.2% ; chi=6.2 ; p<0.05 ) .
furthermore , women who were physically active reported shorter durations of their exercises than men : they worked out for less than 30min / day more often than mendid ( 35.3% and 18.9% , respectively ; chi=69.1 ; p<0.05 ) . however ,
when the durations of more than 60min / day were considered , polish womenstrainings were less frequent than those of men ( 26.8% and 45.2% , respectively ; chi=42.4 ; p<0.05 ) .
it appears that women participated in organised classes more often than men did ( 34.3% , 16.2% , respectively ; chi=42.8 ; p<0.05 ) , whereas men reported using public sports facilities , free ( 19.5% and 19.8% ; chi=8.7 ; p<0.05 ) and paid ( chi=43.1 ; p<0.05 ) , more than women ( 12.5% and 6.1% ) . the proportion of people who met who health recommendations , based on the type of activity ( vigorous , moderate and walking ) undertaken in leisure time , was 58.1% ( in which the proportion of men was 57.7% and of women
despite the lack of significant differences when age , gender and education were taken into account , there was a relatively high proportion of physically active people in the 4049 age group ( 62.7% ) .
on the other hand , the analysis of individual and environmental factors showed that the only factor that determines the duration of sitting of more than 2545 min / week is participation in sport for all ( see table 4 ) , where the value of 2545min / week is a median of the total sitting time declared by the respondents . according to the results , the odds ratios of sitting above the median is higher ( or=1.33 [ 1.031.72 ] ) among those who do not engage in any form of physical activity in their leisure time ( 54.5%vs .
individual factors determining the sitting time , odds ratios ( or ) and the 95% confidence intervals of those sitting below and above 2545min / week the remaining factors , such as the accessibility of sports facilities , the attractiveness of the classes and facilities and the activities of the local authorities within the area of sport and recreation , do not play an important role in the longer durations of sitting ( see table 5 . ) .
it appears that the sitting time of the polish population is not dependent on the who health recommendations being met ( 58.1% ) or not ( 41.9% ) .
environmental factors determining the sitting time , odds ratios ( or ) and the 95% confidence intervals of those sitting below and above 2545min / week
the current investigation conducted on a representative sample of the polish population shows that the duration of sitting ( regardless of participation in any kind of physical activity ) is a very important risk factor for chronic diseases .
it appears that the time spent sitting has increased in comparison with the data from 2009 ( from 343min / day to 401.8min / day ) ( 32 ) . at the moment , polish citizens spend 2812.4159 - 3.9min / week sitting , which is very close to the numbers reported in the developed countries . for instance , british citizens sit 53.823.0h / week ( roughly 3228min / week ) , the dutch 60.021.0h-/week ( roughly 3602min / week ) and the americans from 53.019.8 to 56.023.5h / week , depending on the data sources ( roughly 3361 3860 min / week ) ( 33 ) .
the polish population s results are higher than those of countries such as czech republic ( 2295.5 min / week ) ( 34 ) , portugal ( 23071107 min / week ) ( 35 ) and belgium ( 21911057 min / week ) ( 35 ) .
the median sitting time of polish citizens is 2545 min / week , which is higher than in norway , where , depending on the level of education , men are more likely to spend their time sitting than women ( 3544h / week and 3240h / week , respectively ; these values in minutes per week would be 21002640 for men and
the spanish also spend less time sitting than polish people : 373.1184.6min / day ( roughly 2612min / week ) ( 37 ) .
however , it should be pointed out that the cited data were compared with the total time the polish population devoted to sitting ( during weekdays and weekends ) , including the time spent in vehicles when commuting ( the total without commuting time is 2418.81430.3 min / week ) , whereas the investigations conducted in the czech republic considered only the weekdays and the spanish research excluded the time spent sitting in mechanical vehicles ( when commuting ) .
furthermore , some of the authors ( 35 , 36 ) did not specify what was included in the sitting time .
additionally , it needs to be noted that in the research to date , there is a lack of sufficient evidence that the questions in the surveys were used appropriately ( the met thresholds have not been established ) .
this , in turn , suggests that the sitting - time indicator relies solely on the respondents declarations , which creates problems when comparing the data across different populations and does not seem to be a reliable measure of the total sitting time of any investigated population .
regardless of the methodological differences , it is clear that the duration of sitting among the polish population has increased and their physical activity level is insufficient ( only 9.6% of respondents exercise every day and 13.9% exercise 34 times per week ) .
thesenumbers are alarming and concerning , given that , according to some researchers , prolonged sitting significantly decreases the time people spend on exercising ( 39 ) . furthermore , as mentioned in the introduction , spending leisure time on sitting might decrease the possibility of meeting the who health recommendations ( 2 ) .
additionally , it may correlate with metabolic diseases ( 4043 ) , obesity ( 40 ) and high blood pressure ( 39 ) among those who are not physically active .
the current findings suggest that polish citizens who are not physically active are 1.33[1.031.72 ] times more exposed to sitting durations longer than 2545min / week ( > 6h ) .
it is worrying that within the investigated sample , the authors did not find a relationship between the sitting durations and their healthy behaviours ( implementation of the recommended amount of physical activity ) .
this suggests that polish citizens do not link the need to spend their leisure time actively with preventing asedentary lifestyle .
interestingly , they declare participation in sport for all ( 70% ) , but they do not see the need to exercise on a regular basis .
this may be a result of old habits or a lack of awareness of healthy lifestyle behaviours .
meanwhile , chu and moy ( 39 ) argue that even those who meet these recommendations but sit more than 9.3h per day are in the 3.82 times greater risk of developing metabolic syndrome .
it is therefore expected that this problem may also appear among the most active polish citizens ( those in the 4049 age group ) in the future . at the moment , the majority of the respondents in this particular age group ( 62.7% ) meet who recommendations , but their sitting time is longer than for those in other age groups ( 252.4173.5min / day ) . despite the fact that sedentary behaviours form an independent risk factor for modern diseases , it seems appropriate to ask whether the duration of sitting among the polish population is linked with the opportunities ( or the lack of thereof ) of undertaking leisure physical activity in their place of residence .
unfortunately , the database in the explored topic is rather limited and provides mostly information promoting healthy lifestyle choices ( 44 , 45 ) .
the majority of research focuses on the relationship between the undertaken physical activity and urbanisation ( 46 , 47 ) or pollution ( 48 ) ( particularly in the context of the means of transportation choice ) ( 49 ) .
the research to date also provides analysis of the relationship between environmental factors ( i.e. , access to the sports facilities ) and leisure physical activity ( 50 , 51 ) .
however , the data provided mainly concern the citizens of wealthy countries ( 5254 ) . in general , the health behaviours among people are the results of their individual characteristics ( such as income , age or education ) and the environmental factors ( such as the local authorities activities , sport and recreation infrastructure , physical activity initiatives , or the use of land ) ( 5557 ) . the results of the current study confirm that men ( 48.0% ) , young adults ( aged 1824 : 61.1%;aged 2529 : 52.5% ) and those educatedto a degree level ( 56.7% ) participate in the sport for all initiative more often than the remaining subjects .
these variables appear to be significant factors supporting the healthy behaviours of the respondents ( 58 , 59 ) .
however , many reports indicate that people are more likely to undertake a physical activity if they have the support of their social groups ( 60 ) and the governmental plans ( 61 ) .
the marketing strategies ( 62 ) , access to equipment ( 58 , 63 ) , financing public sport facilities ( 45 ) , promoting active ways of spending leisure time ( 64 ) , safety , andsupport of decision - makers on development and implementation of sport initiatives ( 65 ) strongly influence the development of attitudes and behaviours of those who live in environments that do not actively promote physical activity and present many barriers in accessing sport facilities .
this seems to be an issue for the people living in poor areas : they have to overcome more obstacles when making healthy choices ( 63 , 6668 ) .
this suggests that anincrease in accessibility of free sports facilities and better focus on the safety of public places may encourage those of poorer backgrounds to increase their physical activity ( 69 ) .
it appears that people are more active when they have access to sport facilities , both free and paid ( 70 ) . in rural missouri , for instance , making the hiking trails more accessible created an opportunity to promote physical activity among the groups of people who were at greatest risk of becoming physically inactive , especially among women and those with a lower socio - economic status ( 60 ) .
the closeness of golf courses has increased physical activity among older people ( 71 ) . however , providing free facilities does not itself lead to anincrease in physical activity , as shown in the current study ( 72 ) . despite the fact that 8.4% of the polish population take advantage of free sports facilities , there is no relationship between these activities and the time of sitting .
this yields the effective initiatives that would raise the awareness of the problem and the consequences of the sedentary lifestyle .
the plan for educating people about the ways to fight the sedentary behaviours is also needed .
unfortunately , the results of the current work suggest that a more intense campaign promoting a healthy lifestyle is needed in poland . as revealed by the analysis
, the environmental factors ( with the exclusion of one case of participation in the sport for all initiative ) did not correlate with the sitting time among the polish population .
the results showed that there was no relationship between the duration of sitting longer than 6hrs per day and the recommended by who amount of physical activity , the number and state of free and paid sports facilities , the cost and attractiveness of classes , and the engagement of local authorities in the area of sport and recreation .
this may indicate that local interventions are not effective , polish citizens prefer passive recreation and a lack of awareness in this matter .
it should be underlined that there is a link between lack of physical activity and the risk of prolonged sitting ( or=1.33[1.031.72 ] ) .
moreover , the recent natpol reports reveal that the level of physical activity among the polish population is ( at best ) moderate ( 73 ) .
additionally , the upcoming changes in the age structure within the population and a desperately low fitness level of polish youth may increase the proportion of inactive people .
with all the above in mind , the authors present the following conclusions :
the immediate cooperation with the ministry of health concerning the increase of polish citizens awareness of the risks brought by sedentary lifestyle is necessary.it is necessary to intensify the consistent efforts aiming at the development of the environments promoting anactive lifestyle among the polish population at their place of residence .
the focus of these efforts is also directed towards prevention of the sedentary lifestyle ; these efforts should include planning more effective strategies aiming to engage more local communities in active recreation , partnerships development and the exchange of experiences and information about the obstacles people have overcome.the suggestion that future recommendations forthe amount of physical activity include guidelines intervening in the time people spend on sitting seems well justified.further investigation of the environmental factors with the widest scope possible is advised .
the immediate cooperation with the ministry of health concerning the increase of polish citizens awareness of the risks brought by sedentary lifestyle is necessary .
it is necessary to intensify the consistent efforts aiming at the development of the environments promoting anactive lifestyle among the polish population at their place of residence .
the focus of these efforts is also directed towards prevention of the sedentary lifestyle ; these efforts should include planning more effective strategies aiming to engage more local communities in active recreation , partnerships development and the exchange of experiences and information about the obstacles people have overcome .
the suggestion that future recommendations forthe amount of physical activity include guidelines intervening in the time people spend on sitting seems well justified .
ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors . | abstractbackgroundthe aim of the current investigation was to evaluate the sedentary lifestyle of the polish population and its link with prolonged sitting in light of individual and environmental factors.methodsthe sample consisted of 1505 polish people between 18 and 64 years old .
the computer - assisted telephone interview ( cati ) survey was conducted with the polish version of international physical activity questionnaire ( ipaq - lf ) and the survey of the determinants of a sport activity among the polish population .
the relationships between the time spent sitting and the individual and environmental factors among polish citizens were investigated through log - linear analysis.resultsthe total time polish people spent sitting was 2812.41593.9 min / week . during the weekend , men ( chi2=45.0 ; p=0.04 ) spent significantly more time sitting than women ( 251.1170.1min / week , 230.9155.0 min / week , respectively ) . despite the relatively high proportion of people declaring participation in the sport for all initiative ( 67.8% ) ,
only 9.6% of polish people undertake a physical activity on a daily basis .
the odds ratios above the median ( > 2545 min / week ) are higher ( or=1.33 ; 95% ci=1.031.72 ) for those who are not physically active ( 54.5% vs. 45.5% ) .
there was no relationship between prolonged sitting time and the environmental factors.conclusionit is necessary to increase consistent efforts to create an environment that would successfully promote anactive lifestyle among polish citizens in their place of residence and prevent sedentary behaviours within this population .
the recommendations for physical activity should include the guidelines intervening in the time people spend sitting . | Introduction
Materials and Methods
Statistical Analysis
Results
Discussion
Conclusion
Ethical considerations |
PMC4295041 | the matter between stars , the interstellar medium , varies considerably from region to region in our galaxy .
the sun is inside a very large structure called the local bubble , a region of hot tenuous gas formed by supernova explosions tens of millions of years ago [ 13 ] .
adjacent to the local bubble is a similar but larger bubble , also formed from supernova explosions . inside the local bubble
are smaller , denser clouds which may have broken off from the bubble interaction region .
the h density of the local cloud is about 0.2 cm , the temperature is about 6000 k , and the cloud moves about 23 km / s with respect to the sun
. the magnetic field strength can not be directly measured , but based on models is 35 nt .
the sun is the source of the variable solar wind , with speeds measured near earth ranging from 250 to 2200 km / s , proton densities from 0.01 to > 100 cm , and an average magnetic field strength of 5 nt .
since the solar wind and local interstellar medium ( lism ) plasmas are both magnetized , they can not mix , so the lism flows around the heliosphere .
the boundary between the lism and solar wind is the heliopause ( hp ) , analogous to earth s magnetopause . since the solar wind is supersonic , a shock ( called the termination shock ) forms upstream of the hp . at the ts ,
the solar wind becomes subsonic and begins to turn toward the heliotail , the stretched - out downstream region analogous to earth s magnetotail .
if the lism were supersonic , a bow shock would form in the lism upstream of the hp , but recent data and analysis suggest that the lism flow is subsonic and thus the heliosphere does not have a bow shock .
voyagers 1 and 2 were launched in 1977 and are now exploring the interaction between the lism and the solar wind .
they both have crossed the ts and are in the region of shocked solar wind between the ts and the hp that is called the heliosheath . in late 2011 v1 was 119 au from the sun and v2 was 97 au , moving outward at 3.5 and 3.1 au / yr , respectively .
this paper reviews the observations made by these spacecraft as they enter unexplored regions of space .
the first observed influence of the lism on the solar wind was from the lism neutrals .
the neutrals are unaffected by the magnetic fields and flow into the heliosphere , where they are ionized in the solar wind and form hot , 1 kev , pickup ions .
these pickup ions dominate the thermal pressure outside about 30 au and play a major role in pressure balance structures outside this distance . accelerating the pickup ions to 1
kev slows the solar wind ; this slowdown was first observed near 30 au and by 80 au the solar wind had slowed by about 20% .
some of the energy from the pickup ions is transferred to the thermal protons , causing the temperature of the solar wind to increase with distance .
about 2.5 years before each ts crossing , the voyagers detected a new energetic particle component with proton energies of tens of kev to tens of mev flowing along the magnetic field lines .
this new particle component , called termination shock particles , signified that the voyagers were entering a region analogous to earth s foreshock , with particles accelerated at the ts streaming into the heliosphere along the magnetic field . for these particles to be observed , the ts distance had to be further at the flanks than at the nose so that magnetic field lines at the voyagers would also pass through the ts .
the bluntness alone could not account for all the particle observations ; an additional asymmetry in the heliospheric boundaries due to the interstellar magnetic field was also required .
the realization that the supersonic solar wind must go through a termination shock to become subsonic was first reported by parker .
the location of this shock is determined by the hp location and the upstream plasma parameters .
the hp forms where the solar wind dynamic pressure is balanced by the total lism pressure ; the value of the lism pressure is not well determined the distance to the ts , and thus the scale size of the heliosphere , were determined when v1 crossed the ts at 94 au in 2004 .
it crossed the ts in 2007 at 84 au [ 1720 ] , 10 au closer than v1 .
calculations of the ts motion based on changes in the solar wind dynamic pressure suggested that ts motion was responsible for only 23 au of the distance change .
thus the heliosphere is asymmetric , with the ts closer in the v2 than v1 directions .
models of the interaction of the heliosphere with the lism show that an asymmetry occurs if the lism magnetic field is tilted from the lism flow direction and has a magnitude of > 3 nt .
if these conditions held , the magnetic field would drape around the heliosphere so that the magnetic field strength builds up outside the southern part of the heliosphere , and the increased magnetic pressure would push the boundaries of the southern heliosphere inward .
the ts crossing provided other surprises as well . the ts was a weak shock , with a compression ratio close to two . at voyager 2 ,
the speed decrease started about 80 days before the ts crossing as the speed went from 400 to 300 km / s in three discrete steps .
the last step coincided with a sharp gradient in the energetic particle pressure , with the inward pressure gradient force large enough to produce the observed slowdown . at the v2 ts ( v1 does not have a working plasma instrument ) , the speed decreased from 300 to 150 km / s , the density and magnetic field increased by a factor of 2 , and the ion temperature increased by a factor of 30 . a major surprise ( but see zank et al . ) was that the heating of the thermal ions was much less than the decrease in the flow energy .
about 15% went to heating the energetic ( tens of kev ) ions , but the majority seems to have gone into heating the pickup ions , which are not directly observed .
the ts was the source of the low - energy particles observed in the foreshock ; the intensities of these particles peaked at the ts .
however , the anomalous cosmic ray ( acr ) intensities did not peak at the ts as expected , at least not where crossed by v1 and v2 .
acrs are singly ionized particle with 10100 mev / nuc ; they were observed first near earth and their origin was thought to be pickup ions formed from lism neutrals which were then accelerated at the ts .
the acr intensity did not increase at the ts ; no evidence an acr source at the ts was observed at either of the voyager crossing locations .
the heliosheath was thought to be , in analogy with planetary magnetosheaths , a highly turbulent region and this expectation has been correct [ 2325 ] . figs .
1 and 2 show the daily average plasma parameters obtained by fitting the observed spectra to convected , isotropic proton distributions .
the broad envelope of the data and the 25-day running averages that are superposed show consistent trends . however , the individual sets of spectra very greatly on time scales of tens of minutes .
the magnetic field also varies by factors of 23 over similar time scales , confirming the very dynamic and turbulent nature of this region .
as v2 moves deeper into the heliosheath , these fluctuations decrease slowly in magnitude but remain significant . by the end of 2011 , v2 was 14 au past the ts crossing distance of 84 au .
models suggest that the ts has moved inward 8 au since the ts crossing due the very low solar wind dynamic pressure during the recent solar minimum .
the expectation was that the plasma speed would decrease across the heliosphere and the flow direction would turn tailward .
1 shows that , contrary to these expectations , the average speed at v2 has remained roughly constant at 150 km / s for over 4 years , with a brief dip in speed at 2009.7 followed by a recovery in 2010.5 .
these observations of steady speeds are not predicted by models and are not understood .
although the speed is not slowing , the direction of the flow at v2 is turning as expected .
1 shows that the flow in the rt plane ( the rtn coordinate system has r radially outward , t parallel to the solar equatorial plane and positive in the direction of solar rotation , and n completes a right - handed system ) is about 20 after the ts crossing and increases to about 45 at the end of 2011 .
the flow in the rn plane was toward the south as expected , starting at about 10 after the ts , then oscillating for about a year before it started a monotonic increase to 25 at the end of 2011 .
the initial deflections at the ts must be due to the ts being at an angle to the radial flow . as discussed above
, the ts is blunt near the nose , less curved than a circle , so the flow at the ts is deflected away from the nose of the heliosphere . as the plasma moves across the heliosheath it continues to turn away from the nose , as expected .
the rt angle plot shows a cutoff at about 50. this cutoff is an instrumental effect ; when the flow direction is at too large an angle to the instrument look direction the plasma is not detected . in this case
the distributions of the plasma properties in the heliosheath are well represented by gaussian distributions .
the observed distributions of the plasma parameters are fit to gaussians to find the average properties and standard deviations . for the rt angle , which is cut off at about 50
, we can fit the distribution below 50 with a gaussian and determine the average flow angle .
the flow angles determined from these fits are shown by the dashed line in fig .
1 , which shows the flow is 56 from radial in the t direction in 2011 .
note that the rt angles ( and thus speed ) are greater than the rn angles throughout the heliosheath .
more of the plasma goes around the sides than over the top of the heliosheath , at least in the southern hemisphere where v2 is located , which suggests that the heliosheath is compressed at the southern pole .
2 shows the radial speed vr , the density n and the temperature t in the heliosheath .
although the speed has remained roughly constant as shown above , vr decreased from 130 to 100 km / s as the flow turned tailward . after the ts , the density initially averaged about twice the 0.001 cm value in the solar wind but had large , factor of 34 , fluctuations . by the end of 2008
the cause of the density decrease is likely partially the reduced solar wind flux coming from the sun in the recent solar minimum and partially a heliolatitude effect . at solar
minimum the solar wind flux decreases with heliolatitude , so v2 at 30 s should observe less flux than observed near earth at low - latitudes .
however , a problem with this hypothesis is that these lower fluxes are associated with higher flow speeds , which are not observed in the heliosheath .
the decrease in fluctuations may result from the very quiet solar wind conditions in this solar minimum combined with v2 moving further from the ts .
the density increased by a factor of two during a 6 month period in 2011 , perhaps because of a diminishment of the heliolatitudinal flux gradient as solar minimum ends .
the average density at the end of 2011 is similar to that observed just after the ts crossing , but the fluctuations in daily averages are much smaller .
the temperature decreased from 150,000 k after the ts to about 40,000 k in 2011 .
perhaps it reflects cooler solar wind encountering the ts or less heating at the ts due to differences in the upstream flow parameters .
the temperature increased slightly in 2011 in concert with the increase in density , but the reason is unclear .
although the plasma instrument on v1 does not work , the speeds in the r and t direction can be calculated from the low energy charged particle ( lecp ) instrument observations of tens of kev ion intensities using the compton - getting effect .
the speed after the ts was below 100 km / sand monotonically decreased from 70 km / s in mid-2007 to 0 km / s in early 2011 and has since become negative .
the t component of the speed averaged 40 km / s until mid-2010 , when it started to decrease .
. suggest that the decrease of the speed to near zero signifies that v1 has entered a boundary region in front of the hp in which flow is parallel to the hp .
the v1 spacecraft was recently reprogrammed to do roll maneuvers so that vn could also be determined , and vn is also small < 20 km / s .
thus the v1 has entered a region with nearly stagnant flow which was not predicted .
models show that such a region could be part of the global spatial character of the heliosphere or a time dependent feature near the boundary of the fast and slow solar wind regimes near solar minimum .
since the observed speeds are very low , comparable to the 23 km / s expected in the lism , one might wonder if v1 has already crossed the hp . the magnetic field increased by about a factor of 2 in the stagnation region but that the direction has not changed .
the field is still consistent with the parker spiral direction ; this direction is expected to change in the lism , so v1 likely has not crossed the hp .
the increase in magnetic field magnitude is consistent with predictions that the field will be compressed as it pushes up against the hp boundary .
the most probably explanation for these data is that v1 has entered a boundary layer near the hp but has not yet crossed the hp .
the acr intensity has increased slowly as the voyager spacecraft move deeper into the heliosheath . at v1 ,
the spectra are almost power laws , indicating that v1 is near the source region .
one is that they are accelerated on the flanks of the heliosphere where the particles can interact with the ts longer , then move along the magnetic field lines to the voyager spacecraft .
another hypothesis is that acrs are accelerated by second order fermi acceleration by magnetic islands or ridges near the hp .
a third is that reconnection occurs as the current sheets are compressed near the hp , leading to particle acceleration .
the voyagers may be able to differentiate between these possibilities as they approach and cross the hp .
the voyager spacecraft celebrate their 35th year in space in august 2012 and continue exploring new regions of space .
they should continue to return data until 2025 , when we expect they will be well into the interstellar medium .
this paper describes some of the new discoveries and new mysteries resulting from recent observations .
some of the more intriguing puzzles are the source of the acrs , the very different speed profiles observed in the v1 and v2 directions , and the formation of a boundary layer in front of the hp . | this paper provides a brief review and update on the voyager observations of the interaction of the heliosphere with the interstellar medium .
voyager has found many surprises : ( 1 ) a new energetic particle component which is accelerated at the termination shock ( ts ) and leaks into the outer heliosphere forming a foreshock region ; ( 2 ) a termination shock which is modulated by energetic particles and which transfers most of the solar wind flow energy to the pickup ions ( not the thermal ions ) ; ( 3 ) the heliosphere is asymmetric ; ( 4 ) the ts does not accelerate anomalous cosmic rays at the voyager locations ; and ( 5 ) the plasma flow in the voyagers 1 ( v1 ) and 2 ( v2 ) directions are very different . at v1
the flow was small after the ts and has recently slowed to near zero , whereas at v2 the speed has remained constant while the flow direction has turned tailward .
v1 may have entered an extended boundary region in front of the heliopause ( hp ) in 2010 in which the plasma flow speeds are near zero . | Introduction
Pre-termination shock
Termination shock
Heliosheath
Summary |