Datasets:

karukas
/

pubmed-abstract-matching

Dataset card Viewer Files Files and versions Community

Split (3)

train · 120k rows

sentence1 stringlengths 176 4.35k	sentence2 stringlengths 0 822k
schizophrenia is a brain disorder associated with cognitive deficits that severely affect the patients ' capacity for daily functioning . whereas our understanding of its pathophysiology is limited , postmortem studies suggest that schizophrenia is associated with deficits of gaba - mediated synaptic transmission . a major role of gaba - mediated transmission may be producing synchronized network oscillations which are currently hypothesized to be essential for normal cognitive function . therefore , cognitive deficits in schizophrenia may result from a gaba synapse dysfunction that disturbs neural synchrony . here , we highlight recent studies further suggesting alterations of gaba transmission and network oscillations in schizophrenia . we also review current models for the mechanisms of gaba - mediated synchronization of neural activity , focusing on parvalbumin - positive gaba neurons , which are altered in schizophrenia and whose function has been strongly linked to the production of neural synchrony . alterations of gaba signaling that impair gamma oscillations and , as a result , cognitive function suggest paths for novel therapeutic interventions .	schizophrenia is a severe brain disorder that afflicts 0.51% of the world 's population and that is typically first diagnosed in late adolescence or early adulthood . the illness is manifest as disturbances in perception , attention , volition , inferential thinking , fluency and production of language , and the recognition and expression of emotion that lead to substantial impairments in social and occupational functioning . many affected individuals suffer from comorbid depression , an increased risk of cardiovascular disease , and excessive nicotine , alcohol , and cannabis use . the first domain is positive or psychotic symptoms that include delusions , false beliefs firmly held in the face of contradictory evidence ; perceptual disturbances and hallucinations , which may occur in any sensory modality but are most commonly auditory and experienced as hearing voices distinct from one 's own thoughts ; abnormalities in form of thought that are usually manifest as loose associations , over - inclusiveness , and/or neologisms ; abnormal psychomotor activity that is usually manifest as grossly disorganized behavior , posturing , and/or catatonia . negative symptoms include asociality ( withdrawal or isolation from family and friends ) , avolition ( impaired initiative , motivation , and decision - making ) , alogia ( poverty in the amount or content of speech ) , and anhedonia ( reduced capacity to experience pleasure ) . the third category of symptoms includes a number of cognitive abnormalities such as disturbances in selective attention , working memory , executive control , episodic memory , language comprehension , and social - emotional processing . although positive symptoms are usually the presenting and most striking clinical feature of schizophrenia , disturbances in cognition appear to be the core features of the illness as they are present before the onset of psychosis and are the best predictor of long - term functional outcome for schizophrenia patients . therefore , functional recovery ( e.g. , recovery of the capacity to maintain employment ) is largely dependent on improving cognitive deficits . although schizophrenia was initially characterized over 100 years ago , we still have only a limited understanding of its pathophysiology . moreover , we lack efficient tools for its treatment or prevention . for example , the multicenter , nimh - funded clinical antipsychotic trials in intervention effectiveness project recently found that newer atypical antipsychotics are not significantly more effective for treating psychosis than older typical antipsychotic medications and showed little benefit for improving cognitive symptoms [ 2 , 3 ] . these findings highlight the need to develop novel therapeutic interventions for schizophrenia [ 4 , 5 ] . if functional recovery of patients with schizophrenia depends on improving cognitive deficits , then understanding the neural basis of the normal cognitive operations that are impaired in schizophrenia is crucial to develop new therapies . interestingly , a number of findings from postmortem brain studies suggest that schizophrenia is associated with deficits of gaba - mediated synaptic transmission . furthermore , current hypotheses from cellular and systems neurophysiology suggest that a major role of gaba - mediated transmission is to produce synchronized neural network oscillations [ 7 , 8 ] which by facilitating the processing and flow of information within and between brain regions may be essential for normal cognitive function . here we review convergent findings from schizophrenia research , cellular neurophysiology , and cognitive neuroscience that favor the hypothesis that deficits of cognitive function in schizophrenia result from a dysfunction in gaba - mediated synaptic inhibition that disturbs oscillatory neural synchrony . this paper reviews recent evidence further indicating that in subjects with schizophrenia cognitive dysfunction is associated with alterations of oscillations in the gamma frequency band ( 3080 hz ) , which are normally induced during tasks that engage cognition . in addition , it reviews the cellular and molecular machinery involved in gaba - mediated synaptic transmission and the mechanisms by which gaba - mediated inhibition may synchronize neural activity in cortical circuits , focusing on the role of parvalbumin- ( pv- ) positive gaba neurons , whose function has been increasingly linked to the production of synchronized gamma oscillations . furthermore , data on the postnatal development of pv gaba neurons and their synaptic connections and the developmental trajectories of gene products involved in gaba - mediated synaptic inhibition is reviewed . finally , recent studies further suggesting that schizophrenia is associated with alterations in gaba - mediated synaptic transmission , particularly , but not exclusively , from pv neurons are highlighted . alterations of gaba signaling that impair gamma oscillations and therefore cognitive function in schizophrenia suggest potential paths for therapeutic interventions . schizophrenia is associated with deficits in behavioral tasks that assess perceptual and cognitive processes [ 10 , 11 ] . many such tasks normally increase synchronized oscillatory activity as measured in the electroencephalogram ( eeg ) , and such increase in synchrony is altered in subjects with schizophrenia [ 11 , 12 ] . for example , gamma - band synchrony during tasks that require visual gestalt perception is attenuated in schizophrenia patients . one of the core cognitive deficits in schizophrenia is a dysfunction of working memory , a system to keep information in mind and to manipulate it while performing complex tasks . gamma oscillatory activity ( 3080 hz ) may play an important role in normal working memory , given that gamma band synchrony increases with increasing working memory load [ 15 , 16 ] . in patients with schizophrenia , working memory deficits are accompanied by altered patterns of cortical oscillatory activity , since schizophrenia patients actually fail to enhance gamma activity with increasing working memory load and show overall increased gamma band power during working memory [ 16 , 17 ] . subjects with schizophrenia also have decreased oscillations in various frequency bands during specific phases of the working memory process , including encoding , maintenance , and retrieval . cognitive function , including working memory , implicates an interconnected network of brain regions , many of which show structural and functional abnormalities in schizophrenia . the prefrontal cortex ( pfc ) , which is extensively interconnected with cortical and subcortical regions , is thought to exert top - down control of the flow of neural activity between brain regions to provide cognitive control , coordinating incoming sensory and motor information with representations of internal goals and rules to select a context - appropriate behavioral response . subjects with schizophrenia have significant deficits in cognitive control and attenuated gamma oscillations in pfc during cognitive control tasks . cognitive control - related gamma activity , but not theta activity , is reduced in the frontal cortex of first - episode schizophrenia patients independent of medication status , suggesting a deficit related to the disease process as opposed to medication side effects or the consequences of being chronically ill . interestingly , some studies reported a positive correlation between gamma oscillations and hallucination symptoms score in schizophrenia [ 23 , 24 ] , indicating that the propensity for auditory hallucinations correlates with an increased tendency to enter states of oscillatory synchrony . it remains to be determined whether the decrease in gamma activity associated with cognitive deficits and the positive correlation between gamma activity and psychotic symptoms are due to different underlying mechanisms , manners of eliciting gamma , or cohorts of subjects . if , as suggested by multiple lines of evidence , altered neural synchrony underlies impairment of cognition in schizophrenia , then understanding the neural mechanisms normally involved in production of synchronized oscillations in neocortical circuits is crucial to develop new therapeutic interventions . whereas several mechanisms have been proposed , production of rhythms via gaba - mediated inhibition is currently a leading candidate mechanism , as reviewed in the following . by definition , gaba neurons have the capacity to synthesize gaba from glutamate via the enzymatic activity of glutamic acid decarboxylase ( gad ) , for which there are two gene products of different molecular weight , gad65 and gad67 . whereas the gad isoforms differ in a number of properties , their specific functional roles are not fully understood . interestingly , gad65 and gad67 are differentially expressed in gabaergic terminals in a cell type - specific manner ( see below ) . gad - mediated gaba synthesis occurs in the cytosol , and gaba is transported into synaptic vesicles by the vesicular gaba transporter vgat ( figure 1 ) . shortly after an action potential arrives at the nerve terminal , vesicular gaba release is triggered with a certain probability and in a ca - dependent manner . in pv neuron terminals , pv may act as a ca buffer that binds residual ca after activation of the ca sensor that triggers vesicular gaba release . at all synaptic connections from cortical gaba neurons thus far studied , the effects of synaptically released gaba are mediated by gabaa receptors ( gabaars ) , as the postsynaptic response is abolished by gabaar antagonists in hippocampus [ 2633 ] and neocortex [ 3441 ] . in contrast , gabab receptors mediate the postsynaptic effects of gaba only at connections from gaba neurons of the neurogliaform cell subtype . postsynaptic gabaars are heteropentamers composed of subunits from 7 different families ( 16 , 13 , 13 , , , , and 13 ) typically combined following a 2 : 2 : stoichiometry to form a gaba - activated chloride channel [ 43 , 44 ] . importantly , the subunit composition of the gabaar complex determines many of its functional properties . for instance , gaba - activated chloride currents produced by 1 subunit - containing gabaars ( 1-gabaars ) have much faster decay kinetics than currents mediated by gabaars containing other subunits . benzodiazepines bind to gabaars via a binding site localized at the interface between and subunits [ 45 , 46 ] and may modulate ( potentiate or decrease ) gabaar function in an subunit - selective manner . for example , zolpidem enhances gaba effects preferentially at 1-gabaars , whereas the 3ia and 5ia compounds are inverse agonists preferentially at 3-gabaars and 5-gabaars , respectively . other drugs , including tpa023 ( also named mk0777 ) , tpa023b , tpa123 , and tpa003 have comparable binding affinity at 1- , 2- , 3- , and 5-gabaars but may lack pharmacological effects at certain gabaar subtypes . in particular , tpa023 is a partial agonist at 2- and 3-gabaars but has no agonist efficacy at 1- and 5-gabaars . the magnitude and direction of the ionic current flowing through gabaars depends on its driving force or difference between its electrochemical equilibrium potential ( egabaa ) and the resting membrane potential ( vmr ) in the plasma membrane compartment where gabaars are located . because gabaar channels are largely permeable to chloride , egabaa is close to the chloride equilibrium potential ( ecl ) and therefore egabaa depends on the sodium - potassium - chloride cotransporter 1 ( nkcc1 ) and the potassium - chloride co - transporter 2 ( kcc2 ) , which mediate chloride uptake and extrusion , respectively . importantly , since the active gabaar conductance tends to clamp the membrane potential at egabaa , if egabaa is negative relative to vmr ( vmr > egabaa ) , the gabaar currents are hyperpolarizing , whereas if egabaa is positive to vmr ( egabaa > vmr ) , the gabaar currents produce depolarization . commonly , egabaa is negative to vmr , however , in certain cell types or subcellular compartments ( and in general , early in brain development ) egabaa > vmr . whether the gabaar conductance has inhibitory or excitatory effects depends on the relation between egabaa and the voltage threshold for action potential firing ( vth ) , which is always depolarized relative to vmr ( vth > vmr ) . when vth > vmr > egabaa , the chloride current is hyperpolarizing and clearly inhibitory because it shifts the membrane potential away from firing threshold , thus reducing the probability of firing . in contrast , if egabaa > vth > vmr , the chloride current is excitatory because it tends to depolarize the membrane above vth . however , if vth > egabaa > vmr , the gabaar conductance is inhibitory because , even though egabaa > vmr , shunting by the active gabaar conductance keeps the membrane potential below firing threshold . importantly , gabaar - mediated inputs that depolarize the membrane below vth ( vth > egabaa > vmr ) can have dual , time - dependent inhibitory / excitatory effects . initially , when the gabaar conductance is active , the net effect of the synaptic input is inhibitory because of the shunting effect . however , the depolarizing postsynaptic potential outlasts the duration of the gabaar conductance , thus increasing the firing probability once the gabaar conductance decays . importantly , the depolarizing gabaar - mediated post - synaptic potential may be amplified by voltage - dependent na currents localized perisomatically , possibly in the initial segment of the axon [ 51 , 52 ] , enhancing its excitatory effect . the inhibitory versus excitatory effect of the gabaar conductance may be dynamic , because vmr and vth are subject to modulation and change over time . in addition , egabaa may vary between cell types and subcellular compartments , depending on the nkcc1/kcc2 activity ratio . it has been commonly suggested that uptake of extracellular gaba by plasma membrane transporters could help terminate the synaptic effect of gaba and thus the duration of the inhibitory postsynaptic current ( ipsc ) . in the cns , gaba uptake is largely mediated by the plasma membrane gaba transporter 1 ( gat1 ) which translocates gaba through the neuronal and glial membrane ( figure 1 ) . interestingly , recent experiments indicate that gat1 does not control the time course of gabaar - mediated ipscs , since the duration of ipscs produced at single synapses is not affected by pharmacological inhibition of gat1 , nor in gat1 knockout mice [ 5356 ] . the finding that gat1 does not control ipsc duration may be explained by gat1 's predominantly extrasynaptic localization [ 5762 ] and by the slow gat1-mediated gaba translocation rate [ 63 , 64 ] compared with the rapid gabaar activation by synaptic gaba . other experiments suggest that gat1 's main role is preventing intersynaptic gaba spillover [ 53 , 56 ] , ( e.g. , the unintended activation of gabaars at a given synapse by gaba released at adjacent synapses ) . four different gaba transporters have been cloned : gat1 , gat2 , gat3 , and bgt1 ; therefore , it is possible that some of the gaba transporters different from gat1 have properties consistent with uptake - mediated control of ipsc duration . however , gat2 is only found during very early brain development and bgt1 is not abundant in brain . gat3 is mostly localized in glia , and the effects of gat3 blockade indicate that , similar to gat1 , gat3 's main role is reducing the effects of gaba spillover . the mechanisms by which gabaar - mediated inhibition may synchronize postsynaptic cell activity have been reviewed in detail previously [ 7 , 8 , 67 , 68 ] . figure 2 illustrates a group of pyramidal neurons firing asynchronously in response to some excitatory inputs that receive common gabaar - mediated hyperpolarizing inhibition . if such gaba - mediated hyperpolarizing input is strong enough , then the postsynaptic neurons will be inhibited together during a certain time window and , as the gabaar inhibitory effect decays , will escape from inhibition to resume firing nearly in synchrony ( figure 2 ) . such postinhibition synchronous spiking of pyramidal cells can be elicited by single - gaba neurons and readily generates synchrony throughout large numbers of neurons in computational network models [ 7 , 67 ] . therefore , post - inhibition synchronous spiking is a strong candidate mechanism for production of neural synchrony . alternative synchronization mechanisms , which are not reviewed here , involve gap junctions connecting pyramidal cell axons or noisy but correlated inputs . importantly , neuronal synchrony is commonly observed during episodes of rhythmic / oscillatory network activity , especially in association with cognitive tasks [ 70 , 71 ] . therefore , the circuit mechanisms of synchronized oscillations via gabaar - mediated inhibition must involve rhythmic interneuron firing and trains of ipscs in their postsynaptic target cells . as multiple subclasses of gaba neurons exist , a crucial issue is whether specific subtypes are involved in the mechanisms of synchronized oscillations . synchronized oscillations occur at different frequency bands , including theta ( 410 hz ) , beta ( 1530 hz ) , and gamma ( 3080 hz ) . whether oscillations of all frequency bands depend on gabaar - mediated inhibition and , if so , on particular gaba neuron subtypes is still a matter of investigation . here we focus on models for the mechanisms of gamma oscillations , which are commonly induced during cognitive tasks and seem to be impaired in the cortex of patients with schizophrenia . synchronization by the gabaar - mediated mechanism described in figure 2 requires sufficiently strong gaba synapses activating a relatively large gabaa conductance via inhibitory inputs localized near the site of action potential initiation . in pyramidal cells , action potentials are commonly triggered near the axon initial segment ( ais ) , the axonal compartment that is closest to the soma . therefore , inhibitory inputs onto the perisomatic membrane compartment ( soma , proximal dendrites , and ais ) produce stronger inhibition than inputs onto distal dendrites [ 75 , 76 ] , suggesting that perisomatic - targeting gaba neurons may be crucially involved in production of synchronized oscillations . three main subtypes of perisomatic - targeting gaba neurons exist in neocortex and hippocampus , namely , the parvalbumin - positive and the cholecystokinin - positive basket cells ( pvbcs and cckbcs ) and the pv - positive chandelier cells ( pvchcs ) . both pvbcs and cckbcs innervate pyramidal cells at the soma and proximal dendrites ( figure 3 ) , whereas pvchcs synapse exclusively onto the ais ( figure 3 ) . because a synaptic gabaar conductance has stronger inhibitory effect the closest it is localized to the site of spike initiation , pvchc inputs onto the ais surprisingly , some recent studies suggested that synaptic input from pvchcs is actually excitatory , since stimulation of pvchcs frequently initiates spikes in postsynaptic pyramidal cells via gabaar activation . in addition , electron microscopy studies support the idea that pvchc inputs are excitatory , as they show very low levels of kcc2 in the ais compared to the soma or dendrites [ 40 , 77 ] . since kcc2 extrudes chloride , lower ais levels of kcc2 should produce a more positive egabaa , resulting in a depolarizing gabaar current ( figure 3 ) . in fact , in paired recordings using experimental conditions that preserve the physiological intracellular chloride concentration , pvchc inputs depolarize the postsynaptic pyramidal neurons [ 40 , 78 ] , whereas in identical experimental conditions pvbc inputs are hyperpolarizing [ 40 , 78 ] , consistent with higher levels of kcc2 transporters in the somatic membrane [ 40 , 77 ] . experiments with uncaging of gaba onto gabaars in specific membrane compartments similarly showed a more depolarized egabaa at the ais compared with the soma and dendrites . an excitatory depolarizing gabaar - mediated input by pvchcs is inconsistent with their participation in inhibition - based synchronization . however , although egabaa at the ais is 1020 mv depolarized before vmr , it may be negative relative to vth [ 40 , 78 , 79 ] . as highlighted above , when vth > egabaa > vmr , the gabaa conductance has a dual inhibitory / excitatory effect ( figure 3 ) , which in the case of pvchc inputs may be amplified by their proximity to the spike initiation site . such inhibitory / excitatory effect may contribute to synchronization of postsynaptic cell activity at gamma band frequency [ 7 , 80 ] . specifically , since the postsynaptic potential outlasts the duration of the gabaar conductance ( figure 3 ) , once the conductance is deactivated , the depolarizing postsynaptic potential accelerates the post - inhibition synchronous spiking , facilitating synchronization at gamma frequency . such a mechanism operates at gabaar - mediated synapses onto gaba neurons [ 7 , 80 ] and could also apply at pvchc inputs onto pyramidal neurons . the depolarizing effect of pvchcs described in neocortex [ 40 , 78 ] contrasts with pioneer paired recording experiments showing that either pvbc or pvchc inputs hyperpolarize hippocampal pyramidal neurons . a hyperpolarizing effect of hippocampal pvbcs and pvchcs was also suggested recently by experiments using conditions that preserve the intracellular chloride concentrations , but that are different from the intracellular chloride - preserving conditions employed in studies of neocortical pvchc inputs [ 40 , 78 ] . interestingly , the ais membrane has low levels of kcc2 in neocortical and hippocampal pyramidal cells . the discrepancies between findings for hippocampal versus neocortical pvchcs highlight the need for further research to clarify the enigmatic role of this class of gaba neurons . whether excitatory or inhibitory , the unique properties of pvchc inputs suggest that the reported alterations of these cells must significantly contribute to cortical circuit dysfunction in schizophrenia . in contrast to pvchcs , both pvbc and cckbc inputs are hyperpolarizing ( vmr > egabaa ) , although egabaa is slightly , but significantly , different for pvbc versus cckbc inputs . such differences in egabaa were attributed to the activity of the voltage- and chloride - dependent chloride channel clc-2 , which helps maintaining a low internal chloride concentration ( thus a hyperpolarized egabaa ) at inputs from pvbcs but not from cckbcs . the clc-2-dependent regulation of internal chloride at pvbc inputs is dependent on the extent of gabaar activation . these findings highlight a degree of functional diversity between bc subtypes , showing that pvbc inputs , but not cckbc inputs , possess a mechanism to prevent internal chloride accumulation during high - frequency neuronal activity such as that observed in gaba neurons participating in gamma oscillations . functional diversity between pvbcs , pvchcs and cckbcs is also suggested by recent data demonstrating that the relative levels of gad65 and gad67 proteins in nerve terminals of these cells vary significantly in a cell - type - specific manner . using immunocytochemical labeling combined with a quantitative fluorescence microscopy methodology , the colocalization of gad65 and gad67 proteins in the same terminals was assessed for pvbcs , pvchcs and cckbcs ( figure 4 ) . the latter cells were identified using an antibody that detects the cannabinoid 1 receptors expressed by inhibitory terminals . importantly , in the prefrontal cortex nearly all cannabinoid 1 receptor - expressing cells detected using this antibody are immunoreactive for cck . this assessment showed that the ratio of gad67/gad65 expression varied significantly across each type of terminal , with very high ( 15.42 ) and very low ( 0.18 ) ratios observed in terminals of pvchcs and cckbcs , respectively , and a ratio of 1.49 for pvbc terminals . these data reinforce the idea that synaptic connections from different perisomatic - targeting gaba neuron subtypes are functionally diverse . the impact of different gad67/gad65 ratio on the properties of the gaba synapses studied remains enigmatic because the differential functional roles of gad67 and gad65 are still poorly understood . in mice , gad67 deficiency produces major alterations , as gad67 animals are born with cleft palate ( which is lethal ) and with 90% reduction of bulk gaba concentration in brain tissue [ 88 , 89 ] . in contrast , gad65 mice survive into adulthood , displaying 20% reduction in total brain gaba concentration together with increased susceptibility to seizures [ 90 , 91 ] , increased anxiety , and altered fear conditioning . in synapses of gad65 mice , gabaar - mediated synaptic transmission appears normal during low - frequency stimulation but is strongly impaired at stimulation frequency of 30 hz or higher [ 94 , 95 ] . in mice with cerebellum - specific gad67 deficiency , gabaar - mediated transmission is markedly weaker even at low stimulus frequency , suggesting a crucial role of gad67 in baseline synaptic transmission . whether the effects of gad67 deficiency on cerebellar synapses are observed in cortical synapses as well remains to be determined . importantly , gad67 deficiency markedly impairs the maturation of cortical gabaergic synaptic connections . therefore , testing the role of gad67 in synaptic transmission independent of its role in synapse development requires a paradigm in which gad67 is decreased once gaba synapse maturation has been completed . in addition , gad65 and gad67 both comprise 50% of total gad protein in mouse cortex , while gad65 comprises 70% in the rat cortex . the gad65 and gad67 proportions of the total gad protein in human cortex is unknown ; however , the differences between mouse and rat strongly stress the need to study gad function in molecularly relevant systems when trying to understand their roles in human disease . gabaar - mediated inputs involved in gamma band synchronization must inhibit their postsynaptic neurons for a time compatible with the gamma oscillation period . for instance , long - lasting postsynaptic currents such as those activated by gabab receptors ( which may last hundreds of milliseconds ) are inconsistent with gamma synchrony , as postsynaptic neuron inhibition would be longer than the typical gamma cycle period . whereas all gabaar subtypes produce faster currents than gabab receptors , the gabaa current duration depends on the subunit composition [ 43 , 99 ] . therefore , pvbcs may produce ipscs with faster decay , as 1-gabaars predominate at inputs from pvbcs [ 33 , 41 , 101 , 102 ] whereas 2-gabaars predominate at both pvchc and cckbc inputs [ 41 , 101103 ] . interestingly , pvbcs , cckbcs , and pvchcs elicit in pyramidal cells uipscs with nearly identical kinetics [ 39 , 104107 ] . therefore , in addition to the gabaar subunit composition , the ipsc kinetics are determined by additional factors that may not affect recombinant gabaars . such factors include gabaar subunit phosphorylation and other posttranslational modifications [ 108 , 109 ] , and the time course of the gaba concentration transient to which gabaars are exposed . although pvbcs and cckbcs produce ipscs with similar duration during individual gaba release events , cckbcs distinctively produce multiple asynchronous release events after single - action potentials . multiple gaba release events may prolong the postsynaptic inhibitory effect of each cckbc spike . in contrast , pvbc terminals produce a single synchronous gaba release event per presynaptic action potential [ 104 , 110 ] . asynchronous release from cckbc terminals has been observed in multiple studies [ 104 , 107 , 111115 ] suggesting that it is a fundamental property that prolongs the inhibitory effect of cckbcs on postsynaptic neurons possibly linking their activity with synchronization at frequency bands lower than gamma . the data reviewed above suggest that , relative to cckbcs and pvchcs , pvbcs have unique properties consistent with a crucial role in the mechanisms of gamma band synchrony . however , such data do not directly assess involvement of any of these gaba neuron subtypes in the gamma oscillation mechanisms . interestingly , some electrophysiological studies more directly indicate that among perisomatic - targeting gaba neuron subtypes , pvbcs are most likely to be involved in the production of gamma oscillations . for instance , whereas both bcs and chcs are active during gamma oscillations in vivo [ 116118 ] and in vitro [ 119124 ] , the firing of cckbcs and pvchcs is more weakly coupled with the gamma oscillation cycle than pvbc firing , although cckbc and pvchc firing is strongly coupled with theta oscillations [ 125127 ] . furthermore , gamma oscillations are significantly reduced or abolished by suppressing pv cell activity with optogenetic methods that do not directly affect cckbcs or by stimulation of presynaptic opioid receptors that suppress gaba release from pvbcs but not from cckbcs or chcs . therefore , perisomatic gabaar - mediated currents from pvbcs appear to be the main source of gabaar - mediated synchronization in the gamma frequency band . essential for modeling the circuit mechanisms of gamma synchronization is to understand how pvbcs are normally recruited to fire rhythmically at gamma frequency . recruitment of pvbc firing depends on activation of not only excitatory but also inhibitory synaptic inputs onto them since pvbcs target other gaba neurons , including nearby pvbcs [ 129 , 130 ] , and are also inhibited by inputs from different classes of gaba neurons , including the cckbcs . gamma oscillations are successfully generated in computational model networks that rely on reciprocal inhibition between gaba neurons and are thus called ing , for interneuron network gamma [ 131133 ] . in ing models , gaba neurons are recruited by a strong tonic excitation that drives them to fire at a frequency above gamma , and the reciprocal inhibition synchronizes gaba neuron firing at a frequency inversely related to the ipsc duration , falling within gamma range for durations typical of 1-gabaar - mediated ipscs [ 132 , 133 ] . whereas gamma rhythms possibly induced by ing - like mechanisms have been observed experimentally , the actual source of the strong tonic excitation onto gaba neurons required by ing models is unclear . metabotropic glutamate receptors [ 67 , 134 ] or kainate receptors could provide such a tonic drive , although this possibility is supported , only indirectly , by findings obtained with ampa- and nmda - mediated synaptic transmission blocked . a recent study employing genetically engineered mice with a deletion of gabaar expression in pvbcs directly tested whether inhibition onto pvbcs is necessary to generate gamma oscillations , as predicted by the ing models . in such mice , gabaar - mediated ipscs were abolished exclusively in pvbcs and theta oscillations and their coupling with gamma oscillations were severely disrupted . however , in such mice hippocampal gamma oscillations in vivo were intact as compared with wild - type mice . these data argue against the ing model for gamma band synchrony and suggest that inhibition onto pvbcs , potentially from cckbcs , is crucial for coupling theta and gamma oscillations . such theta - gamma coupling is thought to be important for cognitive function . as the role of ing mechanisms in gamma oscillation production continues to be tested , some studies favor an alternative model , known as pyramidal interneuron network gamma ( ping ) , which depends on recurrent excitatory - inhibitory synaptic interactions . in ping , pvbcs are recruited by phasic glutamate - mediated inputs from the pyramidal cells , and the pvbcs provide strong feedback inhibition that synchronizes pyramidal cell firing [ 67 , 121 , 138 ] . the ping model predicts that during the gamma oscillation cycle pvbcs fire after the pyramidal neurons , with timing consistent with monosynaptic recruitment by pyramidal cells . the spike timing of pyramidal cells and putative bcs during gamma oscillations in awake behaving animals is actually consistent with the ping model , as bcs fire 2 - 3 ms later than pyramidal neurons [ 139 , 140 ] . similar findings were obtained for pyramidal and pvbc spikes during gamma oscillations in hippocampal and neocortical brain slices [ 119 , 120 , 122 , 123 , 141 ] . ping models also predict the presence of trains of gamma frequency ipscs in pyramidal neurons and trains of gamma frequency epscs in pvbcs . evidence consistent with ipsc trains in pyramidal cells was obtained for gamma oscillations in vivo [ 117 , 142 ] and in vitro [ 119123 , 141 , 142 ] . in addition , during gamma oscillations in vitro , pvbcs display rhythmic epscs highly synchronized with the gamma rhythm [ 120122 ] . interestingly , optogenetics experiments show that driving pv neurons by nonrhythmic excitatory inputs is sufficient to generate gamma synchrony via feedback inhibition , a finding also consistent with the ping model of gamma . ping mechanisms rely on recruitment of pvbcs by phasic excitatory input ; therefore , the properties of glutamate synapses onto pv neurons are extremely relevant for models of gamma oscillations . interestingly , schizophrenia has been hypothesized to be associated with a deficit of glutamate transmission , more specifically with hypofunction of nmda receptors , particularly in gaba neurons . moreover , some studies have suggested that nmda hypofunction could especially affect pv cells [ 144146 ] . therefore , an important question is the following : what are the subtypes of glutamate receptors that mediate synaptic activation of pv gaba neurons ? the answer to this question is relevant in the context of alterations of gamma synchrony in schizophrenia , because if nmda receptors are important to recruit pv neurons in a ping mechanism of gamma , then nmda hypofunction could be linked to deficits of gamma synchrony in schizophrenia . data from recent studies showed that systemic administration of nmda receptor antagonists increase the firing rate of putative pyramidal neurons and decrease the firing of putative inhibitory cells in the pfc in vivo , suggesting that nmda receptors may be crucial to recruit inhibition . an important question not directly addressed by such studies is whether the inhibitory neurons dependent on nmda receptors belong to the pv - positive class of gaba neurons . whereas several studies demonstrated that synaptic excitation of pv neurons is relatively nmda receptor independent , until recently no studies directly compared the importance of nmda receptors in synaptically evoked recruitment of pvbcs versus pyramidal neurons in neocortical circuits . recent data from recordings in mouse pfc show that , compared with pyramidal cells , glutamate synapses onto pvbcs have epscs with faster decay and weaker nmda receptor contribution , supporting the idea that the rapid activation of pvbcs is largely dependent on fast ampa receptor - mediated excitation . moreover , in a computational model producing gamma oscillations via ping mechanisms , fast ampa - mediated excitation of pvbcs was critical for gamma band synchronization because the slower decay time course of nmda - mediated epscs disrupted gamma band synchrony . some studies indeed showed that gamma oscillations are not affected or are enhanced by nmda receptor antagonists , whereas ampa receptor antagonism completely abolished them [ 150153 ] . similarly , in mice with ampa receptor deletion genetically engineered to occur exclusively in pv - positive neurons , gamma oscillations are strongly reduced ; however , nmda receptor deletion selectively in pv - positive cells does not decrease and in fact increases gamma oscillation power . the results of recent studies therefore suggest that , in mature cortical circuits , nmda receptors only play a minor role in synaptically evoked excitation of pv - positive neurons and therefore on gamma oscillations produced via ping mechanisms . if indeed pv neuron excitation is normally weakly dependent on nmda receptors , then such data suggest that excitatory synapses onto pv cells are an unlikely target of nmda receptor hypofunction mechanisms in schizophrenia . importantly , although in most cases gamma synchrony is unaffected by nmda receptor blockade , the effects of nmda receptor antagonists on gamma oscillations may vary with cortical region or layer , in some cases ketamine producing a decrease , in others producing an increase in gamma power [ 152 , 156 ] . in addition , some data show that whereas mature pvbcs display a relatively small nmda receptor mediated component in synaptic responses [ 148 , 157 , 158 ] , such nmda component is substantially more prominent in immature pvbcs [ 157 , 158 ] . such findings suggest the interesting possibility that alterations of nmda receptor - signaling during early brain development could alter pvbc function in ways that persist into adulthood , thus changing the role of pvbcs in mature local circuits . interestingly , a recent study showed that a genetically engineered deletion of nmda receptors from pv - positive cells does not have significant effects if the deletion is produced in the brain of adult mice . however , a similar deletion produced during early brain development caused behavioral alterations in adult mice , some of which resemble behavioral dysfunction in patients with schizophrenia . schizophrenia is hypothesized to be a neurodevelopmental disorder based on data linking the disease with adverse events during pre- and perinatal periods and the presence of cognitive and behavioral deficits in childhood many years prior to the onset of psychosis during late adolescence and early adulthood . adolescence , the developmental transition from parent - dependent childhood to independent adulthood , is associated with significant changes in behavior and with marked improvements in cognitive control . moreover , gamma band synchrony emerges during childhood and continues to mature until early adulthood [ 162 , 163 ] . the postnatal developmental trajectory of gaba - synaptic function may therefore suggest critical periods of vulnerability during which the mechanisms producing neural synchrony could become dysfunctional in schizophrenia . in what follows we review developmental studies of the functional properties of pvbcs and their synaptic connections in rodents and of gaba - related gene products studied in the human and nonhuman primate brain . mature pv neurons have a unique fast - spiking ( fs ) firing pattern ( figure 5 ) , which includes very narrow action potentials and high frequency firing without the spike - frequency adaptation typically observed in pyramidal cells and other gaba neuron subtypes . although the nonadapting properties of fs cells are revealed with artificial stimuli ( rectangular currents steps lasting several hundred ms ) , they correlate strongly with the particular ability of fs cells to respond to oscillatory inputs at gamma frequency ( figure 5 ) , which is likely due to the gamma frequency resonance of the fs cell membrane . immature fs neurons , in contrast , have significantly slower action potentials and stronger spike - frequency adaptation , fail to respond efficiently to gamma frequency oscillatory inputs , and show much slower conduction velocity of action potentials along their axon [ 33 , 165167 ] . in rodent hippocampus , as well as in auditory , somatosensory , and prefrontal cortices , maturation of fs neuron electrical properties is complete by postnatal day 25 ( p25 ) after which fs neurons display adult - like electrical properties [ 33 , 157 , 165167 ] . functional properties of the inhibitory connections onto excitatory neurons also differ markedly between developing and mature pvbcs : unitary ipscs ( uipscs ) , from immature pvbcs , are weaker and have slower decay time than uipscs produced by mature pvbcs [ 33 , 168 ] . such acceleration of uipsc decay is explained by an increase in the contribution of 1-gabaars with maturation . a gabaar subunit switch may also contribute to the developmental acceleration of the uipsp decay although developmental changes in the pyramidal cell membrane time constant contribute as well . mature fsbcs produce highly synchronous gaba release , in contrast to asynchronous release from mature cckbcs [ 33 , 104 , 110 ] . however , gaba release is less synchronous and less reliable in synapses from immature fsbcs . postnatal maturation of the fsbc connections takes place relatively rapidly , as uipscs acquire mature properties by p28 [ 33 , 167 ] . inhibition onto fs cells also undergoes significant developmental changes . for example , uipscs at fsbc - to - fsbc connections mature within 3 - 4 weeks postnatally , whereas miniature ipscs ( mipscs , which represent gaba release at single synapses ) recorded from fsbcs acquire adult - like properties by p25 . unitary epsps ( uepsps ) at immature pyramidal cell - to - fsbc connections have slow decay time , which accelerates markedly during development , reaching adult - like values by p22 . similarly , miniature epscs ( mepscs ) recorded from immature fsbcs are slow and show a developmental acceleration of their decay to reach very fast mepsc decay values in mature fs cells [ 148 , 157 , 158 , 165 ] . fast - decaying epscs and epsps in mature fsbcs are largely mediated by ampa receptors [ 148 , 157 , 158 , 167 , 169 ] , suggesting that the rapid epsc decay in fsbcs is due , at least in part , to a weak contribution from the slow - decaying nmda currents [ 148 , 157 ] . actually , the developmental acceleration of epsc decay is accompanied by a marked decrease in the contribution of nmda currents which is still ongoing at p40 to p96 , which in rodents corresponds to the transition from adolescence to adulthood . in contrast to pfc , in hippocampus , auditory , and somatosensory cortices , nmda receptor contribution in fsbcs decreases prior to adolescence [ 167 , 169 , 171 , 172 ] . although the mechanisms controlling postnatal development of pvbc firing properties and their synaptic inputs and outputs are important candidates as the substrate of alterations in schizophrenia , they are still poorly understood . neuregulin-1 is a trophic factor crucial for brain development that is encoded by a schizophrenia susceptibility gene and is highly expressed during late developmental periods and in adulthood [ 173 , 174 ] . among various neuregulin-1 receptors , the erbb4 receptor , whose gene also confers schizophrenia susceptibility [ 173 , 174 ] , is enriched in gaba neurons , particularly in pv - positive cells [ 175 , 176 ] where it facilitates gaba release , possibly mediating neuregulin-1 enhancement of gamma oscillations . neuregulin-1 signaling appears to regulate early development of gaba synapses and , via erbb4 receptors , may control development of pv neuron synapses . interestingly , erbb4-mediated neuregulin-1 effects are crucial for development of excitatory synapses onto pvbcs [ 179 , 180 ] . in parallel to the maturation of their firing pattern and synaptic connections , pvbcs undergo significant developmental morphological changes . for instance , the total length of the dendritic and axonal trees of pvbcs increases significantly from p6 to p25 , the number of axonal branch points increasing five times during this developmental period . the number of postsynaptic neurons innervated by individual pvbcs also increases markedly with postnatal development , resulting in a higher functional connectivity between mature pvbcs and excitatory neurons . a crucial factor regulating development of innervation patterns by pvbcs for instance , gad67 knockdown in single pvbcs dramatically decreases formation of axon branches and synapses , as well as the number of postsynaptic neurons innervated by each pvbc . such effects of gad67 knock - down are observed in organotypic cell cultures and in the primary visual cortex in vivo with gad67 knock - down induced at p13 and the patterns of innervation examined at p20 or p32 . the role of gad67-mediated gaba synthesis in formation and/or stability of pvbc synapses may involve neuroligin - neurexin interactions and modulation of gaba receptor trafficking . because detailed molecular and biophysical analysis of developmental changes in gaba neuron and gaba synapse function is feasible only using animal models , especially rodents , an important question is how developmental time scales translate from animal to human brain . proper translation would require understanding if similar developmental stages are found in rodent and human brains , whether such developmental stages involve similar processes and underlying mechanisms , and whether developmental periods cover similar fractions of the total lifespan . some developmental changes , for instance , excitatory synapse pruning , have similar proportional duration , with only the extent of synaptic pruning differing across mammalian species . also , functional maturation of glutamate synapses onto pyramidal cells occurs prior to adolescence in nonhuman primates , and in rodents [ 185191 ] . whereas some unique studies have assessed functional properties of pvbcs and pvchcs in the cortex of adult humans [ 40 , 192 , 193 ] and nonhuman primates [ 39 , 184 , 194197 ] , we lack information on the developmental trajectory of synaptic inhibition from primate pv neurons . interestingly , functional properties of yet unidentified gaba synapses onto primate pyramidal neurons change during development through adolescence in a manner consistent with changes in the expression of gene products involved in gaba - mediated transmission . specifically , the decay time of gabaar - mediated synaptic potentials accelerates during adolescence in parallel to changes in the protein and/or mrna levels for 1 and 2 gabaar subunits that would predict such acceleration . the synaptic connections from pvchcs onto the pyramidal cell ais form vertical arrays of multiple synaptic boutons that are usually easy to distinguish and typically called cartridges . developmental properties of inputs from pvchcs onto the ais can be studied using immunocytochemistry to detect biochemical markers that are concentrated at the cartridges in the ais . in monkey pfc , the density of chandelier neuron axon cartridges immunoreactive for either pv or gat1 changes markedly during postnatal development . although the precise time course differs for the two markers , the density of labeled cartridges is low in the newborn , increases to reach a peak prior to the onset of puberty , and then declines markedly to adult levels . because cartridges are readily visualized with the golgi technique over this same time period , the changes in pv- and gat1-immunoreactive cartridges may reflect developmental shifts in the concentration of these proteins , rather than in the number of axon terminals , but this remains to be experimentally assessed . the detectability of gabaa 2 subunits in ais is very high in the early postnatal period and then steadily declines through adolescence into adulthood . immunoreactivity for ankyrin - g , iv spectrin , and gephyrin ( a scaffolding protein that regulates the clustering of gabaars containing 2 subunits at ais ) [ 202204 ] also exhibit substantial changes during postnatal development . the densities of ankyrin - g and iv spectrin immunoreactive aiss are greatest at birth and then sharply decline to reach relatively stable values by one year of age . in contrast , the relative density of gephyrin - immunoreactive ais did not appear to change through the two postnatal years but then sharply decline through adolescence and into adulthood . the high density of ais with detectable levels of ankyrin - g immunoreactivity in the first three postnatal months may reflect the recruitment to this location of a portion of the large number of gaba synapses that are formed in the monkey dlpfc during this developmental epoch . binding to ankyrin - g is also essential for the localization of many other membrane proteins to the ais , including the voltage - gated na channels that are required for action potential generation . thus , the high levels of ankyrin - g immunoreactivity may also indicate an increased capacity of pyramidal neurons for repetitive firing that parallels their increase in excitatory inputs during early postnatal development [ 206 , 209 ] . the parallel relative densities of ankyrin - g - ir and iv spectrin - sd - ir ais likely reflect that ankyrin - g is required for the recruitment of iv spectrin to ais . although iv spectrin is not essential for the formation of the ais , it does appear necessary for maintenance of membrane structure and molecular organization , and thus the stability , of the ais . given the general role of spectrins in maintaining membrane integrity and elasticity , high levels of iv spectrin during early postnatal development might insure the stability of ais structure while pfc thickness is increasing . interestingly , in human pfc the levels of gad67 mrna increase progressively during prenatal and postnatal development through childhood until around the peripubertal period , followed by a plateau or mild decline during aging . a similar pattern was reported for gad67 mrna expression during mouse and monkey cortical development , suggesting a highly conserved developmental trajectory of gad67 expression across mammals . studying protein expression by immunoblotting , a recent study found that gad67 protein levels did not change across the lifespan in human visual cortex . in contrast , gad65 showed a progressive 60% increase until teenage years and young adulthood , followed by slight decline in older adults . two other presynaptic proteins involved in gaba transmission , the cannabinoid receptor 1 and vgat , showed higher levels in infants and young children , which declined to adult - like levels in preteenage years . the levels of pv mrna increase markedly in postnatal human pfc , from very low perinatal levels until adult - like levels are reached by 25 years of age , an early developmental trajectory which is similar to that reported for pv mrna and protein in rodent neocortex [ 217 , 218 ] . interestingly , a comparative analysis using immunolocalization of the chloride transporters nkcc1 and kcc2 revealed a very similar developmental trajectory in rat and human cortex . nkcc1 levels peaking during perinatal development and decaying rapidly thereafter reach adult - like levels during childhood ; conversely , kcc2 is undetectable perinatally and increases until reaching adult levels during childhood . since the nkcc1/kcc2 activity ratio determines whether gabaar - mediated ipscs depolarize or hyperpolarize the postsynaptic membrane , these data suggest that the very early developmental switch from excitatory to inhibitory effects at most gaba synapses is highly conserved between rodent and human neocortex . analysis of gabaar subunit proteins during postnatal development in human visual cortex showed that 1 gabaar subunit levels increase from < 1 years until reaching adult levels at 13.5 years of age , whereas 2 gabaar subunits decreased significantly with age to reach adult levels by ~10 years and 3 gabaar subunit levels do not change significantly with age . consequently , the 1/2 subunit protein ratio increased markedly with development attaining adult - like ratios at 4.5 years of age . remarkably , very similar developmental trajectories were found for the levels of gabaar subunit mrnas in postmortem samples of human pfc . for example , 1 gabaar subunit mrna levels are very low perinatally and increase markedly until toddler ages , thereafter remaining consistently high through to adulthood . in contrast , 2 subunit mrna increased during the first postnatal months , decreasing subsequently until reaching mature levels at teenage years or young adulthood . the mrnas for 4 and 5 gabaa subunits in human pfc showed a developmental pattern similar to that of 2 mrna , whereas 3 subunit mrna did not change significantly with age . postnatal expression of mrna for and gabaar subunits similarly shows significant age - dependent changes , with 1 subunits showing a very early developmental decrease between neonate and infant ages , remaining constant thereafter , and 2 increasing somewhat later , between toddler and teenage years . on the other hand , 1 and 3 subunit mrna levels decrease with age during childhood and teenage the developmental trajectories reviewed above suggest that similar processes underlie developmental changes in gabaar - mediated synapses across various areas of human and rodent cortex , although further studies are necessary to properly compare developmental trajectories across species . a major difference between species is that the maturation of gaba - related markers in humans involves progressive change over one to two decades , whereas in rodents gaba synapse maturation appears to be complete within 3 - 4 postnatal weeks . such difference suggests that the absolute time window during which activity and experience may influence gaba synapse development is markedly expanded in primate versus rodent brains . the prolonged period that may be necessary for the normal developmental tuning of the more complex circuitry of the primate cortex probably also prolongs the time window during which environmental factors can produce subtle developmental alterations that may contribute to the pathophysiology of schizophrenia . the hypothesis that a deficit in gabaar - mediated transmission underlies cortical circuit dysfunction in schizophrenia is supported by convergent lines of evidence from post - mortem studies of the brain of subjects with schizophrenia . furthermore , such hypothesis is strengthened by the fact that one of the most reliably replicated findings in schizophrenia research is the decrease in gad67 mrna ( for review , see ) . interestingly , equivalent measurements of gad65 levels thus far failed to reveal alterations , suggesting that the role of gad65 in gaba - mediated transmission maybe intact in schizophrenia . a remarkable recent study found that in schizophrenia several gaba - related transcripts , including those for gad67 , pv , gat1 , somatostatin , and the gabaar subunits 1 and , show decreased levels in dorsolateral pfc as well as in the anterior cingulate , primary motor , and primary visual cortices . such conserved regional pattern of gaba alterations suggests that dysfunctional gaba neurotransmission contributes to multiple clinical features of schizophrenia including perceptual and motor deficits that could contribute to impaired cognitive function . the disruption of pv expression across cortical areas confirms the previous findings and , moreover , suggests that alterations of pv - positive cells are central to the schizophrenia disease process , although the consequences of such decrease in pv are not completely understood . pv is a ca buffer that is present in nerve terminals of pv - positive neurons ( figure 1 ) . due to its slow kinetics of ca binding , pv is unable to bind intracellular ca before ca influx activates the ca sensor that triggers gaba release , because in pv - positive terminals ca influx is tightly coupled with gaba release [ 110 , 225 ] . interestingly , gaba release by single stimuli does not differ between pv - deficient and wild - type mice , but pv deficiency facilitates repetitive gaba release [ 226228 ] . in synapses from pv - deficient mice , the amplitude of intracellular ca transients in nerve terminals is not affected , but their decay is slowed , indicating that pv normally accelerates such decay [ 227 , 228 ] . therefore , one possibility is that the decrease of pv in schizophrenia , instead of contributing to deficits , is a compensatory response to enhance gaba release in the face of decreased gaba synthesis . alternatively , reduced pv levels may produce synaptic dysfunction via loss of some asynchronous release normally produced when caunbinds from pv , well after the presynaptic action potential ended . a pathological loss of asynchronous gaba release by decreased pv levels , however , requires the existence of asynchronous release when pv is intact , a feature that is not observed in cortical pv - positive cells [ 104 , 110 ] , although it is found in pv - positive cerebellar interneurons . whereas schizophrenia may be associated with an increased density of 2-gabaars at the ais synapses from pvchcs , one study failed to detect significant changes in total tissue levels of 2-gabaar mrna . one possibility is that changes in 2-gabaars are synapse- or layer - specific and perhaps found exclusively at ais synapses in superficial cortical layers , as initially reported . consistent with this interpretation , laminar analysis of mrna levels for gabaar subunits in the cortex of subjects with schizophrenia revealed significantly increased levels of mrna for 2 gabaar subunits exclusively in layer 2 . moreover , the same study revealed lower levels of 1 gabaar subunit mrna in layers 3 and 4 , which is consistent with a decrease in total tissue levels of 1 gabaar subunit mrna observed using quantitative pcr . given that 1 subunit - containing gabaars constitute about 60% of the total gabaars in adult brain , it is possible that 1 subunit mrna is significantly more abundant than that for 2 subunits , thus increasing the chance of detecting changes of total tissue 1 mrna levels in schizophrenia . importantly , a decrease in 1 subunits in schizophrenia is consistent with weaker synaptic transmission from pvbcs , since 1 subunit - containing gabaars are predominant at synapses from mature pvbcs [ 33 , 41 , 101 , 102 ] . in addition to gabaar levels , the strength of the postsynaptic response to gaba depends on the driving force for the gabaar current which is determined by its reversal potential egabaa . as egabaa depends on chloride extrusion by kcc2 and chloride uptake by nkcc1 , a recent study examined mrna expression for both chloride transporters in the cortex of subjects with schizophrenia . interestingly , kcc2 and nkcc1 transcript levels were not altered in subjects with schizophrenia ; however , transcripts for two kinases ( oxsr1 and wnk3 ) that strongly regulate kcc2 and nkcc1 activity in opposite directions , are overexpressed in schizophrenia . if increased levels of oxsr1 and wnk3 mrna actually represent increased kinase activity , then the chloride gradient across the postsynaptic membrane may be decreased , resulting in an egabaa significantly more depolarized than normal . since normally egabaa varies with cell type and subcellular compartment , understanding the consequences of alterations in chloride transport requires a detailed quantitative analysis of protein localization and activity , a challenging task in this case , given that postmortem interval effects alter the integrity of some of these proteins . direct demonstration that gaba - mediated synaptic inhibition is decreased in the cortex of subjects with schizophrenia is challenging . interestingly , magnetic resonance spectroscopy ( mrs ) was recently applied to noninvasively measured gaba concentration in human neocortex and determined whether a decrease of gaba is observed in schizophrenia . mrs does not distinguish extracellular gaba from transmitter stored in particular cellular compartments or cell types and also lacks adequate temporal resolution but nevertheless reveals activity - dependent changes in gaba levels . for example , acute psychological stress which elevates subjective anxiety produces a short - term decrease in gaba concentration in human dorsolateral pfc that can be detected with mrs . combining mrs and eeg in the same subjects , the relations between brain gaba content and oscillatory neural activity in schizophrenia may be explored . interestingly , in normal human subjects , gaba concentration measured in visual cortex with mrs was positively correlated with the strength of gamma oscillations induced by visual stimulation . moreover , interindividual variation in gaba concentration determined by mrs in visual cortex was correlated with variability of performance in a visual stimulus orientation discrimination task that induces gamma oscillations . measurements of tissue gaba concentration with mrs may help in clarifying the relations between gad67 levels , gamma oscillations , and cognitive performance in schizophrenia . one such study did not detect differences in gaba concentration in the anterior cingulate cortex of schizophrenia versus control subjects , whereas gaba concentration was apparently decreased by antipsychotic medications . another mrs study reported reduced gaba concentration in basal ganglia but not frontal cortex of schizophrenia patients . in patients with relatively low antipsychotic exposure , mrs revealed a significant reduction of gaba concentration in visual cortex that did not covary with medication dosage but was correlated with behavioral abnormalities in a visual surround - suppression task thought to depend on gaba - mediated inhibition . moreover , a longitudinal study of early - stage first - episode schizophrenia patients showed that 6 months of treatment with atypical antipsychotics did not change gaba concentrations measured with mrs in frontal and parietal lobe cortices nor in basal ganglia . in contrast , mrs revealed elevated gaba concentration in anterior cingulate and parietal cortex of subjects with chronically treated schizophrenia compared to control subjects . the mrs findings suggesting that antipsychotics may change brain gaba concentration highlight the importance of addressing the effects of confounding factors such as medications . significantly , both postmortem studies in humans and experimental studies in animals have failed to show an effect of antipsychotic medications on gad67 mrna levels [ 224 , 242 , 243 ] . the data from mrs studies therefore underscore the importance of combining neurochemical , electrophysiological , and behavioral assessment , given the large interindividual variability in bulk gaba concentration , gamma activity levels , and behavioral performance . instead of or in addition to medication effects , the large variability in cortical gaba content measured with mrs in human cortex may be explained by the effects of genetic variants in the gad1 gene that may differentially confer risk of schizophrenia . the findings reviewed here suggest that alterations of gaba transmission produce cognitive deficits in schizophrenia by altering the circuit mechanisms of gamma oscillations . these observations suggest a molecular and cellular basis for the development of new therapeutic interventions . importantly , the proposal that gaba alterations are linked to altered gamma oscillations and cognition is supported , at least in part , by animal model studies showing that producing a functional loss of gaba - mediated inhibition diminishes gamma oscillations and impairs cognitive function [ 247 , 248 ] . whereas work in animal models is essential , the difficulty of capturing in animals the complexity of behavioral alterations in a uniquely human disorder may explain the relative lack of success in developing drugs to treat schizophrenia compared with other disease areas [ 4 , 5 , 245 ] . interpretation of studies in human subjects based on comparisons between healthy controls and patients is complicated as well , given that schizophrenia versus control differences may actually reflect pathogenesis but also could represent compensatory changes or effects of confounding factors [ 4 , 241 ] . for example , the effects of producing a transient deficit in gabaar - mediated signaling were tested recently in human subjects using iomazenil , a compound that binds to the benzodiazepine site of gabaars and negatively modulates the effects of gaba ( i.e. , an inverse agonist ) . consistent with dysfunctional gabaar signaling in schizophrenia , iomazenil produced perceptual deficits and psychotic symptoms in schizophrenia patients at doses that did not affect healthy control subjects . however , the schizophrenia patients in such study chronically received antipsychotics and anxiolytics , raising the question of whether an interaction between acute iomazenil and chronic medications influenced such findings . preliminary tests of the idea that enhancing gabaar signaling improves behavioral and electrophysiological measures in subjects with schizophrenia were conducted in two recent studies evaluating the effects of mk-0777 , an 2/3 gabaar - preferring positive allosteric modulator . in one study , randomized administration of mk-0777 or placebo in double - blind fashion improved performance of schizophrenia patients in a cognitive control task , simultaneously increasing gamma oscillation power in frontal cortex . in contrast , the second study did not find significant effects of mk-0777 compared with placebo in the performance of patients in a battery of tests designed to assess cognitive function in schizophrenia . the inconsistent beneficial effects of mk-0777 administration might be explained by the fact that mk-0777 is a partial agonist at 2/3 subunit - containing gabaars with only about 1020% potency compared to a full agonist . thus , one possibility is that more potent 2/3 benzodiazepine site agonists need to be employed . such drugs should also be more selective because , compared with placebo , mk-0777 had a tendency to produce sedation and somnolence [ 250 , 251 ] , which could mask improvements in cognitive performance . importantly , the 2/3 gabaar modulator mk-0777 was selected based on the compelling rationale that inputs from pvchcs onto the ais show important alterations in schizophrenia and that , depending on cortical layer , such inputs involve 2-gabaars or 3-gabaars [ 101 , 201 , 252 ] . however , whether or not pvchcs play a role in production of gamma band synchrony remains unclear , and so it is possible that pvchc alterations in schizophrenia produce cognitive deficits unrelated to dysfunctional gamma band synchrony . therefore , further information from both basic and clinical research studies is necessary to further assess the effectiveness of 2/3 gabaar modulators for treatment of gamma synchrony - related cognitive deficits in schizophrenia . indeed , basic research studies continue to provide insight into the role of specific subtypes of gaba neurons on inhibition - mediated cortical network oscillations , and molecular pharmacology studies are identifying novel compounds acting at different sites within the gabaar receptor complex [ 44 , 47 , 253 ] . importantly , a potential role of gaba - mediated neural synchrony in cortical circuits is to enable spike - timing - dependent plasticity , indirectly modifying the strength and stability of excitatory synaptic connections . whether spike - timing - dependent plasticity at glutamate synapses is impaired in schizophrenia is not yet clear , but it is possible that the decrease of dendritic spine density in pyramidal neurons in schizophrenia is due to glutamate synapse loss produced by altered plasticity mechanisms . if neural synchrony - dependent glutamate synaptic plasticity is dysfunctional in schizophrenia , then cognitive enhancement behavioral therapies that involve learning paradigms may help in preventing or reversing the consequences of altered circuitry on the induction of synaptic plasticity . interestingly , cognitive enhancement behavioral therapy was recently shown to improve cognition and prevent gray matter loss in schizophrenia . potentially , combining cognitive therapies with pharmacological treatment that boosts otherwise weakened neural synchrony may constitute an effective treatment intervention in schizophrenia , as for other psychiatric disorders .
human infection with avian influenza ( h5n1 ) virus raises concern for the possibility of a pandemic . we report 20 cases , which ranged from asymptomatic to fatal , in pakistan in 2007 . these cases indicate human - to - human - to - human transmission of this virus , and the number of cases may be higher than realized .	records were examined from all hospitals that treated patients with influenza ( h5n1 ) virus in northwest frontier province during 2007 . data were rendered anonymous and entered into a secure database with predetermined clinical and epidemiologic fields . cases matching predefined criteria ( table 1 ) were classified as laboratory confirmed , likely , or possible . we slightly modified who criteria to resemble criteria that clinicians might adopt during an actual outbreak , especially in a resource - poor setting ( 3 ) . we identified 20 cases4 laboratory confirmed , 7 likely , and 9 possible resulting in a ratio of 4 likely / possible cases for each laboratory - confirmed case . median age was 29 years ( range 760 years ) for all patients and 30 years ( range 2335 years ) for confirmed case - patients ; 16 ( 80% ) patients were male . the infecting exposure could not be established for all patients because multiple exposures , human and avian , were recorded for some . of the 4 patients with laboratory - confirmed cases , 3 were treated with oseltamivir ( 2 [ 67% ] of whom survived ) , and 1 had asymptomatic disease and received no antiviral treatment . signs and symptoms were mainly those of a febrile influenza - like illness ( table 2 ) , although 1 patient with a laboratory - confirmed case was asymptomatic ( microneutralization titer 320 , western blot positive , throat swab positive for h5 by reverse transcription pcr ) ; this case - patient was also described in a previous epidemiologic investigation ( 2 ) . the index case - patient ( patient 1 ) had culled influenza ( h5n1 ) virus infected poultry . after becoming febrile ( 38c ) while in abbottabad , he traveled by public transportation to his family home in peshawar . his illness progressed and on november 5 , 2007 , he was admitted to khyber teaching hospital , where the diagnosis of influenza ( h5n1 ) infection was made . infection appeared to spread initially from household family contacts ( patients 26 ) to medical staff ( patient 7 , who had positive pcr but negative microneutralization test results ) and to a frequent visitor to the intensive care unit ( patient 8) . path of infection of influenza ( h5n1 ) , pakistan , 2007 . during october 2230 , patient 1 worked culling infected chickens ; on november 2 , he moved home and had contact with 4 brothers ( patients 25 ) and possibly a cousin ( patient 6 ) . he was hospitalized on november 5 and transferred to an intensive care unit the next day . his cousin cared for him and became patient 6 ; his attending doctor became patient 7 . on november 23 , patient 3 was hospitalized and on november 28 was transferred to an intensive care unit ; during this time , patient 8 frequently visited his wife in the same intensive care unit . as previously noted ( 2 ) , the extended period from the time persons were exposed to the index case - patient , during which family members became ill , points to human - to - human - to - human transmission ; patient 2 probably accounted for intermediary or second - generation infection . the chain of infection illustrated in figure 2 suggests that further human - to - human - to - human transmission might have occurred and suggests nosocomial transmission . of note , patient 6 ( a cousin of the index case - patient ) had a microneutralization titer of 80 but a negative western blot result . although 4 contacts of patient 6 exhibited no signs or symptoms of influenza , they did have positive h5 microneutralization titers ranging from 80 to 160 . no evidence epidemiologically links the remaining 12 patients to the 8 patients in the cluster ; each of the 12 either had direct contact with influenza ( h5n1 ) virus infected poultry or was near healthy or diseased poultry before symptom onset . three patients worked on poultry farms : 1 had taken a sample from an influenza ( h5n1 ) virus infected chicken , 1 was directly involved in culling , and 1 was indirectly exposed to live poultry . eight patients had negative test results for influenza ( h5n1 ) virus , and 3 had positive results from the national institute of health islamabad but negative confirmatory - testing results from who ; 1 patient died before samples could be taken . different laboratories reported conflicting results with respect to confirmation of infection , possibly because of the difficulties of complying with specimen - handling requirements in resource - poor settings . the preponderance of male patients is probably explained by sociocultural factors ; the index case - patient was a poultry culler , a male - dominated task , and shared accommodation with male family members . the human - to - human transmission from the index case - patient to at least some household contacts seems clear , and the extended period over which these contacts became ill supports subsequent human - to - human transmission . figure 2 supports the conclusion that patient 2 initiated a chain of infection in which further human - to - human transmission to patients 7 and 8 occurred . possible nosocomial transmission is of concern because full implementation of isolation procedures in resource - poor settings may be problematic . although virologically supported probable human - to - human transmission of influenza ( h5n1 ) virus has been documented , it has been thought to occur only with prolonged and close contact ( 4 ) . household clustering and the difficulty of establishing exact virus exposures have encumbered efforts to investigate possible human - to - human transmission ( 5 ) . modeling has ( 6 ) suggested human - to - human transmission in indonesia , but the utility of statistical modeling unsupported by field data has been questioned ( 7 ) . although the index case - patient traveled by public transportation from abbottabad , where he acquired his infection , no infections were reported for anyone other than household contacts , who were all related and exposed at his family home at peshawar . in contrast , patients 2 and 6 might have spread infection through less intimate contact , which raises 2 questions . might some persons shed virus more efficiently than others , possibly in greater quantity ? and what role might host factors play in susceptibility to influenza ( h5n1 ) virus infection and disease ? a degree of virus adaptation to humans might also have occurred , although absence of sustained community transmission argues against this possibility . of concern is the 4:1 ratio of likely / possible to laboratory - confirmed cases , suggesting that official tallies understate true incidence of infection . factors that may contribute to undercounting are the difficulty of obtaining virologic confirmation or of storing and transporting samples in resource - poor settings and reluctance by relatives to consent to autopsy . another reason to believe that less fulminant cases may go unreported is the occurrence in pakistan , and elsewhere , of clinically mild and asymptomatic cases ( 5,814 ) , indicating that influenza ( h5n1 ) virus may cause a spectrum of illness . the demonstration during the 1997 hong kong outbreak of influenza ( h5n1 ) with seroconversion in apparently asymptomatic health care workers and social contacts suggests human - to - human transmission , although in hanoi , no transmission to health care workers was detected ( 8,13,15 ) . also contributing to underreporting are the predominant clinical signs of undifferentiated influenza - like illness observed in pakistan and elsewhere , which , unless clinical deterioration occurred , would be unremarkable in many tropical settings . although the survival rate was greater for patients who received oseltamivir , the small number of patients and the inclusion of those with mild and asymptomatic illness prevent meaningful statistical comparison . several features of the outbreak are unusual or give cause for concern : human - to - human - to - human transmission , possible nosocomial transmission , occurrence of mild and asymptomatic cases , and difficulties of establishing laboratory confirmation of likely and possible cases ( which also prevented genotypic matching of specimens from primary and putative secondary cases ) . taken together , these features suggest that current surveillance might undercount the extent of human infection with influenza ( h5n1 ) virus and that human - to - human transmission might possibly be associated with less severe disease .
[ purpose ] the purpose of this study was to investigate the effect of the timing of leg support elevation on the horizontal force acting on the buttocks in a reclining wheelchair . [ subjects and methods ] the participants were 17 healthy men . two experimental conditions were tested : the leg - down and leg - up conditions . the back support was reclined at increasing angles , from the initial upright position ( iup ) , proceeding to the fully reclined position ( frp ) , and returned to the upright position ( rup ) . the posterior inclination phase was from iup to frp , and the returning inclination phase was from frp to rup . [ results ] the horizontal force under the leg - up condition was significantly higher than that under the leg - down condition in all positions of back support . [ conclusion ] the leg supports should be positioned downward before reclining the back support of a wheelchair .	wheelchairs with reclining back supports are often used for individuals with leg and trunk disorders , including those with post - stroke hemiplegia and spinal cord injuries . individuals who have difficulty sitting in the hospital can sometimes be more easily transported sometime in wheelchairs with reclining back support . in a previous study of reclining wheelchairs , individuals with flaccid hemiplegia were often found to often slide forward when returning to a seated position from a reclined position1 . many wheelchair users who need reclining back support can not correct this slouching posture unassisted . this leads to a sacral sitting posture which results in increased shear force on the sacrum , predisposing the individual to a sacral decubitus ulcer2 . to address these problems , reclining wheelchairs with tilting seats jan et al.3 suggested that wheelchairs that can tilt and recline can enhance skin perfusion over ischial tuberosities without reducing sacral skin perfusion when moving the person from an upright to a tilted and reclined position . however , wheelchairs with tilting seats remain uncommon . therefore , it is important to evaluate the reduction in the shear force acting on the buttocks when such a wheelchair s back support is reclined . in our previous research4,5,6 , we investigated the functions that reclining wheelchairs should possess to reduce the shear force acting on the buttocks when reclining the back support . the results obtained suggested that reclining wheelchairs should be adjustable with the rotational axis position of the back support closer to the hip joint . carlson and payette7 described other techniques for minimizing friction and shear forces in wheelchair seating through orientation of the seat surface , positioning of the foot supports , and the use of low - friction seat cover materials . when patients are transported in wheelchairs with a reclining back support , their lower legs are elevated by leg supports . elevated leg support has been described as being important for easy patient transfer and wheelchair stability8,9,10 . elevated leg supports are generally used to augment venous circulation and reduce dependent edema , or to fix the knee in extension because of orthopedic deformity , surgical immobilization , or severe hypertonicity in the extensors11 . however , the influence of any of these factors on the shear force acting on the buttocks has not yet investigated . the rationale behind footplate adjustment usually appears to be empirically based and differs from researcher to researcher . hobson12 reported that the foot plates should be adjusted to support approximately 10% of the body weight . gilsdorf et al.13 found that the lowest ischial interface pressures on any cushion occurred with the legs dangling . the results of these studies suggested that the position of the lower extremities affects the forces acting on the buttocks . thus , we hypothesized that the forces acting on the buttocks , which result in the formation of decubitus ulcers , are influenced by the elevation of the leg supports . aissaoui et al.14 investigated the effects of elevated leg support on posture and pressure distribution in healthy subjects sitting in a wheelchair . their results showed that the elevating the leg support induces an increase in pressure under ischial tuberosities . no studies exist , however , on the relationship between the elevation of leg support and the shear force acting on the buttocks when a wheelchair s back support is reclined . moreover , the timing of leg support elevation , i.e. , whether leg support elevation before or after reclining of the back support is better , has not been investigated . therefore , the purpose of this study was to investigate the influence of elevating wheelchair leg supports and the timing of the leg support elevation on the shear forces acting on the buttocks in wheelchairs with reclining back support . it was presumed that the size and shape of the pelvis would affect the results of this study . the participants were 17 healthy adult men without leg or trunk disease : mean age , 22.6 6.6 years ; mean height , 170.1 4.4 cm ; mean body weight , 62.4 8.9 kg . exclusion criteria were : the participants that had back pain , a history of surgery , rheumatism , or neurologic disorders . this study was conducted with the approval of the research ethics committee of kawasaki university of medical welfare ( no . 415 ) , and informed consent was obtained from all the participants prior to their participation . we used an experimental chair with electric controls for reclining the back support ( hashimoto artificial limb manufacturer , okayama , japan ) . the dimensions of the experimental chair were as follows : back support height , 104 cm ; seat depth , 40 cm ; backward angle of the seat , 0 ; reclining angle of the back support , 1040 ; angular velocity at which the back support reclined , 3/s . the rotational axis of the back support , the joint between the seat and back support , were located at the same height as the seat . the elevation angle of the leg supports could be adjusted between 10 and 80 backward from the vertical line . the rotational axes of the leg supports , which the joint between the seat and the top edge of the leg support frame , were located at the same height as the seat ( fig . 1fig . the dimensions of the experimental chair were as follows : back support height , 104 cm ; seat depth , 40 cm ; backward angle of seat , 0 ; reclining angle of back support , 1040 ; angular velocity at which the back support reclined , 3/s . the elevation angle of the leg supports could be adjusted between 10 and 80 backward from the vertical . a : level goniometer , b : leg - support , c : rotational axis of back support , c : rotational axis of leg support ) . the dimensions of the experimental chair were as follows : back support height , 104 cm ; seat depth , 40 cm ; backward angle of seat , 0 ; reclining angle of back support , 1040 ; angular velocity at which the back support reclined , 3/s . the elevation angle of the leg supports could be adjusted between 10 and 80 backward from the vertical . a : level goniometer , b : leg - support , c : rotational axis of back support , c : rotational axis of leg support measurements were obtained which each participant sat comfortably with bilateral symmetry and rested on the back support and force plate located on the chair seat . hirose15 reported that inclinations of the sternum and abdominal line are correlated with inclinations of the thoracic and lumbar spine in both the frontal and sagittal planes . therefore , the posture of each participant was checked , by visually and manually inspecting the sternum and abdominal line , to ensure that the thoracic and lumbar spine in the frontal plane did not lean laterally . in addition , to maintain constant friction between the clothing of each participant and the seat surface , all participants wore 100% cotton clothing during the experiment . because the smooth metal surface of the force plate was conducive to the participant sliding forward in the chair , a rubber net was laid over the plate to minimize sliding and the risk of postural collapse . the coefficients of friction were calculated on the basis of the maximum static friction force , measured using a pull - tension gauge and weight . the measured coefficients of friction between the clothing and the rubber net , between the rubber net and the surface of the force plate , and between the surface of the back support and the clothing were 0.9 , 0.8 , and 0.4 , respectively . the participants were instructed to fold their arms in front of their chests in a relaxed state and to not intentionally change their body position during the experiment . kemmoku et al.16 reported that the vertical and horizontal forces acting on the sacrococcygeal and ischial tuberosity areas increase in a seated posture as the angle of pelvic tilt increases . thus , to ensure consistency in the pelvic tilt angle , each participant s buttocks were positioned so that the back support and dorsal surface were in contact . , the positions of the lower legs were adjusted to be perpendicular to the feet on the floor , and the horizontal thigh angle was then adjusted by stacking wooden boards under the experimental chair . furthermore , to reduce the resistance of the lower extremities , a roller board was placed under the participants feet . in contrast , under the leg - up condition , the positions of the lower legs were elevated upward to an 80 incline backward from the vertical line by elevating the leg supports . aissaoui et al.14 reported that the angls of lower leg supports can affect the posture of a sitter s hip if the angular modification changes the distance from the seat front to the foot plates . this change results from the fact that the leg support s axis of motion is not aligned with the knee joint axis . this interdependency between leg support angles and the distance between the footplate and the seat requires the linear placement of the lower leg and foot plate to be adjusted in synchrony with the leg support s angular modification . in addition , the soles of the feet did not counteract the horizontal force applied to the buttocks by applying pressure to the foot plates during the experiment . thus , under the leg - up condition , the foot plates were not used in this study ( fig . measurements were made while each participant sat comfortably with bilateral symmetry and resting on the back support and force plate located on the chair seat . ) . measurements were made while each participant sat comfortably with bilateral symmetry and resting on the back support and force plate located on the chair seat . to investigate the cause of the increased shear force acting on the buttocks , we measured the horizontal and vertical forces when the back support was reclined . in addition , we also measured the center of pressure ( cop ) on the force plate and the trunk sliding distance along the back support to examine how the shear force increased . shear as an action or stress resulting from applied forces which causes or tends to cause two contiguous internal parts of the body to deform in the transverse plane . as measuring shear force is difficult , we measured the horizontal and vertical forces to determine the shear force as described by kemmoku et al16 . the horizontal and vertical forces acting on the buttocks were measured using a force plate ( 400 400 mm ; sampling frequency , 100 hz ; kyowa electronic instruments , tokyo , japan ) that measured the reaction force in the posterior direction , which is equivalent to the horizontal force in the anterior direction when the back support was inclined . in addition , the anterior - posterior position of the cop on the force plate was measured . the point of origin of the measured cop was the center of the plate . thus , a positive value indicated that the cop was located farther forward than the center of the plate . furthermore , we measured the trunk sliding distance along the back support ( bs ) was using a video camcorder . videos of the trunk and back support were taken from the left side using a digital video camera ( panasonic , osaka , japan ) for the duration of the back support movement . dartfish teampro data 6.0 video analysis software ( dartfish , fribourg , switzerland ) was used to measure the trunk sliding distance along the back support . the distance was defined as follows : bs = va vs ( 1 ) where va and vs correspond to the distances between the acromion and the reference base point ( b ) , projected on the back support plane , from a position of back support after reclining ( a ) and at each start position ( s ) , respectively18 . a positive value indicates that the trunk was slid farther downward from the start position . to correct for the influence of each participant s postural collapse during measurement , measurements were performed 10 seconds after the posture was set . with respect to the angle of back support inclination , park et al.19 reported that decubitus ulcers may be prevented or diminished in tetraplegia patients when the back support angle of the wheelchair is more than 120 , which is similar to 30 from the vertical line . accordingly , the experimental back support was reclined at increasing angles , beginning at 10 from the vertical ( the initial upright position , iup ) , proceeding to a fully reclined position ( frp ) of 40 from the vertical , and then returning to the upright position ( rup ) . in this study , the reclining cycle of back support was divided into two phases ( i.e. , the posterior inclination phase and the returning inclination phase ) . the posterior inclination phase was from iup to frp , and the returning inclination phase was from frp to rup . the position of the lower legs was set according to the experimental condition before the back support was reclined . between each phase , the participants were asked to stand up and relax for one minute to release the residual forces on the back support and the seat . in both inclination phases , the time required to measure the forces in each position of back support was 5 seconds . for each experimental condition , we used the average of the forces acting on the buttocks and the position of the cop after measuring 201 stable samples for each participant . the two conditions were measured in a random order , with three trials for each experimental condition . if the participant could not continue sitting because of intolerance of the position or danger of sliding out of the wheelchair , the experiment was stopped for safety reasons . between each trial , the participants were asked to reset and relax for one minute . to correct for the effects of body weight , the measured horizontal and vertical forces acting on the buttocks were normalized by body weight ( percent body weight ; % bw ) , on using of the raw data measured by the force plate . preliminary analysis of the forces acting on the buttocks and the distance sliding along the back support was performed using the shapiro - wilk normality test . to investigate the influence of elevating the leg supports , the forces acting on the buttocks , the position of the cop , and the sliding distance were compared between the two experimental conditions in each of the reclining positions in the inclination phase . in addition , to investigate the influence of the reclining the back support , the forces and the position of cop were compared between the two positions of the back support in each inclination phase . the statistical analyses were performed using the statistical package for the social sciences ( spss ) version 16.0 j for windows ( spss , chicago , il , usa ) , and a significance level of p < 0.05 . tables 1 and 2table 1.horizontal force acting on the buttocks in the various positions of back support ( n=17)phasepositionthe leg - down conditionthe leg - up conditionthe posterior inclinationiup10.2 1.015.5 2.8frp10.3 1.014.9 1.5 phasepositionthe leg - down conditionthe leg - up conditionthe returning inclinationfrp13.2 0.916.4 1.7rup17.4 2.022.8 3.4mean sd ( % bw ) . * * p < 0.01 ( significant differnce between two experimental conditions ) . p < 0.01 ( significant differnce between the two positions of back support)table 2.vertical force acting on the buttocks in the various positions of back support ( n=17)phasepositionthe leg - down condition the leg - up condition the posterior inclination iup71.8 2.974.8 4.1frp60.3 5.068.0 5.6the returning inclination frp60.4 3.367.3 3.0rup79.4 4.687.5 3.6mean sd ( % bw ) . * * p < 0.01 ( significant differnce between the two experimental conditions ) . p < 0.01 ( significant difference between the two positions of back support ) show the horizontal and vertical forces acting on the buttocks , table 3table 3.position of the cop on the force plate in the various positions of back support ( n=17)phasepositionthe leg - down condition the leg - up condition the posterior inclination iup87.3 14.346.1 11.8frp114.7 14.569.8 12.4the returning inclination frp111.8 13.767.1 10.5rup82.7 16.649.5 12.3mean sd ( mm ) . * * p < 0.01 ( significant differnce between the two experimental conditions ) . p < 0.01 ( significant differnce between the two positions of back support ) . a negative value indicates that the cop was located farther backward than the center of the plate . shows the position of the cop on the force plate , and table 4table 4.trunk sliding distances of the back support in the various inclination phases ( n=17)phasethe leg - down conditionthe leg - up conditionthe posterior inclination78.8 10.481.2 12.9the returning inclination66.8 13.268.8 13.1mean sd ( mm ) . a positive value indicates that the trunk was slid farther downward than the start position . mean sd ( % bw ) . * * p < 0.01 ( significant differnce between two experimental conditions ) . < 0.01 ( significant differnce between the two positions of back support ) mean sd ( % bw ) . * * p < 0.01 ( significant differnce between the two experimental conditions ) . < 0.01 ( significant difference between the two positions of back support ) mean sd ( mm ) . * * p < 0.01 ( significant differnce between the two experimental conditions ) . p < 0.01 ( significant differnce between the two positions of back support ) . a negative value indicates that the cop was located farther backward than the center of the plate . a positive value indicates that the trunk was slid farther downward than the start position . regarding the horizontal force acting on the buttocks , there were significant differences in the horizontal forces measured under the two experimental conditions in all the reclining positions of the back support . in addition , there were not significant differences between the two positions of back support in the posterior inclination phase , but there were significant differences for that in the return - to - the - upright phase ( table 1 ) . regarding the vertical force acting on the buttocks , there were significant differences in the vertical forces between the two leg elevation conditions in all reclining positions of the back support . in addition , there were significant differences between the two leg elevation positions in each inclination phase ( table 2 ) . regarding the anterior - posterior position of cop on the force plate , there were significant differences in the anterior - posterior positions of the cop between the two leg elevation conditions in all reclining positions of the back support . in addition , there were significant differences between the two leg elevation positions in each inclination phase ( table 3 ) . regarding the trunk sliding distance along the back support , the differences in the sliding distances of the back support under the two conditions were not significant ( table 4 ) . this study examined the influence of elevating leg support elevation on the shear force acting on the buttocks during reclining of the back support to aid in the prevention of decubitus ulcers in supposed individuals who use wheelchairs with reclining back supports . in the rup , under both experimental conditions , the forward horizontal force acting on the buttocks increased significantly in comparison with that in the frp as the back support reclined . in previous research4,5,6 , we investigated the mechanism of the increase in the horizontal force acting on buttocks from the frp to rup . the results suggested that the increase in horizontal force was produced by the friction force on the back support and the difference in the position of the rotational axes of the back supports and the trunk - pelvis . with respect to the influence of elevating leg support elevation , in the iup as the start position of the posterior inclination phase and the rup as the end position of the returning inclination phase , the horizontal forces acting on the buttocks under the leg - up condition were significantly greater than under the leg - down condition . kemmoku et al.16 reported that the horizontal forces acting on the sacrococcygeal and ischial tuberosity areas increased in a seated posture as the angle of pelvic tilt increased . in the present study , the angle of backward pelvic tilt was increased by extending the hamstrings as a result of elevating the leg support . moreover , aissaoui et al.14 reported that the pressure on the back support during sitting on a chair was increased by elevating leg support . in addition , there is a strong relationship between the reaction force on the back support and the horizontal force acting on the buttocks20 . the pressure , i.e. , the reaction force , on the back support is increased by increasing the angle of backward pelvic tilt . with respect to the present study , therefore , it might be suggests that the horizontal force acting on the buttocks under the leg - up condition was also increased significantly in the iup and rup . incidentally , with the frp as the start and end positions of each inclination phase , the hamstrings should not be extended by elevating the leg support so that the pelvis is inclined backward by reclining the back support . however , the horizontal and vertical forces acting on the buttocks under the leg - up condition were significantly higher than under the leg - down condition . gilsdorf et al.13 reported that the anterior - posterior position of the cop is located forward on the seat , so that the mass of the thighs produces a load on the front of the seat that can be adjusted by adjusting the thigh angle downward from the horizontal plane when sitting on a chair . under the leg - up condition in the present study , the cop was located significantly more forward on the seat than under the leg - down condition , even although the differences in the sliding distances of the back support of the two conditions were not significant . the lower legs were elevated by the leg supports under the leg - up condition . the angle of the lower legs was 10 less than the horizontal plane at the maximum elevating on angle of the leg supports . the inclination of the leg support and the mass of the lower legs resulted in rotated the thighs forward . under the leg - up condition , the vertical force on the seat was significantly increased , and the position of the cop was shifted forward as a result of the thighs pressing the seat due to the rotation . these findings suggest that the leg support used in this study did not sufficiently support the mass of the lower legs . the vector of the lower leg weight was separated into parallel and vertical components for analysis of the leg support inclination . the resistance force in the parallel direction was the static friction force between the leg support surface and the lower legs . the maximum static friction force just before sliding of the lower legs occurs is defined as the product of the friction coefficient and the vertical force on the surface of the leg support . this implies that in regions where the vertical force is relatively high , the static friction can become high as well , suggesting that the static friction value was low , and thus that the vertical force on the leg support was low because the vector of the lower leg mass was separated into two components for the leg - up condition in this study . therefore , in the frp , the horizontal force acting on the buttocks under the leg - up condition , which did not sufficiently support the lower legs , was significantly higher than that under the leg - down condition , in which sufficient support was perpendicularly provided to the lower legs through the soles of the feet . the main limitation of this study was that it included only healthy adult males . in addition , because the measurement times were short , the effect of delayed postural collapse was not evaluated . however , we did not measure body alignment in this study . furthermore , the form ( i.e. , the rotational axis position of the leg support ) , material , and coefficient of friction of the experimental wheelchair s seat differed from those used to measure the horizontal forces . for example , the foot plates were not used in this study so that the soles of the feet did not resist the horizontal force acting on the buttocks by pressing against the foot plates . if the foot plates had been used in this study , it might be presumed that the fluctuation of the horizontal force applied to the buttocks under the leg - up condition was decreased by resistance to the parallel force forward and downward on the leg support . moreover , we did not consider factors that interact with the friction force , such as urinary incontinence and sweat , which affect many wheelchair users . therefore , the present results should be extrapolated to actual wheelchair users with great caution . the results of this study suggest that leg supports should be positioned downward before reclining the back support of a wheelchair to prevent decubitus ulcer formation . we plan to investigate the influences of the seat material and the friction force of the back support on the horizontal force acting on the buttocks while the wheelchair is reclining . these results will aid in the development of reclining wheelchairs and ultimately reduce the occurrence of decubitus ulcers .
background : the purpose of this study was to quantify the volume of the kinetic visual field with a single unit that accounts for visual field area and differential luminance sensitivity.methods:kinetic visual field perimetry was performed with a goldmann perimeter using i4e , i3e , i2e , and i1e targets . the visual fields of 25 normal volunteers ( 17 women , eight men ) of mean age 33.9 10.1 ( range 1764 ) years were obtained and digitized . isopter areas were measured with a method devised to correct cartographic distortion due to polar projection inherent in perimetry and are expressed in steradians . the third dimension of each isopter represents sensitivity to target luminance and was calculated as log ( target luminance1 ) . if luminance is expressed in cd / m2 , the values for the third dimension are 0.5 for i4e , 1.0 for i3e , 1.5 for i2e , and 2.0 for i1e . the resulting unit is a steradian ( log 103 ( cd / m2)1 which is referred to as a goldmann . in addition , the visual fields of four patients with representative visual defect patterns were examined and compared with normal subjects.results:mean isopter areas for normal subjects were 3.092 0.242 steradians for i4e , 2.349 0.280 steradians for i3e , 1.242 0.263 steradians for i2e , and 0.251 0.114 steradians for the i1e target . isopter volumes were 1.546 0.121 goldmanns for the i4e target , 1.174 0.140 goldmanns for i3e , 0.621 0.131 goldmanns for i2e , and 0.126 0.057 goldmanns for i1e . the total mean visual field volume in our study for the i target was 3.467 0.371 goldmanns.conclusion:the volume of the island of vision may be used to quantify a visual field with a single value which contains information about both visual field extension and differential luminance sensitivity . this technique may be used to assess the progression or stability of visual field defects over time . a similar method may be applied to static perimetry .	the kinetic visual field has been classically interpreted by measuring one or more isopter field areas . areas are expressed in degrees squared , centimeters squared , average diameter , or steradians . however , these units are usually limited to one isopter field area , and no information about the sensitivity to target luminance is included . this study presents a method that quantifies the kinetic visual field as a three - dimensional form by expressing both isopter area and sensitivity to target luminances of a particular target size . this quantification of a visual field can be used clinically to follow changes in the visual fields of patients over time.1 kinetic perimetry was performed with a goldmann perimeter ( haag - streit inc , bern , switzerland ) using target sizes of i4e , i3e , i2e , and i1e . twenty - five normal subjects , comprising eight males and 17 females of mean age 33.9 10.1 ( range 1764 ) years were selected . one subject was under 20 years of age , 10 were aged 2029 years , nine were 3039 years , and five were 40 years or older ( table 1 ) . the study was approved by the national eye institute institutional review board , and was performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki . best corrected visual acuity using the early treatment of diabetic retinopathy study lighthouse chart was measured and a complete dilated ophthalmologic examination was performed on each volunteer . all patients had best corrected visual acuity of 20/20 or better in the tested eye , and there were no media opacities or other abnormal findings on dilated slit lamp and fundus examination that would affect perimetric testing . in addition , visual fields of four patients with representative visual defects obtained during the course of their normal clinical visits were chosen to illustrate this method . these were concentric contraction ( gyrate atrophy ) , temporal hemianopia ( craniopharyngioma ) , arcuate scotoma ( glaucoma ) , and central scotoma ( macular dystrophy ) . one steradian is the area on the surface of a sphere that is subtended by a solid angle emanating from the center of a sphere.2 the two - dimensional kinetic visual field is an equidistant polar projection of the interior of the perimetry bowl onto a flat surface ( azimuthal ) . such projections contain equally spaced circles that are equidistant only along meridians.3 units such as degrees squared , centimeters squared , or average diameter , that are measured on a planar surface , distort the true size of peripheral field regions by making them appear larger than equivalent central field regions . tangential azimuthal deformation increases greatly with eccentricities beyond 50 from the center ( 20% at 60 and 57% at 90).4 small distortions using the solid angle to measure visual fields still exist with regard to retinal eccentricity , and require certain assumptions and approximations in order to be corrected mathematically.2,68 despite these , the steradian is a more appropriate unit to quantify visual field area than other units . one steradian is one radian squared and since one radian subtends 57.3 , one steradian equals ( 57.3 ) or 3283.3 squared . the visual fields obtained were scanned and each of the four isopters was then digitized . the area of each isopter was calculated in steradians using a method described by weleber and tobler that corrects for cartographic distortion due to polar projection.2 the series of formulae is listed in table 2 . the digitized x and y coordinates for each isopter are listed in columns a and b. the formulae in each of the columns can be copied and pasted into a spreadsheet application , such as microsoft excel . the total area of an isopter in steradians is the sum of all of the values in column p. it should be noted that an x coordinate of 0.000 will be read as infinity by the formula in column c (= atan b1/a1 ) , therefore an x coordinate of 0.000 is converted to 0.001 . thus , a dim target luminance , such as 1e , will require greater sensitivity to be perceived and will have a greater vertical height . we utilized cd / m , ie , si units , rather than apostilbs , to describe luminance per area . apostilbs can be converted to si units ( cd / m ) by dividing apostilbs by . the inverse of the target luminance values is used to describe target luminance sensitivity . the log of the sensitivity compresses the axis scale and the units are multiplied by 1000 to keep the log units positive . the volume of each visual field isopter is the product of area and sensitivity to target luminance . the resulting units are steradians log 10(cd / m ) , referred to as goldmanns . the volume of the visual field of the four luminances for the size i target is calculated by summing the products of area and height for each isopter . in this study , the same target size ( i ) for each isopter measurement is used , so the only variable change is target luminance . in order to avoid duplication of visual field volume between isopters , the height of each larger target sizes can be used by multiplying the area by the appropriate target luminance factor . this would increase the height of the i target because of its decreased luminance relative to larger target sizes ( table 3 ) . if there is a visual field deficit present , the volume of the scotoma is similarly calculated for each isopter and the total is subtracted from the total visual field volume . the area of the blind spot is 0.012 steradians or 0.048 goldmanns and may or may not be subtracted from the total volume measurement as long as the measurements between studies are consistent . this method of calculating the volume of the visual field can also be applied to automated static perimetry . in threshold static perimetry , differential luminance sensitivity units are expressed in decibels of target luminance attenuation . each threshold value already represents the height of a specific area of the visual field . the humphrey field analyzer ( carl zeiss , meditec , dublin , ca ) 30 - 2 program contains 76 threshold measurements , each one representing 36 squared degrees ( or 0.011 steradians ) . thus , there are 76 columns of visual space in the humphrey field analyzer 30 - 2 program . the volume of each column is the product of the area of the base and its height , and the total volume ( db ) is the sum of all the column volumes . the foveal threshold is not included in this calculation because it overlaps with the four surrounding threshold columns . the total volume can be converted to goldmann units by multiplying the decibels of luminance attenuation with a conversion factor . the humphrey field analyzer 30 - 2 represents a total area of ( 36 ) 76/(3283.3)/steradians or 0.8333 steradians . the height of the visual field is determined by converting the mean target luminance attenuation in decibels ( dbmean or log ( cd / m ) ) to apostilbs using a conversion chart . in static perimetry , thus , to keep the log units positive , ( cd / m ) is multiplied by a factor of 10,000 instead of the 1000 used in kinetic perimetry . the relationship between decibels of target luminance attenuation and log 10(cd / m ) is linear , and is described by the following equation : log 10(cd / m ) = 0.1 ( dbmean ) + 0.5 . for the humphrey field analyzer 30 - 2 , the sensitivity to luminance can be converted to goldmanns by multiplying by 0.8333 steradians . thus : gn = 0.8333 [ ( 0.1 ( dbmean ) ] + 0.5 ) . because of inherent differences , units obtained by static perimetry can not be interchanged with the volumetric measurements obtained from kinetic perimetry . the assignment of a vertical dimension for luminance sensitivity reinterprets the island of vision for a particular target size from the classical planar figure that is used in kinetic perimetry to a three - dimensional form ( figures 1a and 1b ) . isopter areas and volumes , as well as total volume obtained with goldmann perimetry for each subject and their age , are listed in table 1 . the mean total volume of the 25 normal subjects for the i isopter was 3.467 0.37 goldmanns , but these decreased in the older age groups . the mean for the 10 subjects aged 2029 years was 3.615 0.36 goldmanns , that of the nine subjects aged 3039 years was 3.459 0.407 goldmanns , and for the five subjects older than 40 years , was 3.176 0.200 goldmanns . table 4 depicts the isopter volumes and total volume of the four representative visual field defects illustrated in figures 2a d . the field with concentric contraction ( figure 2a ) demonstrates a large decrease in the total isopter i volume ( 0.212 goldmanns ) . in addition , the differential sensitivity to target luminances dimmer than i4e decreases abruptly in this particular patient , resulting in a volume that is mostly comprised of the i4e isopter ( 62.3% of total i isopter volume versus 44.7% for age - matched controls ) . the patient with temporal hemianopia ( figure 2b ) demonstrates a proportional decrease in volume between isopters . although the i-2e isopter area of the central scotoma is 0.2605 steradians , or 5.9 times larger than that of the arcuate scotoma ( 0.044 steradians ) , the central scotoma contains greater depth because it extends across three isopters and is 11.5 times the volume of the arcuate scotoma ( 0.254 goldmanns versus 0.022 goldmanns ) . figure 3 illustrates three - dimensional examples of 30 - 2 humphrey visual field measurements in a normal subject . the average differential luminance sensitivity over 76 points represented in the 30 field was 30.54 db or 2.96 goldmanns . this report presents a quantitative method to interpret kinetic visual fields by using a single volumetric measurement that incorporates all isopter areas of one target size as well as differential luminance sensitivity . this can be used as a global index comparable with mean sensitivity in static perimetry to describe quantitatively the hill of vision as assessed by kinetic perimetry . this measurement can be used to follow the visual field of a patient over time or to compare between patients . although it can be cumbersome to scan and digitize each isopter using a manual kinetic perimeter , the process can be greatly ameliorated with the use of more recent semiautomated perimeters , such as the octopus 101 ( haag - streit , kniz , switzerland ) , that can digitally store the coordinates at each measured isopter point and calculate the isopter areas.913 the area of each isopter and total field volume can then be calculated with appropriate software . in static perimetry , conversion to a volume unit simply involves multiplying the sum of the decibels of target luminance attenuation with a conversion factor . because current static perimeters are automated , they could be programmed to perform such a function . three - dimensional modeling of the hill of vision using both semiautomated kinetic and automated static perimetry has been previously described.11,1416 these offer the possibility of modeling visual field volumes using normal age - dependent local reference values , thereby generating age - normalized mean defect values.17 in goldmann perimetry , the volume of the island of vision that is measured is an underestimate of the true volume . first , target differential luminance sensitivity for an isopter target size is often well below threshold levels . second , a finite number of points for each isopter is collected by the perimetrist . third , a small number of target luminances ( usually four ) are used in clinical kinetic perimetry . thus , the illustration in figure 1b representing a normal visual field depicts a four - stepped pyramid , when a cone - like structure would be a closer approximation of the true island of vision for a particular isopter target . based on the stepped structure from the measured isopter volumes , a smoother hill of vision could be modeled with interpolation software . nevertheless , standard goldmann perimetry provides a method of kinetic visual field testing that is consistent , practical , and requires few assumptions for measuring the size of a visual field . the use of a volumetric parameter in kinetic perimetry can be used to evaluate more accurately the amount of field contraction or scotoma enlargement over time than the area of one isopter alone . in patients with dense scotomas or , in conditions such as retinitis pigmentosa or gyrate atrophy with reduced light sensitivity , kinetic visual fields contain smaller i4e isopter areas when compared with normal fields , but field volumes that are often proportionally much smaller than the isopter area . in the patient with gyrate atrophy , the i4e isopter volume was 8.5% compared with the mean of our age - matched controls , while the volume was 6.1% because of decreased differential luminance sensitivity to dimmer targets . by contrast , in a patient with a proportionate decrease in visual field across all isopter luminances for a particular target size , such as in hemianopia , the visual field demonstrates a more consistent decrease between area and volume ( 27.6% and 26.2% , respectively , in the patient with craniopharyngioma ) . the use of semiautomated kinetic perimetry can be used to measure scotoma size and to relate scotoma depth with age - related normal values as mean defect , a function not available with standard goldmann perimetry.17 we chose one target size ( i ) to represent the island of the visual field because this limits the number of assumptions in constructing the volume . it avoids the added variable of spatial summation that occurs when using different target sizes with similar luminances , in goldmann perimetry , dimmer luminances ( 3e , 2e , and 1e ) are most frequently used with i target rather than iii or v target size . however , use of the i target alone truncates the true island of vision considerably , in essence measuring the top portion of the island of vision . nevertheless , the use of one target size requires few assumptions about the luminance levels of other target sizes and avoids variance seen with far peripheral field measurements using larger target sizes . larger target sizes , such as v4e and iii4e , can be added by multiplying the isopter by the appropriate luminance attenuation factor . although standard luminance levels for larger target sizes can be found in table 1,18 it would be useful to calibrate the luminance of each target to obtain a more accurate conversion factor for luminance sensitivity . however , considerable differences in its size between individuals has been recently demonstrated with semiautomated kinetic perimetry.16 whether the physiologic blind spot is included in isopter size calculations or whether one or more target sizes are used , it is consistency in visual field volume formulation that allows for more accurate evaluation of visual field changes over time . in our study , 20 of 25 subjects were under the age of 40 years , so our results do not represent normative data from a widely ranged age group . the decrease in differential luminance sensitivity in the peripheral portions of the visual field and decreased sensitivity to smaller and dimmer targets has been eloquently demonstrated to correlate with increasing age , particularly after the age of 40 years.12,1316,19 in addition , semiautomated kinetic perimeters , such as the octopus 101 , allow for measurement of reaction time or the interval between stimulus onset and patient response . isopter and scotoma sizes derived by semiautomated kinetic perimetry can be corrected for variable response times reducing variance of isopter area.12,17,20 although not available with standard goldmann perimetry in this study , reaction time corrected isopters could be used with newer automated perimeters to derive visual field volume . the 30 - 2 humphrey visual field analyzer measures the central 30 core of the island of vision . there is a small amount of cartographic distortion inherent with two - dimensional projection from a curved surface . for the central 30 surface , the volumetric increase is small , approximately 2% of the entire central 30 surface , and was not accounted for in this study.4 it should be noted that volumetric measurements between static and kinetic perimetry can not be compared because of inherent differences between the two methods . physiologic dissociation between kinetic and static stimuli in perimetry is well known , particularly with achromatic target perception.21 moreover , the sensitivity for kinetic stimuli has been shown to be greater than for static stimuli independent of age , stimulus size , and eccentricity.22 static perimetry may leave areas within a measured visual field untested , especially with the use of small target sizes and scattered grids with limited coverage . thus , direct comparison of static and kinetic visual field volumes is inaccurate , and extrapolation between the two methods by a simple conversion factor is inaccurate . finally , unlike manual perimetry , differences in individual local differential luminance sensitivity values in static perimetry are related to age - corrected normal differential luminance sensitivity values , and the resulting deviations are used to determine global indices , such as mean defect . nevertheless , description of a visual field as a volume represents a useful index of quantifying the visual field for each of these perimetric techniques . this study presents a method to quantify the volume of the island of vision using a method to correct for cartographic distortion due to polar projection , with a single value containing information about both visual field area and sensitivity to luminance . this technique can be applied to assess the progression or stability of visual field defects quantitatively over time in both kinetic and static perimetry . newer semiautomated kinetic perimeters may be able to incorporate this type of volumetric measurement in the assessment of the visual field .
the internal rotation in ammonia borane ( ab ) was studied on the basis of natural orbitals for chemical valence ( nocv ) and eigenvectors for pauli repulsion ( nopr ) . we found that the total hyperconjugation stabilization ( ca . 5 kcal mol1 ) , based on the charge transfer from the occupied ( b h ) orbitals into the empty *(n h ) , slightly favors the staggered conformation over the eclipsed one ; however , the barrier to internal rotation in ammonia borane can be understood predominantly in a classical way , as originating from the steric ( pauli ) repulsion contributions ( of the kinetic origin ) that act solely between n h and b h bonds . repulsion between the lone pair of ammonia and the adjacent b h bonds was found to be dominant in absolute terms ; however , it does not determine the rotational barrier . similar conclusions on the role of chhc repulsion appeared to be valid for isoelectronic ethane.figurepauli ( kinetic ) repulsion acting between the n - h and b - h bonds of ammonia boraneelectronic supplementary materialthe online version of this article ( doi:10.1007/s00894 - 014 - 2272-y ) contains supplementary material , which is available to authorized users .	the online version of this article ( doi:10.1007/s00894 - 014 - 2272-y ) contains supplementary material , which is available to authorized users . one of the most important goals of theoretical chemistry is to understand the origin of conformational changes in molecules [ 1 , 2 ] . in order to achieve this goal many methods can be applied to the description of electronic structures : molecular orbitals ( mos ) [ 3 , 4 ] , localized molecular orbitals ( lmos ) [ 57 ] , bond orders [ 817 ] , atoms in molecules ( aim ) , fermi hole , kinetic energy and information theory based quantities [ 2022 ] , and various charge and energy decomposition schemes [ 2328 ] . a useful and elegant approach suitable for description of energy profiles of chemical reactions was proposed by torro - labbe and coworkers based on the reaction force concept . bickelhaupt and zeist proposed the activation strain model , which also appears to be very useful in the analysis of chemical reactions . carbon bond is one of the most important conformational transitions in organic chemistry [ 31 , 32 ] . a typical example is ethane , which exhibits staggered and eclipsed conformations ; the former minimum energy structure is more stable than the transition state eclipsed structure by 3.0 kcal mol . the classical and intuitive explanation of the barrier suggested in organic textbooks is based on the steric repulsion between c an alternative explanation is based on hyperconjugation stabilization , which is stronger in the staggered conformation [ 3538 ] . however , as pointed out by mulliken , hyperconjugation effect should have only a minor influence on the barrier . . stated that the barrier to rotation in ethane can be related to the polarization of charge density along the carbon carbon bond . goodman and coworkers have shown , based on the natural bond orbitals ( nbo ) method , that ethane s staggered conformation is the result of hyperconjugation . goodman s results based on the nbo method have been challenged by the work of bickelhaupt and baerends based on the model of a chemical bond originating from fragmented molecular orbitals ; according to these results the internal rotational barrier in ethane is due to pauli repulsion acting between the ch bonds of opposite ch3 units . subsequent calculations by mo and coworkers [ 44 , 45 ] and then by pendas et al . , confirmed the classical , steric - based interpretation of the barrier ; in addition , the former authors have shown that hyperconjugation stabilizes the staggered conformer only by about 4 kj mol relative to the eclipsed form . a very elegant recent paper by mo and gao provided a compact overview of the most important studies on this subject ; the main conclusion is that the internal rotational barrier in ethane is due predominantly to steric effects acting between c we have recently developed the ets - nocv scheme [ 4750 ] by combining the extended transition state ( ets ) [ 25 , 26 ] energy decomposition approach with the natural orbitals for chemical valence ( nocv ) method [ 5157 ] . ets - nocv has proved suitable for qualitative and quantitative description of the crucial components ( , , , etc . ) in addition , the energy profiles of some chemical reactions can be also characterized [ 62 , 63 ] . more importantly for this study , it was shown that nocv representation allows for qualitative and quantitative description of hyperconjugation effects [ 47 , 48 ] . furthermore , the ets energy decomposition scheme provides quantitative information on the pauli repulsion effects [ 2 , 25 , 26 ] . therefore , the main goal of this article was to apply for the first time the ets - nocv charge and energy decomposition scheme to analysis of the internal rotation in ammonia borane . hyperconjugation and steric factors will be discussed in a detailed way . it should be noted that ammonia borane is considered nowadays as one of the most promising hydrogen storage materials . in addition , it was already proven that ammonia borane exhibits dissimilar features as compared to isoelectronic ethane . furthermore , the present study sheds additional qualitative and quantitative light on the steric repulsion in ammonia borane by decomposition of total pauli repulsion into specific contributions stemming from different symmetry ( and ) . in order to achieve this goal , we defined for the first time the eigenvectors for pauli repulsion ; in this representation , one can thus discuss the pauli repulsion components originating from different symmetries . all dft calculations presented here were based on the amsterdam density functional ( adf 2009.01 ) program [ 2 , 6568 ] in which the ets - nocv scheme was implemented [ 4763 ] . the becke - perdew exchange - correlation functional [ 69 , 70 ] was applied ( bp86 ) . a standard triple - zeta sto basis containing two sets of polarization functions ( tz2p ) our analysis is based on the ets - nocv approach , which is a combination of the extended transition state ( ets ) [ 25 , 26 ] method with the natural orbitals for chemical valence ( nocv ) scheme [ 5157 ] . the basic concept of the ets scheme involves partitioning of the total bonding energy etotal between interacting fragments into four components:1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \vardelta { e}_{\mathrm{total}}=\vardelta { e}_{\mathrm{dist}}+\vardelta { e}_{\mathrm{elstat}}+\vardelta { e}_{\mathrm{pauli}}+\vardelta { e}_{\mathrm{orb } } $ $ \end{document}etotal=edist+eelstat+epauli+eorb the first component , edist , referred to as the distortion term , represents the amount of energy required to promote the separated fragments from their equilibrium geometry to the structure they will take up in the combined molecule ; it can also be seen as strain energy . the second term , eelstat , corresponds to the classical electrostatic interaction between the promoted fragments as they are brought to their positions in the final complex . the third term , epauli , accounts for the repulsive pauli interaction between occupied orbitals on the two fragments in the combined molecule . it is calculated as the difference between the energies of orthogonalized and non - orthogonalized fragments [ 2 , 25 ] . finally , the last stabilizing term , eorb , represents the interactions between the occupied molecular orbitals of one fragment with the unoccupied molecular orbitals of the other fragment as well as the mixing of occupied and virtual orbitals within the same fragment ( inner - fragment polarization ) . this energy term , eorb , may be linked to the electronic bonding effect coming from the formation of a chemical bond ( eq . 2 ) . the nocv are eigenvectors that diagonalize deformation density matrix p = pmolecule p0 , where p0 corresponds to the sum of density matrices for orthogonalized fragments ; it has been shown that the natural orbitals for chemical valence pairs ( -k,k ) decompose the deformation density orb into nocv - contributions , orbk:1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \vardelta { \rho}_{orb}(r)={\displaystyle \sum_{k=1}^{m/2}{v}_k\big[-{\uppsi}_{-k}^2(r)+{\uppsi}_k^2}(r)\big]={\displaystyle \sum_{k=1}^{m/2}\vardelta { \rho}_{orb}^k } $ $ \end{document}orbr=k=1m/2vk[k2r+k2r]=k=1m/2orbkwhere k and m are the nocv eigenvalues and the number of basis functions , respectively . visual inspection of deformation density plots ( orbk ) helps to attribute symmetry and the direction of the charge flow . in addition , information gained from the analysis of deformation density plots can be enriched by providing the energetic estimations , eorbk , for each orbk within ets - nocv scheme:2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \vardelta { e}_{orb}={\displaystyle \sum_k\vardelta { e}_{orb}^k=}{\displaystyle \sum_{k=1}^{m/2}{v}_k\big[-{f}_{-k ,- k}^{ts}}+{f}_{k , k}^{ts}\big ] $ $ \end{document}eorb=keorbk=k=1m/2vk[fk,kts+fk , kts]where fi , its are diagonal kohn - sham matrix elements defined over nocv with respect to the transition state density ( at the midpoint between density of the molecule and the sum of fragment densities ) . the above components eorbk provide the energetic estimation of orbk that may be related to the importance of a particular electron flow channel for the bonding between the considered molecular fragments . in the present study , in analogy to nocvs , we defined for the first time the natural orbitals ( eigenvectors ) for pauli repulsion , k , that diagonalize the pauli deformation density matrix , p = p0pisolated , where pisolated is the sum of density matrices for non - orthogonalized fragments , whereas p0 correspond to the sum of density matrices for orthogonalized fragments . such eigenvectors decompose the total pauli deformation density , =0 ( orthogonalized - fragments) ( non - orthogonalized - fragments ) , into the nocv - like contributions ( kpauli ) ( in analogy to eq . 1):3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \vardelta { \rho}^{pauli}(r)={\displaystyle \sum_{k=1}^{n/2}{v}_k^{pauli}\big[-{\phi}_{-k}^2(r)+{\phi}_k^2}(r)\big]={\displaystyle \sum_{k=1}^{n/2}\vardelta { \rho}_k^{pauli}(r ) } $ $ \end{document}paulir=k=1n/2vkpauli[k2r+k2r]=k=1n/2kpaulir the total charge transferred in this channel can be considered as:4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \vardelta { q}_k^{pauli}={\nu}_k^{pauli } $ $ \end{document}qkpauli=kpauli the present study characterized not only the total values of pauli repulsion ( epauli ) in ammonia borane based on the original ets scheme ( eq . 1 ) but , in addition , provided a more detailed picture by analyses of both the pauli repulsion contributions kpauli ( eq . 3 ) and the corresponding quantitative charge estimations qkpauli ( eq . 4 ) . this approach ( eqs . 3 , 4 ) was implemented by one of us in the home version of adf2009.01 . at present , the energetic pauli repulsion contributions ( epaulik ) from kpauli ( calculated in an analogous way to eq . 2 ) hence , we focused our attention on the quantitative measures of kpauli based on eq . 4 . red areas of deformation density channels correspond to charge depletion , whereas blue indicates charge accumulation upon bond formation . due to the fact that the steric interaction , which is a non - observable quantity , is very often attributed in the literature to pauli repulsion quantum effect [ 2 , 43 , 46 ] , we use both terms interchangeably throughout the text . finally , we should note that pauli repulsion is one of the bonding components in various energy decomposition schemes ; hence , we believe that a more detailed description of this term based on eqs . 3 , 4 , could be of wide interest . it is very important to point out that the main source of the pauli repulsion is related to an increase in the kinetic energy contribution ; so we could also refer to the pauli repulsion term as kinetic repulsion due to the pauli exclusion principle . such a concept , which relates the steric repulsion to the kinetic energy pressure has already been put forward by various authors [ 73 , 74 ] . in addition , the pauli repulsion contribution appears to qualitatively correlate very well with the experimental taft s steric parameters . all dft calculations presented here were based on the amsterdam density functional ( adf 2009.01 ) program [ 2 , 6568 ] in which the ets - nocv scheme was implemented [ 4763 ] . the becke - perdew exchange - correlation functional [ 69 , 70 ] was applied ( bp86 ) . a standard triple - zeta sto basis containing two sets of polarization functions ( tz2p ) our analysis is based on the ets - nocv approach , which is a combination of the extended transition state ( ets ) [ 25 , 26 ] method with the natural orbitals for chemical valence ( nocv ) scheme [ 5157 ] . the basic concept of the ets scheme involves partitioning of the total bonding energy etotal between interacting fragments into four components:1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \vardelta { e}_{\mathrm{total}}=\vardelta { e}_{\mathrm{dist}}+\vardelta { e}_{\mathrm{elstat}}+\vardelta { e}_{\mathrm{pauli}}+\vardelta { e}_{\mathrm{orb } } $ $ \end{document}etotal=edist+eelstat+epauli+eorb the first component , edist , referred to as the distortion term , represents the amount of energy required to promote the separated fragments from their equilibrium geometry to the structure they will take up in the combined molecule ; it can also be seen as strain energy . the second term , eelstat , corresponds to the classical electrostatic interaction between the promoted fragments as they are brought to their positions in the final complex . the third term , epauli , accounts for the repulsive pauli interaction between occupied orbitals on the two fragments in the combined molecule . it is calculated as the difference between the energies of orthogonalized and non - orthogonalized fragments [ 2 , 25 ] . finally , the last stabilizing term , eorb , represents the interactions between the occupied molecular orbitals of one fragment with the unoccupied molecular orbitals of the other fragment as well as the mixing of occupied and virtual orbitals within the same fragment ( inner - fragment polarization ) . this energy term , eorb , may be linked to the electronic bonding effect coming from the formation of a chemical bond ( eq . 2 ) . the nocv are eigenvectors that diagonalize deformation density matrix p = pmolecule p0 , where p0 corresponds to the sum of density matrices for orthogonalized fragments ; it has been shown that the natural orbitals for chemical valence pairs ( -k,k ) decompose the deformation density orb into nocv - contributions , orbk:1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \vardelta { \rho}_{orb}(r)={\displaystyle \sum_{k=1}^{m/2}{v}_k\big[-{\uppsi}_{-k}^2(r)+{\uppsi}_k^2}(r)\big]={\displaystyle \sum_{k=1}^{m/2}\vardelta { \rho}_{orb}^k } $ $ \end{document}orbr=k=1m/2vk[k2r+k2r]=k=1m/2orbkwhere k and m are the nocv eigenvalues and the number of basis functions , respectively . visual inspection of deformation density plots ( orbk ) helps to attribute symmetry and the direction of the charge flow . in addition , information gained from the analysis of deformation density plots can be enriched by providing the energetic estimations , eorbk , for each orbk within ets - nocv scheme:2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \vardelta { e}_{orb}={\displaystyle \sum_k\vardelta { e}_{orb}^k=}{\displaystyle \sum_{k=1}^{m/2}{v}_k\big[-{f}_{-k ,- k}^{ts}}+{f}_{k , k}^{ts}\big ] $ $ \end{document}eorb=keorbk=k=1m/2vk[fk,kts+fk , kts]where fi , its are diagonal kohn - sham matrix elements defined over nocv with respect to the transition state density ( at the midpoint between density of the molecule and the sum of fragment densities ) . the above components eorbk provide the energetic estimation of orbk that may be related to the importance of a particular electron flow channel for the bonding between the considered molecular fragments . in the present study , in analogy to nocvs , we defined for the first time the natural orbitals ( eigenvectors ) for pauli repulsion , k , that diagonalize the pauli deformation density matrix , p = p0pisolated , where pisolated is the sum of density matrices for non - orthogonalized fragments , whereas p0 correspond to the sum of density matrices for orthogonalized fragments . such eigenvectors decompose the total pauli deformation density , =0 ( orthogonalized - fragments) ( non - orthogonalized - fragments ) , into the nocv - like contributions ( kpauli ) ( in analogy to eq . 1):3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \vardelta { \rho}^{pauli}(r)={\displaystyle \sum_{k=1}^{n/2}{v}_k^{pauli}\big[-{\phi}_{-k}^2(r)+{\phi}_k^2}(r)\big]={\displaystyle \sum_{k=1}^{n/2}\vardelta { \rho}_k^{pauli}(r ) } $ $ \end{document}paulir=k=1n/2vkpauli[k2r+k2r]=k=1n/2kpaulir the total charge transferred in this channel can be considered as:4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \vardelta { q}_k^{pauli}={\nu}_k^{pauli } $ $ \end{document}qkpauli=kpauli the present study characterized not only the total values of pauli repulsion ( epauli ) in ammonia borane based on the original ets scheme ( eq . 1 ) but , in addition , provided a more detailed picture by analyses of both the pauli repulsion contributions kpauli ( eq . 3 ) and the corresponding quantitative charge estimations qkpauli ( eq . 4 ) . was implemented by one of us in the home version of adf2009.01 . at present , the energetic pauli repulsion contributions ( epaulik ) from kpauli ( calculated in an analogous way to eq . hence , we focused our attention on the quantitative measures of kpauli based on eq . 4 . red areas of deformation density channels correspond to charge depletion , whereas blue indicates charge accumulation upon bond formation . due to the fact that the steric interaction , which is a non - observable quantity , is very often attributed in the literature to pauli repulsion quantum effect [ 2 , 43 , 46 ] , we use both terms interchangeably throughout the text . finally , we should note that pauli repulsion is one of the bonding components in various energy decomposition schemes ; hence , we believe that a more detailed description of this term based on eqs . 3 , 4 , could be of wide interest . it is very important to point out that the main source of the pauli repulsion is related to an increase in the kinetic energy contribution ; so we could also refer to the pauli repulsion term as kinetic repulsion due to the pauli exclusion principle . such a concept , which relates the steric repulsion to the kinetic energy pressure has already been put forward by various authors [ 73 , 74 ] . in addition , the pauli repulsion contribution appears to qualitatively correlate very well with the experimental taft s steric parameters . we will start with a brief description of the bonding situation in the most stable staggered conformation ( s ) of ammonia borane ( fig . 1 ) . it can be seen from table 1 that the bond dissociation energy ( etotal ) amounts to 31.94 kcal mol ( bp86/tz2p ) . this value fits well to the experimental enthalpy estimated by haaland ( 31.1 1 kcal mol [ 76 , 77 ] ) as well as to other theoretical estimations [ 48 , 7881 ] . in line with previous studies [ 48 , 78 , 8285 ] , we found a slight dominance ( by 0.7 kcal mol ) of the electrostatic stabilization over the orbital interaction term ( table 1 ) . decomposition of the latter stabilizing term into nocv - based deformation density channels leads to the conclusion that donation ( orb ) from the lone electron pair of ammonia to the lowest unoccupied orbital of bh3 is by far most dominant ( eorb = 66.32 kcal mol ) as compared to the two hyperconjugation contributions , orbhyp1 , orbhyp2 ; the corresponding orbital interaction stabilizations are eorbhyp1 = eorbhyp2 = 2.30 kcal mol ( fig . 2 ) . the latter two degenerated contributions stem from charge transfer from the occupied ( b h ) orbitals into the empty (n h ) ( fig . 2 ) . it is noteworthy that , in the isoelectronic ethane , the sum of stabilization arising from the two orthogonal hyperconjugation components was found to be significantly stronger ( 10 kcal mol ) [ 46 , 85].fig . b n bond lengths ( in ) are indicatedtable 1extended transition state ( ets ) energy decomposition results describing the h3n bh3 bond in various isomers of ammonia borane . charge estimates for pauli repulsion contributions are indicated s e es e s e total 31.9430.011.9329.87e dist 12.6513.020.3712.65e elstat 77.3273.184.1477.8e pauli 109.39102.127.27111.5e orb 76.6671.974.6976.22 q globalpauli(q 1pauli + q 2pauli + q 3pauli)1.18331.16930.0141.2135 q 1pauli 0.72610.70490.02120.7267 q 2pauli 0.22860.23250.00390.2434 q 3pauli 0.22860.23190.00330.2434 e total = e orb + e pauli + e elstat + e dist [ kcal mol ] labels assigned in fig . 1 ; e s corresponds to the eclipsed structure in the staggered geometry see eqs . 3 , 4 in computational methods and fig . 2dominant natural orbitals for chemical valence ( nocv)-based deformation density channels , orb , orbhyp1 , orbhyp1 , with the corresponding orbital interaction energies for the alternative isomers of ammonia borane . s fully optimized staggered isomer , e s eclipsed structure in the staggered geometry , e fully optimized eclipsed isomer energy profile for internal rotation in ammonia borane . n bond lengths ( in ) are indicated extended transition state ( ets ) energy decomposition results describing the h3n bh3 bond in various isomers of ammonia borane . charge estimates for pauli repulsion contributions are indicated e total = e orb + e pauli + e elstat + e dist [ kcal mol ] labels assigned in fig . 1 ; e s corresponds to the eclipsed structure in the staggered geometry see eqs . 3 , 4 in computational methods and fig . 5 dominant natural orbitals for chemical valence ( nocv)-based deformation density channels , orb , orbhyp1 , orbhyp1 , with the corresponding orbital interaction energies for the alternative isomers of ammonia borane . the contour value is \|\| = 0.005 a.u . for orb , whereas for remaining hyperconjugation contributions 0.001 a.u . was applied . s fully optimized staggered isomer , e s eclipsed structure in the staggered geometry , e fully optimized eclipsed isomer it is clear from fig . 1 that rotation from the staggered to the eclipsed form leads to a change in energy , by 1.93 kcal mol . this barrier agrees quite well with the experimental value of 2.07 kcal mol determined based on microwave spectra , and with other high level computations . it is very important to point out that when going from the staggered ( s ) to the eclipsed isomer ( e ) , one observes a notable stretch of the b such elongation leads expectedly to a significant decrease in pauli repulsion , by 7.27 kcal mol ; at the same time the electrostatic ( eelstat ) and orbital interaction ( eorb ) contributions become less stabilizing , by 4.14 kcal mol and 4.69 kcal mol , respectively ( see table 1 and the blue line in fig . 3 ) . from the examples of ethane or biphenyl , it is known that this type of elongation when going from one isomer to the other is due to the steric ( pauli ) repulsion . as indicated in a series of recent works [ 4246 , 88 , 89 ] , in order to estimate and characterize the forces leading to such elongation , one must first consider rigid rotation from the staggered to the eclipsed conformation ; we have labeled such eclipsed conformation ( in the staggered geometry ) as es . we can clearly see now from table 1 and fig . 3 ( the orange curve ) , that an increase in the pauli repulsion contribution , by 2.11 kcal mol , is noted when going from s to es a similar trend , i.e. , the maximum pauli repulsion in ammonia borane with the dihedral angle (h b n h ) = 0.0 , is noted when considering the rigid rotation from the geometry of the eclipsed structure to the staggered one ( se ) ( gray curve in fig . thus , the pauli ( steric ) repulsion contribution is responsible for stretching of the b n bond and , accordingly , for the rotational barrier in ammonia borane ; the analogous situation holds true for the ethane molecule , as demonstrated first by bickelhaupt et al . and then by others [ 4346 ] . an increased kinetic repulsion ( the main source of the pauli term ) in the es geometry is related through the virial theorem to the existence of repulsive forces acting predominantly on nitrogen and boron nuclei . it must be added that hypercongutation stabilizations stemming from the charge transfer from the occupied ( b h ) orbitals into the empty (n h ) ( orbhyp1 , orbhyp2 ) , favors the staggered conformation ( fig . 2 ) , although the effect is minor ( 0.4 kcal mol ) compared to changes in the remaining bonding contributions ( table 1 ) . a quantitatively similar effect is observed for the change in the energy distortion contribution ( edist ) ( table 1).fig . s fully optimized staggered isomer , e s eclipsed structure in the staggered geometry , s e staggered structure in the eclipsed geometry , e fully optimized eclipsed isomer pauli repulsion energies in the alternative isomers of ammonia borane . s fully optimized staggered isomer , e s eclipsed structure in the staggered geometry , s e staggered structure in the eclipsed geometry , e fully optimized eclipsed isomer let us now focus our attention on detailed changes in the pauli repulsion contributions in the three ammonia borane isomers , s , es and e. figure 4 presents the total pauli deformation density contours ( ) together with the corresponding energy values ( epauli).fig . in addition , charge - based estimations are presented based on eq . 4 . the blue / red contours correspond to accumulation / depletion of electron density due to the pauli exclusion principle contours of the total pauli deformation density together with the corresponding energies . the blue / red contours correspond to accumulation / depletion of electron density due to the pauli exclusion principle as already stated , the s es transition leads to a jump in the pauli term that it is then the important question that arises at this point is how the total pauli repulsion is an analysis of function leads to the observation that electrons are removed from the n b binding region ( in fact , it is a manifestation of the pauli exclusion principle [ 2 , 90 ] ) ; although one can see that the red lobes extend also to the areas of nhhb interaction . however , such contours do not allow us to extract information on whether the total changes in pauli repulsion are determined by the repulsive interaction between the lone electron pair of ammonia with the occupied ( b h ) orbitals or directly by n - hh - b repulsion [ an interaction between the occupied ( b h ) orbitals with the occupied ( n h ) ] . in order to obtain such separated information , we have decomposed total pauli repulsion into the contributions ( kpauli ) according to eq . the three leading pauli deformation density channels , 1pauli , 2pauli , 3pauli , together with the corresponding quantitative charge estimations ( eq . 4 ) are presented in fig . it should be noted that the total charge , qglobalpauli = q1pauli + q2pauli + q3pauli , that is removed from the h3n bh3 binding region correlates well with the trend based on the pauli repulsion energy ( table 1 , fig . qualitative inspection of the contours ipauli leads to the important observation that the first channel ( 1pauli ) corresponds solely to the interaction between the lone electron pair of ammonia with the b h bonds , whereas the two latter orthogonal contributions ( 2pauli , 3pauli ) show nhhb repulsion ( fig . more importantly , quantitative analysis of the charge depletion , based on the eigenvalues ( eq . 3 ) , leads to the conclusion that , when going from s es , the major changes ( by 0.0148 a.u . ) are within the second and third values of q2pauli , q3pauli . these results show that an increase in the total repulsion in the eclipsed conformation compared to staggered ( s es ) is determined solely by the nhhb repulsion ( of the kinetic origin ) due to the pauli exclusion principle ( an interaction between the electrons with the same spin as within the b h and n the repulsive contribution from the interaction between the lone electron pair of ammonia with the electrons of b h bonds ( 1pauli ) is dominating in absolute terms ; however , it does not influence the barrier . classical view that the internal rotational barrier in ammonia borane can be understood solely in terms of nhhb steric ( pauli ) effects , with minor participation stemming from the hyperconjugation ( fig . 2 ) and geometry distortion term . it must be further noted that we performed a detailed study of the changes in qipauli values ( based on various sets of molecules ) and have found that differences in the second decimal place are quantitatively meaningful.fig . 5dominant pauli repulsion deformation density channels , 1pauli , 2pauli , 3pauli , together with the corresponding charge estimations , q 1pauli , q 2pauli , q 3pauli in the selected ammonia borane conformations . the blue / red contours correspond to accumulation / depletion of electron density . s fully optimized staggered isomer , e s eclipsed structure in the staggered geometry , e fully optimized eclipsed isomer dominant pauli repulsion deformation density channels , 1pauli , 2pauli , 3pauli , together with the corresponding charge estimations , q 1pauli , q 2pauli , q 3pauli in the selected ammonia borane conformations . s fully optimized staggered isomer , e s eclipsed structure in the staggered geometry , e fully optimized eclipsed isomer finally , we performed similar ets - nocv and pauli repulsion analyses for ethane and found that chhc pauli ( kinetic ) repulsion channels are responsible for the rotation of methyl groups ( see supplementary electronic material ) ; this is in line with the conclusions reached first by bickelhaupt and subsequently by other researchers [ 4246 ] . it is important at this point to cite another important work in the field explaining the origin of rotation in ethane . it is possible to decompose the barrier into changes in the kinetic ( t ) and potential energy contributions : nuclei-nuclei ( vnn ) , nuclei-electrons ( vne ) and electrons - electrons ( vee ) , as done by bader and others for ethane . such an approach , while very physical and compelling , does not allow , for example , separate discussion of the role of the hyperconjugation effect , which is well rooted in chemistry . it was shown that rigid rotation s es leads to a rise in all of the destabilizing terms ( t + 9 kcal mol , vnn+vee + 16 kcal mol ) , whereas the electron - nuclei stabilization is vne 22 kcal mol . the rise in kinetic energy is related , through the virial theorem , to the repulsion force acting on the nuclei . accordingly , in the next step , ese , the cc bond elongates , which leads to weakening of the attraction ( vne + 207 kcal mol ) and decrease in the repulsion ( vnn+vee 201 kcal mol ; t 3 kcal mol , the values are provided with respect to ethane in staggered geometry ) . one should also note that various authors have combined the above contributions in different ways . finally , liu and govind , defined in an elegant way at dft level , the steric contribution ( equal to the kinetic weizscker term ) from a difference between the total electronic energy and the sum of electrostatic ( vne+vee+vnn ) and quantum energy terms eq ( comprising the sum exc+epauli ) ; the change in the kinetic term due to the pauli exclusion principle is incorporated in epauli . it was shown that rigid rotation s es results in the appearance of destabilizing forces originating from the fermionic quantum contribution eq ; closer inspection of the author s data shows that this change is due entirely to a rise in the kinetic energy term . finally , one should cite the separate work of nagy , who discussed the fisher information based on the kinetic term ; the role of kinetic energy and the information origin of the chemical bonding have been studied by nalewajski . the present work studied for the first time the internal rotation in ammonia borane based on our recently developed charge and energy decomposition scheme , ets - nocv , as well as the eigenvectors for pauli repulsion . detailed analyses of the electronic and the steric factors were performed in order to understand the origin of the barrier to rotation in ammonia borane . we found that the barrier to rotation , staggered eclipsed , is only 2 kcal mol . it was demonstrated using the ets - nocv scheme that the hyperconjugation , originating from the charge transfer from the occupied ( b h ) orbitals into the empty *(n h ) , favors the staggered isomer , although , quantitatively it leads to only a slight stabilization ( 5 kcal mol ) . for ethane , we have found , based on our newly proposed scheme , the natural orbitals for pauli repulsion , that rigid rotation from the staggered to the eclipsed conformation causes predominantly the enhancement of steric ( pauli ) repulsion acting solely between n h and b h bonds ; this is subsequently relieved , leading to elongation of the b accordingly , the barrier to rotation in ammonia borane can be understood in a classical way ; namely , as originating from the steric ( pauli ) repulsion contributions that act solely between n h and b h bonds . h bonds is dominant in absolute terms ; however , it does not influence the barrier .
abstractpositron emission tomography ( pet)-computed tomography ( ct ) is emerging as a valuable tool for assessing a wide variety of pediatric malignancies , including lymphomas , soft - tissue tumors , and bone sarcomas . pet - ct may provide information that is not apparent on conventional imaging performed to stage these diseases and monitor their response to treatment . the use of pet - ct in children requires an awareness of the technical and logistical issues unique to this patient population . in addition , interpretation of pediatric pet - ct imaging requires familiarity with aspects of pediatric anatomy and physiology that differ from those of adults . in this article , the technical considerations in performing pediatric pet - ct , pitfalls in the diagnostic use of pet - ct in children , and current and emerging applications of pet - ct in pediatric oncology are reviewed .	the value of positron emission tomography ( pet ) and pet combined with computed tomography ( ct ) imaging in pediatric lymphomas has been fairly well described . however , because pediatric solid tumors are rare , the role of pet in their management is less well established . there is a growing body of literature about the use of pet - ct imaging in the diagnosis and management of pediatric sarcomas , the most common of which are the bone tumors , osteosarcoma ( os ) and ewing sarcoma ( ews ) , and the soft - tissue malignancy , rhabdomyosarcoma ( rms ) . therefore , this review focuses on the value of pet - ct imaging in the assessment of these malignancies . when pet - ct is performed in children , challenges unique to pediatric patients these include the need for sedation , pregnancy screening of patients and caregivers , and potential artifact caused by metabolically active brown fat . several pitfalls in interpretation of pet - ct in children with sarcomas are also presented . accurate anatomic coregistration of pet and ct images requires that the patient remain completely still throughout the procedure . because pet - ct examinations can be lengthy , sedation or general anesthesia may be required if children are unable to cooperate . when a sedation team is used , its members should rotate through the pet - ct area to minimize their radiation exposure . the need for sedation or anesthesia will influence patient scheduling and the timing of oral contrast administration when a diagnostic ct is also being performed . before sedation or anesthesia , ( npo ) period including 2 h of clear liquids only immediately before the procedure . therefore , when pet - ct patients must ingest oral contrast material for diagnostic ct , appointments are scheduled to allow adequate time between examinations so that sedation or anesthesia , if needed , can be safely administered . pregnant guardians of young patients must not be allowed in the room where radionuclide tracer ( usually the glucose analogue fluorine-18 fluorodeoxyglucose [ fdg ] ) administration and uptake occur . further , before administration of any radioisotope , young female patients must be questioned about the possibility of pregnancy . this delicate subject is best handled by a technologist who is experienced in working with young girls . before fdg is administered at our institution , a nuclear medicine technologist questions female patients who are10 years of age or older , and all female guardians , about the possibility of pregnancy . when a patient or guardian is unsure of her pregnancy status , a pregnancy test is performed and must be negative before fdg is administered . brown adipose tissue , or brown fat , is metabolically active in pediatric patients , women , and persons with a low body mass index . the primary function of brown fat is the production of heat through an anaerobic , glycolytic pathway that results in the uptake of glucose and hence of fdg . intense fdg activity is often observed within brown fat in the supraclavicular regions , axillae , paraspinal regions of the posterior mediastinum , and adjacent to the adrenal glands . intense fdg avidity in brown fat can be difficult to distinguish from cervical , supraclavicular , or axillary pathology ( which are commonly seen in lymphoma ) . in such cases , we administer an anxiolytic agent , such as diazepam , the night before and the morning of pet - ct to reduce fdg activity in brown fat and improve the prominence of lesions ( fig . ( a ) this maximum intensity projection ( mip ) pet image shows intense fdg activity in metabolically active brown fat in the neck , supraclavicular areas and axilla . ( b ) the examination was repeated after administration of diazepam the evening before and on the morning of the pet - ct scan . brown fat fdg activity resolved and the site of pathologic uptake in the right neck ( arrow ) became more apparent . an 8-year - old boy with hodgkin ( a ) this maximum intensity projection ( mip ) pet image shows intense fdg activity in metabolically active brown fat in the neck , supraclavicular areas and axilla . ( b ) the examination was repeated after administration of diazepam the evening before and on the morning of the pet - ct scan . brown fat fdg activity resolved and the site of pathologic uptake in the right neck ( arrow ) became more apparent . because the value of pet imaging in lymphoma has been well described , this review focuses on the emerging role of pet - ct in the assessment of children with sarcomas . the conventional staging evaluation of these children includes magnetic resonance imaging ( mri ) or ct of the primary tumor and regional nodal beds , chest ct for detection of pulmonary metastases , and technetium-99 m methyldiphosphonate ( [ tc]mdp ) bone scan for detection of bone metastases . the response evaluation criteria in solid tumors ( recist ) , introduced in 2000 as an alternative to the world health organization ( who ) criteria , provide a standard , reproducible , and objective method of assessing the efficacy of solid tumor therapies . these criteria incorporate important advances in imaging technology and simplify the who scheme , but they still rely on changes in unidimensional tumor measurements to define tumor response or progression . unfortunately , solid malignancies , such as bone sarcomas and cystic soft - tissue sarcomas , may respond well to chemotherapy without substantially changing in size . further , sarcomas do not shrink or grow in a uniform manner ; therefore , unidimensional measurements may not accurately reflect response or progression . tumors that respond poorly to therapy may be followed for months before a unidimensional measurement increases significantly . meanwhile , patients are exposed to toxic but ineffective chemotherapy and are likely to have a diminished probability of survival . fdg - pet has the advantage of revealing viable tumor tissue , which is highly metabolically active and therefore accumulates fdg . a decrease in tumor glucose uptake after treatment is correlated with a reduction in the percentage of viable tumor cells . therefore , fdg - pet may offer a more sensitive evaluation of the response or progression of some pediatric solid malignancies than do conventional , anatomic imaging modalities . in children with bone and soft - tissue sarcomas , the lung is the first site of distant spread and is involved in 2025% of patients at diagnosis . the prognosis is poor for children with os and pulmonary metastases unless all metastases are surgically resected . the survival of patients with ews and pulmonary metastases may be improved by whole - lung irradiation . therefore , chest ct is the gold standard for detection of pulmonary metastases ; however , in a retrospective study of 41 children with solid malignancies who underwent biopsy of pulmonary nodules , we found that it has limited accuracy in distinguishing benign from malignant nodule histology . we also found only slight to moderate interreviewer agreement among 3 experienced pediatric radiologists who classified nodules as benign or malignant on the basis of ct features . there is clearly a need to improve the imaging distinction of benign from malignant pulmonary nodules to reduce the frequency of unnecessary thoracotomy . to assess the ability of pet alone to detect pulmonary metastases of os or ews , franzius and colleagues compared pet to ct , clinical follow - up , and histologic interpretation of resected nodules . among 30 pet scans performed at diagnosis or recurrence ( before initiation of salvage therapy ) , pet had a sensitivity of 14% , specificity of 91% , and accuracy of 73% . the ability of pet to detect malignant nodules increased with nodule size : 12 of 16 malignant nodules 10 mm were identified , whereas none measuring < 5 mm were detected ( fig . their results were supported by the recent work of vlker et al . , who investigated the value of pet alone for staging 46 cases of childhood ews ( n = 23 ) , os ( n = 11 ) , and rms ( n = 12 ) . they found that 21 of 28 lung metastases detected by ct were missed by pet ( sensitivity 25% ) and that pet - positive nodules were 8 mm in diameter whereas pet - negative nodules were < 7 mm . gertha and colleagues showed that the fusion modality pet - ct detected pulmonary ews metastases better than pet alone in 53 patients . however , because pet - ct is generally performed with a low milliampere - second technique for ct scanning , image quality may be insufficient to detect very small pulmonary nodules . increasingly smaller nodules can now be detected by using current - generation , multislice helical scanners coupled with picture archiving and communication systems ( pacs ) , whose window and level adjustment features and magnification tools improve image interpretation . we have shown that small pulmonary nodules ( < 5 mm ) are as likely to be malignant as larger nodules in children with solid malignancies . given the need to aggressively treat pulmonary metastatic disease in children with sarcomas , pet - ct is preferred over pet alone as a potential adjunct to distinguish benign from malignant nodules , but the inherent limitation of spatial resolution of the pet and ct components must be taken into account . we have also found that benign processes , such as active infection , can result in substantial fdg uptake within pulmonary nodules , mimicking that seen in metastases ( fig . 3 ) . figure 2(a ) these 0.8-cm nodules ( arrows ) , metastatic from ewing sarcoma , seen on ct , show ( b ) no fdg activity on correlative pet ( arrows indicate approximate location of nodules ) . in contrast , ( c ) this 1.1-cm nodule ( arrow ) , metastatic from rhabdoid tumor , shows ( d ) intense fdg activity on correlative pet imaging ( arrow ) . in children , nodule size affects the detection of fdg activity but not the likelihood of malignant histology . figure 3this 14-year - old boy was suspected of having lymphoma and underwent pet - ct . ( a ) ct shows an ill - defined , 1.8-cm pulmonary nodule ( arrow ) that on ( b ) pet showed intense fdg activity ( arrow ) . ( a ) these 0.8-cm nodules ( arrows ) , metastatic from ewing sarcoma , seen on ct , show ( b ) no fdg activity on correlative pet ( arrows indicate approximate location of nodules ) . in contrast , ( c ) this 1.1-cm nodule ( arrow ) , metastatic from rhabdoid tumor , shows ( d ) intense fdg activity on correlative pet imaging ( arrow ) . in children , nodule this 14-year - old boy was suspected of having lymphoma and underwent pet - ct . ( a ) ct shows an ill - defined , 1.8-cm pulmonary nodule ( arrow ) that on ( b ) pet showed intense fdg activity ( arrow ) . about 10% of os patients develop bone metastases , with or without concurrent pulmonary metastases . as many as 25% of patients with ews or rms experience bone or bone marrow involvement . because ews is radiosensitive , targeted radiation can be beneficial if these metastases are few and well defined . however , when more than 50% of the marrow is involved , irradiation can cause significant myelosuppression and compound the myelosuppressive toxicity of chemotherapy . unfortunately , the prognosis of patients with rms and bone or bone marrow metastases is not improved by surgical metastatectomy or targeted radiotherapy . because os metastases often contain calcification or ossification , they can typically be detected by the bone - seeking radioisotope tc mdp . franzius and colleagues directly compared pet to tc mdp bone scintigraphy for the detection of bone metastases in 32 patients with os . they found 5 bone metastases in 2 patients ; all foci were evident on tc mdp bone scan , whereas none were demonstrated by pet . found 4 of 12 patients to have 31 bone metastases at the time of diagnosis of os ; the sensitivity of detection was 90% for pet , compared with 81% for tc mdp scintigraphy . pet is more convincingly superior to tc mdp scintigraphy for the detection of osseous metastases . found that on 66 paired pet and tc mdp bone scans performed before or after initiation of therapy for ews , osseous metastases were detected by pet with a sensitivity of 100% ( 19/19 ) , specificity of 96% ( 45/47 ) , and accuracy of 97% ( 64/66 ) , compared with 68% ( 13/19 ) , 87% ( 41/47 ) and 82% ( 54/66 ) , respectively , for tc mdp scintigraphy . 6 of whom had 49 osseous metastases , they found that the sensitivity of pet was 88% , compared with 37% sensitivity for tc bone scan ( p < 0.01 ) . in our practice , we have found pet - ct to be more sensitive than tc bone scan for the detection of osseous metastases in several children with rms or ews ( fig . the greater sensitivity of pet to tc mdp scintigraphy in detecting osseous metastases of ews and rms is probably multifactorial . fdg - pet is postulated to detect the increased glucose metabolism of bone marrow metastases before an osteoblastic reaction is appreciable . in contrast , detection of bone metastases by tc bone scintigraphy depends on ossification within the metastatic deposit , the degree of associated cortical destruction , and the intensity of osteoblastic activity , all of which are greater in os bone metastases than in those of ews or rms . it is also possible that cellular glucose uptake and metabolism differ in these sarcomas . ( b ) this mip pet anterior image , obtained 1 day later , shows widely metastatic disease throughout the axial and appendicular skeleton . the primary tumor is the focus of intense soft - tissue activity in the lower left calf ( arrow ) . ( b ) this mip pet anterior image , obtained 1 day later , shows widely metastatic disease throughout the axial and appendicular skeleton . the primary tumor is the focus of intense soft - tissue activity in the lower left calf ( arrow ) . ( reprinted with permission from ajr 2005 ; 184 : 1293304 . ) in assessing children with malignancies that metastasize to bone , it is important to consider benign processes that can mimic metastatic disease on pet imaging . akoi and colleagues assessed the value of pet in distinguishing benign ( n = 33 ) from malignant ( n = 19 ) bone tumors in 52 children and adults . they measured the mean standardized uptake value ( suv ) within a region of interest ( roi ) in the area of tumor with maximum fdg accumulation and found no significant difference between os and giant cell tumors ( p = 0.171 ) , os and fibrous dysplasia ( p = 0.127 ) , or fibrous dysplasia and chondrosarcomas ( p = 0.667 ) . further , they found no suv threshold value that reliably distinguished benign from malignant bone lesions . we reviewed pet - ct imaging performed to evaluate underlying cancers ( n = 13 ) or aggressive fibromatosis ( n = 1 ) in 14 children who also had benign fibrocortical defects , nonossifying fibromas , or cortical desmoids that were discovered as foci of fdg avidity . in some cases , the intense fdg avidity of these benign lesions mimicked the appearance of bony metastatic disease ( fig . these common , benign lesions are often found incidentally on radiographs of children and young adults . they undergo spontaneous regression over time and are rarely seen after the second decade of life . because they have characteristic radiographic features that are well described in the literature , the information gained from correlative ct ( when pet - ct is performed ) or plain radiographs is invaluable in determining whether further imaging or biopsy is necessary . ( a ) mip pet image shows a focus of intense activity above the left knee ( arrow ) that on ( b ) correlative ct and ( c ) axial pet images localized to a benign fibrocortical defect ( arrows ) . ( a ) mip pet image shows a focus of intense activity above the left knee ( arrow ) that on ( b ) correlative ct and ( c ) axial pet images localized to a benign fibrocortical defect ( arrows ) . local - regional lymph node metastasis is uncommon in os and ews but is present at diagnosis in as many as 20% of children with rms . nodal spread is present at diagnosis in approximately half of children with extremity rms and occurs more frequently in older boys ( 10 years ) with paratesticular rms and in association with the alveolar rms subtype . when lymph node metastasis is present , the likelihood of survival can be improved by intensification of chemotherapy and irradiation of the affected region . therefore , identification of sites of lymph node involvement is crucial to the management and outcome of childhood rms . klem et al . investigated the value of pet in detecting metastases during the baseline staging of 24 patients with rms by comparing it to ct , mri , clinical assessment , and histology of resected lesions . the measured suv of suspicious foci was considered abnormal if it exceeded that of background tissue and did not reflect a normal physiologic process . although the nodes were seen on conventional imaging , klem et al . did not report node size or the presence or absence of pathologic enlargement . an additional patient in the study had a lower extremity tumor and a pet interpretation a ct of the inguinal area was uninformative , and therefore chemotherapy was initiated after resection of the primary tumor , without inguinal node biopsy . tumor progression in the involved inguinal node subsequently became apparent on physical examination , pet , and mri and was confirmed by biopsy . the authors concluded that had the nodal involvement been confirmed at diagnosis , the patient might have benefited from more aggressive therapy . described 20 presumed lymph node metastases identified in 8 of 46 children with bone and soft - tissue sarcomas ( 1 with os , 1 with ews , and 6 with rms ) . a lesion - based analysis showed a sensitivity of 95% ( 19/20 ) for pet but only 25% ( 5/20 ) for ct and mri . this study was limited by the absence of size criteria defining abnormal lymph nodes on conventional imaging ; further , 16 of the 20 presumed nodal metastases were not confirmed by pathology studies . in a small case series by ben arush and colleagues , 3 children with alveolar rms underwent pet - ct and conventional imaging for baseline staging ; 2 had moderately fdg - avid regional lymph nodes from which biopsies were taken . the third patient had mild fdg avidity within a regional node but no biopsy was taken . the patient was started on therapy for high - risk disease ; at the time of scheduled follow - up , adenopathy was palpable in the area of the previously mildly fdg - avid node . we have found that benign processes , such as reactive hyperplasia , can cause regional node enlargement and fdg avidity on pet imaging in children with sarcomas ( fig . the available evidence suggests that although pet may identify sites of nodal disease not appreciated on conventional imaging , care should be used in interpreting pet findings . metastatic lymph nodes may demonstrate only mild fdg avidity , whereas nonmetastatic nodes may show intense avidity . therefore , biopsy of suspicious lymph nodes should be considered when there is discordance between the findings of different imaging modalities or between imaging and clinical findings and when identification of nodal disease will affect patient management and outcome . figure 6a 20-year - old woman with malignant peripheral nerve sheath tumor in the left thigh , underwent baseline pet - ct . ( a ) ct shows enlarged iliac nodes ( arrows ) ipsilateral to thigh tumor that on ( b ) pet showed intense fdg activity . biopsy of these nodes revealed reactive hyperplasia . a 20-year - old woman with malignant peripheral nerve sheath tumor in the left thigh , underwent baseline pet - ct . ( a ) ct shows enlarged iliac nodes ( arrows ) ipsilateral to thigh tumor that on ( b ) pet showed intense fdg activity . patients with nonmetastatic os and ews are treated with neoadjuvant therapy before surgical resection of the primary tumor and subsequent adjuvant therapy . chances of survival are improved when 90% or more of the resected tumor is necrotic and resection margins are void of tumor . factors that predict the histologic tumor response to neoadjuvant therapy might also predict patient outcome and help to identify candidates for limb - sparing surgery versus amputation or early resection . a reliable , noninvasive method for assessing tumor metabolism and viability , such as pet , could allow the oncologist and surgeon to individualize management of tumors that are aggressive , respond poorly , or arise in surgically challenging sites . osteogenic sarcoma and ews are metabolically heterogeneous tumors . because tumor histologic grade and predicted patient outcome are determined by the percentage of viable tumor in the resected specimen , the maximum tumor suv is believed to provide the most accurate noninvasive assessment of tumor grade . several investigators have compared the maximum tumor suv before and after neoadjuvant therapy with tumor grade and patient outcome . in 2002 , hawkins and colleagues reported a comparison of the histologic response of resected tumors ( 18 os and 13 ews ) to tumor suv at diagnosis ( suv1 ) and at the end of neoadjuvant therapy ( suv2 ) . in these 31 patients , histologic response was significantly associated with the suv2 ( p = 0.01 ) and the suv2/suv1 ratio ( p = 0.01 ) . an suv2 < 2 had a positive predictive value ( ppv ) of 93% for identifying tumors that were 90% or more necrotic and a 75% negative predictive value ( npv ) for identifying tumors that were < 90% necrotic ( unfavorable response ) . when an suv2/suv1 cutoff point of 0.5 was used , the ppv and npv were 78% and 63% , respectively . in 2005 , hawkins and coworkers reported similar findings in a follow - up study of 36 patients with ews who had pet imaging before and after neoadjuvant therapy . among the 32 patients who underwent resection of the primary tumor , an suv2 < 2.5 had a ppv of 79% for a favorable response and an npv of 40% for an unfavorable response . the ppv and npv for an suv2/suv1 ratio 0.5 were 77% and 33% , respectively . an suv2 < 2.5 was also associated with greater 4-year progression - free survival ( pfs ) in all 36 patients ( p = 0.006 ) and in the 24 patients who had no metastatic disease at diagnosis ( p = 0.036 ) . these findings and those of others suggest that the maximum suv of primary os and ews after neoadjuvant therapy can predict tumor response and patient outcome . further studies are needed to determine whether the suv can be used to identify unresponsive or poorly responsive tumors earlier in the course of neoadjuvant therapy , so that therapy can be appropriately modified . pet imaging of children requires an expert team of professionals to manage the unique technical challenges encountered in this age group . further , the interpreting physician must be aware of the aspects of pediatric anatomy and physiology that differ from those in adults . whereas the role of pet in the assessment of lymphomas is fairly well established , the growing body of evidence supports the continued investigation of pet imaging in the management of pediatric sarcomas . pet may provide valuable information about pulmonary nodules in children , but accurate interpretation requires awareness of its limitations . small malignant pulmonary nodules may not be appreciable on pet imaging , whereas large benign nodules may show intense fdg avidity . pet may detect osseous metastases of childhood ews and rms earlier than [ tc]mdp bone scintigraphy , because bone marrow infiltration precedes cortical destruction and an osteoblastic reaction . on the other hand , the bone - seeking radiotracer [ tc]mdp may offer superior detection of lytic and ossifying bone metastases often associated with os . when pet is used to detect bone metastasis in children , an awareness of the common benign bone lesions in this age group is essential , as they can mimic metastatic disease . in such cases the correlative ct imaging obtained during pet - ct , or plain - film radiography , can preclude the need for further imaging or biopsy . pet imaging that shows sites of local - regional nodal fdg uptake in children with bone and soft - tissue sarcomas should be interpreted with caution . biopsy of suspicious lymph nodes is probably indicated when the findings of different imaging modalities or of imaging and physical examination are inconsistent . because the size of bone tumors does not change substantially in response to neoadjuvant therapy , ct and mri have limited value for assessment of tumor response . because fdg - pet reflects the metabolic activity of tumors , which can be semi - quantitatively measured as the suv , pet holds promise as an alternative method of assessing response in these tumors . additional studies are needed to better define the optimal timing of pet during neoadjuvant therapy to detect poorly responsive tumors and allow early intervention .
summarybackgroundthe authors performed a long term outcome analysis of minimal brain damage in children delivered in breech presentation , and related the results to the mode of delivery ( vaginal or by cesarean section).material / methodsin the department of obstetrics at the medical university of gdansk ( poland ) , 917 breech deliveries took place between 1981 and 1990 . excluding stillbirths and multiple pregnancies , 874 deliveries were analyzed . we received positive responses from 232 mothers , who provided us with considerable information about the children s further development and problems that had arisen during their school years . all the respondents were contacted by telephone , and 83 of them agreed to visit our department with their children to undergo a psychological examination the following tests were performed : 1 ) the bender - kopitz test ( bkt ) , and 2 ) the benton visual retention test ( bvrt).resultsthe mode of delivery for all groups and subgroups had no influence on the incidence of organic brain disorders in later childhood , assessed by the benton visual retention test and by the bender - kopitz test.conclusionsvaginal breech deliveries are safe in both primiparous and multiparous mothers .	even metaanalyses do not give a clear answer to the question as to which mode of delivery is better . a recent study by hannah ( toronto ) has proven that cesarean section ( c - section ) seems to be safer for the infant . when weighing the pros and cons of c - section , however , one should be aware that these results may be due to the safety of low segment surgery and the safer anesthesia now available . on the other hand , the problem of the uterine scar has to be considered , especially in families that want to have more children . there have also been some suggestions that babies delivered by c - section more frequently suffer from respiratory problems . two radically different approaches to ceserean section become apparent among mothers . while some actually this kind of delivery , others regard it as a last resort , not only in view of possible complications for mother and child , but also because of the delayed return to professional activity as a results of surgical complications . the last follow - up performed in this study was done at two years of age . the question may fairly be asked as to whether or not it suffices to do the follow - up at this age , since most babies are doing well at this time . some of them may be suffering from various kinds of damages that present only at a later age , and we do not know how they cope during the school years or in later life . this was the main reason we decide to analyze our results by extending the follow - up study to late childhood . the aim of our study was to assess the presence of signs of organic disorders , using psychological examination tools , in children delivered vaginally versus c - section , separately for primiparas and multiparas . in the department of obstetrics at the medical university of gdansk , 917 breech deliveries took place between 1981 and 1990 . all the mothers in question received a questionnaire , sent to the address we found in the hospital records ; they were asked to answer some questions and to indicate whether they might be interested in a follow - up psychological and neurological examination of their breech delivered child . we received positive responses from 232 mothers , who provided us with considerable information about the children s further development and problems that had arisen during their school years . all the respondents were contacted by telephone , and 83 of them agreed to visit our department with their children to undergo a psychological examination . the ages of the children included in this study ranged from 9 to 18 years . the whole study was carried out with the consent of our university bioethical committee . in this paper we analyze the results of psychological tests intended to assess the possibility of organic brain disorders that could have been due to the breech delivery . the following tests were performed : the bender - kopitz test ( bkt ) ; the benton visual retention test ( bvrt ) . the bkt is a modified version of the lauretta bender test , a familiar method for detecting organic brain damage in adult patients . the version used in our study was developed by elisabeth kopitz to examine children , in order to detect organic brain disorders . it is also used to investigate minimal brain dysfunction , the level of development of visuo - motor integration , and emotional disorders . in order to classify the results as normal , almost normal , or pathological they were assessed on the basis of polish norms , where a score between 090 is treated as relatively normal , 91100 is borderline normal , and a score of 101 or higher indicates organic lesions of the central nervous system . it allows us to assess memory disorders , concentration disorders , and orientation and motor disorders in the case of many patients suffering from psychiatric and neurological disorders . the subject draws an image from memory immediately after having looked at it for 10 seconds , so that the test detects the abnormal decay of visual information . there are 10 images , each of which contains one or more figures ( usually three ) . the results of the bvrt were assessed both on the basis of the number of mistakes made by the patent , and the number of correct representations . in both cases , depending on the level of the patient s intelligence and his age , both the expected number of mistakes and the expected number of correct representations were calculated on the basis of the instruction . if the score obtained in our study in terms of the number of correct answers was 2 points lower than expected , this was treated as a result somewhere between normal and pathological . when the result was 3 points lower , it was treated as pathological . as for the number of mistakes made by the patient , the result was treated as a borderline when it was higher than expected by 3 points , while a result 4 points higher than expected was considered to be pathological . the results were analyzed statistically using the test , with the level of significance established at p<0.05 . since delivering for the first time is thought to have an impact on the prognosis in breech delivery , we analyzed the results separately for primiparas and multiparas . we analyzed each group as a whole and with respect to the presenting part , dividing each group into complete breech , frank breech and footling breech cases . nearly 90% of children born by primiparas presented with no organic brain damage assessed by the bvrt . half of these ( 3 cases ) were in the vaginal delivery group , and the other half in the c - section group . only one abnormal result from the bender - kopitz test was noted in the vaginal delivery group . statistical analysis of results from both tests revealed no statistically significant differences between groups . in only 1 child with complete breech at delivery abnormal bvrt results were found in 4 cases , 2 cases each in the vaginal delivery and c - section groups , with frank breech presentation at delivery . only 1 abnormal bender - kopitz test result was found in the vaginal delivery group . statistical analysis revealed no influence of the mode of delivery on the incidence of abnormal test results . in all deliveries from the footling presentation at delivery in primiparas a c - section was performed , thus no statistical analysis could be carried out . although no statistical analysis was possible for the footling presentation in primiparas , we mention our results here to stress that abnormal results were also found in the c - section group . we may therefore safely assume that the protective value of the c - section is disputable . 81.3% of babies born by multiparas from the breech presentation presented no signs of organic brain disorders as assessed by the benton test . statistical analysis showed no influence of the mode of delivery on the incidence of brain damage in these children ( table 8) . abnormal results from the bender - kopitz test were found in only one child born from the breech presentation . statistical analysis did not reveal any difference between the mode of delivery groups . in the complete breech delivery group in multiparas , thus no comparison of the mode of delivery was possible , and our results have only a purely informative function . one 8-year - old child in this group had abnormal results on both tests ( 12.5% ) . although comparative analysis was not possible , we can assume that most children born vaginally from complete breech presentation by multiparas presented no signs of organic brain disorders . 78.6% of the children born vaginally from frank breech by multiparas and 85.7% of those born by c - section presented no signs of brain damage in the benton visual retention test . all children delivered from frank breech presentation by multiparas had normal results on the bender - kopitz test regardless of the mode of delivery . the results presented above show no influence of the mode of delivery on the incidence of organic brain dysfunction in multiparas with frank breech presentation at delivery . we were unable to perform a comparison of the mode of delivery in this group . even though we had no deliveries by c - section in this group , all results were found normal in the bender - kopitz test , and only 1 was abnormal in the benton visual retention test , so we can consider the vaginal mode for breech delivery to be a safe one . a normal result on the bender - kopitz test is an indirect indicator of a correct , or at least satisfactory level of development of visual perception and visual coordination . since many authors , e.g. losiowski et al . , connect the score in the apgar scale in the fifth minute of life with , among other things , the level of psychomotor development and neurological status , this relation was also investigated in our research . no correlation was found between the number of points scored by a child on the apgar scale and the results of the benton test ( r=0.001 , f=0.000025 , p<0.996 ) or the bender test ( r=0.206 , f=2.084 , p<0.156 ) . nearly 90% of children born by primiparas presented with no organic brain damage assessed by the bvrt . half of these ( 3 cases ) were in the vaginal delivery group , and the other half in the c - section group . only one abnormal result from the bender - kopitz test was noted in the vaginal delivery group . statistical analysis of results from both tests revealed no statistically significant differences between groups . in only 1 child with complete breech at delivery abnormal bvrt results were found in 4 cases , 2 cases each in the vaginal delivery and c - section groups , with frank breech presentation at delivery . only 1 abnormal bender - kopitz test result was found in the vaginal delivery group . statistical analysis revealed no influence of the mode of delivery on the incidence of abnormal test results . in all deliveries from the footling presentation at delivery in primiparas a c - section was performed , thus no statistical analysis could be carried out . although no statistical analysis was possible for the footling presentation in primiparas , we mention our results here to stress that abnormal results were also found in the c - section group . we may therefore safely assume that the protective value of the c - section is disputable . nearly 90% of children born by primiparas presented with no organic brain damage assessed by the bvrt . half of these ( 3 cases ) were in the vaginal delivery group , and the other half in the c - section group . only one abnormal result from the bender - kopitz test was noted in the vaginal delivery group . statistical analysis of results from both tests revealed no statistically significant differences between groups . in only 1 child with complete breech at delivery did we find abnormal bvrt and bender - kopitz test results . abnormal bvrt results were found in 4 cases , 2 cases each in the vaginal delivery and c - section groups , with frank breech presentation at delivery . only 1 abnormal bender - kopitz test result was found in the vaginal delivery group . statistical analysis revealed no influence of the mode of delivery on the incidence of abnormal test results . in all deliveries from the footling presentation at delivery in primiparas a c - section was performed , thus no statistical analysis could be carried out . although no statistical analysis was possible for the footling presentation in primiparas , we mention our results here to stress that abnormal results were also found in the c - section group . we may therefore safely assume that the protective value of the c - section is disputable . 81.3% of babies born by multiparas from the breech presentation presented no signs of organic brain disorders as assessed by the benton test . statistical analysis showed no influence of the mode of delivery on the incidence of brain damage in these children ( table 8) . abnormal results from the bender - kopitz test were found in only one child born from the breech presentation . statistical analysis did not reveal any difference between the mode of delivery groups . in the complete breech delivery group in multiparas , thus no comparison of the mode of delivery was possible , and our results have only a purely informative function . one 8-year - old child in this group had abnormal results on both tests ( 12.5% ) . although comparative analysis was not possible , we can assume that most children born vaginally from complete breech presentation by multiparas presented no signs of organic brain disorders . 78.6% of the children born vaginally from frank breech by multiparas and 85.7% of those born by c - section presented no signs of brain damage in the benton visual retention test . all children delivered from frank breech presentation by multiparas had normal results on the bender - kopitz test regardless of the mode of delivery . the results presented above show no influence of the mode of delivery on the incidence of organic brain dysfunction in multiparas with frank breech presentation at delivery . we were unable to perform a comparison of the mode of delivery in this group . even though we had no deliveries by c - section in this group , all results were found normal in the bender - kopitz test , and only 1 was abnormal in the benton visual retention test , so we can consider the vaginal mode for breech delivery to be a safe one . a normal result on the bender - kopitz test is an indirect indicator of a correct , or at least satisfactory level of development of visual perception and visual coordination . since many authors , e.g. losiowski et al . , connect the score in the apgar scale in the fifth minute of life with , among other things , the level of psychomotor development and neurological status , this relation was also investigated in our research . no correlation was found between the number of points scored by a child on the apgar scale and the results of the benton test ( r=0.001 , f=0.000025 , p<0.996 ) or the bender test ( r=0.206 , f=2.084 , p<0.156 ) . 81.3% of babies born by multiparas from the breech presentation presented no signs of organic brain disorders as assessed by the benton test . statistical analysis showed no influence of the mode of delivery on the incidence of brain damage in these children ( table 8) . abnormal results from the bender - kopitz test were found in only one child born from the breech presentation . in the complete breech delivery group in multiparas , all the children were delivered vaginally . thus no comparison of the mode of delivery was possible , and our results have only a purely informative function . one 8-year - old child in this group had abnormal results on both tests ( 12.5% ) . although comparative analysis was not possible , we can assume that most children born vaginally from complete breech presentation by multiparas presented no signs of organic brain disorders . 78.6% of the children born vaginally from frank breech by multiparas and 85.7% of those born by c - section presented no signs of brain damage in the benton visual retention test . all children delivered from frank breech presentation by multiparas had normal results on the bender - kopitz test regardless of the mode of delivery . the results presented above show no influence of the mode of delivery on the incidence of organic brain dysfunction in multiparas with frank breech presentation at delivery . we were unable to perform a comparison of the mode of delivery in this group . even though we had no deliveries by c - section in this group , all results were found normal in the bender - kopitz test , and only 1 was abnormal in the benton visual retention test , so we can consider the vaginal mode for breech delivery to be a safe one . a normal result on the bender - kopitz test is an indirect indicator of a correct , or at least satisfactory level of development of visual perception and visual coordination . since many authors , e.g. losiowski et al . , connect the score in the apgar scale in the fifth minute of life with , among other things , the level of psychomotor development and neurological status , this relation was also investigated in our research . no correlation was found between the number of points scored by a child on the apgar scale and the results of the benton test ( r=0.001 , f=0.000025 , p<0.996 ) or the bender test ( r=0.206 , f=2.084 , p<0.156 ) . controversies surrounding breech deliveries have engaged many researchers and considerable financial resources . a new approach to this subject was achieved by mary hannah in a multicentre study coordinated by the university of toronto . the results achieved in this study have indicated that the c - section is safer for breech delivered babies , but there has been much discussion on its reliability [ 2,3,1619 ] . many obstetricians remained unconvinced generally , not due to other , contradictory results published afterwards , or doubts about the methodology , but usually on the basis of their own experience in breech deliveries . the idea of terminating each breech delivery by c - section has led to the loss of obstetrical skills among older obstetricians and the lack thereof in the younger practicioners . after many years of such a policy in obstetrics in the developed countries , nowadays , the lack of the requisite skill on the part of the physician has become yet another indication for doing a c - section routinely . we decided to analyze our own data and to present them in the present study . despite the general division into the group of primiparas and multiparas , each group of deliveries was analyzed in total and in subgroups of complete breech , frank breech and footling breech presentation . we were motivated to perform this study after the appearance of papers suggesting that babies delivered by c - section more often present respiratory disorders in comparison to those delivered vaginally . in all groups and subgroups we found no influence of the mode of delivery on the incidence of organic brain disorders in later childhood , as assessed by the benton visual retention test and by the bender - kopitz test . it should be emphasized , however , that not every brain damage results in a worse mastery of the visual memory tasks measured by the benton visual retention test and by the bender - kopitz test . many factors come into play , including the size , kind and location of the damage and its persistence . it is also worth pointing out that mistakes in the drawing tests are connected first of all with the dysfunction of the posterior ( occipital , parietal and temporal ) lobes . this results in difficulties in so called organic tests , which could be a genuine indicator confirming organic brain dysfunction ; nevertheless , the lack of such difficulties can not be the basis for excluding organic brain damage . these children also underwent examination for neurological and other psychological disorders for other studies , and no differences between groups were found . some researchers have investigated the relation between the number of points on the apgar scale and intellectual - cognitive functioning in early childhood . it should nevertheless be stressed that methods of assessing infants are not definite prognosticators of the eventual intellectual level , if they are considered in separation from other risk factors [ 20,21 . the vaginal route is a safe method of delivery in breech presentations in both primiparas and multiparas .
the german research network on schizophrenia ( grns ) is a nationwide network currently comprising 16 psychiatric university departments and 14 state and district hospitals , as well as six local networks of psychiatric practices and general practitioners collaborating on about 25 interrelated , multicenter projects on schizophrenia research . the grns aims to intensify collaboration and knowledge exchange betvi / een leading research institutions and qualified routine care facilities , both within ( horizontal network ) and between ( vertical network ) the two levels of research and care , in order to create the scientific preconditions for optimization of the management of schizophrenia . the concept and the first results of studies aiming at the investigation of(i ) strategies for early detection and early intervention in the prodromal stage of psychosis ; ( ii ) treatment in first - episode schizophrenia ; ( iii ) quality management ; and ( iv ) destigmatization , are described as examples of this effort .	the german research network on schizophrenia ( grns ) is one of 17 existing medical research networks funded by the german federal ministry of education and research ( bmbf ) since 1999 in order to improve care of patients with illnesses characterized by high morbidity and/or mortality . each of these networks is funded for a maximum of 8 years , with up to 2.5 million euro per year during the first 5 years and up to 0.5 million euro per year for the last 3 years . additional sponsoring ( about 5% of the budget ) by the industry is provided for research projects or public relations activities of the network . one of the main reasons that the bmbf established such networks originates from the fact , that new knowledge from research is only insufficiently transferred to practice , and problems in everyday care worth being scientifically investigated are only insufficiently recognized by researchers . thus , the structural aim of the grns - in a way to be taken as a precondition - consists of the establishment of long - term communication structures between research , services , consumers , and the public . a means for attaining these aims is the creation or expansion , intensified utilization , and routine application of collaboration and knowledge exchange within ( horizontal networking ) and between ( vertical networking ) the two levels of research and care . with regard to the funding by the federal ministry of research , the framework for attaining these objectives is that , of research projects , which constitute the means for intensified collaboration between institutions and optimized care for patients with schizophrenia . the network comprises about 25 interrelated research studies and projects on health care education with high practice relevance . a current total of 16 psychiatric university departments and 14 state and district hospitals , as well as six local networks of psychiatric practices and general practitioners throughout germany , participate in these studies . as regards content , the major objective of the grns is to create the scientific preconditions for the implementation of strategics for early detection and early intervention in the prodromal stage of the first episode ( project network i ) , for the optimization of acute and long - term treatment in first - episode patients and for the rehabilitation in patients with residual symptoms ( project network ii ) . quality of care in hospitals and practices is evaluated and improved by quality assurance programs implementing the existing guidelines for outand inpatient treatment . basic research on structural and functional brain imaging and genetic markers investigates underlying determinants of the manifestation and re - manifestation of the illness as well as the individual response to drug treatment ( special network on molecular and pharmacogenetics ) . a number of more general projects on fighting stigma and discrimination , health care economy , postgraduate training , quality assurance , and methodology complete the spectrum of network projects ( see ref 1 and www.kompetenznetz-schizophrenie.de for more information ) . generally , the studies are multicenter studies designed in such a manner that vertical and horizontal networking is forced , essential , or at least , supported . in order to create synergy and added value as important criteria for successful networking , most , of the projects are strongly interrelated regarding conceptual background , method , and organization . the superordinate aim of these studies is to allow for an improvement of the course and the outcome of schizophrenia along with considering cost - benefit aspects . since some of the studies are long - term studies which finished recruitment only recently , reliable results will only be available later in 2006 . thus , the description will mainly focus on the concept of the studies with regard to the improvement of the management of schizophrenia . it is known that the first , treatment , contact of people suffering from schizophrenia is preceded by a period of manifest , psychotic symptoms , on average lasting for 1 year , and a prepsychotic prodromal period of about 5 years with increasing negative and unspecific symptoms and functional impairment . at , the same time , it has been shown that a delayed treatment , is associated with significant disadvantages for schizophrenia patients , often resulting in functional and social decline . thus , in order to optimize outcome it seems essential to recognize and treat at - risk persons and schizophrenia patients as early as possible . for this purpose awareness programs are being carried out as a first step in several german cities within the grns , in order to improve utilization of newly founded early - recognition centers by at - risk persons . as a second step , two early recognition and intervention studies are carried out ; these are also used to prospectively validate a two - step early - recognition inventory ( eri ) and a set , of cognitive tests developed for early detection of at - risk persons . for the two early intervention studies , two groups of atrisk persons are selected from the larger group of persons referred to the early - recognition centers , according to their presumed prodromal stage . early initial prodromal stage is assumed in case subjects report predictive basic symptoms in the eri or in case they have a first - degree relative with schizophrenia and show a marked decline in global functioning . late initial prodromal stages are defined by the occurrence of brief limited intermittent psychotic symptoms ( blips ) or by attenuated positive symptoms . persons at risk for psychosis in the early prodromal state are included into an early intervention study examining the effects of a newly developed cognitivebehavioral therapy ( cbt ) strategy for prodromal persons , which is compared with clinical management , within a randomized control design over a 24-month period . persons in the late prodromal state of psychosis are included into a second early intervention study , which compares the effects of atypical antipsychotic medication with amisulpride in combination with clinical management ( supported by crisis intervention or family counseling in case of need , but no regular psychotherapy ) to such clinical management alone . this is a phase - ill study with an open - label , randomized parallel design , with a treatment period of 2 years . effects of both studies will be evaluated with regard to improvement of prodromal symptoms , prevention of social decline , and suppression , or at least , delay , of progression to psychosis . preliminary results of both studies are encouraging , indicating a benefit for at - risk persons treated with cbt or amisulpride , respectively , compared with the control treatments with regard to these outcome variables . should these trends be validated in the final analyses , the use of early recognition and early intervention strategies as developed within the grns would be an important , step in the management , of developing psychosis . though a number of studies have shown advantages of atypical second - generation antipsychotics compared with conventional antipsychotics in acute treatment ( for review see ref 12 ) as well as in long - term treatment , of schizophrenia ( for review see ref 13 ) it is still under debate whether these results may be biased by the high dosages of conventional antipsychotics usually used in these studies . low - potency neuroleptics even might not induce more extrapyramidal side effects under a lowdose strategy than second - generation drugs and the potential advantage of atypical antipsychotics may be compromised by their side effects , such as weight , gain and metabolic effects . nevertheless , atypical antipsychotics are recommended as first - choice treatment for both first- and multiple - episode schizophrenia or for first - episode schizophrenia preferentially . however , independent , long - term studies in first - episode patients substantiating these recommendations are lacking or are still under way , such as the clinical antipsychotic trials of intervention effectiveness ( catie ) trial in the us and the european first episode schizophrenia trial ( eufest ) study in europe ; beyond this uncertainty regarding the best kind of antipsychotic treatment for the special group of first - episode patients , it is furthermore unclear how long treatment should be continued after cessation of the first , acute phase . published guidelines recommend treatment durations of minimum 1 year ; the appropriate duration of further treatment in case of symptom remission , however , has not been adequately specified . in order to contribute to these open questions , a comprehensive acute and long - term treatment , study in patients with first - episode schizophrenia is currently been conducted in up to 13 german university hospitals within the grns . the study comprises a prospective doubleblind , randomized , parallel - group comparison of risperidone as a new - generation antipsychotic with halopcridol as a conventional antipsychotic . both drugs are administered in rather low daily dosages of 2 to 8 mg per day during the 8 weeks of acute treatment , and thereafter in a reduced dosage - where possiblc - of 2 to 4 mg per day during a 2-year long - term treatment period . to investigate the necessary duration of long - term treatment in first - episode patients , patients completing the first treatment year without , relapse are randomly allocated to either maintenance treatment , or stepwise drug discontinuation in the second treatment year . in case of impending re - exacerbations , prodrome - based early intervention , either by means of resumption or augmentation of neuroleptic treatment ( depending on the basic treatment strategy of discontinuation or maintenance treatment ) or by means of treatment / additional treatment , with the benzodiazepine lorazepam is applied in the second treatment year to prevent relapses . this randomized , double - blind comparison shall contribute to the open question of whether prodromes are unspecific consequences of stress experience , treatable with benzodiazepines , or have to be regarded as more specific , prepsychotic symptoms requiring neuroleptic treatment . preliminary findings so far suggest that the treatment , with low dosages of antipsychotics is feasible and effective , and leads to a significant improvement , in positive , negative , and prodromal symptoms in first - episode schizophrenia patients . none of the patients has fulfilled the criteria for relapse within the first year of treatment . because the medication is only about , to be unblinded , comparison of differential treatment or side effects between both drugs is not yet available . schizophrenia patients often exhibit , impairments in facial affect recognition ( see ref 31 for review ) , which is already present in first - episode patients , and even in unaffected siblings of schizophrenia patients . such impairments are strongly associated with more global social dysfunctions characteristic of schizophrenia and may have adverse effects on psychosocial functioning independent of the presence and severity of positive and negative symptoms and cognitive deficits . thus , these impairments represent , a core feature of the disorder and are of high relevance for the psychosocial functioning of the patients . the traditional drug and psychological treatment usually administered to schizophrenia patients seem to be ineffective in this regard , as indicated by the stability of the impairment , across different stages of the disorder despite treatment . against , this background , a new training program for the remediation of such impairments has been developed within the grns . ( tar ) have been compared with a cognitive remediation program ( crt ) primarily aiming at improving attention , memory , and executive functioning , and with treatment , as usual ( tau ) without participation in a specific remediation program within a randomized three - group pre - post design . results indicated that , patients on tar significantly improved in facial affect recognition , with recognition performance after training approaching the level of healthy controls from former studies . patients on crt and those without special training ( tau ) did not improve in affect recognition , though patients on crt improved in verbal memory functions . according to these results , remediation of disturbed facial affect recognition in schizophrenia patients is possible , but not achievable with a traditional cognitive rehabilitation program such as the crt . instead , functional specialized remediation programs such as the newly developed tar arc a more suitable option . whether these promising training effects of the tar endure across time and pervade into everyday social functioning has to be investigated by future studies . if these effects can be validated , such training programs could become an important , module of psychosocial rehabilitation programs in future . optimizing treatment , of schizophrenia through implementation of guidelines is essential for early and acute as well as long - term and chronic phases of schizophrenia . such measures of quality assurance shall guarantee optimal care in accordance with the state - of - the - art knowledge under consideration of available resources . at present , it is estimated that only 40% to 50% of schizophrenia patients arc treated according to scientific standards and treatment guidelines . although several treatment , guidelines for schizophrenia have been published in recent , years , it has been supposed that only a case - focused implementation of these guidelines will lead to an improvement in outcome quality measures . therefore , two projects targeting quality assurance , either in inpatient , care or in outpatient care , have been performed within the grns . the first of these projects targeted the systematic development , implementation , and evaluation of specific measures of quality management in inpatient treatment of 597 schizophrenia patients at seven psychiatric hospitals , mostly district hospitals . using an experimental control group design with preand post - assessments , quality - orientated interventions according to the concept of total quality management , ( tqm ) and with reference to the german treatment guidelines were compared in four experimental hospitals with documentation of structural parameters ( hospital and patient characteristics ) , of treatment , and of outcome in three control hospitals . experimental hospitals received feedback by means of comparative benchmarking , and were guided in implementing quality circles for specific problem areas identified from the benchmarking process . results indicated that , poorer average clinical outcome was associated with lower guideline conformity in a variety of treatment domains . after case - mix adjustment , benchmarking proved to be an opportunity to improve quality of treatment and promote guideline conformity . the main focus was to implement guidelines , but also other elements of internal ( documentation system , monitoring ) and external ( benchmarking ) quality management , in four hospital - associated networks of private psychiatric practices in three different , german cities ( dsseldorf , freiburg , and munich ) . one of the three experimental groups used a computer - based documentation system with implemented treatment , guidelines and decisionsupport , and received comparative benchmarking . this computerized documentation system draws the attention of the physician to the treatment guidelines by means of a pop - up window showing the relevant guideline algorithm whenever the entered data indicate critical changes in the patient 's clinical status . two further experimental groups used either the computer - based documentation system without , implemented guidelines and benchmarking , or papcr - and - pencil documentation with additional organization in quality circles . a control group used paper - and - pencil documentation without additional organization in quality circles . results in 583 patients with schizophrenia treated by 55 psychiatrists for at least 16 months demonstrated a significantly better outcome in patients in the experimental practices , either using the decision support , system or working with quality circles , as compared with those practices merely documenting their treatment , either computer - based or with paper and pencil , but without , further measures of quality assurance . the stigma associated with mental illness and psychiatric treatment , and the discrimination toward people with mental illnesses that frequently results from this , are the main obstacles preventing early and successful treatment . to reduce such stigma and discrimination , especially towards people with schizophrenia , the world psychiatric association 's ( wpa ) global anti - stigma program fighting stigma and discrimination because of schizophrenia - open the doors is currently being implemented in 27 countries . since august 1999 , the campaign has also been carried out in seven cities in germany , partly within , and with funding of , the grns . a survey of attitudes towards people with mental illness was conducted at the beginning of this campaign in 7246 persons in six german cities by telephone using a standardized questionnaire . the respondents were asked about , their knowledge with regard to schizophrenia , their social distance from people with schizophrenia , and estimations of the social stigmatization of mental patients in general . thereafter public information programs and educative measures aimed at selected target groups were performed , and the opportunity for personal contact with mentally ill people was promoted in two of the cities in order to improve the public 's knowledge regarding symptomatology , causes , and treatment options for schizophrenia . the first results of a recently executed second survey of the same persons indicate that such improvement , could indeed be partly obtained in these two cities , whereas no comparable changes occurred in the cities not participating in the antistigma campaign . the next step to be performed is to investigate whether improved knowledge in turn also contributes to abolishing prejudice and negative perceptions and facilitates the social reintegration of those suffering from mental illness . significant structural improvements regarding intensified collaboration between and within the research and care levels have already been achieved . moreover , significant contributions to improved management of schizophrenia have already been obtained , for instance in the area of quality assurance in inpatient , and outpatient treatment . several studies regarding early detection and early intervention , as well as treatment , of first - episode schizophrenia , were initially designed as long - term studies lasting up to 5 years , which only recently reached the phase of analysis . due to the comprehensive design of these carefully coordinated studies targeting a number of important and open questions in schizophrenia the next essential task will be to transfer these results into health care . based on a successful midterm evaluation , further funding has recently been granted by the bmbf until mid2008 in order to promote this transfer process . thus , development , and implementation of measures for early recognition and intervention , for treatment of firstepisode schizophrenia , for quality management , and for destigmatization will be in the focus of this last funding period before the grns has to finance itself by other resources . these measures will comprise development , of manuals and brochures , and continued medical education measures , as well as the setting up of special competence centers for each of these topics . nevertheless , an ongoing aim of the grns will still be to offer a research platform , particularly for clinical studies , in order to continue successful horizontal networking between the institutes of research . maintenance and extension of the existing dna and clinical data banks will be an important part , of this effort . the complexity of psychiatric disorders on the one hand , and the progressive specialization in research , especially when using complex biological methods , on the other hand , results in an increasing necessity for inter- and intradisciplinary collaboration organized into larger networks like the grns . primarily , such a strategy seems promising to find answers to the urgent and complex questions regarding schizophrenia that are still unresolved .
purposethe superior image quality of 3 tesla ( 3 t ) magnetic resonance ( mr ) imaging in cervical cancer offers the potential to use a single image set for brachytherapy . this study aimed to determine a suitable single sequence for contouring tumour and organs at risk , applicator reconstruction , and treatment planning.material and methodsa 3 t ( skyra , siemens healthcare ag , germany ) mr imaging system with an 18 channel body matrix coil generated hdr cervical cancer brachytherapy planning images on 20 cases using plastic - based treatment applicators . seven different t2-weighted turbo spin echo ( tse ) sequences including both 3d and contiguous 2d scans based on sagittal , axial ( transverse ) , and oblique planes were analysed . each image set was assessed for total scanning time and usefulness in tumour localization via inter- and intra - observer analysis of high - risk clinical target volume ( hr ctv ) contouring . applicator reconstruction in the treatment planning system was also considered.resultsthe intra - observer difference in hr ctv volumes between 2d and 3d axial - based image sets was low with an average difference of 3.1% for each observer . 2d and 3d sagittal image sets had the highest intra- and inter observer differences ( over 15% ) . a 2d axial double oblique sequence was found to produce the best intra- ( average difference of 0.6% ) and inter - observer ( mean sd of 9.2% ) consistency and greatest conformity ( average 0.80).conclusionsthere was little difference between 2d and 3d - based scanning sequences ; however the increased scanning time of 3d sequences have potential to introduce greater patient motion artifacts . a contiguous 2d sequence based on an axial t2-weighted turbo - spin - echo ( tse ) sequence orientated in all planes of the treatment applicator provided consistent tumour delineation whilst allowing applicator reconstruction and treatment planning .	three - dimensional ( 3d ) image - based planning approaches to high dose rate ( hdr ) brachytherapy of cervical cancer have only been introduced in most departmental brachytherapy programs in the past decade , with magnetic resonance ( mr ) imaging now recommended as the imaging modality of choice . current international recommendations state that t2-weighted , fast - spin - echo ( fse ) or turbo - spin - echo ( tse ) sequences to enable gross tumour to appear as high signal intensity masses should be taken in the para - axial , para - coronal , and para - sagittal planes when possible , for the ease of clinical target volume ( ctv ) visualisation [ 14 ] . ideally , ctv contouring and applicator reconstruction should be performed on the same image sequence to reduce uncertainties in image fusion and patient / organ motion between different sequences . however , it has been acknowledged that there are often difficulties accurately visualising the applicator in t2-weighted mr images [ 3 , 4 ] , particularly those with large slice thicknesses . in those instances , additional sequences such as a t1-weighted mr scan , a ct scan or even x - ray images may be required [ 3 , 4 ] . these recommendations are based on studies conducted in brachytherapy treatment centres using low ( 0.1 - 0.5 tesla [ t ] ) and high ( 1.0 - 1.5 t ) magnetic resonance imagers , with acknowledgement that these studies contained no experience with higher ( 3 t or higher ) magnetic resonance imagers . several diagnostic imaging studies have shown that 3 t mr image of the female pelvis provides better image contrast and signal to noise ratio in the uterine cervix and vagina [ 5 , 6 ] , and there is continuing work in the evaluation of 3 t mr scans for brachytherapy imaging [ 79 ] . brachytherapy , along with surgery and external beam radiotherapy ( with or without chemotherapy ) remains a significant part of the pattern of care for stage i - iva cervical cancer . hdr brachytherapy is generally performed post external beam irradiation , which provides greater initial tumour shrinkage , allowing the brachytherapy boost to sterilise any residual tumour while sparing bladder , rectum and small bowel from excessive dose . cervical cancer has a high - signal intensity on t2 weighted mr images [ 11 , 12 ] . evaluation of t2 weighted mr images , taken at diagnosis , during treatment ( external beam ) , after external beam ( for brachytherapy ) , and up to 6 months follow up are often a good indication of how the tumour is responding to treatment . unfortunately , any inflammation , fibrosis and oedema resulting from the external beam irradiation may also exhibit intermediate to high signal intensity on t2 weighted images [ 13 , 14 ] . gec - estro have recommended these areas of intermediate to high signal intensity be incorporated into either the high or intermediate risk clinical target volume ( ctv ) [ 1 , 2 , 15 ] . this makes mr image quality an important consideration in brachytherapy planning images and image resolution a major issue in accurate brachytherapy applicator and ctv determination . in mr imaging , some important considerations are the signal - to - noise ratio ( snr ) and image resolution . a 3 t mr image has approximately double the snr of a 1.5 t mr image . by reducing slice thickness of the 3 t mr scan , it is possible to maintain similar snr to a 1.5 t mr image , whilst increasing the resolution . snr is a central factor in hr ctv contouring and whilst resolution is critical in both hr ctv contouring and applicator reconstruction . the purpose of this study was to determine a suitable 3 t mr t2-weighted image sequence for 3d planning of hdr cervical cancer brachytherapy treatments . the increased image quality from a 3 t mr image was hoped to produce a single image sequence for contouring tumour and organs at risk , applicator reconstruction , and treatment planning . a single image set will be more useful not only for treatment planning purposes but also a time - advantage to those departments that do not have immediate access to an mr scanner and may need to transfer a patient from theatre to radiology for treatment imaging . the study was performed on 20 cases referred to the radiation oncology department for the treatment of cervical cancer with brachytherapy . all patients had mr imaging performed for brachytherapy planning after plastic - based , mr compatible ring applicator ( ring ct / mr applicator , nucletron , an elekta company ) placement under ultrasound guidance and general anaesthesia . the age range of patients was between 31 - 88 years with a mean age of 54.3 years and all patients were rated figo ( international federation of gynaecology and obstetrics ) stage iib to iiib . the range of contoured hr ctv for all patients was between 47.9 and 9.7 cm with a mean of 22.6 cm . the range of hr ctv dimensions ( l - r , s - i and a - p ) were between 4.5 , 4.6 and 5.5 cm and 1.9 , 1.7 , and 1.8 cm , respectively . all patients underwent external beam radiation therapy ( ebrt ) between 45 and 52.2 gy in 1.8 or 2 gy fractions . hdr brachytherapy ( 24 gy in 8 gy fractions prescribed to the 100% isodose line ) was commenced either within the final week or within a week of finishing ebrt . each insertion was given 1 week apart , with the applicator removed after each insertion and mr - based treatment planning occurring for each fraction . all patients had cervical hdr brachytherapy applicators inserted under general anaesthetic , underwent mr scanning in radiology after leaving recovery and were then transported to the department of radiation oncology for treatment . a 3 t ( skyra , siemens healthcare ag , germany ) mr imaging system with combination of an 18 channel body matrix and 32 channel phase array spine coil was used for generating hdr cervix brachytherapy planning images . patients were positioned in supine head - first orientation with a body matrix coil placed over the pelvis . a small foam wedge was placed between the pelvis and coil to help reduce anterior abdominal wall motion . the pre - emptied bladder was filled with 100 ml of saline for adequate distension of the bladder during imaging and reproducibility of bladder volume at treatment . an anti peristalsis agent was administered intravenously ( hyoscine butylbromide 20 mg in 1 ml ) to minimise ghosting artefacts from gross peristaltic movements during data acquisition . scanning sequences that were part of the standard radiation therapy scanning practice in our department were considered for assessment . in collaboration with a radiologist and mr imaging specialist , a parallel imaging technique , generalized auto - calibrating partially parallel acquisition ( grappa ) algorithm ( siemens healthcare ag , germany ) with r factor of 2 spatially selective saturation bands were used superiorly and anteriorly on all sequences to suppress the signal from inflowing spins and reduce motion related artefacts . t2 weighted turbo spin echo ( tse ) sequences were selected without the application of iv contrast media in keeping with international recommendations . both 3d t2-weighted sampling perfection with application - optimised contrast and different flip - angle evolutions ( space , siemens healthcare ag , germany ) ( 3d t2 ) sequences and contiguous 2d t2-weighted tse sequences ( 2d t2 ) scans were performed for comparison in sagittal , axial , and oblique planes tilted to correspond to the treatment applicator . firstly , a 3-plane half - fourier acquisition single - shot turbo spin - echo ( haste , siemens healthcare ag , germany ) localizer was taken . this haste scan was used for planning the axial and oblique - axial sequences oriented to correspond to the treatment applicator . initially , a 2d t2 weighted tse ( 2d t2 ) sequence was obtained in the sagittal plane between the obturator muscles , and a 2d t2 sequence was positioned and acquired in a true axial plane and reviewed for both applicator and tissue delineation . a 3d t2 volumetric sequence was also performed in a true axial plane with an isotropic 3d t2 volumetric sequence acquired in the sagittal plane . both a 2d t2 sequence and a 3d t2 volumetric sequence were planned and acquired para - axially with the scanning plane aligned parallel to the ring surface from the sagittal haste scan ( axial oblique , see fig . another 2d t2 sequence was then obtained with the scanning plane parallel to the ring surface , based on both the sagittal and the coronal reconstructions of the haste localiser ( axial double oblique , see fig . 2 ) . axial - oblique plane , aligned parallel to the plane of the ring in the sagittal scan axial double - oblique additional plane , aligned parallel to the plane of the ring in the coronal localizer scan all scan volumes included the uterine fundus superiorly and the distal end on the treatment applicator inferiorly . all scanning sequences were designed to have slice thicknesses less than 2 mm to aid in 3d reconstruction in the brachytherapy treatment planning system . turbo spin echo , space sampling perfection with application - optimised contrast and different flip - angle evolutions , tr relaxation time , te echo time , fov field of view , nex number of excitations after importation into the treatment planning system ( oncentra brachy version 4.2 and 4.3 , nucletron , an elekta company , sweden ) , the images were initially evaluated by an experienced medical physicist with regards to the ability to reconstruct the treatment applicators . an applicator library ( applicator modelling module in oncentra version 4.2 and 4.3 , nucletron , an elekta company , sweden ) was used in the reconstruction of each applicator . in this module , a minimum of 3 pre - defined points ( anchor points ) are required in order to reconstruct the applicator . successful if , after placement of the anchor points and any additional manual manipulation , the reconstructed applicator was clearly seen to lie within 2 mm of the visualised applicator in the mr images . this value was chosen as it represents the tolerance placed on applicator reconstruction defined by the aapm task group 56 . each image set was then assessed for total scanning time and usefulness in tumour localization via inter- and intra - observer analysis of high - risk clinical target volume ( hr ctv ) contouring . hr ctv contours were generated based on gec - estro recommendations [ 1 , 2 ] for each data set by three radiation oncologists experienced in gynaecological brachytherapy , who were blinded to the scanning technique . anatomical changes and geographical shifts between imaging sequences were also examined , along with visual differences between the contouring and planning modules of the brachytherapy treatment planning system . intra - observer analysis ( comparing contours from a single radiation oncologist between different imaging sequences ) was performed by determining the relative volume difference from the mean in hr ctvs between image sets for the same patient . these values were then averaged across all patients in the study . inter - observer analysis ( comparing contours from all three radiation oncologists for a single image sequence ) was performed not only by determining the relative volume difference in hr ctvs , but also by applying a conformity index ( ci ) based on common and encompassing volume for all 3 observers . the ci was determined based on the work by kouwenhoven et al . that allowed a ci to be determined independent of the number of observers . the ci is given by the following equation ( 1):1ci=k=1kk(k-1)vk2(k-1)k=1kkvk-k=1kk(k-1)vk where : k total number of observers , v k volume encompassed by k observers . the study was performed on 20 cases referred to the radiation oncology department for the treatment of cervical cancer with brachytherapy . all patients had mr imaging performed for brachytherapy planning after plastic - based , mr compatible ring applicator ( ring ct / mr applicator , nucletron , an elekta company ) placement under ultrasound guidance and general anaesthesia . the age range of patients was between 31 - 88 years with a mean age of 54.3 years and all patients were rated figo ( international federation of gynaecology and obstetrics ) stage iib to iiib . the range of contoured hr ctv for all patients was between 47.9 and 9.7 cm with a mean of 22.6 cm . the range of hr ctv dimensions ( l - r , s - i and a - p ) were between 4.5 , 4.6 and 5.5 cm and 1.9 , 1.7 , and 1.8 cm , respectively . all patients underwent external beam radiation therapy ( ebrt ) between 45 and 52.2 gy in 1.8 or 2 gy fractions . hdr brachytherapy ( 24 gy in 8 gy fractions prescribed to the 100% isodose line ) was commenced either within the final week or within a week of finishing ebrt . each insertion was given 1 week apart , with the applicator removed after each insertion and mr - based treatment planning occurring for each fraction . all patients had cervical hdr brachytherapy applicators inserted under general anaesthetic , underwent mr scanning in radiology after leaving recovery and were then transported to the department of radiation oncology for treatment . a 3 t ( skyra , siemens healthcare ag , germany ) mr imaging system with combination of an 18 channel body matrix and 32 channel phase array spine coil was used for generating hdr cervix brachytherapy planning images . patients were positioned in supine head - first orientation with a body matrix coil placed over the pelvis . a small foam wedge was placed between the pelvis and coil to help reduce anterior abdominal wall motion . the pre - emptied bladder was filled with 100 ml of saline for adequate distension of the bladder during imaging and reproducibility of bladder volume at treatment . an anti peristalsis agent was administered intravenously ( hyoscine butylbromide 20 mg in 1 ml ) to minimise ghosting artefacts from gross peristaltic movements during data acquisition . scanning sequences that were part of the standard radiation therapy scanning practice in our department were considered for assessment . in collaboration with a radiologist and mr imaging specialist , a parallel imaging technique , generalized auto - calibrating partially parallel acquisition ( grappa ) algorithm ( siemens healthcare ag , germany ) with r factor of 2 was incorporated in all sequences to reduce scan times to tolerable limits . spatially selective saturation bands were used superiorly and anteriorly on all sequences to suppress the signal from inflowing spins and reduce motion related artefacts . t2 weighted turbo spin echo ( tse ) sequences were selected without the application of iv contrast media in keeping with international recommendations . both 3d t2-weighted sampling perfection with application - optimised contrast and different flip - angle evolutions ( space , siemens healthcare ag , germany ) ( 3d t2 ) sequences and contiguous 2d t2-weighted tse sequences ( 2d t2 ) scans were performed for comparison in sagittal , axial , and oblique planes tilted to correspond to the treatment applicator . firstly , a 3-plane half - fourier acquisition single - shot turbo spin - echo ( haste , siemens healthcare ag , germany ) localizer was taken . this haste scan was used for planning the axial and oblique - axial sequences oriented to correspond to the treatment applicator . initially , a 2d t2 weighted tse ( 2d t2 ) sequence was obtained in the sagittal plane between the obturator muscles , and a 2d t2 sequence was positioned and acquired in a true axial plane and reviewed for both applicator and tissue delineation . a 3d t2 volumetric sequence was also performed in a true axial plane with an isotropic 3d t2 volumetric sequence acquired in the sagittal plane . both a 2d t2 sequence and a 3d t2 volumetric sequence were planned and acquired para - axially with the scanning plane aligned parallel to the ring surface from the sagittal haste scan ( axial oblique , see fig . another 2d t2 sequence was then obtained with the scanning plane parallel to the ring surface , based on both the sagittal and the coronal reconstructions of the haste localiser ( axial double oblique , see fig . axial - oblique plane , aligned parallel to the plane of the ring in the sagittal scan axial double - oblique additional plane , aligned parallel to the plane of the ring in the coronal localizer scan all scan volumes included the uterine fundus superiorly and the distal end on the treatment applicator inferiorly . all scanning sequences were designed to have slice thicknesses less than 2 mm to aid in 3d reconstruction in the brachytherapy treatment planning system . magnetic resonance scanning parameters used for image acquisition tse turbo spin echo , space sampling perfection with application - optimised contrast and different flip - angle evolutions , tr relaxation time , te echo time , fov field of view , nex number of excitations after importation into the treatment planning system ( oncentra brachy version 4.2 and 4.3 , nucletron , an elekta company , sweden ) , the images were initially evaluated by an experienced medical physicist with regards to the ability to reconstruct the treatment applicators . an applicator library ( applicator modelling module in oncentra version 4.2 and 4.3 , nucletron , an elekta company , sweden ) was used in the reconstruction of each applicator . in this module , a minimum of 3 pre - defined points ( anchor points ) are required in order to reconstruct the applicator . successful if , after placement of the anchor points and any additional manual manipulation , the reconstructed applicator was clearly seen to lie within 2 mm of the visualised applicator in the mr images . this value was chosen as it represents the tolerance placed on applicator reconstruction defined by the aapm task group 56 . each image set was then assessed for total scanning time and usefulness in tumour localization via inter- and intra - observer analysis of high - risk clinical target volume ( hr ctv ) contouring . hr ctv contours were generated based on gec - estro recommendations [ 1 , 2 ] for each data set by three radiation oncologists experienced in gynaecological brachytherapy , who were blinded to the scanning technique . anatomical changes and geographical shifts between imaging sequences were also examined , along with visual differences between the contouring and planning modules of the brachytherapy treatment planning system . intra - observer analysis ( comparing contours from a single radiation oncologist between different imaging sequences ) was performed by determining the relative volume difference from the mean in hr ctvs between image sets for the same patient . inter - observer analysis ( comparing contours from all three radiation oncologists for a single image sequence ) was performed not only by determining the relative volume difference in hr ctvs , but also by applying a conformity index ( ci ) based on common and encompassing volume for all 3 observers . the ci was determined based on the work by kouwenhoven et al . that allowed a ci to be determined independent of the number of observers . the ci is given by the following equation ( 1):1ci=k=1kk(k-1)vk2(k-1)k=1kkvk-k=1kk(k-1)vk where : k total number of observers , v k volume encompassed by k observers . all sequences were able to be imported into the brachytherapy treatment planning system , however , those sequences that contained oblique planes required any automatic image fixing to be disabled . due to the use of plastic - only treatment applicators , image distortion effects that are known to be present particularly in 3 t mr imaging were negligible . visualization and eventual reconstruction of the applicator by the medical physicist resulted in agreement between the applicator model and the mr images of 4 mm at first pass , and less than 2 mm after manual manipulation for all imaging sequences . table 1 indicates the scanning times for each of the sequences tested . a double oblique axial t2 3d scan was not used in this study as the scanning time the double oblique axial t2 2d scan had the fastest scan time after the t2 2d sagittal scan , however the sagittal scan had a much larger slice thickness ( 2.5 mm compared with 1.8 mm ) . results for intra- and inter - observer hr ctv contouring are shown in table 2 . for intra - observer hr ctv analysis , both 2d and 3d sagittal image sequences had the worst consistency across all radiation oncologists with an average difference of 19.8% and 15.1% , respectively . the best volume consistency across all radiation oncologists was for 2d axial double oblique and 3d axial oblique image sequences , with an average difference of less than 1% , and there was an average difference of 3.1% across all 2d and 3d axial image sequences . intra- and inter - observer differences for different 3 t t2 mr scanning sequences when comparing volume sizes only for inter - observer hr ctv analysis , the mean relative sd across all cases and sequences was 24.2% . the 2d axial double oblique image sequence had the best consistency with a mean relative sd of 9.2% and the 2d sagittal the worst with a mean relative sd of 37% . the 2d axial double oblique image sequence had the highest conformity index ( mean 0.80 , range 0.72 - 0.91 ) , while the 2d axial image had the worst ( mean 0.55 , range 0.54 - 0.75 ) . problems may arise with reconstruction from mr imaging scans as it is very difficult to visualize the internal structure of the applicator to confirm the source path . whilst markers and applicator libraries can be effective in detailing the source path , there are uncertainties in reconstruction that relate directly to the slice thickness of the imaging scans used . reconstruction uncertainties have been noted to be greater in the direction perpendicular to slice orientation which is commonly linked to the large pixel size or slice thickness in this direction [ 15 , 19 ] . it is recommended that slice thickness be as small as possible without increasing mr image acquisition time too greatly as this increases the risk of patient motion during the scan . reducing the mr slice thickness to less than 2 mm can help minimize random error in applicator reconstruction . this type of scan is only achievable in high strength ( 3 t ) mr scanners , as the loss of snr , increased scanning time , and increased potential for patient motion [ 15 , 19 ] would be too great for lower strength mr scanners . the gec estro recommendations for 3d image guided brachytherapy of the cervical cancer are acknowledged as being a well - defined method for creating target and organ at risk volumes with which to optimize a patient treatment [ 13 ] . even with these guidelines , there are still uncertainties and different interpretations of clinical and pathological disease , implying that it is not possible to have complete agreement between observers . there have been several studies into inter - observer differences for target volume delineation in cervical cancer brachytherapy . ( 2013 ) performed a multi - institutional study with 10 observers and 6 cases . the inter - observer relative sd for the hr ctv was up to 42% with an average conformity index of 0.74 . in 2008 , petri et al . investigated hr ctv contour differences between axial and para - axial ( oblique axial ) image sets for a 0.2 t mr scanner . the inter - observer conformity index for the axial and para - axial sequences was given as 0.79 and 0.78 , respectively . there was a large difference between the 2d t2 axial oblique and 2d t2 axial double oblique , 30.6% mean relative sd dropping to 9.2% mean relative sd . this indicates that all scanning planes must be carefully and individually designed before a scan takes place and highlights the importance of testing multiple imaging planes , when setting up a mr imaging program for cervical cancer brachytherapy . the dosimetric impact of inter - observer variability has been studied , with levels of uncertainty in hr ctv contouring causing variation of 5 gy in d90 over the total course of radiotherapy ( external beam and brachytherapy ) or an uncertainty of 9% for a single intracavitary brachytherapy fraction . this study used images from mr scanners with magnet strength 0.2 t and 1.5 t . the mean relative sd of the contoured hr ctv was 19% ( across 10 observers and 6 cases ) . interestingly , the patients scanned on the 0.2 t mr scanner had lower mean sd than the patients scanned on the 1.5 t ( 17% vs. 21% ) . this is compared to 9.2% for 3 t mr 2d axial double oblique scans in the present study . in any case , it is ideal to eliminate as much as possible any potential causes of observer contouring variation . there have been several studies that have looked into ways to reduce this variation [ 24 , 25 ] . in these they recommended multiple imaging modalities , the latter two recommendations can be addressed using the gec - estro contouring criteria [ 13 ] , while the second recommendation can be achieved by optimizing the choice of scanning sequence and the quality of the imaging system . ( 2012 ) studied the differences in contours between 2d multi - planar scanning sequences ( as recommended by gec estro ) and a 3d single scanning sequence for a 1.5 t mr scanner and found little difference in target contours for 2 observers and 14 patients . the results of our study also indicate that there is minimal difference in hr ctv contours when completed on 2d or 3d image sets using a 3 t mr scanner . several diagnostic studies have also indicated little difference in image quality between 2d t2 weighted and 3d space t2 weighted images . the main benefit of using a 3d space t2 image set was defined as the time saving over using multi - planar 2d t2 image sequences [ 2729 ] . if the 2d multi - planar image set could be reduced to a single image set without compromising image quality in 3-dimensional reconstructions , the reduction in scanning time could be even further improved over the 3d t2 sequence . this study has examined 7 different 3 t mr scanning sequences in order to determine which single image set provides the best consistency for target delineation , both intra- and inter - observer . whilst a limitation to this study is the size of the number of observers ( n = 3 ) , this work still highlights the variability that the choice of scanning sequence can produce . the organ at risk ( oar ) contour variation was not performed in this study . further work investing the possible changes to oar volume definition for varying 3 t mr sequences will be carried out . patient shifts sometimes seen between scanning sequences can vary the position of the applicator relative to the ctv and oar , resulting in inconsistencies if using multiple scanning sequences to contour and plan . this would be a particular concern if one sequence was used for contouring and another sequence used for planning as the relationship between source position and ctv or oar may have changed between scanning sequences . sagittal and coronal images as recommended by gec estro [ 13 ] are currently mostly used for verification purposes as treatment planning software does not allow users to contour a single structure across multiple imaging planes . a standard sagittal 2d t2 tse sequence scan can also be performed if time permits , and there appears to be little patient motion during the para - axial scan . this scan can then be a useful tool in single plane verification of the ctv and oar contours if required . using the 2d axial double oblique scanning sequence , the total scanning time for each patient can be reduced to less than 7 minutes . even with a bladder filling protocol and administration of anti - peristalsis agent in the scanning room , patients can be in and out of the mr scanning room in less than 15 minutes . this has multiple benefits to the patient with less time isolated inside the scanning room and less time required to be lying compliant on the scanning table . this is particularly important in smaller centres that do not have the capability to generate treatment planning images , whilst the patient is under a general anaesthetic . the rapid turnaround of patients also allows a more efficient use of staff time and equipment resources . different mr scanning sequences can greatly affect the disparity in hr ctv contours between radiation oncologists . intra and inter - observer variation in hr ctv contouring suggests a 2d t2 tse axial sequence oriented in all planes to the treatment applicator ( double oblique ) on a 3 t mr scanner , allows more accurate and uncomplicated tumour determination in brachytherapy treatment planning of cancer of the cervix . applicator visualization is sufficient for planning requirements and the thinner slice thickness reduces the expected uncertainty in applicator reconstruction . the ability to contour , plan , and optimize the brachytherapy treatment using a single image set that is orientated in the most useful plane for these tasks is a great benefit . this imaging sequence was tested on 20 cases , but has since been successfully applied to another 35 cases , including patients using an interstitial / intracavitary applicator ( interstitial ring ct - mr applicator set , nucletron , an elekta company , sweden ) . minimizing scanning time through elimination of excess scanning sequences implies greater patient throughput via reduced demand on the mr scanner and lessens patient discomfort via reducing the time required to be on the mr scanning table . this is a critical factor in departments that do not have access to an mr unit whilst the patient is under general anesthetic .
imatinib , a specific tyrosine kinase inhibitor is a newer anticancer agent , which has shown excellent efficacy in managing chronic myeloid leukemia . it is generally well tolerated with few side effects . most commonly reported adverse events are maculopapular eruptions and periorbital edema . severe adverse reactions are seen in 5% of patients . exfoliative dermatitis has been very rarely reported with this drug . we report a case of a 52-year - old male who initially presented with a maculopapular rash and developed erythroderma on continuation of the drug .	imatinib , a specific tyrosine kinase inhibitor has shown excellent efficacy in management of chronic myeloid leukemia ( cml ) . there may be almost complete remission hematologically in the chronic phase of management of cml due to this drug . most commonly reported adverse events are maculopapular eruptions , periorbital edema , and the less commoner ones include steven johnson syndrome - toxic epidermal necrolysis ( sjs / ten ) , acute generalized exanthematous pustulosis , hypopigmentation , lichenoid reaction , pityriasis rosea , and sweet 's syndrome . a 52-year - old male presented to the medical outpatient department with complaints of low grade fever and lump on the left side of abdomen since one month duration . bone marrow examination revealed 2% blast cells , serum lactate dehydrogenase was 1134 iu / l , and an ultrasound of the abdomen revealed splenomegaly with span of 19 cm . the case was diagnosed as cml in chronic phase and patient was prescribed imatinib mesylate 400 mg once daily . ten days later , he developed redness and scaling involving dorsum of both hands which increased to involve his entire body over a period of 20 days . there was no history of any other drug intake , or history of fever , jaundice , chest pain , palpitation and dyspnoea on exertion . imatinib was continued and the rash worsened . when the patient was referred to us , he had generalized skin rash of 40 days duration . general physical examination was unremarkable while systemic examination revealed splenomegaly , 3 cm below costal margin . dermatological examination revealed generalized involvement of the body in form of dusky , blanchable erythema , and diffuse fine scaling . lichenification was noted in flexures viz groin , axillae and neck [ figures 1 and 2 ] . investigations revealed hemoglobin of 14.5 gm% , total leukocyte count 11,500/mm , and differential count was polymorphs- 50 , lymphocytes- 8 , monocytes- 2 , and eosinophils 40 . urine examination , blood sugar , renal , and liver function tests were within normal limits . extensive scaling of ( a ) neck ( b ) axillae and upper chest involvement of ( a ) lower limbs and ( b ) both forearms imatinib was immediately stopped and patient was prescribed on tablet prednisolone 40 mg / day , which was maintained at full dose for two weeks , tapered by 10 mg every week , and stopped after a total of five weeks . supportive measures including high protein diet , appropriate temperature control , fluid , and electrolyte balance were provided . improvement was noted with decreased erythema on fifth day and significant reduction in scaling by day 11 . the patient was changed to an alternative anti - cml medication ( cyclophosphamide based ) and rechallenge or desensitization was not resorted to , as erythroderma is considered a severe form of drug reaction . despite common occurrence of cutaneous adverse event with imatinib , severe adverse cutaneous drug reactions are uncommon and seen in 5% of cases . acute generalized exanthematous pustulosis , epidermal necrolysis , and steven johnson syndrome have been reported previously . other rare reactions mentioned include mycosis fungoides like eruption , follicular mucinosis , porphyria cutanea tarda , neutrophilic eccrine hidradenitis , eccrine squamous syringometaplasia , and panniculitis . there are only six probable cases of exfoliative dermatitis due to imatinib reported in literature . exfoliative dermatitis generally occurs 1 - 3 wks after starting therapy on initial exposure and within hours to days on rechallenge . mechanism of development of rash after imatinib administration is not known , however , hypersensitivity reaction as a mechanism has been postulated . in this case causality assessment using naranjo scale showed that imatinib was the probable cause for the adrs ( score 7 ) . most of the rashes due to imatinib are self limiting and do not require discontinuation of treatment . desensitization therapy can be used by administering increasing doses of drug in cases of mild reactions . erythroderma necessitating stoppage of therapy is considered as grade 4 toxicity according to national cancer institute / national institutes of health ( nci / nih ) common toxicity criteria . it is important to recognize the cutaneous adverse effects of this drug , so that severe and potentially life threatening adverse reaction can be identified early and remedial action can be taken early . it is equally important to continue therapy in minor cutaneous adverse events so that patient can get benefit from treatment with this gold standard drug for cml .
the prevalence of childhood - onset type 2 diabetes mellitus has increased dramatically over the past two or three decades in japan , but epidemiological and clinical data remain limited . this survey was conducted to elucidate the current use of antidiabetic medications and the efficacy , safety and problems associated with the use of these agents . clinical data on 259 children ( younger than 18 yr of age ; 121 boys and 138 girls ) with type 2 diabetes treated at 42 medical centers throughout japan between june and september 2003 were analyzed . sixty - nine percent of all the type 2 diabetic patients ( 78% of the boys , 63% of the girls ) were obese ( percent overweight 20% ) at the time of diagnosis . overall , 172 subjects ( 66% ) were treated using anti - hyperglycemic agents , including -glucosidase inhibitors ( -gi ) , insulin , metformin and sulfonylureas ( sus ) . many patients who were initially treated with a single medication eventually required insulin alone or in combination with an additional agent , suggesting that their diabetic control had deteriorated during the course of treatment . the hba1c level of the 14 subjects who received only metformin decreased significantly without an improvement in obesity . three cases with adverse events were reported , but causal relations with anti - hyperglycemic agents were not clear . in conclusion , mainly -gi , insulin and metformin have been prescribed for childhood - onset type 2 diabetes patients in japan . the results of this survey suggest that metformin is safe and effective for the treatment of type 2 diabetes with obesity in children and adolescents .	the prevalence of childhood - onset type 2 diabetes mellitus has increased dramatically over the past two or three decades in japan ( 1 ) . several nation - wide surveys have been conducted , but epidemiological and clinical data remain limited ( 2 ) . it is well known that life style modification and weight loss are the most important interventions that may realistically be expected to prevent disease progression and complications in type 2 diabetes mellitus in adults ( 3 , 4 ) . if the treatment goals with nutrition education and exercise are not met , pharmacologic therapy is indicated for children with type 2 diabetes ( 5 ) . indeed , many pediatricians realize the necessity of medical therapy for type 2 diabetes in children , and research on oral anti - hyperglycemic agents for children and adolescents is warranted . the uk prospective diabetes study ( ukpds ) reported that intensive blood - glucose control with sulfonylureas ( sus ) or insulin substantially reduced the risk of complications but not macrovascular disease in adult type 2 diabetes patients ( 6 ) . the ukpds also reported that , since intensive glucose control with metformin appears to decrease the risk of diabetes - related endpoints in overweight diabetic patients , and is associated with less weight gain and fewer hypoglycemic attacks than are insulin and su , it may be the first - line pharmacological therapy of choice for these patients ( 7 ) . metformin has been recommended as a first choice for the treatment of type 2 diabetes in children and adolescents by the american diabetes association ( ada ) ( 5 ) . metformin improves glycemic control by reducing hepatic glucose production , increasing insulin sensitivity , and reducing intestinal glucose absorption , without increasing insulin secretion ( 8 , 9 ) . in the u.s . , metformin was approved by the fda in 2000 for the treatment of diabetes in children based on results from a randomized controlled trial ( 10 ) . in japan , no oral anti - hyperglycemic agents , including metformin , have been approved for children with type 2 diabetes by the ministry of health , labour and welfare . furthermore , information on the efficacy , safety and problems associated with the use of anti - hyperglycemic agents for children is insufficient . this survey was conducted to elucidate the current use of antidiabetic medication and the efficacy , safety and problems associated with these agents in children and adolescents with type 2 diabetes mellitus in japan . a questionnaire survey targeted councilors of the japanese society for pediatric endocrinology ( jspe ) and members of the japanese study group of insulin therapy for childhood and adolescent diabetes ( jsgit ) in february 2003 , in advance of our main survey of current medical treatments for childhood - onset type 2 diabetes mellitus . seventy - two doctors currently managed childhood - onset type 2 diabetes patients , and the total number was estimated to be 530 patients . clinical data sheets were sent to the 70 members of the jspe and jsgit , who signed up to participate in this survey , in june 2003 . clinical data on 259 children ( younger than 18 yr of age ; 121 males and 138 females ) with type 2 diabetes treated at 42 medical centers throughout japan were received between june 2003 and september 2003 , and were then analyzed . the percent overweight was determined on the basis of the japanese standard body weights for height by age and sex : ( actual body weight the study was approved by the institutional ethics review board of tokyo women s medical university and informed consent was obtained from the patients and/or their parents . the mann - whitney u , and fisher exact probability tests were used for statistical analysis . the mann - whitney u , and fisher exact probability tests were used for statistical analysis . the ages of the 259 recruited patients were 11.9 2.1 yr ( 616 yr ) at diagnosis and 14.4 2.0 yr ( 917 yr ) at survey . their hba1c were 8.8 2.8% ( 4.418.4% ) at diagnosis and 7.0 2.2% ( 4.114.2% ) at survey . sixty - nine percent ( n=174 ) of 253 patients had a family history of diabetes : father , mother , siblings and/or grandparents . 1 the percent overweight distribution of patients at the time of type 2 diabetes mellitus diagnosis . the percent overweight was determined on the basis of the japanese standard body weights for height by age and sex : ( actual body weight standard weight)/ standard weight 100 ( % ) . seventy - eight % ( n=93 ) of the boys and 63% ( n=86 ) of the girls were obese ( percent overweight 20% ) at the time of diagnosis . shows the distribution of percent overweight at diagnosis of type 2 diabetes . seventy - eight percent ( n=93 ) of the boys and 63% ( n=86 ) of the girls were obese ( percent overweight 20% ) at the time of diagnosis . thirty - nine percent ( n=47 ) of the boys and 23% ( n=31 ) of the girls had percent overweight values 50% . the percent overweight distribution of patients at the time of type 2 diabetes mellitus diagnosis . the percent overweight was determined on the basis of the japanese standard body weights for height by age and sex : ( actual body weight standard weight)/ standard weight 100 ( % ) . seventy - eight % ( n=93 ) of the boys and 63% ( n=86 ) of the girls were obese ( percent overweight 20% ) at the time of diagnosis . one - hundred and seventy subjects ( 66% ) were diagnosed through school - age screening for glycosuria . hba1c levels of patients identified by school - age glycosuria screening were 8.4 2.6% and most were asymptomatic . eighty - nine patients were found by means other than school - age screening , and their hba1c levels were 9.5 3.0% . hba1c levels of patients identified by school - age glycosuria screening were significantly lower than those of the others ( p<0.005 ) . eighty - seven ( 34% ) of 259 subjects were treated without medication by adjusting their diet and physical activities . one - hundred and seventy - two subjects ( 66% ) were treated using anti - hyperglycemic agents , including -glucosidase inhibiter ( -gi ) , insulin , metformin , sulfonylureas ( sus ) and nateglinide . table 2table 1 prescribed medication for 172 type 2 diabetes patients in this surveytable 2 clinical characteristics of medicated or unmedicated patients with type 2 diabetes mellitus shows clinical characteristics of medicated and unmedicated subjects with type 2 diabetes . percent overweight at diagnosis was higher in unmedicated ( 46.0 32.4% ) than in medicated ( 34.9 31.8% ) patients ( p<0.005 ) . hba1c levels of unmedicated patients were lower than those of medicated patients at both diagnosis ( 7.3 2.1% vs. 9.5 2.8% , p<0.0001 ) and survey ( 5.9 1.2% vs. 7.4 2.4% , p<0.0001 ) . the frequency of a family history of diabetes was significantly higher in medicated ( 75.9% ) than in unmedicated ( 55.2% ) patients ( p<0.005 ) . -gi was the most commonly used anti - hyperglycemic agent , followed by insulin , metformin , sus , nateglinide and a combination of -gi and metformin ( table 3table 3 age , hba1c and percent overweight at the time of diagnosis according to prescribed medications ) . figure 2fig . 2 distribution of hba1c at the time of diagnosis according to prescribed medication . mainly -gi was prescribed for children with a lower hba1c level , while mainly insulin was prescribed for children with a higher hba1c level . shows the distribution of hba1c at the time of diagnosis according to the prescribed medication . -gi was prescribed mainly for children with a lower hba1c level ( 8.1 2.5% ) , while insulin was prescribed mainly for subjects with a higher hba1c level ( 11.3 2.6% ) at the time of diagnosis ( table 3 , fig . 2 ) . metformin and sus were given mainly to subjects with moderate levels of hba1c , while metformin was prescribed mainly for subjects with a higher percent overweight ( 49.9 29.9% ) in contrast to sus in subjects with lower percent overweight ( 21.4 28.1% ) at the time of diagnosis ( table 3 , fig . 3fig . 3 distribution of percent overweight at the time of diagnosis according to prescribed medication . metformin was prescribed mainly for subjects with a higher percent overweight , while sus was given to those with a lower percent overweight at the time of diagnosis . ) . mainly -gi was prescribed for children with a lower hba1c level , while mainly insulin was prescribed for children with a higher hba1c level . metformin and sus were mainly prescribed for children with a moderate hba1c level . metformin was prescribed mainly for subjects with a higher percent overweight , while sus was given to those with a lower percent overweight at the time of diagnosis . table 4table 4 age , hba1c and percent overweight at the time of survey according to prescribed medications shows age , hba1c and percent overweight at the time of survey according to the prescribed medication . many patients who were initially treated with a single agent eventually required combination therapy with an additional medication . twenty - three ( 38% ) of 61 patients who started with -gi alone remained on monotherapy with -gi . however , 10 ( 16% ) of 61 patients who started with -gi were given insulin therapy , 14 ( 23% ) were also given metformin , 9 ( 15% ) had sus added to their treatments , and for 8 ( 13% ) nateglinide was added ( fig . 4fig . twenty - three ( 38% ) of 61 patients who started on -gi alone remained on -gi monotherapy . however , 10 ( 16% ) of 61 patients who started on -gi were also given insulin therapy , metformin was added in 14 ( 23% ) , cases sus in 9 ( 15% ) , and nateglinide in 8 ( 13% ) . ) . the addition of another agent to -gi treatment for 59% of patients suggests that their diabetic control had not changed for the better or had deteriorated during the course of treatment . twenty - three ( 38% ) of 61 patients who started on -gi alone remained on -gi monotherapy . however , 10 ( 16% ) of 61 patients who started on -gi were also given insulin therapy , metformin was added in 14 ( 23% ) , cases sus in 9 ( 15% ) , and nateglinide in 8 ( 13% ) . two ( 8% ) of 24 patients who started with metformin alone were given insulin therapy ( fig . two ( 8% ) of 24 patients started on metformin alone , and insulin therapy was later added . ) . on the other hand , 13 ( 25% ) of 51 patients who started with insulin alone were able to discontinue insulin therapy , suggesting recovery of endogenous insulin secretion ( fig . thirteen ( 25% ) of 51 patients who started on insulin alone were able to discontinue insulin therapy , suggesting the recovery of endogenous insulin secretion . ) . two ( 8% ) of 24 patients started on metformin alone , and insulin therapy was later added . thirteen ( 25% ) of 51 patients who started on insulin alone were able to discontinue insulin therapy , suggesting the recovery of endogenous insulin secretion . fourteen subjects from 8 centers were treated with metformin alone ( table 5table 5 changes in hba1c and percent overweight in 14 type 2 diabetes patients treated with metformin alone ) . regarding the metformin dosage , 7 of the 14 received 500 mg or less , 5 were given 750 mg , and 2 subjects received 1000 mg . the hba1c level of the 14 subjects who received only metformin decreased significantly from 9.1 1.9% to 6.7 1.3% ( p=0.001 ) without a significant improvement in obesity ( table 5 ) . three cases with adverse events were reported ( table 6table 6 three cases with adverse events reported in this survey ) , but causal relations with metformin were not clear . school - age screening for glycosuria has proven useful for finding diabetes mellitus in japan , but a follow - up system for the diabetic patients identified has not yet been established in many regions of japan . furthermore , the precise number of children and adolescents with childhood - onset type 2 diabetes in japan is unknown . the nation - wide survey in 1999 by ohki et al . recruited 812 patients less than 18 yr of age with type 2 diabetes mellitus in japan ( 2 ) . they estimated that the prevalence of type 2 diabetes in japan was 3.25 : 100,000 children . in 2000 , 1,019 patients with type 2 diabetes and 3,740 with type 1 diabetes were registered with the official fund for chronic diseases in children and adolescents in japan ( 12 ) . in our survey , about 530 patients with type 2 diabetes were estimated to be under the management of pediatric endocrinologists in 2003 in japan and clinical records of 259 patients were obtained and analyzed . the present survey may represent one half to one quarter of the population with childhood - onset type 2 diabetes in japan . this survey revealed the clinical characteristics of childhood - onset type 2 diabetes and current medical treatment for type 2 diabetes by pediatric endocrinologists in japan . this survey also revealed that 78% of the boys and 63% of the girls were obese ( percent overweight 20% ) at the time of their diagnosis . obesity was confirmed to be one of the main factors associated with a childhood - onset type 2 diabetes in japan . it was notable that the percent overweight distribution was similar between male and female patients except for a high frequency in non - obese female patients with 010% of percent overweight ( fig . no individual in this non - obese female population ( n=25 ) had positive gadab titers . the frequency of a family history of diabetes in this non - obese female population was slightly higher ( 76% ) than that of the obese population ( 68% ) , suggesting that this non - obese female population may be associated with stronger genetic factors including mody . in total , 172 subjects ( 66% ) were treated with anti - hyperglycemic agents . the frequency of anti - hyperglycemic agent usage has doubled , as compared to the approximately one third of patients who were on medication in the survey of 1999 by ohki et al . mainly -gi , insulin and metformin are currently prescribed for childhood - onset type 2 diabetes patients . -gi is used mainly for children with a lower hba1c level , in contrast to insulin which is mostly prescribed for subjects with a higher hba1c level at the time of diagnosis ( table 3 , fig . 2 ) . metformin and sus were prescribed mainly for subjects with moderate levels of hba1c , metformin mainly for those with a higher percent overweight in contrast to sus given to subjects with a lower percent overweight at the time of diagnosis ( table 3 , fig . the choice of these medications in this survey is considered to be reasonable , given the functional mechanism of each agent ( table 1 ) . after the start of treatment with a single medication , combination therapy with an additional agent increased in many patients , suggesting that their diabetic control had not changed for the better or had deteriorated during the course of treatment . further study to clarify the clinical characteristics of patients with poor diabetic control during the course of treatment may be necessary . medication for children with type 2 diabetes has not been approved by the ministry of health , labour and welfare . anti - hyperglycemic agents except for insulin are all off - label pharmaceuticals for children with diabetes in japan . the american diabetes association consensus statement in 2000 was , of note is the fact that efficacy and safety data are not available for children nor are any of the oral drugs fda approved for use in children . if treatment goals with nutrition education and exercise are not met , pharmacologic therapy is indicated . the first oral agent used should be metformin ( 5 ) . in the u.s . , metformin was approved in 2000 by the fda for use in children ( older than 10 years of age , maximum dose : 2000 mg / day ) , based on the results of a randomized controlled trial ( 10 ) . in the ukpds report , metformin similarly improved hba1c level as did sus and insulin therapy but did not induce weight gain ( 7 ) . in meta - analysis metformin lowers blood glucose and hba1c significantly , compared with placebo , and the body weight is significantly lower after metformin treatment compared with su treatment because of an increase in body weight after su treatment ( 13 ) . our survey reveals that the hba1c level of the 14 subjects who received only metformin decreased significantly without a remarkable change of percent overweight ( table 5 ) . lactic acidosis is a life - threatening condition characterized by low arterial ph ( < 7.35 ) and elevated arterial lactate levels ( > 5.0 meq / l ) . lactic acidosis is precipitated by phenformin , a biguanide formerly used to control hyperglycemia in type 2 diabetes . metformin is now the only biguanide in general use , since it has a 1020-fold lower risk of lactic acidosis than phenformin , and is regarded as a safe drug provided it is not used in at - risk patients , such as those with renal failure ( 14 ) . there is no evidence to date that metformin therapy is associated with an increased risk of lactic acidosis or with increased levels of lactate compared with other antihyperglycemic treatments if the drugs are prescribed under study conditions , taking into account contraindications ( 15 ) . three cases with adverse events were reported ( table 6 ) , but causal relations with metformin were not clear , and there were no reports of lactic acidosis among 51 patients treated with metformin in this survey . these results provide very useful and important information for us . in conclusion , mainly -gi , insulin and metformin have been prescribed for childhood - onset type 2 diabetes patients in japan . this survey suggests that metformin is safe and effective for the treatment of type 2 diabetes with obesity in children . additional studies on the efficacy and safety associated with a higher dosage ( 10001500 mg ) of metformin in japanese children may be required . furthermore , we hope that a medication for children with type 2 diabetes will be approved by the ministry of health , labour and welfare in the near future .
lung cancer remains the leading cause of cancer - related mortality worldwide . early detection of lung cancer is problematic due to the lack of a marker with high diagnosis sensitivity and specificity . to determine the differently expressed proteins in the serum of lung cancer and figure out the function of the proteins , two - dimensional electrophoresis ( 2de ) and matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof - ms ) were used to screen the serum proteins of lung cancer model induced by 3-methylcholanthrene ( mca ) . from optimized 2de image , 455 spots in the normal sera and 716 spots in the lung cancers sera were detected . among them , 141 protein spots were differentially expressed when comparing the serum from normal rat and serum from lung cancer model , including 82 overexpressed proteins and 59 underexpressed proteins . changes of haptoglobin , transthyretin , and tnf superfamily member 8 ( tnfrs8 ) were confirmed in sera from lung cancer by maldi - tof - ms . proteomics technology leads to identify changes of haptoglobin , transthyretin , and tnfrs8 in serum of rat lung cancer model and represents a powerful tool in searching for candidate proteins as biomarkers .	statistics demonstrate that , in the past 20 years , with the development of industry and aggravation of air pollution , the morbidity rate and mortality rate of lung cancer are increasing most quickly in all malignant tumors in china . in cities , the mortality of lung cancer is 27.5/1 000 000 , accounting for 22.4/100 of all death in malignant tumors . the five - year survival rate is 14/100 in america , less in china . besides the reasons of environment pollution , smoking , heredity , lack of early diagnostic tools is also the important factor which contributes to that bad situation . in the recent years , with the development of biology and imageology , for example , spiral ct , cytological examination of sputum and endoscopy of bronchus , and so on , positive rate of early diagnosis is increasing but there are related limitations in all kinds of methods . the sensitivity of fine needle aspiration biopsy ( fnab ) in diagnosis of malignant nodes is 70/100100/100 , but it may destroy some normal tissues and bring about some complications , for instance , pneumatothorax and hemoptysis . blood plasma is the most easily controlled and acquired specimen . so making use of serum sample to detect special markers is the most perfect method in diagnosis of clinical lung cancer . the reasons why we choose the blood plasma as the pathfinder of proteome research are as follows . ( i ) serum proteome is the most complicated proteome , including inferior proteome in different tissues . ( ii ) it is easy to obtain enough serum proteins as research sample and it is easy to be standardized . ( iii ) there is a large range of dynamic state in property of serum proteins . ( iv ) there is no strict linear relationship between the expression levels of the mrnas and proteins . the modification after translation , such as glycosylation and phosphorylation , is more and more popular , so it is necessary to globally analyze the secreted proteins expressed by cells or tissues . in the research of neoplastic serum proteomics , the related proteomics technology is principally used to fathom the change of serum proteins in the course of forming tumor , to screen and discover the tumor markers and potential drug targets which could be used for classification , early diagnosis , therapy , and intervention of the tumor . nowadays , two technical modes are often applied in the research of serum proteomics : ( i ) surface - enhanced laser desorption and ionization take - of - flight mass spectrometry ( seldi - tof - ms ) , ( ii ) matrix assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof - ms ) . the new generation of maldi - tof - ms could quickly , exactly identify polypeptides and proteins with high sensitivity and high throughput . there are many advantages : ( i ) small needed quantity ( fmol ) , ( ii ) short time of analysis ( 35 minutes ) , ( iii ) high accuracy of quality ( 1/10000 ) , ( iv ) the immunity to the effect of n - terminal close , ( v ) making use of situ - enzymolysis - technology could directly identify the protein spots which are cut from gels . so it reduces the loss of specimen , ( vi ) it could identify a single protein in protein mixture and directly identify the modification after translation [ 3 , 4 ] . so it develops quickly and becomes the preferred way of analyzing polypeptides and proteins in laboratory . in our research , we made the rats our lung cancer model which was induced with mca , used the method of 2de combined with maldi - tof - ms , made compared serum proteomics research in lung cancer , analyzed the changes of serum proteins before and after the formation of lung cancer in same body , identified serum proteins which related to lung cancer for offering serum proteomic evidence to probe the pathogenesis of lung cancer as well as providing new style to probe hematological diagnostic method of lung cancer . 20 wistar rats ( 10 male , 10 female ) were purchased from experimental zoology department at tongji medical college in huazhong technology university . they ranged in weight from 180 g to 200 g. 3-methylcholanthrene ( mca ) was the product of sigma ( sigma - aldrich , inc , saint louis , missouri , usa ) , immobilized ph gradient dry strip was purchased from amersham pharmacia biotech company , sodium dodecyl sulfate ( sds ) , dithiothreitol ( dtt ) , tris(hydroxymethyl ) aminomethane ( tris ) , bromophenol blue ( bpb ) , ammonium persulfate ( aps ) , glycine , acrylamide , methylene bisacrylamide ( bis ) , tetramethylethylenediamine ( temed ) , ipg - buffer , drystrip cover fluid , agarose , carbamide , protein - markers were purchased from a - pharmacia co. , coomassie brilliant blue r-250 , 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate ( chaps ) were purchased from sigma co. , glacial acetic acid , methanol , glycerol , paraform , alcohol were national analytical pure , sequencing - grade modified trypsin was purchased from american roche co. , -cyano-4-hydroxycinnamic acid ( cca ) ( sigma co. ) , acetonitrile ( acn ) ( fisher chromatographic pure ) , tri - fluoro - acetic - acid ( tfa ) ( sigma co. ) . ipgphor isoelectric focusing electrophoretic apparatus was purchased from swedish amersham pharmacia biotech co. , proteanxi cell vertical electrophoretic apparatus was purchased from bio - rad co. , image scanner was purchased from swedish amersham pharmacia biotech co. , centrivap centrifugal concentrators and cold traps were purchased from labconco co. , spotcutter apparatus was purchased from bio - rad co. , and autoflex maldi - tof - ms mass spectrometry apparatus was purchased from germany bruker co. image master ide software and mascot software are found in http://www.matrixscience.com/search_form_select.html . the rats were kept in cozy ( 1525c ) , quiet and avoided of highlight for 58 hours before making the experiment . the rats were kept for 14 days after the whole blood was collected by heart puncture . then we made the model of lung cancer induced with mca in iodized oil by intrabronchial installation [ 5 , 6 ] with minor modifications . the details were as follow : iodized oil 0.1 ml containing mca 10 mg was dissolved at 7678c for 24 hours . as the basal anesthesia , ketamine hydrochloride 0.12 ml was injected into the muscle of right thigh ( 0.6 ml / kg ) , at the same time , penicillin 20 000 u and phytomycin 50 mg per rat were injected into the muscle of left thigh once a day for a week for infection prevention . after about 2 - 3 minutes , early drugged state was showed up , and then the rats were anaesthetized with diethylether for relaxing the muscle of laryngeal . the anaesthetized rats were put on a consple with 45c inclined plane and the upper jaw incisor teeth were fixed by a retention suture on superior extremity of table top . the throat was exposed with gripping tweezers and bended tongue spatula with elastic metals . in a frontal mirror , at the moment of inspiration , the injection needle with blunt end in 1 ml syringe was inserted in glottis until reached the crotch of trachea , then turned left bottom to lobar bronchi in inferior lobe of left lung , and then iodized oil 0.1 ml with mca 10 mg was injected . take pictures with dsa after installation for a day to find the location of iodized oil with mca . sacrificed the animals after installation for 180 days dissected the rats systematically and took the tissues where pathological changes were obvious and all organs , metastasis , lymph nodes with suspected of invasion , fixed them with paraform , embeded them with paraffin wax , sliced them , and stained with he . the rats were raised with skins of hind neck in order that the limbs could downward sagging after they were anesthetized with diethylether . the injector that contains heparin was used to pump blood ( 13 ml ) on the left chest wall where the heart beats most powerfully ( 1.52 cm higher than the lower margin of ensiform cartilage ) . the blood was kept in centrifuge tube for 30 minutes at room temperature , centrifuged at 2000 rpm for 15 minutes . the plasma was taken out and aliquoted into sterile ep tubes , stored at 70c until used . 50 ul plasma was taken out , respectively , before and after the formation of lung cancer , then combined and quantified with bradford method , followed by taking 1 mg protein used for isoelectric focusing . all measurements were performed as described previously with minor modification . in the first dimension , proteins were separated by ief with ipg strip ( ph310 , nonlinear gradient and ph47 , linear gradient , both 180 mm 3 mm 0.5 mm ) . of the total proteins , 1 mg was dissolved in lysis buffer ( 8 m urea , 0.02 chaps , 0.02 m dtt , 0.05 ipg buffer ) to obtain a total volume of 350 ul per strip . focused ipg were rehydrated at 30 v for 6 hours , at 500 v for 1 hour , at 1000 v for 1 hour , and at 8000 v for 10 hours . after ief , ipg strips were washed with milli - q water and then incubated in equilibration buffer ( 30% glucerin , 6 m urea , 2% sds , 50 mm tris - hcl ph 8.8 , trace bromophenol blue ) containing 20 mm dtt on rocking bed for 15 minutes , followed by 15 minutes incubation in the same equilibration buffer containing 100 mm - iodoacetamide on rocking bed . equilibrated ipg strips were placed on top of a 13% homogeneous sds - polyacrylamide gel . a scrap of filter paper was immersed in 10 ul protein marker and put on top of one side of the gel . 13% sds - page gels 75 ml contained 30% acr/0.8% bis 33.5 ml , 4 tris - hcl 19.5 ml ph 8.8 , milli - q water 24.5 ml , 10% aps 0.25 ml , temed 0.05 ml . gels were run in parallel at constant power ( 40 ma for 40 minutes , 60 ma for 5 hours ) in running buffer until bpb reached the bottom line of each gel . the gels were put into 0.1% coomassie brilliant blue solution ( methanol 484 ml , glacial acetic acid 92 ml , milli - q water 454 ml , coomasie brilliant blue r-250 1 g ) and stained on rocking bed for 3 hours . after pouring out the coomassie brilliant blue solution , we decolored them in depigmenting agent solution ( methanol 50 ml , glacial acetic acid 75 ml , milli - q water 875 ml ) for 24 hours until the background of gel pieces became clear and transparent . spots detection , quantification , matching and comparison of 2de protein pattern were done with image master ide software . in the protein matching , serum protein gel maps of normal controls were regarded as referenced gel , artificial matched spots were built up , and the analysis of different expression was performed by applied software . the individual spot volumes were normalized by dividing their intensity values by the total intensity values of all the spots present in the gel and expressed as % vol . if the ratio of relative content in different group was more than 2 or less than 0.5 , we could assess there were differences . it was carried out by the procedure described in detail previously with minor modification . of all the spots , 7 protein spots were overexpressed , 4 protein spots were underexpressed , and 2 protein spots were new in the plasmas of lung cancer . the chosen protein spots in 2de maps were excised with spot - cutter apparatus and transferred into 1.5 ml ep tube , followed by washing them thrice with 1 ml water for 10 minutes . gel pieces were discolored in 50100 ul solution ( 50% acetonitrile , 25 mmol / l ammonium bicarbonate ) for 20 minutes with constant oscillation , followed by discarding the solution , repeated 1 - 2 times until blue color disappeared , dried them in vacuum centrifugal dryer for 30 minutes . then 510 ul of 0.01 ug / ul trypsin diluted in 25 mmol / l ammonium bicarbonate was added to each sample tube for digesting the proteins and kept the tubes in freezer at 4c for 2030 minutes . after all digestion solution was ingested , 510 ul of 25 mmol / l ammonium bicarbonate was added to it . stored it at 37c for 15 hours . the digested polypeptides were eluted twice with 5% tfa 50100 ul at 40c for 1 hour and 2.5% tfa , 50% acn 50100 ul at 30c for 1 hour , supernatants were collected , combined , vacuum - dried by centrifugation until used for mass spectrographic analysis . 5 ul of 0.5% tfa was added to dried sample tubes for dissolving the peptides mixture . the sample and ground substances ( cca added to 30% acn or 10% aceton to obtain saturated solution ) were combined with 1:1 volume and spotted onto the target plate , then air - dried . peptide mass fingerprinting spectrum was evaluated with a reflector positive ion model in following setting : accelerating voltage , 20 kv ; reflected voltage , 1.12 kv ; nitrogen laser wavelength , 337 nm ; pulse width , 3 nanoseconds ; delayed extraction time , 100 nanoseconds ; the degree of vacuum , 4 10 torr ; each spectrum was obtained by accumulating 50 laser shots and averaging them . a matrix peak and the peptide mass fingerprinting of each protein was searched from ncbinr database using mascot software . the conditions of searching are as follows ( i ) the peptides mass were controlled between 800 da and 4000 da . ( ii ) the sphere of error in apparent pi and apparent mr was + 0.5ph and 20% . ( iii ) the largest error in molecular weight of peptide fragment was controlled in the range of 0.3 da to + 0.3 da . ( iv ) the trypsin was chosen as the enzyme and the incomplete choice of enzymolysis fragment was 1 or 2 . lung cancer models were made successfully after installing mca in iodized oil . it was demonstrated that iodized oil was present in the inferior part of left pulmonary lobes by scissors shadow with x - ray . only in ten rats of 18 rats ( two rats died naturally in 20 rats ) , tubercles in bottom left of pulmonary lobes were observed in naked eye . by pathological examination , it was proved that they were squamous cell carcinomas . after he staining , by light microscope , we could observe that carcinoma nests were different in size and shape , the horny pearl was obvious , cell nucleus were very big , part of them were vacuous , the chromatins in cell nucleus were rough , there were many karyokinesis , and nucleoli were big and localized to the edge ( figure 1 ) . the electrophoresis of plasma in 10 animal models was performed before and after forming lung cancer , respectively . the protein spots were separated better using ph47 ipgief ( figure 2(b ) ) than ph310 ipgief ( figure 2(a ) ) . the protein spots in the range of pi46 and mr1065 kd were distributed most intensively . when making an analysis of 2de patterns which were separated with ph47 ipg strip ( figure 3 ) , we found that 455 spots on the normal plasmas 2de patterns and 716 spots on the lung cancer plasmas 2de patterns were detected . when we matched the lung cancer plasmas 2de patterns with the normal plasmas 2de patterns , it was demonstrated that the number of matched protein spots was 295 and matched rate was 50.38% . 141 protein spots were differently expressed significantly in plasmas 2de patterns , 82 spots overexpressed in the lung cancer and 59 spots overexpressed in normal controls . we chose 13 differently expressed proteins spots with clear margin and high quantity of proteins for maldi - tof - ms analysis . of all the protein spots , 7 protein spots were overexpressed ( numbers 1 , 2 , 3 , 4 , 7 , 8 , 9 ) , 4 protein spots were underexpressed ( numbers 10 , 11 , 12 , 13 ) , 2 protein spots were new in the plasmas of lung cancer ( numbers 5 , 6 ) ( figures 4 and 5 ) . the peptide mass fingerprinting maps were obtained successfully by analysis of maldi - tof - ms after digesting the chosen protein spots . finally , 3 proteins were identified through the ncbinr database search by mascot software in chosen 13 protein spots . the 3 proteins were heptoglobin ( overexpressed protein ) , transthyretin ( underexpressed protein ) , and tumor necrosis factor receptor superfamily member 8 ( new protein ) . the figures were the mass spectrum of identified proteins spots and detailed results of database retrieval ( figures 6 , 7 , 8 , 9 , 10 , and 11 ) . mca is an indirect chemical carcinogen , which belongs to polycyclic aromatic hydrocarbon with 3 , 4-benzpyrene , and is responsible to air pollution . its major effect is to make epithelium mucosae cell of bronchiole , respiratory bronchiole squamous metaplasia and gradually progress to squamous carcinoma cell . we had proved that there were a series of changes of genetic materials and related genes in the process of variation from normal bronchi epithelium to lung squamous carcinoma cell by immunohistochemistry staining and molecular pathology [ 8 , 9 ] . for example , the different positive expression rate of p53 protein was 69.57% in rat lung cancer tissue . the positive expression rate of proliferative cell nuclear antigen ( pcna ) took up 20% in normal bronchi epithelium tissue , and it advanced to 82.61% in infiltrating lung cancer tissue . in addition , the number of strong positive expressed cells was increasing obviously , even all carcinoma nests were positive expressed , it hinted the changes of activity of cell proliferation during the formation of lung cancer . thus making use of the model of rat lung cancer to carry out serum proteome research was effective and feasible . haptoglobin , one of acute phase proteins , is an acid glycoprotein , which belongs to 2-globulin and extensively exists in human plasmas and other body fluid . most of hp is synthesized in liver . in addition , hp mrna is also expressed in human endometriotic lesions , intestinal epithelium and mouse liver , lung , adipose tissue cell , skin . friedrichs et al . found that hp mrna was detected in some special cells of adernal gland , submandibular gland , ovary , ureter . further proved that hp was considered as a secretory protein , however , hp synthesized in thp-1 monocytic cells was largely retained within cells . and the content of hp in plasmas often greatly increases in pathologic state , for example , carcinoma , inflammation , infection , myocarditis , and so on . the main factor stimulates the composition of hp is il-6 . on the condition of inflammation , injury , il-1 , tnf are secreted by macrophage , leading to the increase of il-6 , resulting in the composition of hp . in addition , darmiento proved that lps also could stimulate the expression of hp mrna . proved protein kinase - c - delta could stimulate haptoglobin secretion as well . by forming the high - affinity complexes , , these hp - hb complexes could bind to the cd163 scavenger receptor on macrophages with high specificity , leading to endocytosis and subsequent intracellular degradation . so hp - hb complex formation is considered to lessen the loss of free hb through glomerular filtration and it is helpful for supporting the recycling of iron . moreover , because free hb is a source of iron , which may otherwise enhance bacterial growth and virulence , so it is important that hb released from erythrocytes could be cleared promptly . finally , free hb could release heme and iron which may participate in generating reactive oxygen species ( fenton reaction ) and promote the injury of tissue as well . in a word , hp works indirectly as a bacteriostatic agent and an antioxidant in the way of facilitating the immediate clearance of free hb by macrophages . haptoglobin polymorphism correlates to the prevalence and clinical evolution of many inflammatory diseases , for example , infections , atherosclerosis , and autoimmune disorders and that leads to the generation of two distinct alleles , hp2 - 1 , hp1 - 1 , and hp2 - 2 . hp2 - 2 has been reported to be associated with the risk of atherosclerosis and coronary heart disease . vormittag et al . proved that hp2 - 2 was a potentially new risk factor for spontaneous venous thromboembolism as well . there are many researches about hp correlate with injury , inflammation , metabolic disease [ 1618 ] . more and more documents report that the level of hp in plasmas goes up in many malignant tumors , such as ovarian cancer , breast cancer , renal cell cancer , colon carcinoma , acute leukemia , and lung cancer . our research results were consistent with them . in our research , we found hp greatly increased in lung cancer ( the content of protein adds up to 8.328 times ) . though maybe it does not belong to specific protein in lung cancer , owning to greatly increase level of hp in the active stage of carcinoma and high sensitivity of immunology method , it could become an important check index for lung cancer diagnosis and monitoring and improve the sensitivity and specificity of early diagnosis in lung cancer when combining with other diagnostic indexes . as for its function , ttr could transport the thyroid hormone t ( 4 ) and retinol through binding to the retinol binding protein . in addition , ttr has been established as a cryptic protease able to cleave apoa - i in vitro . some scholars have reported that ttr is involved in preventing a - beta fibrillization by inhibiting and disrupting a - beta fibrils , with consequent abrogation of toxicity . costa et al . further confirmed that as a protective mechanism in alzheimer 's disease ( ad ) , ttr could serve as a protective molecule in this disease and prompted a - beta proteolysis . it may be proved that ttr is a useful therapeutic agent in preventing or retarding the formation of cerebral amyloid plaque implicated in ad pathology . in the past , someone confirmed that in familial amyloidotic polyneuropathy ( fap ) which was associated with the abnormal extracellular tissue deposition of mutant transthyretin ( ttr ) , inflammatory and apoptotic pathways both are triggered in the presymptomatic stages of the disease on the condition that nonfibrillar ttr deposits are present . meistermann et al . also reported that transthyretin could serve as a biomarker for gentamicin - induced nephrotoxicity in rat . conversion of ttr tetramer to monomer may relate to the development of beta - cell failure / destruction in type 1 diabetes . so we could see that ttr expression was associated with diabetes . at the same time , bai et al . gussed that the concentration of ttr maybe relates to the level of blood pressure , however , the concrete mechanism is still unclear . the content of ttr quickly decreased when the proteins were insufficient and increased obviously when the amount of proteins gone up . therefore , ttr was often used as an index of evaluating nutritional state , especially the nutrition and immunity state after operation [ 33 , 34 ] . so far , reports about ttr relate to tumors mainly concentrate on liver cancer , ovarian cancer , leukemia . gu et al . first reported that the gene of ttr had close correlation to liver cancer , they found that the gene of ttr was expressed most highly in normal liver . the level of its mrna greatly went down in liver cancer which the parenchyma was destroyed little . ttr was also considered as potential serum marker in early ovarian cancer [ 3538 ] . in children with leukemia , the content of ttr and rbp in plasmas was greatly less than that in normal children . that difference also existed in different race , the melanoderm children were much less , and the lower level related to bad prognosis . some scholars reported that ttr was overexpressed in blood serum in lung squamous cell carcinoma compared with normal lung . our research also demonstrated that the content of ttr in plasmas greatly declined in the formation of lung cancer , it was consistent with their results . the amount of proteins in normal ones was 14 times bigger than that in lung cancer . tnfrs8 also named cd30 , because ki-1 monoclonal antibody was used in identifying it , therefore it named ki-1 as well . as a marker of activation of t and b lymphocytes , cd30 is a 120 kda surface phosphorylated glycoprotein and it is predominantly overexpressed by the tumor cells ( hodgkin 's and reed - sternberg cells ) of classical hodgkin 's lymphoma ( chl ) and those of anaplastic large cell lymphoma ( alcl ) in classical hodgkin lymphoma . it is well known that cd30 expression is not unique to hodgkin lymphoma and anaplastic large cell lymphoma ( alcl ) because of its expression in mediastinal large b cell lymphoma , a subset of nodal diffuse large cell lymphoma , large cells in follicular lymphoma , nodal and cutaneous diffuse large b cell lymphoma , peripheral t cell lymphoma , embryonal carcinoma , and other nonlymphoid cells and neoplasms . the expression of cd30 was also associated with pregnancy . kusanovic found that the content of maternal serum soluble cd30 increased in normal pregnancy , however , it decreased in preeclampsia and it is small for gestational age pregnancies . at the same time , lambropoulou et al . proved that during the second trimester , cd30 was expressed highly in many medullary thymic epithelial cells ( tecs ) and hassall 's corpuscles , but small numbers of cd30 tecs were showed during the late first trimester . large number of researches proved that if cd30 crosslinked with cd30l , activated signal may be issued . so it promoted the transmission of intracellular information . because that information could be positive or negative signal , consequently , it resulted in cellular growth was inhibited or cellular apoptosis , cellular hyperplasia or differentiation , secretion of cytokine , and so on . wright et al . further confirmed that in alcl cells , the stimulation of cd30 elicits p21 ( waf1)-mediated arrest through the canonical but not the alternative nf - kappab pathway . reported that there were many cd30cd4 , cd30cd8 cell in joint synovial fluid of patient with ra , large amount of scd30 existed in peripheral blood and affected joint synovial fluid , especially in active stage , it had direct relation with the titre of raf . it hinted that there was relationship between cd30 cell , scd30 cell , and th2 reactive disease . in addition , the increase of cd30 cells and scd30 cells was detected in acute stage of th2 reactive illness , such as sle , scleroderma , specifical scytitis . many researches showed the amount of scd30 increased in active stage of chronical viral hepatitis b and there were striking differences compared with normal controls and virus carries , hinting the activity of th2 cytokine in active stage of hepatitis [ 47 , 48 ] . in hodgkin 's disease , the level of serum scd30 abnormally increased in active stage in nontreated patients . the extent of increase related to the stage of illness and general symptoms . it could be an independent prognosis index , the higher of scd30 , the worser of prognosis . in alcl , the great increase of serum scd30 there was not scd30 in normal controls , indicating that the amount of scd30 went up indeed in forming lung cancer , so far , the mechanism of increase is not clear . hargreaves et al . guessed that it was possible that the function of scd30 brought into full play by blocking the cross - link of cd30 and cd30l . recently , researches in lung cancer demonstrated that a series of complicated molecular events involved in carcinogenesis , such as activation of oncogene and inactivation of antioncogene . proteomics aim at the large - scale analysis of protein in tissues , cell , and plasmas , including protein expression , posttranslational modification , and interaction of proteins . proteomics has become a hot spot in lung cancer research . by applying the proteomics technology , many researchers had found a large number of proteins as the candidate tumor markers from blood plasmas in lung cancers . by comparing protein expression levels among 93 lung adenocarcinomas and 10 uninvolved lung samples , chen et al . found that nine different enzyme proteins were identified by the method of 2d - page and maldi - ms or peptide sequencing . all of them increased obviously in the lung adenocarcinomas , including the antioxidant enzyme aoe372 , atp5d , b4galt , cytosolic inorganic pyrophosphatase ( ppase ) , grp58 , gstm4 , p4hb , tpi , and ubiquitin hiolesterase . identify proteins that commonly induce an antibody response in lung cancer by making use of proteomic approach ; protein gene product 9.5 ( pgp 9.5 ) was detected in serum . when they took advantage of a549 lung adenocarcinoma cell line , they have proved that pgp 9.5 existed at the cell surface as well as secreted . therefore , the discovery of pgp 9.5 antigen and/or antibodies in serum of patients with lung cancer showed that pgp 9.5 may be used in lung cancer screening and diagnosis . chatterji and borlak found that eight proteins were also identified in serum though they were identified in tissue of lung tumor bearing mice . a total of 50 proteins were identified in serum , some of which were specifically regulated or exclusively expressed either in tumor bearing or wild - type mice . what is more , they revealed that alpha-1-antitrypsin ( a1at ) and alpha-2-macroglobulin ( a2 mg ) were upregulated in the serum proteome of 12 months old mice . in cancer tissue , hemoglobin subunit alpha went up in serum samples of lung tumor bearing mice ( 12 months ) . they discovered the expression of transthyretin to be gone up in 1 month and repressed in 12 months old tumor bearing mice . thus they suggested that their in - depth validation could become biomarker candidates for the identification of lung adenocarcinomas . serological proteome analysis ( serpa ) of ten hlsc tissues was performed by li et al . to identify the tumor - associated antigens . the results showed 36 8 differential proteins reactive with patients ' autologous serum , 14 proteins of them were identified . in addition , six of the 14 proteins , alpha enolase , pre - b cell - enhancing factor precursor , triosephosphate isomerase , phosphoglycerate mutase 1 , fructose - bisphosphate aldolase a , and guanine nucleotide - binding protein beta subunitlike protein were also increased in hlscs in the comparative proteomic research , therefore , it indicated that these 6 hlsc - associated antigens can be applied in diagnosis and therapy of hlsc . autoantibodies against triosephosphate isomerase ( tim ) and superoxide dismutase [ mn ] ( mnsod ) were detected by yang et al . in serum from over 20% patients with lsc but none from the normal controls . their results suggested that autoantibody and antigen of tim and mnsod may also be potential application for screening and diagnosis of the lung squamous carcinoma . at the same time , lisa et al . found that five truncation forms of serum amyloid a protein precursor and serum amyloid a1 isoform 2 as a part of the components of the serum proteomic biomarkers of lung cancer . these results indicated that it was possible that the serum proteomic signature of lung cancer was the part of the result of specific proteolytic activity in the serum and/or in lung tumors . built up the protein configuration in the plasmas of lung adenocarcinoma using the method of the seldi - tof - ms technology and discovered 5 special protein peaks which highly expressed in lung adenocarcinoma . in addition , liao et al . captured serum proteins in lung adenocarcinoma by the technology of wcx2 chip and found 10 different expressed proteins in which 6 overexpressed proteins and 4 underexpressed proteins . however , a large number of lung cancers and normal people were essential to prove these differently expressed proteins and special expressed spectral patterns , to identify and show their functions in the formation of tumor . due to direct examination of the molecular machinery of cell physiology , including protein expression , sequence variations and isoforms , posttranslational modification , and protein - protein complexes , proteomics has the self - evident advantages over genomic - based assays . nonetheless , there are certain disadvantages as well , for instance , ( i ) sample collection , preparation , and analysis need rigorous requirement , ( ii ) there are no amplification procedures which similar to pcr that could enable assay to improve making use of the limited biological starting material , ( iii ) we are short of purification strategies which could enrich samples for intended work ( e.g. , phosphoprotein analysis ) , and ( iv ) necessary costs are insufficient for staffing and equipping shared resources or clinical laboratory which can be able to perform the required assays . though there are some limitations in the technology of proteomics , with the maturation of proteomic technique and improvement of sample pretreatment and preparation technique . we believe its application in analysis of blood plasmas will become more and more popular and become a new method for clinical diagnosis and illness monitoring . in addition , the screening and identification of tumor markers will step into a new stage . in a word , the research tactics in proteomics of tumor markers will bring about far - reaching extensive effects for lung cancer diagnosis and treatment . it will quicken the process of researches in lung cancer as well as making a great contribution to win a victory in human being 's flight with tumor . in our experiment , we made the rat plasmas derived from lung cancers and the normal controls to be our targets of observation and comparison . by regulating and optimizing all conditions of 2de technique , we finally obtained satisfactory 2de patterns in which the 700 serum proteins were detected from the lung cancers and the normal controls . in addition , the 141 differently expressed protein spots were acquired by image screen and image analysis . the 82 spots were overexpressed in lung cancer and the 59 spots were overexpressed in normal controls . at the same time , we successfully identified three proteins including hp , ttr , and tnfrs8 by making use of the maldi - tof - ms to analyze the 13 differently expressed protein spots . the increase of hp , which belongs to app , hinted the appearance of stress reaction in the process of forming lung cancer . in addition , the decrease of ttr , which shows the nutritional state , hinted the changes of nutritional state . as a member of tumor necrosis factor super family , tnfrs8 could regulate the function of immunity and hinted a series of changes of immunity state in the process of forming lung cancer . in a conclusion , ( i ) the plasmas proteins were separated successfully for proteome research of lung cancer by the optimized 2de technique . ( ii ) there were the differently expressed proteins between the normal plasmas and the lung cancer plasmas . ( iii ) the differentially expressed proteins in plasmas of rat lung cancer provided the evidence for keeping searching the markers for lung cancer diagnosis and therapy .
many cam modalities afford relief from pain , each in its own way , or according to its own terminology . comparison of different cam modalities results in a simple phenomenology of pain centered around the idea that pain may be associated with blockages of the flow of energy in the system of nadis / acupuncture meridians .
human papillomavirus ( hpv ) , a sexually transmitted infection and the etiologic cause of genital warts and cervical cancer , is highly prevalent in sexually active men and women . although cervical screening procedures have significantly reduced the disease burden associated with hpv infection , they are expensive and abnormal results cause significant emotional distress . therefore , prevention may be an effective strategy for reducing the economic , psychosocial , and disease burden of hpv infection . multivalent vaccines are now in clinical development . a bivalent vaccine that protects against hpv 16 and 18 , and a quadrivalent vaccine which protects against hpv types 6 , 11 , 16 , and 18 , have been shown to significantly reduce the occurrence of incident and persistent hpv infections in phase 2 clinical trials ; phase 3 trials are currently underway . hpv vaccines will be most effective when administered prior to initiation of sexual activity , and vaccination campaigns should aggressively target preadolescent and adolescent populations .	human papillomavirus ( hpv ) is the most common newly acquired sexually transmitted infection ( sti ) in the united states , with an estimated 20 million people infected . furthermore , incidence of hpv infection has increased during the past two decades , with approximately 6.2 million newly diagnosed cases annually [ 1 , 2 ] ( figure 1 ) . hpv infection has a very high prevalence rate in adolescent girls and young women . one study showed that 36% of women 25 years of age or younger are hpv - positive compared with less than 3% of women 45 years of age and older . hpv is the etiologic agent of several genital epithelial lesions including genital warts ( condylomata acuminata ) , cervical intraepithelial neoplasia ( cin ) , and cervical cancer . more than 100 different types of hpv have been identified [ 4 , 5 ] ( table 1 ) . low - risk types , hpv 6 and hpv 11 , are the most common types implicated in causing genital warts . furthermore , due to their ability to cause low - grade cervical lesions , infection with low - risk hpv types is often associated with abnormal papanicolaou ( pap ) test results . although genital warts are medically benign , and low - grade cin can spontaneously regress , diagnosis of genital warts or an abnormal pap smear result can cause emotional distress and impose an economic burden . in contrast to infection with hpv types 6 and 11 , infection with high - risk hpv types 16 and 18 can lead to anogenital cancers . hpv types 16 and 18 cause 70% of all cases of cervical cancer , with half of all cervical cancers caused by type 16 alone . persistent infection with high - risk hpv types is implicated in 99.7% of cervical cancers . preventing infection with the most common low - risk and high - risk hpv types would prevent the majority of cases of genital warts and cervical cancer , respectively . the transmission typically occurs through the skin - to - skin anogenital contact . increased risk for acquiring hpv has been associated with multiple sexual partners , younger age of sexual debut , failure to use condoms , and sex with uncircumcised males . however , one study reported that 20% of women became infected with only one lifetime sex partner , suggesting that both partners must be sexually nave to prevent infection . several studies have shown that the risk of infection increases substantially when initiating a new sexual relationship [ 5 , 1013 ] . the transmission of hpv infection can be blocked by latex condoms if the infected area is physically covered . nonetheless , hpv often manifests on external anogenital sites not covered by a condom , and so the latter does not prevent all infections . however , the use of a condom may reduce hpv persistence and therefore aid in the regression of hpv - associated lesions . annual cervical cancer screening is expensive . in a study of women enrolled in a usa health care plan , it was estimated that an average of $ 26,415 per 1000 women was spent on annual cervical screening and treatment for hpv - related diseases . when these data are extrapolated to the general usa population , it can be estimated that $ 3.4 billion is spent annually on diagnosis and treatment of hpv infection and its associated cervical diseases . approximately 90% of the estimated cost can be attributed to strategies for prevention of cervical cancer , such as treatment of precancerous lesions and routine pap tests , whereas the other 10% is spent on treatment of cervical cancer ( figure 2 ) . hpv infection is most prevalent in adolescents and young adults , and this group also incurs the majority of hpv - associated health care costs . when it comes to hpv - related health care , women in the 2029 year age group incurred an annual cost of $ 51,863 per 1000 women . the estimated lifetime total medical cost of hpv infection for men and women aged 1524 is $ 2.9 billion , which makes hpv the second most expensive sti after hiv . in addition , when the cost of treating genital warts is analyzed by itself , it becomes apparent that this too causes a substantial economic burden . based on an incidence of 500,000 cases of hpv infection per year , the annual total direct medical cost for treatment of anogenital warts in all age groups for the year 2000 was $ 167.4 million . assembly of infectious virus , a necessary step in the hpv life cycle , involves the formation of the capsid , or outer layer , of the virion . the capsid is composed of two proteins , l1 and l2 , which are expressed later in infection . the major capsid protein l1 comprises the outermost layer of the capsid and is necessary for virus structure . hpv vaccine development has been considerably advanced due in part to the production of virus - like particles ( vlps ) . hpv - like vlps , which mimic the structure of the hpv virion but do not contain genetic material and can be manufactured by exogenous expression of l1 in a variety of cell types , including bacterial , yeast , insect , and mammalian cells [ 18 , 19 ] . vlps are noninfectious and nononcogenic , making them ideal candidates for use in hpv vaccine production . vlps are purified , concentrated , distributed into aliquots , and combined with an adjuvant . early studies with a monovalent vaccine against hpv 16 have shown that vlp vaccines induce a strong immune response against l1 in animal models [ 21 , 22 ] and humoral immunity in humans . other trials demonstrated that booster doses of vlp vaccines induced protective levels of antibodies [ 2325 ] . furthermore , in a proof - of - principle study , the hpv 16 vlp vaccine was safe , well tolerated , and induced antibody titers to levels significantly higher those produced in response to natural infection . although this study was not sufficiently powered to assess vaccine efficacy in preventing clinical disease , vaccine recipients developed fewer cervical lesions than placebo recipients . in addition , the vaccine was 100% effective in preventing persistent infection , suggesting that vlp vaccines may help reduce the incidence of cervical cancer precursors and invasive cervical cancer . similar results have been reported with other monovalent vlp vaccines [ 2729 ] . immune responses to hpv infection are type - specific ; therefore , vaccine efficacy can be greatly improved by combining vlps from several types of hpv into one multivalent vaccine . multivalent vaccines that offer protection against the most common disease - causing hpv types are in late stages of clinical development . currently , a bivalent vaccine that protects against 2 high - risk hpv types , and a quadrivalent vaccine that protects against 2 high - risk and 2 low - risk hpv types are being tested . to determine the efficacy , immunogenicity , and safety of a bivalent vaccine containing hpv types 16 and 18 , a double - blind , placebo - controlled phase 2 trial was conducted on 1113 women ( 1525 years old ) with no prior history of hpv infection and normal cervical cytology . in this study , women received intramuscular injections of vaccine ( 20 g of each vlp plus adjuvant ) or placebo ( adjuvant alone ) on day 1 , at month 1 , and at month 6 , and then followed for at least 17 months . the bivalent hpv 16/18 vaccine was well tolerated , produced no vaccine - related serious adverse events , and induced a major humoral immune response to both hpv types . furthermore , the vaccine was 90% efficacious at reducing incident infection and 100% efficacious at preventing persistent infection . including additional hpv types in vaccines would be expected to cumulatively reduce hpv - associated disease burden by preventing additional hpv infections . a quadrivalent vaccine has been developed to protect against hpv types 6 , 11 , 16 , and 18 . a double - blind , placebo - controlled phase 2 safety and efficacy trial women who were enrolled in the trial received either the quadrivalent vaccine ( 20 g each of hpv 6 and 18 vlp , and 40 g each of hpv 11 and 16 vlp plus adjuvant ) or placebo ( adjuvant alone ) on day 1 , month 2 , and month 6 , and then were followed for 36 months . results of this trial showed that the quadrivalent vaccine was well tolerated , produced few serious adverse events ( none of which were judged to be related to the vaccine ) , and stimulated the production of antibodies directed against all four hpv types . furthermore , the vaccine reduced persistent infection in vaccine recipients by 89% and prevented 100% of clinical disease associated with hpv types 6 , 11 , 16 , and 18 ( figure 3 ) . similarly high efficacy results were reported for a cohort of women who did not adhere completely to the study protocol . recently , data from the phase 3 females united to universally reduce endo - ectocervical disease ( future ii ) clinical trial were presented . the quadrivalent hpv vaccine was 100% effective at preventing cin 2/3 , ais , and cervical cancer associated with hpv 16 or 18 infection during two years of follow - up . the vaccine was well - tolerated and there were no vaccine - related serious adverse events . vaccines that provide protection against the most common disease - causing hpv types would be expected to significantly reduce the incidence of hpv - associated diseases . reducing hpv - associated disease burden may also reduce the health care costs associated with these diseases . although hpv vaccines are not currently available to the public , their potential public health benefits have been reported in several mathematical modeling studies [ 3335 ] . sanders et al reported that if a hypothetical vaccine that was 75% efficacious at preventing high - risk hpv infection were administered to approximately two million 12-year - old girls , it would prevent 224,255 hpv infections , 3317 cases of cancer , and 1340 cervical cancer - associated mortalities in the girls ' lifetimes . another study predicted that an hpv 16/18 vaccine would reduce the number of cervical cancer cases associated with the hpv 16 and hpv 18 by 95% . mathematical modeling and sexual transmission data have also suggested that both sexes should be vaccinated to provide the greatest reductions in hpv infections . for example , one population - level study predicted that a female - only hpv vaccination program would be only 68% as effective in reducing hpv prevalence as a program aimed at vaccinating both men and women . in fact , diagnosis with genital warts is often the most anxiety - provoking outcome of hpv infection . because vaccination has shown promising results in the reduction of the disease burden associated with hpv infection , it would be expected to reduce some of the psychosocial and emotional burden as well . a number of obstacles will need to be overcome to maximize the public health benefits of hpv vaccination . vaccination programs must identify appropriate candidates for vaccination , establish booster requirements , and overcome potential individual , parental , and social barriers to hpv vaccine acceptance . for example , individuals may view acceptance of hpv vaccines as admitting to risky sexual behavior . furthermore , research has shown that knowledge about hpv , an infection that many people know little about , is directly correlated to vaccine acceptance [ 3739 ] . parents may feel that their child is not at risk , or that vaccination would support teenage sex or encourage risky sexual behavior ( ie , reduced condom use ) . societal and cultural issues may include beliefs that sexually transmitted diseases are a deterrent or punishment for non - marital sexual behavior . each of these obstacles can be overcome by widespread efforts to educate individuals and society about the prevalence of hpv and the risks associated with forgoing vaccination . administering hpv vaccines to populations prior to initiating sexual activity will yield the greatest health benefit . because preadolescent and adolescent children are generally sexually nave and develop robust immune responses to vaccines , vaccinating young adolescents is predicted to significantly reduce the incidence of hpv infection and hpv - associated diseases . in order to foster broad acceptance of hpv vaccine , public health initiatives will need to educate parents / caregivers as well as health care professionals about the risks associated with hpv infection and the benefits of vaccination . vaccination has been widely accepted as an effective means by which infectious diseases can be prevented or eliminated . vlp vaccines that protect against infection with the most common disease - causing hpv types are currently in clinical development and early reports have suggested that hpv vaccines are highly efficacious in preventing hpv infection and hpv - associated disease . hpv vaccines will be most effective when administered prior to initiation of sexual activity and vaccination initiatives will most likely target preadolescent and adolescent populations . * average age adjusted to the 1998 us female population ; all cost estimates were converted to 2002 dollars ; asc = atypical squamous cells ; agc = atypical glandular cells ; lsil = low - grade squamous intraepithelial lesion ; hsil = high - grade squamous intraepithelial lesion . incidence of infection or disease associated with hpv 6 , 11 , 16 , or 18 after vaccination with a quadrivalent vaccine versus placebo ( * reported as incidence per 100 women - year at risk ) .
background : obesity is common in patients with cardiovascular disease ( cvd ) and the vast majority of patients entering into cardiac rehabilitation program ( crp ) are obese . regarding the gender differences , the risk of developing coronary heart disease ( chd ) is recognized to be different between obese men and women . so , the purpose of this study was to explore the effect of crp in functional capacity ( fc ) and risk factors , such as obesity indexes , lipid profiles , and fasting blood sugar ( fbs ) in obese men and women with chd.marterials and methods : in an observational study between 2000 and 2011 , we evaluated a total of 156 obese men and women patients with chd who were referred to cardiac rehabilitation of isfahan cardiovascular research institute . before and after crp , fc and risk factors were assessed and all the participants completed this period . data were analyzed with spss software version 15 . for comparing the mean of outcomes , independent t tests and paired sample t tests were used.results:data revealed , after crp , obese women had significant improvement in most evaluated risk factors except total cholesterol ( p = 0.05 ) and fbs ( p = 0.09 ) ; and obese men had favorable changes in weight ( p = 0.00 ) and body mass index ( p = 0.00 ) , fc ( p = 0.00 ) and total cholesterol ( p = 0.02 ) ; in spite of no significant differences in other lipid profiles . comparing the 2 groups did not show any significant differences unless high - density lipoprotein cholesterol ( p = 0.01 ) and low - density lipoprotein cholesterol / high - density lipoprotein cholesterol ratio ( p = 0.02 ) had greater improvement in obese women.conclusion:we concluded that crp is an important step initiating the process of risk reduction and restoration of fc in obese men and obese women with chd under attendance and supervision of physician , nurse , and exercise physiologist .	raised body mass index ( bmi ) is common in patients with cardiovascular disease ( cvd ) and obesity ( bmi 30 ) has been known as a risk factor for hypertension , type 2 diabetes , functional capacity ( fc ) , dislipidemia , and generally for coronary heart disease ( chd ) that is the most frequent cause of death . the prevalence of obesity in patients with coronary artery disease ( cad ) moves toward 40% . noticeably overall 80% of patients that referred to cardiac rehabilitation program ( crp ) were overweight and obese . crp is known as a way for enhancement and maintenance of cardiovascular health through individualized programs , designed to optimize physical , psychologic , social , vocational , and emotional status by improving coronary risk factors management . regarding the gender differences , the risk of developing cvd is recognized to be obviously different between men and women . according to previous studies , cad presents in women approximately 10 years later than in men ; and , the incidence of coronary events was 60% higher in men than in women . nevertheless , some studies revealed that women have similar improvements in fc and risk factors compared with men after crp . also , there are many contradictory results about similarity and dissimilarity between men and women . kazuhiro et al confirmed gender differences in physiologic outcomes , whereas in another study both the groups benefited in a similar way from crp in most aspects . by paying attention to these investigations , so , the purpose of this survey was to explore the effect of crp on fc and risk factors , such as obesity indexes , lipid profiles , and fasting blood sugar ( fbs ) in obese men and women patients with chd ; also , to know whether obese men and obese women equally achieve the benefits of crp or not . in an observational study between 2000 and 2011 , a total of 156 eligible patients were identified as obese patients with chd who were referred to phase ii cardiac rehabilitation of isfahan cardiovascular research institute . it should be mentioned that phase ii has taken the form of a structured and supervised exercise program in a hospital setting with educational and psychologic support and risk factors modification . the patients cardiac diagnoses were defined as a history of at least one of the following : myocardial infarction , coronary artery bypass grafting , percutaneous coronary intervention , and chronic stable angina . for patients to enter a crp , written informed consent was obtained from all the patients and then their heights and weights ( after they had fasted overnight ) were measured by a nurse in the morning and bmi was calculated by weight / height(kg / m ) . also , blood samples were taken after 12 - 14 h of fasting for measuring fbs , serum lipids , including triglycerides ( tg ) , total cholesterol ( tc ) , high - density lipoprotein ( hdl ) using enzymatic methods , and low - density lipoprotein ( ldl ) were calculated . these patients used usual angina treatment and the dose of those medications were not altered during the program . exercise test which was performed by each patient under the supervision of a cardiologist and a nurse , provided an opportunity to identify body reaction by monitoring hr and blood pressure and observing the electrocardiogram . in fact , it determined fc by indicating the changes in hemodynamic responses and metabolic equivalent . all the patients were divided into 2 groups : obese men and obese women ; and they began crp for 2 months , which consisted of 24 sessions and 3 times a week . crp included exercise training sessions , nutritional and psychologic consultation , and risk factor management . exercise sessions were similar in each group and exercise training consisted of combined aerobic and resistance training and it was performed in cardiac rehabilitation center under the supervision of a physician , a nurse , and an exercise physiologist by using treadmill , ergometer , stair climbing , rowing , step , jogging , and by using some resistance devices . each session lasted 90 min , 10-min warm - up followed by 60 min aerobic and resistance training , and finally 20 min cooldown and relaxation . the intensity of training was established according to the clinical condition and calculated between 60% and 85% of maximum heart rate . in addition , personal information of each patient and their history of disease were registered by a nurse and were archived . also , each patient had individual dietary instruction by a visiting dietitian during the program . after 2 months and completing the rehabilitation program , the tests were accomplished by each patient , again . the research protocol was taken under the medical ethics standards and approved by the isfahan cardiovascular research centre ethics committee , which is a member of the office for human research protections , u.s . independent sample t tests were used to identify baseline differences and comparing the changes between the 2 groups . for investigation about the changes between baseline and completion of crp , we used paired t tests . results were expressed as mean and standard deviation ( sd ) and the level of significance was set at p < 0.05 . they were separated into obese men group [ n = 72 , age = 54.47 ( 9.63 ) years ] and obese women group [ n = 84 , age = 57.69 ( 7.94 ) years ] . independent sample t tests were used to identify baseline differences among obese men and obese women patients [ table 1 ] . at baseline obese men had higher fc ( p = 0.001 ) and weight ( p = 0.00 ) ; but , tc was higher in women ( p = 0.01 ) . both groups did not have significant differences in other risk factors , such as tg , ldl cholesterol ( ldl - c ) , hdl cholesterol ( hdl - c ) , and fbs . comparison between obese women and obese men at baseline also , for examination of the changes between baseline and completion of crp , we used paired sample t tests . obese women had significant improvements in all risk factors except tc and fbs , and obese men had positive improvement in weight , bmi , fc , and tc but we did not see any significant improvement in other risk factors . again , for comparing the changes between the 2 groups , independent sample t tests were used . these data did not show any significant differences in fc and most evaluated risk factors except hdl - c and ldl - c / hdl - c , which showed more significant improvement in obese women [ table 2 ] . it is proved that obesity is a major cause of mortality and morbidity and it is associated to cvd and its risk factors , including hyperlipidemia and diabetes mellitus . because the vast majority of patients entering into cardiac rehabilitation are overweight and limited data are available regarding to the effects of crp and exercise training in obese men and women with chd , the present study was aimed at finding out the benefits of crp in obesity indexes , fc , and some lipid profiles on obese men and women . our baseline finding showed women who participated in the crp were 3 years older than men in comparison with 10 years in another study ; and , bmi was greater in women than in men , in spite of women s lower weight ; in this regard we can explain that heavier skeleton mass in men can be a cause of men being heavier compared with women . after crp , both groups attained significant improvement in weight and bmi ; but comparison of the 2 groups did not show any significant differences after intervention . considerably , there are many studies which implied that crp and exercise training had significant effects in obesity indexes in obese patients with chd.[1417 ] on the contrary , kiat et al . observed no weight reduction in patients with cad after 2 years of crp . on the other hand , obesity is associated with lower exercise performance , which results in reduced fc after coronary events . in the present study , obese women had lower fc than obese men at baseline , but similar relative improvements were seen ( 32.49% and 29.41% , respectively ) as a result of crp . the difference between the 2 groups was not significant but data showed greater improvement in women . in this regard , it can be noted that lower baseline fc values may interpret this greater improvement in obese women . like our results several studies suggested that women had lower fc than men and ; also , there were statistical similarities in improvement between men and women . generally we can explain that weight reduction after crp may be a cause for affirmative effects on fc and fitness level in both the groups . in our results , at baseline , no considerable differences were seen in lipid profiles between two groups except tc , which was higher in obese women than in obese men . after crp , obese women had favorable changes in tg , hdl - c , ldl - c , and ldl - c / hdl - c ; but , tc did not have any significant differences in them . according to a pervious study , there was a significant relationship between tc and obesity , which is due to an increase in cholesterol production with increased weight . conversely , in obese men improvement was seen just in tc but other lipid factors did not have any significant improvement . also , comparing the 2 groups showed that among lipid profiles , substantial differences were seen only in hdl - c and ldl - c / hdl - c . in relation to tg , we can note that obese women had higher tg value at baseline that was the strongest predictor of improvement in tg level when compared with men after crp . in addition , there are many investigations that have similarity or dissimilarity to our finding . lavie and milani showed significant improvement in hdl - c and ldl - c / hdl - c in obese patients but other lipid profiles did not have any changes during crp . also , another study suggested improvement only in hdl - c in women after comprehensive crp . williams et al . did not demonstrate significant reductions in ldl - c levels with obesity intervention , exercise training , and dietary therapy . some studies proposed weight reduction with exercise training , and dietary limitation has significant effects on increasing levels of hdl - c and reducing tg . another study showed that patients had improvement in tc , tg , hdl - c , ldl - c , and ldl - c / hdl - c after cardiac rehabilitation and exercise training . also , sarrafzadegan et al . reported that neither men nor women patients experienced a significant overall improvement in tc , ldl - c , and tg after the 3-month exercise training alone . the present study did not show significant improvement in fbs in obese men and women after crp . brochu et al . confirmed our result , but ades et al . mentioned exercise and weight reduction prevent type ii diabetes and increasing fbs . about some disparities between our study and other investigations , we can mention that lower age difference between our groups and also the range of patients age and even the kind and level of exercise training and finally variation in their diets may be the cause for dissimilarities . in general , it is understood that at baseline women had worse cvd risk factors than men . after crp , both the groups achieved significant improvements in fc and most evaluated risk factors . and comparing the 2 groups demonstrated no significant differences between them except hdl - c and ldl - c / hdl - c so , it should be noted that both obese men and obese women can benefit from crp without any attention to gender differences . the first limitation was short - term intervention , which consisted of 24 sessions of crp . it would be desirable to document long - term intervention changes in physiologic outcomes and risk profiles in patients with chd . the second limitation was the lack of control group who did not participate in crp after cardiac events , and comparing them to our groups . the third limitation was that a detailed evaluation of dietary adherence and exercise training outside of the formal crp was not available . we can suggest for future researches to study , whether considerable weight reduction after crp results in improved clinical outcomes in patients with chd or not . we conclude that crp , which is accomplished under attendance and supervision of a physician , a nurse , and an exercise physiologist is an important step initiating the process of risk reduction and restoration of fc in obese men and women with chd . it is an acceptable management for enhancement and maintenance of cardiovascular health through individualized programs designed to optimize physical condition and manage related risk factors , such as obesity indexes and lipid profiles .
middle ear acquired cholesteatoma is a pathological condition associated with otitis media , which may be associated with temporal bone resorption , otorrhea and hearing loss , and occasionally various other complications . cholesteatoma is characterized by the enhanced proliferation of epithelial cells with aberrant morphologic characteristics . unfortunately , our understanding of the mechanism underlying its pathogenesis is limited . to investigate its pathogenesis , different animal models have been used . this paper provides a brief overview of the current status of research in the field of pathogenesis of middle ear acquired cholesteatoma , four types of animal models previously reported on , up - to - date cholesteatoma research using these animal models , our current studies of the local hybrid ear model , and the future prospect of new animal models of middle ear cholesteatoma .	middle ear acquired cholesteatoma is a pathological condition associated with otitis media [ 1 , 2 ] , and as the cause of otorrhea , hearing loss , and occasionally complications such as facial palsy , brain abscess , and meningitis . middle ear acquired cholesteatoma is morphologically characterized by epithelial cell proliferation and granulation tissue formation . unfortunately , despite many studies our understanding of the mechanisms underlying the pathogenesis of cholesteatoma is limited . ( 1 ) metaplasia theoryin 1873 , wendt suggested that metaplasia of the mucosa of the middle ear into the keratinizing epithelium led to cholesteatoma . [ 7 , 8 ] proposed that chronic irritation can cause the mucosal lining of the middle ear to convert to a keratinizing epithelium . in 1873 , wendt suggested that metaplasia of the mucosa of the middle ear into the keratinizing epithelium led to cholesteatoma . [ 7 , 8 ] proposed that chronic irritation can cause the mucosal lining of the middle ear to convert to a keratinizing epithelium . ( 2 ) immigration theorybezold in 1890 and habermann described immigration theory . friedmann and tumarkin have been more contemporary supporters of this aspect , which proposed that squamous epithelium migrates through a defect in the tympanic membrane in an effort to cover areas of inflammation in the middle ear . friedmann and tumarkin have been more contemporary supporters of this aspect , which proposed that squamous epithelium migrates through a defect in the tympanic membrane in an effort to cover areas of inflammation in the middle ear . ( 3 ) hyperplasia theoryredi presented evidence that supported the basal cell hyperplasia ( papillary proliferation ) theory first published by manasse et al . in 1917 . as the result of inflammation of the middle ear , the proliferation of epithelial cones in the basal layers of the keratinizing epithelium of shrapnell 's membrane leads to cholesteatoma formation . redi presented evidence that supported the basal cell hyperplasia ( papillary proliferation ) theory first published by manasse et al . in 1917 . as the result of inflammation of the middle ear , the proliferation of epithelial cones in the basal layers of the keratinizing epithelium of shrapnell 's membrane leads to cholesteatoma formation . ( 4 ) retraction pocket theorybezold in 1890 first described this currently most - accepted theory that proposes that acquired cholesteatoma develops from retraction pockets . a retraction of shrapnell 's membrane as a result of chronic dysfunction of the eustachian tube might progress into cholesteatoma formation.4(a)habitual sniffing theory was described as under the heading of retraction theory . habitual sniffing associated with closing failure of the eustachian tube is believed to be closely related to the etiology of retraction - type cholesteatoma [ 1618 ] . it seems that such sniffing induces a high negative pressure in the middle ear and may sometimes promote the development of cholesteatoma or its recurrence after surgery [ 19 , 20 ] . bezold in 1890 first described this currently most - accepted theory that proposes that acquired cholesteatoma develops from retraction pockets . a retraction of shrapnell 's membrane as a result of chronic dysfunction of the eustachian tube might progress into cholesteatoma formation.4(a)habitual sniffing theory was described as under the heading of retraction theory . habitual sniffing associated with closing failure of the eustachian tube is believed to be closely related to the etiology of retraction - type cholesteatoma [ 1618 ] . it seems that such sniffing induces a high negative pressure in the middle ear and may sometimes promote the development of cholesteatoma or its recurrence after surgery [ 19 , 20 ] . habitual sniffing associated with closing failure of the eustachian tube is believed to be closely related to the etiology of retraction - type cholesteatoma [ 1618 ] . it seems that such sniffing induces a high negative pressure in the middle ear and may sometimes promote the development of cholesteatoma or its recurrence after surgery [ 19 , 20 ] . currently , the retraction pocket theory has many supporters following clinical observation , and there is clinical evidence for the retraction and proliferation theory on the pathogenesis of cholesteatoma [ 21 , 22 ] . sudhoff and tos suggested the proliferation of epithelial cells in the retraction pocket was altered by inflammatory stimuli of the subepithelial connective tissue and that this excessive proliferation may finally lead to cholesteatoma formation . they proposed a four - step concept for the pathogenesis of cholesteatoma that combined the retraction and proliferation theories : ( a ) the retraction pocket stage , ( b ) the proliferation stage of the retraction pocket , subdivided into cone formation and cone fusion , ( c ) the expansion stage of attic cholesteatoma , and ( d ) bone resorption ( figure 1 , ) . but , there was a lack of explanation for the transition from a retraction pocket to cholesteatoma . animal models are very important for studying the pathogenesis of acquired cholesteatoma [ 2527 ] . chinchillas , guinea pigs , mongolian gerbils , meriones unguiculatus , and rats have been used to make animal models of cholesteatoma . in several studies , chinchillas were used because their auditory apparatus is similar to that of humans . on the other hand , pigs were used for temporal bone pneumatization models because they have a mastoid ( figure 2 ) . the gerbil is the only nonhuman animal known to spontaneously develop aural cholesteatomas [ 25 , 30 ] . the ultrastructure of the epithelial and subepithelial linings of the gerbilline middle ear are similar to that of the human , and destructive characteristics of the gerbilline cholesteatoma closely mimic human cholesteatoma . chole et al . indicated that aural cholesteatoma were found to arise spontaneously in 45.7% of gerbilline ears studied . henry et al . indicated that both the prevalence of spontaneous cholesteatoma and the resultant peripheral auditory evoked potential threshold increased from 6 to 18 months of age . stage i is an accumulation of keratin debris on the outside surface of the tympanic membrane ; stage ii has a medial displacement of the tympanic membrane into middle ear without contact with the middle wall of the bulla ; stage iii , the cholesteatoma is in contact with the prominence of the cochlea ; in stage iv , cholesteatoma fills the bulla ; in stage v , the cholesteatoma extends intracranially . most of the affected 6- and 12-month - old gerbils ( younger gerbils ) had stage i or ii cholesteatomas . more than half of the 18- and 24-month - old gerbils ( older gerbils ) had stage iii or stage iv cholesteatomas . some of the oldest gerbils were at stage v , with the cranial cavity being invaded . also , in 2006 , tinling and chole investigated the migration rate and patterns for keratin on the tympanic membrane of the gerbil and guinea pig in comparison to human data and indicated that the gerbil was an appropriate model for cholesteatoma because gerbils quite closely resemble humans in rate and pattern of epithelial migration . in this paper , although the spontaneous gerbilline cholesteatoma is useful for studying the pathogenesis of cholesteatoma , the experimental ligation technique of the external ear canal in gerbils is more valuable in predicting the stage of the cholesteatoma [ 26 , 34 ] . mongolian gerbils between 2 and 6 months of age were used in this experiment . under anesthesia , some 69 months after ligation of the external auditory canal , these cholesteatomas are in contact with the bone of the middle ear . these induced cholesteatomas were seen to erode bone and displace soft tissue structures , as is typical of human aural cholesteatomas ( figure 4 , ) . from a surgical point of view , this model is easy to handle , with a high percentage of success . this animal model has been proved to be useful and informative regarding retraction pocket formation and cholesteatoma development . kim and chung examined the distribution of cytokeratin and the binding patterns of lectin in experimental cholesteatoma specimens . they concluded that the origin of aural cholesteatoma may be the external auditory canal epidermal cells , and the characteristics of these cells do not change once the cholesteatoma develops . they also suggested that cholesteatoma have a different biological nature from that of normal epithelial cells , especially basal cells . larsson et al . performed the acoustic admittance measurements and morphological analysis in experimental cholesteatoma and tympanic membrane . they indicated that the thickness of the fibrous layer was almost doubled , mostly because of an increased amount of collagen fibers , and the acoustic stiffness was significantly increased in all cholesteatoma ears . park et al . investigated the immunohistochemical study of cell proliferation using brdu labeling and the expression of plg - gamma1 , ligand - mediated signal transduction for cell proliferation on the tympanic membrane , external auditory canal , and induced cholesteatoma in gerbils . according to the results , the induced aural cholesteatoma showed a more active proliferation center of the epithelial cell and more intense immunolabeling of plg - gamma1 protein than the eardrum and external ear canal of the normal gerbil . tinling and chole also indicated the hyperproliferation of kertinocytes to be a causative factor in the development and progression of spontaneous and experimental cholesteatomas in this gerbilline model . the cornerstone of the retraction pocket theory is that eustachian tube obstruction leads to negative middle ear pressure , middle ear effusion , and retraction of the pars flaccida into the epitympanum , and subsequent cholesteatoma . to evaluate the evidence of this theory , the eustachian tube blocking model was developed by wolfman and chole in 1986 . bilateral eustachian tube obstruction by electrocauterization of the nasopharyngeal portion was performed in gerbils . mongolian gerbils between 6 to 10 weeks of age were used in this experiment . after each animal was anesthetized , they attempted to gain exposure of the tubal orifice by slitting the soft palate in the midline ( figure 5(a ) , ) . a cautery tip was then inserted through the soft palate in the midline and rotated so as to contact the right and left nasopharyngeal walls , respectively ( figure 5(b ) , ) . the results of their study were as follows ( figure 6 , ) . at two weeks , all animals had bilateral serous effusions and retraction pockets . at four weeks , four of eight ears had middle ear fluid , retractions , and cholesteatomas . after eight weeks , five of eight ears had middle ear effusions , and four of these had cholesteatomas ; one ear had total atelectasis with a cholesteatoma filling the bulla . by 16 weeks this study provides experimental evidence that aural cholesteatomas may arise by retraction of the tympanic membrane . in 2001 , they suggested that the expression pattern of cytokeratin in retraction pocket cholesteatoma is different from that in normal skin and that the transmigration and hyperproliferation process of squamous epithelium occurs in areas adjacent to the aural cholesteatoma . using this model , wilmoth et al . suggested that the elevation of tumor necrosis factor - alpha and matrix metalloproteinases associated with progressive tympanic membrane atelectasis indicated a possible role for these inflammatory mediators in the pathogenesis of cholesteatoma it was known that chemical injection into the middle ear led to the occurrence of inflammatory change and cholesteatoma formation . an animal model of cholesteatoma was made by using these chemicals in guinea pigs or rats [ 42 , 43 ] . placing a mixture of talcum powder and fibrin in the bulla of a guinea pig results in a typical cholesteatoma . it originates from the epidermal basal cells of the tympanic membrane and migrates into the middle ear . schmid and hellstrom induced the cholesteatoma formation in rats by using dimethyl - benzanthrancene ( dmba ) . after perforating the upper quadrant of the tympanic membrane of a rat , the perforation was exposed to dmba four times at weekly intervals . propylene glycol ( pg ) has been shown to cause epithelial migration and cholesteatomatous chronic otitis media in north - american chinchillas using optic drops ( cortisporin ) containing pg ( 10% ) . increasing the dose of pg in topical preparations injected transtympanically yields cholesteatomas [ 46 , 47 ] in a progressively higher percentage of rats , reaching 100% at a concentration of 90% . several studies were done in another to establish new therapies as alternative to surgery for middle ear cholesteatomas using these models . after producing a cholesteatoma in chinchillas by using pg ( 60% ) , 5-fluorouracil was used to inhibit growth of the cholesteatoma with satisfactory results . hyaluronic acid was applied to the external ear of chinchillas in an attempt to inhibit pg - induced cholesteatoma development with poor results . the local use of the transretinoic acid is effective in inhibiting the induced formation of cholesteatomas in guinea pigs . cholesteatoma is a squamous cell cyst , characterized by keratinizing epidermal tissue that can migrate and erode to adjacent structures frequently found in temporal bones . an animal model of cholesteatoma was made by using dermal tissues in the middle ears of animals [ 5254 ] . after each animal was anesthetized , a retroauricular skin incision was made following abrasion of the graft in the opposite ear . the dorsal bulla was opened , and the whole of the mucosal lining was carefully removed . the graft , taken from the external ear canal or from other parts of the body , was then placed so that the subepithelial plane faced the exposed bone . the opening of the bulla was covered with a piece of temporalis muscle and the wound sutured . according to the results , full - thickness skin grafts transplanted into the middle ear with superimposed infections induced expansive growth and cholesteatoma development [ 5254 ] . to circumvent limitations of the previous models and their unpredictable degree of bone resorption , chole et al . developed a new model of bone resorption in mouse calvarias using keratin particles . under anesthesia , murine keratin collected from the nails and fur of mice was implanted onto the dissected calvarium of mature mice . the caused an activation of osteoclasts in the adjacent bone in a manner similar to that seen in human cholesteatoma and in particle - induced osteolysis ( figure 7 ) . this model was useful in investigating the pathological bone remodeling related to cholesteatoma in a genetically well - defined animal . many of the genes and their products that control the inflammatory process are well characterized in this mouse model . based on their findings , sudhoff et al . [ 56 , 57 ] also used this dermal implanting model to investigate bone resorption observed in middle ear cholesteatoma . they concluded that the dermal implant tissue remained viable and produced a robust , localized inflammatory osteolytic response on the adjacent calvarial surface and that osteoclasts were predominantly found on the surface of the calvarium with the greatest osteoclast density under the increased expression of osteoprotegerin ( opg ) , opg ligand , and macrophage - colony stimulating factor . to investigate the origin of the epithelial cells of cholesteatoma , whether from the epithelial cells of the external auditory canal or the tympanic membrane , in a previous study , we described a new animal model named local hybrid ear model and used in situ pcr , which can detect a few copies of genes within a cell in the section by amplifying the target gene [ 58 , 59 ] . cholesteatomas were induced in gerbils with transplanted tympanic membranes using the ear auditory canal ligation method . after the pars flaccida of the tympanic membranes were completely removed from male gerbils , corresponding portions of tympanic membranes obtained from the ear of female gerbils were transplanted to the area of defect in the tympanic membranes . we then ligated the external auditory canal of the hybrid - model group . as a control group , the ear auditory canal of normal male and female gerbils was ligated without previous myringoplasty . the origin of cholesteatoma cells was analyzed by the identification of male ( xy ) or female ( xx ) cells in the tissue section . thus , in situ pcr was performed to detect the mouse x - chromosome - linked phosphoglycerate kinase-1 ( pgk-1 ) gene on the paraffin sections [ 6062 ] . as a result , one pgk-1 spot in the epithelial nuclei was detected in male cholesteatoma ( figure 8(a ) ) , and one or two pgk-1 spots were detected in female cholesteatoma ( figure 8(b ) ) . on the other hand , in the hybrid - model group , we detected not only one but two pgk-1 spots in the epithelial nuclei of cholesteatoma ( figure 8(c ) ) and one pgk-1 spot was detected in the cells of the ear auditory canal ( figure 8(d ) ) . the percentage of the number of cells having one pgk-1 spot or two pgk-1 spots of cholesteatoma in the hybrid model was almost the same as that of female cholesteatoma . these results indicated that all cholesteatoma cells in the hybrid model have xx chromosomes that were female tissue origin . the results strongly demonstrated evidence that the origin of epithelial cells in cholesteatoma is the tympanic membrane , not residential middle ear epithelial cells or the skin of the external ear canal , in this hybrid model of cholesteatoma . animal model studies on the pathogenesis of cholesteatoma have led to an improved comprehension of this disease . mongolian gerbils have a remarkable propensity for the development of aural cholesteatoma ; canal cholesteatomas develop spontaneously in aged animals . cholesteatomas were produced by five different methods of induction : ( 1 ) ligation of the external ear canal , ( 2 ) eustachian tube blocking , ( 3 ) chemical or free skin graft injection into the middle ear , ( 4 ) the autologous dermal implantation model , and ( 5 ) a local hybrid - ear model of experimentally induced cholesteatoma . as shown in table 1 , we summarized the advantages and disadvantages of each model . depending upon the purpose of studying the pathogenesis of cholesteatoma gerbilline cholesteatomas induced by these three methods ( method 1~method 3 ) were compared by kim and chole . these animal models proved to be useful and informative regarding retraction pocket formation and cholesteatoma development . as a result , the appearance rates of cholesteatoma in each group were almost the same , but the patterns of epithelial hyperplasia , keratin accumulation , thickening of the tympanic membrane , and adhesions of the tympanic membrane were different among the three groups . also , in 2002 , kim et al . detected prominent changes in the expression of markers for migration and hyperproliferation in gerbilline cholesteatomas produced by three methods compared with that in the tympanic membrane , and their results supported the epidermal migration theory . as in their previous study , the expression patterns of epithelial markers in gerbilline cholesteatomas produced by the three methods were not similar . they concluded that each of the three methods of inducing cholesteatoma may be helpful in investigating different clinical aspects of this disease . on the other hand , in 2007 , choufani et al . performed the quantitative comparison of eight biological markers involved in inflammation , cell differentiation , and cell adhesion / apoptosis between sections of the ligated external ear canal animal model ( method 1 ) and clinical specimen . their results indicated that the majority of staining parameters was statistically significantly different between sections of the animal model and clinical specimen from the panel of the above markers . they concluded that from a surgical point of view , this model was easy to handle , with a high percentage of success , thereby allowing one to quickly obtain an external auditory duct cholesteatoma . however , these data did not support the concept of complete validity of the popular animal model . the problem with the previous method 1 to method 3 animal models was that the morphological aspects were almost the same as human cholesteatoma , but the immunohistochemical results were not strictly the same . in the recent study , we modified the auditory canal ligation model to investigate the origin of the cholesteatoma cells . we analyzed spontaneously occurring cholesteatomas associated with a new transplantation model in gerbils , and using pgk-1 as a tracer provided evidence that transplanted tympanic membranes were the origin of the epithelial cells associated with the development of auditory canal - ligated cholesteatomas in gerbils . the results strongly demonstrated that the origin of epithelial cells in cholesteatoma is the tympanic membrane , but not residential middle ear epithelial cells or the skin of the external ear canal in this hybrid model of cholesteatoma . accordingly , other studies revealed different protein and cytokeratin profiles between cholesteatoma keratinocytes and epithelial cells of the middle ear [ 66 , 67 ] . habitual sniffing has clinically been observed to increase the risk of developing retraction pockets and cholesteatoma [ 68 , 69 ] . recently , the habitual sniffing - simulating model in gerbils was made by von unge and dircks . the retraction of the tympanic membrane was found , but cholesteatoma formation did not appear . the retraction and proliferation theory is a pathogenesis that combines the retraction caused by otitis media or habitual sniffing and the proliferation of the epithelial cells in the retraction pocket altered by inflammatory stimuli of the subepithelial connective tissue . therefore , some improvement will be needed to investigate the retraction and proliferation theory using this habitual sniffing model . based on the results of inflammatory changes in clinical cholesteatoma specimens , we would like to develop a new animal model in the future . in our previous study , we indicated that keratinocyte growth factor ( kgf)/fibroblast growth factor-7 plays an important role in cholesteatoma formation . kgf is a mesenchymal cell - derived paracrine growth factor that specifically stimulates epithelial cell growth and is supposed to be secreted from fibroblasts mainly in stroma binding to kgf receptor , which has only been detected on the surface of epithelial cells [ 72 , 73 ] . on the other hand , direct in vivo plasmid dna transfer to the skin via injection has been reported previously [ 74 , 75 ] , and transfer of dna using electroporation has been demonstrated as a useful procedure for the short - term delivery of gene therapy . we will use kgf - cmv-14 vector and cause overexpression of kgf by electroporatively transfected kgf cdna in the cells of epithelial tissues in vivo . a single injection of kgf cdna - expressive vector coupled with electroporation will enhance inflammatory reaction and increase keratinization of epithelium in the auditory canal . this model can possibly be used to investigate the effects of various cytokines and growth factors in cholesteatoma formation in vivo . the goal of the experiments using animal models is to analyze the pathogenesis of cholesteatoma . of course , we know that biological differences between animal models and human cholesteatoma would make it difficult to understand the pathogenesis of human cholesteatoma .
background . relatives who are bereaved by suicide likely consult their gp when they feel the need for professional help . gps may play a key role in establishing who is at risk for adverse consequences of the loss as they are familiar with relatives possible psychiatric vulnerabilities . the availability of evidence - based services for relatives of suicide victims is limited . successful implementation of services needs analysis of key factors considered critical in the achievement of changes . we investigated gps management of help requests of relatives bereaved by suicide and examined determinants of gps willingness to refer for evidence - based follow - up care.methods . a cross - sectional survey among 488 gps in the northern part of the netherlands.results . a 44% response was achieved ( n = 214 ) during the last 3 years , 38 ( 18% ) were exposed to suicide , 21 ( 10% ) to help requests without being exposed to suicide and 52 ( 24% ) to both suicide and help requests . out of 106 requests , 69 ( 65% ) were handled by the gp ; 60 ( 57% ) were either directly or additionally referred , principally for mental health care . suicide exposure and female gender were associated with the doctor 's perception that follow - up care following a loss through suicide is useful . the perception that help is useful increased the likelihood of gps referral for evidence - based follow - up care.conclusions . gps support the availability of evidence - based follow - up care for relatives of suicide victims . to modify gps key role in referring relatives for it , gps should be well informed of its usefulness and to whom .	a loss through suicide is a dreadful experience to those who are left behind.1 these persons likely consult their gp when they feel the need for help . appropriate care meets an unmet need ; 2688% feel that help is required2,3 while evidence - based services are scarce . relatives of suicide victims are at increased risk of adverse mental health consequences of the loss , e.g. complicated grief ( cg).35 cg is characterized by avoidance , disbelief , numbness , detachment and excessive irritability and anger . it is strongly associated with suicidal behaviour , physical morbidity and other mental morbidity.6,7 moreover , suicidal behaviour8 and completed suicide9 cluster in families , posing relatives of suicide victims at increased suicide risk . it may be effectuated by referring these high - risk individuals for effective follow - up care.10 family - based cognitive behaviour therapy ( fgt ) is more helpful to reduce feelings of guilt and maladaptive grief reactions than usual care.11 among relatives with suicidal feelings , fgt seems to be more effective to decrease the risk of cg confirming the benefits of grief interventions for high - risk individuals.12 gps active engagement in suicide prevention strategies is acknowledged.13 gps are identified as key persons to initiate14and ensure follow - up care for people bereaved by suicide.15 gps attitudes towards referral of suicide bereaved relatives for follow - up care are unknown . knowledge of these factors is of interest since acceptance and implementation of effective interventions need careful analyses of key factors critical in the achievement of changes in practice.16 in order to study the proposed key role of gps in referring relatives for follow - up care , we investigated the gps attitudes and management of help requests following a suicide within the framework of experimentally implementing fgt . factors associated with gps willingness to refer relatives were examined . among these factors was the gps gender as female gps have shown to be more attentive to psychosocial factors than male gps.17 the findings from the present study may be of interest for policy makers and mental health care providers deliberating the availability of effective suicide prevention measures . a cross - sectional , semi - structured questionnaire survey was sent to a random sample of 488 gps of the total number of 895 in the northern part of the netherlands18 gps exposure to patient suicide , bereaved relatives help requests , management of help requests , preference as to the type of care and gps contentment with their interventions were assessed . gps involvement in patient suicide was assessed by asking have you been exposed to a patient 's suicide and if so how many times? exposure to help requests was assessed by the following item : have you been exposed to help requests for bereavement by suicide , not necessarily concerning suicide of one of your patients . if so , how many times? gps were asked to report suicides and help requests over the past 3 years . of at most the last two help requests , the time lag ( in months ) between the suicide and the request was assessed . gps reported whether they counselled the relatives themselves and/or referred for additional help . in case of referral , gps indicated the type of health care workers . further , it was assessed what kind of health care setting is generally preferred : mental health care , primary health care or other kinds of care . multiple services could be indicated . in the questionnaire , the aims , method , benefits and potential risks of fgt were briefly presented . gps were asked if they think fgt is useful ( yes , no , i do n't know ) . gps were also asked if they would be willing to refer patients for fgt if it were available ( yes , characteristics of the gps , their exposure to suicide and help requests , management of help requests , preferences as to types of care and gps contentment with the outcome of their proceedings were descriptively analysed . next , bivariate associations between gps exposure to suicide , exposure to help requests , gps gender , perceptions of usefulness and willingness to refer for fgt were calculated using logistic regression . using multivariable logistic regression models , it was examined whether female gender and gps exposure to suicide were independently associated with gps exposure to help requests . further , we investigated whether female gender , gps exposure to suicide and exposure to help requests are independently associated with the perception that follow - up care would be useful . finally , the associations between the aforementioned determinants as well as perception that help is useful were examined in independent association with gps willingness to refer relatives for follow - up care . results were expressed as odds ratios as measures of relative risk , indicating the magnitude of associations . characteristics of the gps , their exposure to suicide and help requests , management of help requests , preferences as to types of care and gps contentment with the outcome of their proceedings were descriptively analysed . next , bivariate associations between gps exposure to suicide , exposure to help requests , gps gender , perceptions of usefulness and willingness to refer for fgt were calculated using logistic regression . using multivariable logistic regression models , it was examined whether female gender and gps exposure to suicide were independently associated with gps exposure to help requests . further , we investigated whether female gender , gps exposure to suicide and exposure to help requests are independently associated with the perception that follow - up care would be useful . finally , the associations between the aforementioned determinants as well as perception that help is useful were examined in independent association with gps willingness to refer relatives for follow - up care . results were expressed as odds ratios as measures of relative risk , indicating the magnitude of associations . results are presented with a 95% confidence interval . the two - sided level of significance of the 488 gps who were approached , 214 ( 44% ) returned the questionnaire . the proportion of female responders was equal to the proportion in the study region ( 31%).18 female doctors had fewer mean ( sd ) years of practice experience than male doctors ( 11 ( 7.9 ) years versus 17 ( 9.6 ) years ) . sample characteristics and the exposure to suicide and help requests are shown in table 1 . characteristics , suicide and help request exposure of gps representing the samplea stated in n ( % ) unless indicated otherwise . sixty - two per cent ( n = 176 ) indicated that follow - up care for relatives following a suicide pertains to primary health care , 47 gps ( 27% ) to mental health care , 31 ( 22% ) indicated that bereavement support is not a professional health care occupation ; 9 ( 4% ) did not know . seventy - four gps reported 128 help requests ; 106 could be explored ; 81 ( 76% ) were presented within 6 months following the suicide ; 13 ( 12% ) were > 1 year after the suicide . table 2 shows how gps dealt with the help requests ( n = 106 ) . in 61 cases ( 58% ) , the gp was content with the results of their interventions , 33 gps ( 31% ) indicated partial and 7 gps ( 7% ) were not content with the outcomes . gps management of help requests ( n = 106 ) relatives were occasionally referred for multiple services . one hundred thirty - nine gps ( 66% ) indicated that fgt might be useful ; 146/214 ( 68% ) stated that they would refer patients for fgt if it were actually available ; 43 ( 21% ) would conditionally refer patients . most noted conditions were when fgt has additional effects and when the patient wants to. bivariate relationships between determinants of gps management of help requests after a suicide are displayed in table 3 . bivariate associations between determinants of gps management of help requests by relatives bereaved by suicidea values are univariable odds ratios with 95% confidence intervals . table 4 shows that gps who were exposed to a patient suicide were six times more likely to be consulted by relatives . more gps who had recently been exposed to suicide believed that follow - up care for the bereaved would be useful than those who were not . female gender was strongly associated with the perception that help is useful , independent of exposure to patient suicide and exposure to relative 's help request . however , the perception that follow - up care is useful was less likely when gps had been confronted with help requests in the previous 3 years . the willingness to refer patients for follow - up care did not depend on gps gender or suicide exposure . when gps were , perceiving that follow - up care is useful , referring patients for it was four times more likely . adjusting the outcomes for the number of years of practice experience did not change the results . although not statistically significant , exposure to help requests was associated with a two times reduced likelihood of the perception that follow - up care is useful and gps willingness to refer relatives for it . factors associated with gps willingness to refer relatives bereaved by suicide for follow - up care adjustment for years of practice experience of the gp did not change the results . or , odds ratio ; ci , confidence interval . this study demonstrates that gps think that effective follow - up care for relatives bereaved by suicide ( e.g. fgt ) is useful and they are willing to refer relatives if it were available . it is in line with halligan and corcoran ( 2001)19 who reported a 62% proportion who thinks that services to rely on in the aftermath of suicide would be helpful . although it has been suggested that female gps are more involved in patients who present with psychosocial problems than male colleagues,20 it apparently does not hold for help seeking following a suicide . the high impact of a patient suicide on gps19 may be an overriding factor in this respect . bereaved families believe that the gps should initiate contact with the family briefly after the suicide.14,15 in this study , it remained unclear whether relatives or gps were the initiators . therefore , the findings do not allow the suggestion that gps meet relatives needs sufficiently , taking into account that 57% of the help requests were referred for mental health care ( table 2 ) . possibly , gps recognize the psychiatric vulnerability of relatives of suicide victims,21 whereas an estimated 20% of the bereaved are actually at increased risk of psychiatric consequences . it implicates that the high rate of referral for mental health care may , in part , be unnecessary , but probably come along with relatives persisting need for help.2 possibly , gps may be inclined to refer relatives anyway due to gps feelings of guilt or due to a disrupted relationship with the bereaved family14,19 or when the gp is blamed for the suicide.11 however , unnecessary referral for mental health care may stigmatize relatives and should be prevented . the response rate ( 44% ) is low , but not uncommon in studies on subjects of sensitive nature ( e.g.14,22,23 ) . probably , gps who were recently involved in suicide might be somewhat over represented in the current sample . the explanation that higher exposure is causing higher response rates in this research area is supported by a previous finding of hilligan and corcoran ( 2001 ) . they achieved a 79% response in the sample of gps , of which 86% was confronted with a patient suicide over a 10 years period , whereas dutch gps are only four times involved in a patient suicide during their professional life.24 responders in the current study may have been implicated in a randomised controlled trial ( rct ) to the effectiveness of fgt.11 possibly , this has affected their attitudes and proceedings regarding relatives help requests . this assumption is supported by the large proportion of gps ( 65% ) who counselled the bereaved themselves ; more often than a 50% rate found in previous studies.25 unfortunately , it remained unclear to what extent the sample represents gps who were actually involved in the rct . overall , the extrapolation of the results should be applied cautiously ; however , issues related to follow - up care for those bereaved by suicide principally concern gps who were actually involved and this may , in turn , support the findings validity . in countries with self - employed doctors and a referral system , gps are in a better position to provide psychosocial care.26 this may moderate the generalizabiliy of the findings to countries without a referral system . gps should be well informed of the effectiveness of follow - up care for bereaved relatives as when gps are convinced of the benefits , they are more inclined to refer relatives for it , especially male gps who previously dealt with help seeking relatives of suicide victims . however , gps are pragmatic and cautious of change27 ; their sense of competence regarding the management of help requests of relatives bereaved by suicide may discourage the implementation of evidence - based services.16,28 given the need for help and its effects for relatives who are liable to adverse health consequences and feelings of guilt , fgt should be available for vulnerable relatives . fgt might be provided in primary care , for instance by consultation of trained mental health nurses . this may give easier access to this kind of help than when provided in mental health care . the availability of fgt should be recommended in guidelines on the prevention of suicidal behaviours . in view of the key role for gps in suicide prevention , especially in referring people bereaved by suicide for effective follow - up care , deliberations of gps in the management of these help requests might be explored in future qualitative research . the response rate ( 44% ) is low , but not uncommon in studies on subjects of sensitive nature ( e.g.14,22,23 ) . probably , gps who were recently involved in suicide might be somewhat over represented in the current sample . the explanation that higher exposure is causing higher response rates in this research area is supported by a previous finding of hilligan and corcoran ( 2001 ) . they achieved a 79% response in the sample of gps , of which 86% was confronted with a patient suicide over a 10 years period , whereas dutch gps are only four times involved in a patient suicide during their professional life.24 responders in the current study may have been implicated in a randomised controlled trial ( rct ) to the effectiveness of fgt.11 possibly , this has affected their attitudes and proceedings regarding relatives help requests . this assumption is supported by the large proportion of gps ( 65% ) who counselled the bereaved themselves ; more often than a 50% rate found in previous studies.25 unfortunately , it remained unclear to what extent the sample represents gps who were actually involved in the rct . overall , the extrapolation of the results should be applied cautiously ; however , issues related to follow - up care for those bereaved by suicide principally concern gps who were actually involved and this may , in turn , support the findings validity . in countries with self - employed doctors and a referral system , gps are in a better position to provide psychosocial care.26 this may moderate the generalizabiliy of the findings to countries without a referral system . gps should be well informed of the effectiveness of follow - up care for bereaved relatives as when gps are convinced of the benefits , they are more inclined to refer relatives for it , especially male gps who previously dealt with help seeking relatives of suicide victims . however , gps are pragmatic and cautious of change27 ; their sense of competence regarding the management of help requests of relatives bereaved by suicide may discourage the implementation of evidence - based services.16,28 given the need for help and its effects for relatives who are liable to adverse health consequences and feelings of guilt , fgt should be available for vulnerable relatives . fgt might be provided in primary care , for instance by consultation of trained mental health nurses . this may give easier access to this kind of help than when provided in mental health care . the availability of fgt should be recommended in guidelines on the prevention of suicidal behaviours . in view of the key role for gps in suicide prevention , especially in referring people bereaved by suicide for effective follow - up care , deliberations of gps in the management of these help requests might be explored in future qualitative research . funding : the impress study ( primary prevention of cg in the aftermath of suicides . region - wide implementation of a brief , nurse - led , cognitive behavioural , family - oriented , primary care and evidence - based approach ) was funded by zon - mw ( the netherlands organisation for health research and development ) ( 24200027 ) ; http://www.zonmw.nl/en . funding to pay the open access publication charges for this article was provided by the department of general practice medicine of the university medical center groningen , the netherlands . ethical approval : the study was approved by the university medical center groningen ethics committee ( metcumcg 2002/137 ) .
introductionrasagiline is a selective , irreversible monoamine oxidase b inhibitor that ameliorates the symptoms of parkinson s disease ( pd ) by inhibiting striatal dopamine metabolism . there is also evidence that monoamine oxidase b inhibitors increase melatonin levels in the pineal gland and may have a beneficial effect on sleep disorders , which are a common feature in patients with pd.methodsthis single - center , prospective , observational , 12-week study compared the effect of combination therapy with levodopa 200300 mg / d + rasagiline 1 mg / d ( n=19 ) with levodopa 200300 mg / d alone ( n=19 ) in the treatment of sleep disorders in patients with idiopathic pd.resultsafter 12 weeks treatment , mean sleep latency was significantly ( p<0.001 ) lower and the improvement in sleep latency from baseline was significantly ( p=0.001 ) greater in patients receiving levodopa + rasagiline than in patients receiving levodopa alone . similarly , at the end of the study , the mean total sleep time was significantly ( p=0.002 ) longer and the improvement from baseline in mean total sleep time was significantly ( p=0.026 ) greater in patients receiving levodopa + rasagiline than levodopa alone . there were no significant differences between treatment groups for the mean number of awakenings reported at week 12 nor the change from baseline to week 12 in mean number of awakenings.conclusionadding rasagiline to levodopa improved sleep outcomes and may be an appropriate option for patients with pd experiencing sleep disorders .	idiopathic parkinson s disease ( pd ) is often associated with sleep disorders , the most common of which are insomnia , increased daytime sleepiness , restless legs syndrome , and rapid eye movement ( rem ) sleep behavior disorder.1 polysomnographic studies have demonstrated that patients with pd experience increased sleep latency , reduced sleep efficiency , and reduced duration of rem sleep.2 sleep disorders affect approximately two - thirds of patients with pd,3 and these patients are likely to experience a greater severity of nonmotor pd symptoms than those without sleep disorders.4 unsurprisingly , sleep disorders in patients with pd have a negative impact on quality of life.2 assessing sleep using traditional instruments ( polysomnography ) is both costly and time consuming and requires specialized hospital - based settings . consequently , sleep scales are widely employed in clinical practice . in patients with pd , individual items of the parkinson s disease sleep scale ( pdss ) accurately distinguish patients from healthy controls , and items of the pdss have been shown to correlate with polysomnography.5 the pdss is also useful to assess the severity of sleep disorders in pd , and individual sleep scales can be used to evaluate response to treatment . the most frequently used pd medications ( eg , levodopa and dopamine agonists ) show little efficacy in controlling sleep disturbances.6,7 in fact , dopaminergic medication appears to adversely affect sleep continuity ; low - dose dopamine agonists are associated with insomnia while higher doses are associated with day - time sleepiness.1,6 rasagiline is a selective , irreversible inhibitor of monoamine oxidase b ( maob - i ) that ameliorates the symptoms of pd by inhibiting striatal dopamine metabolism.8 maob - is also increase melatonin levels in the pineal gland and may therefore contribute to modulation of wakefulness / sleep patterns and circadian rhythms.9 rasagiline has been used for many years in the european union and usa as a first - line treatment for early forms of pd , as well as in combination with levodopa and dopamine agonists in more advanced forms of the disease . clinical studies with rasagiline have demonstrated that the drug has a good safety profile as monotherapy10 and in combination regimens.11 the aim of this observational study was to compare the efficacy of levodopa + rasagiline with that of levodopa alone in the treatment of sleep disorders in patients with pd . this single - center , prospective , observational study evaluated 38 outpatients with pd experiencing sleep disturbances . the study was performed over 12 weeks from january to june 2015 at the clinic of movement disorders , ii division of neurology , second university of naples , italy . the study was approved by the medical ethical committee of second university of naples and security board , and all participants provided written informed consent . patient privacy was maintained and only the investigator was able to link data to an individual via the identification number assigned at enrollment . eligible patients were aged 18 years with a diagnosis of idiopathic mild - to - moderate pd ( hoehn and yahr stage 12 ) , according to the united kingdom parkinson s disease society brain bank diagnostic criteria . patients were also required to be experiencing sleep disturbances ( including insomnia , hypersomnolence , vivid dreaming , restless legs , and other sleep - related movement disorders ) , as reported by the patient ( pdss mean score 100 ) and to be receiving treatment with levodopa ( 200300 mg / d ) with or without rasagiline ( 1 mg / d , administered in a single dose at 7 pm ) . exclusion criteria included the presence of any primary sleep disorder ; any neurodegenerative disease other than pd or any type of dementia ( according to diagnostic and statistical manual of mental disorders , 4th edition criteria ) , by physician assessment ; pregnancy and/or breastfeeding ; and comorbidities that could result in secondary sleep disorders , including congestive heart failure , other serious heart disease , severe liver disease / cirrhosis , severe renal failure / dialysis , severe respiratory failure , severe anemia , hypothyroidism , and diabetes . cutoff values for comorbidity signs and symptoms were not defined ; inclusion or exclusion of a given patient was at the investigator s discretion . patients were randomly assigned to 12 weeks treatment with levodopa ( 200300 mg / d ) or levodopa + rasagiline ( 1 mg / d ) . patient demographics and characteristics were collected at baseline and included family history , age of onset of pd , stage of disease ( hoehn and yahr staging ) , motor symptoms ( unified parkinson s disease rating scale iii scores ) , cognitive function ( mini - mental state examination and beck depression inventory scale scores ) treatments received , and the presence of comorbidities . the primary end point was improvement in sleep quality based on sleep latency ( hours ) , number of awakenings , and total sleep time ( hours ) as recorded in autonomously compiled patient sleep diaries . patients were required to record , within 30 minutes of waking , information on their sleep clinical evaluations , performed at baseline and at the end of the 12-week study period , included the following : general physical , neurological and psychological examination , assessment of adherence to therapy , administration of the hoehn and yahr scale , and administration of the pdss ( a decrease in pdss scores indicates improvement ) . given the observational nature of the study , an expected difference between the two treatment groups was not assumed a priori , and so sample size was not calculated . descriptive statistical analyses were performed using a general linear model ( glm ) to correct for observed variation in baseline features . data are expressed as means with standard deviation and mean standard error ( se ) for the continuous variables . two - tailed t - tests were applied to evaluate statistical significance within groups and significance of between - group difference was based on analysis of variance models ( p<0.05 ) . this single - center , prospective , observational study evaluated 38 outpatients with pd experiencing sleep disturbances . the study was performed over 12 weeks from january to june 2015 at the clinic of movement disorders , ii division of neurology , second university of naples , italy . the study was approved by the medical ethical committee of second university of naples and security board , and all participants provided written informed consent . patient privacy was maintained and only the investigator was able to link data to an individual via the identification number assigned at enrollment . eligible patients were aged 18 years with a diagnosis of idiopathic mild - to - moderate pd ( hoehn and yahr stage 12 ) , according to the united kingdom parkinson s disease society brain bank diagnostic criteria . patients were also required to be experiencing sleep disturbances ( including insomnia , hypersomnolence , vivid dreaming , restless legs , and other sleep - related movement disorders ) , as reported by the patient ( pdss mean score 100 ) and to be receiving treatment with levodopa ( 200300 mg / d ) with or without rasagiline ( 1 mg / d , administered in a single dose at 7 pm ) . exclusion criteria included the presence of any primary sleep disorder ; any neurodegenerative disease other than pd or any type of dementia ( according to diagnostic and statistical manual of mental disorders , 4th edition criteria ) , by physician assessment ; pregnancy and/or breastfeeding ; and comorbidities that could result in secondary sleep disorders , including congestive heart failure , other serious heart disease , severe liver disease / cirrhosis , severe renal failure / dialysis , severe respiratory failure , severe anemia , hypothyroidism , and diabetes . cutoff values for comorbidity signs and symptoms were not defined ; inclusion or exclusion of a given patient was at the investigator s discretion . patients were randomly assigned to 12 weeks treatment with levodopa ( 200300 mg / d ) or levodopa + rasagiline ( 1 mg / d ) . patient demographics and characteristics were collected at baseline and included family history , age of onset of pd , stage of disease ( hoehn and yahr staging ) , motor symptoms ( unified parkinson s disease rating scale iii scores ) , cognitive function ( mini - mental state examination and beck depression inventory scale scores ) treatments received , and the presence of comorbidities . the primary end point was improvement in sleep quality based on sleep latency ( hours ) , number of awakenings , and total sleep time ( hours ) as recorded in autonomously compiled patient sleep diaries . patients were required to record , within 30 minutes of waking , information on their sleep clinical evaluations , performed at baseline and at the end of the 12-week study period , included the following : general physical , neurological and psychological examination , assessment of adherence to therapy , administration of the hoehn and yahr scale , and administration of the pdss ( a decrease in pdss scores indicates improvement ) . given the observational nature of the study , an expected difference between the two treatment groups was not assumed a priori , and so sample size was not calculated . descriptive statistical analyses were performed using a general linear model ( glm ) to correct for observed variation in baseline features . data are expressed as means with standard deviation and mean standard error ( se ) for the continuous variables . two - tailed t - tests were applied to evaluate statistical significance within groups and significance of between - group difference was based on analysis of variance models ( p<0.05 ) . all 38 patients completed the 12 weeks treatment , and key baseline demographics / clinical characteristics are described in the supplementary information ( table s1 ) . patients had a mean age of 70.310.6 years , 56.8% were male , and mean disease duration was 55.95.5 months . almost all the patients reported difficulties in initiating and maintaining sleep , with correspondingly high pdss ( 104.721.5 and 103.921.8 in the monotherapy and combination therapy groups , respectively ) . the mean levodopa dose was 267.41 mg / d in the combination group vs 281.7 mg / d in the monotherapy group . sleep latency ( figure 1a ) , number of awakenings ( figure 1b ) , and total sleep time ( figure 1c ) all improved significantly from baseline to week 12 in patients receiving levodopa + rasagiline . in contrast , sleep latency was the only measure to show a significant improvement from baseline to week 12 ( figure 1a ) with levodopa alone . at 12 weeks and compared with levodopa alone , patients receiving levodopa + rasagiline had a significantly lower mean sleep latency time ( figure 1a ) , while the improvement from baseline was significantly greater with levodopa + rasagiline ( 0.54740.69472 h vs 1.67891.20627 h ; p=0.001 ) . there was no significant difference between treatment groups for the mean number of awakenings reported at week 12 ( figure 1b ) or change from baseline to week 12 ( 0.21050.41885 vs 0.15790.50146 ; p=0.728 ) . at week 12 , mean total sleep time was significantly greater in patients receiving combination treatment than monotherapy ( figure 1c ) , and the improvement from baseline to week 12 was significantly greater with levodopa + rasagiline than levodopa alone ( 1.26421.62450 h vs 0.32110.69648 h ; p=0.026 ) . applying a glm to correct for baseline features , the treatment received and the baseline value of each end point were significantly related to variation at week 12 . the estimated marginal mean of delta sleep latency was 1.57 ( se : 0.166 ; 95% ci : 1.906 , 1.225 ) in the levodopa + rasagiline group vs 0.58 ( se : 0.181 ; 95% ci : 0.954 , 0.208 ) in the levodopa alone group ( p=0.001 ) ; the estimated marginal mean of delta number of awakenings was 0.36 ( se : 0.086 ; 95% ci : 0.535 , 0.182 ) and 0.004 ( se : 0.101 ; 95% ci : 0.203 , 0.211 ) , respectively ( p=0.013 ) , while the estimated marginal mean of delta total sleep hours was 1.35 ( se : 0.315 ; 95% ci : 0.701 , 1.995 ) vs 0.30 ( se : 0.362 ; 95% ci : 0.449 , 1.041 ) , respectively ( p=0.044 ) . improvements in pdss at 12 weeks were greater with combination treatment compared with levodopa alone ( figure 2 ) . a significant reduction in pdss scores ( ie , an improvement in sleep ) from baseline to week 12 was observed in six of 15 items in the levodopa alone group ( distressing dreams , urine incontinence , limb paresthesia , cramps , tremor , and feeling tired and sleepy ) and ten of 15 items ( restless limbs , fidgeting in bed , distressing dreams , distressing hallucinations , nycturia , urine incontinence , limb paresthesia , cramps , tremor , and feeling tired and sleepy ) in the levodopa + rasagiline group . pdss scores at 12 weeks were significantly lower with levodopa + rasagiline vs levodopa alone for nycturia ( p=0.012 ) and limb paresthesia ( p=0.032 ) , while the change from baseline to week 12 significantly favored levodopa + rasagiline vs levodopa alone for nycturia ( p=0.002 ) . applying a glm to correct for baseline features , the baseline value of each item was significantly related with variation for 12 of 15 items at week 12 . this observational study indicates that the addition of rasagiline to levodopa improves sleep quality in patients with pd experiencing sleep disorders . after 12 weeks treatment , sleep latency and total sleep time were significantly more improved in patients receiving combination therapy than in those receiving levodopa alone . our findings support those of the randomized , double - blind actor study in patients with pd , which demonstrated that physician - assessed sleep disorders and daytime sleepiness decreased significantly more with rasagiline 1 mg / d than with pramipexole 1.5 mg / d.10 in animal models , it has been shown that inhibition of monoamine oxidase ( mao ) also increases melatonin levels in the pineal gland,9 and it is hypothesized that this is how rasagiline may improve sleep disorders in patients with pd . furthermore , rasagiline lacks amphetamine - like metabolites , and so may confer advantages over other maob - is in the treatment sleep disorders in patients with pd.12 although the study was prospective , it had a number of limitations including the relatively small number of patients recruited . furthermore , we restricted our study to subjective symptoms and perception of restfulness using patient sleep diaries and pdss scores . we accept that detailed assessment of the complex sleep architecture in patients with pd requires additional studies using instrumental techniques that currently are not in common clinical practice ( eg , polysomnography ) , expanding our observations on rem behavior disorder , and periodic limb movement syndrome . this small monocentric study suggests that rasagiline added to chronic levodopa therapy might provide relevant benefit to patients with pd experiencing sleep disorders . given the high prevalence of sleep disorders in this population , prospective , randomized studies in more patients and over a longer time period are warranted . baseline characteristics and demographics note : all values are mean sd unless otherwise stated . abbreviations : bdi , beck depression inventory ; mmse , mini - mental state examination ; pdss , parkinson s disease sleep scale ; updrs iii , unified parkinson s disease rating scale part iii ; sd , standard deviation .
purposeto compare grading of goniophotographic images and gonioscopy in assessing the iridocorneal angle.methodsin a population - based , cross - sectional study , participants underwent gonioscopy and goniophotographic imaging during the same visit . the iridocorneal angle was classified as closed if the posterior trabecular meshwork could not be seen . a single masked observer graded the goniophotographic images , and each eye was classified as having angle closure based on the number of closed quadrants . agreement between the methods was analyzed by calculating kappa ( ) and first - order agreement coefficient ( ac1 ) statistics and comparison of area under receiver operating characteristic curves ( auc).resultsa total of 4149 chinese americans ( 3994 eyes ) were included in this study . the agreement for angle closure diagnosis between gonioscopy and eyecam was moderate to excellent ( = 0.60 , ac1 0.90 , auc 0.760.80).conclusionsdetection of iridocorneal angle closure based on goniophotographic imaging shows moderate to very good agreement with angle closure assessment using gonioscopy .	details of the study design and procedures are described in detail elsewhere . in brief , the examination clinic is located in the city of monterey park , california , in los angeles county , which has a large chinese - american population . the study enrollment began in 2010 and included a total of 4582 individuals of chinese descent , aged 50 years and older . ethics committee approval was obtained from the institutional review board of the university of southern california health sciences . exclusion criteria included prior intraocular surgery ( e.g. , history of cataract extraction , corneal transplant , incisional glaucoma surgery , and retina surgery ) , penetrating eye injury , or the presence of corneal disorders such as corneal endothelial dystrophy , pterygium , or corneal scars that may preclude satisfactory imaging . after a detailed ophthalmic history was obtained , participants received a complete ocular examination , including visual acuity assessment , pupil assessment , visual field exam ( sita standard 24 - 2 ) , slit - lamp biomicroscopy , goldmann applanation tonometry , gonioscopy , imaging with the eyecam , and dilated fundus examination of the optic nerve , macula , and periphery . clinical examinations were performed by two glaucoma - trained ophthalmologists ( dw , cl ) . indirect gonioscopy was performed with a posner - type 4-mirror lens ( model odpsg ; ocular instruments , inc . , the slit was minimized to a 1-mm height and width to prevent light from entering the pupil . the light was also minimized to a level that was necessary to adequately observe the angle . grade 1 was recorded for angles in which the width was judged to be 5 to 15. grade 2 was assigned for angles with a width of 15 to 25. grade 3 was used for 25 to 35 angles , and grade 4 for greater than 35 angles . subjects were placed in a supine position , and the room was darkened during testing . imaging with the eyecam was performed by a single trained technician . images were obtained from all four quadrants ( inferior , superior , nasal , and temporal quadrants sequentially ) of both eyes . topical anesthetic drops ( proparacaine hydrochloride 0.5% ; alcon laboratories , inc . , fort worth , tx , usa ) were applied , followed by a coupling gel . if the view of the angle was blocked by a convex lens curvature , the technician was allowed to move the tip approximately 10 anteriorly along the cornea , similar to slightly tilting a gonioscopy lens . evaluation of the angle structures was accomplished by adjusting the distance of the hand piece tip from the limbus , and the illumination was adjusted by the foot pedal control . the observer was blinded to the patient 's age , sex , and clinical information , including gonioscopy results . parameters assessed included : ( 1 ) image quality , graded as 1 to 3 , ( 2 ) angle grade by structures identified , ( 3 ) pigmentation level , ( 4 ) presence or absence of sampaolesi 's line , and ( 5 ) any abnormalities such as pigmented tumors . image quality was graded between 1 and 3 , with grade 1 representing the best quality score in which the angle image was clear , grade 2 indicating a slightly blurred image in which angle structures remained distinguishable , and grade 3 a blurred image in which angle structures are difficult to identify . angle grading was according to the following : ( 1 ) grade 4 ( wide open angle ) in which ciliary body band is observed for the majority of the image , ( 2 ) grade 3 ( open angle ) in which scleral spur is observed for the majority of the image , ( 3 ) grade 2 ( narrow but open angle ) in which the posterior tm is the most posterior structure seen for the majority of the image , ( 4 ) grade 1 ( narrow occludable angle ) in which only the anterior tm is seen for the majority of the image , and ( 5 ) grade 0 ( closed / appositional angle ) in which no angle structures are seen for most of the image . these angle grading categories matched the shaffer classification system used to grade the eyes clinically on gonioscopy . for both eyecam and gonioscopy , angle closure was defined as grade 0 in the classification scheme described . sampaolesi 's line was graded as positive if a linear pigmented deposition was observed anterior to the schlwabe 's line . abnormalities that were specifically denoted included focal peripheral anterior synechia , iris processes , patchy tm pigmentation , iris elevation , iris lesion ( mass with increased pigmentation ) , and neovascular vessels . the right eye of each patient was used for analysis unless that eye met one or more of the exclusion criteria , in which case the left eye was used ( provided it met no exclusion criteria ) . the mcnemar test was used to test the null hypothesis of marginal homogeneity ( rater agreement ) in 2 2 cross classifications of paired responses to dichotomous items ( quadrant / angle closure ) . the simple kappa ( ) statistic was used to assess the strength of agreement between dichotomized ( open / closed ) variables , while the weighted was used to measure agreement for ordinal variables ( the polychotomous grade variable used to quantify angle width ) , and to measure intra- and interrater reliability . the first - order agreement coefficient ( ac1 ) statistic was used in addition to , as the latter statistic can yield coefficients that are paradoxically low relative to overall agreement under conditions where high trait prevalence causes a marked imbalance in contingency table marginals . receiver operating characteristic curves with calculations of area under the curve ( auc ) and 95% confidence intervals ( cis ) were used as an index of performance for identification of eyes with angle closure , using gonioscopy as the reference standard . statistical analyses were performed using sas version 9.4 ( sas institute , inc . , cary , nc , usa ) . after a detailed ophthalmic history was obtained , participants received a complete ocular examination , including visual acuity assessment , pupil assessment , visual field exam ( sita standard 24 - 2 ) , slit - lamp biomicroscopy , goldmann applanation tonometry , gonioscopy , imaging with the eyecam , and dilated fundus examination of the optic nerve , macula , and periphery . clinical examinations were performed by two glaucoma - trained ophthalmologists ( dw , cl ) . indirect gonioscopy was performed with a posner - type 4-mirror lens ( model odpsg ; ocular instruments , inc . , bellevue , wa , usa ) under dark ambient lighting . the slit was minimized to a 1-mm height and width to prevent light from entering the pupil . the light was also minimized to a level that was necessary to adequately observe the angle . grade 1 was recorded for angles in which the width was judged to be 5 to 15. grade 2 was assigned for angles with a width of 15 to 25. grade 3 was used for 25 to 35 angles , and grade 4 for greater than 35 angles . subjects were placed in a supine position , and the room was darkened during testing . imaging with the eyecam images were obtained from all four quadrants ( inferior , superior , nasal , and temporal quadrants sequentially ) of both eyes . topical anesthetic drops ( proparacaine hydrochloride 0.5% ; alcon laboratories , inc . , fort worth , tx , usa ) were applied , followed by a coupling gel . if the view of the angle was blocked by a convex lens curvature , the technician was allowed to move the tip approximately 10 anteriorly along the cornea , similar to slightly tilting a gonioscopy lens . evaluation of the angle structures was accomplished by adjusting the distance of the hand piece tip from the limbus , and the illumination was adjusted by the foot pedal control . the observer was blinded to the patient 's age , sex , and clinical information , including gonioscopy results . parameters assessed included : ( 1 ) image quality , graded as 1 to 3 , ( 2 ) angle grade by structures identified , ( 3 ) pigmentation level , ( 4 ) presence or absence of sampaolesi 's line , and ( 5 ) any abnormalities such as pigmented tumors . image quality was graded between 1 and 3 , with grade 1 representing the best quality score in which the angle image was clear , grade 2 indicating a slightly blurred image in which angle structures remained distinguishable , and grade 3 a blurred image in which angle structures are difficult to identify . angle grading was according to the following : ( 1 ) grade 4 ( wide open angle ) in which ciliary body band is observed for the majority of the image , ( 2 ) grade 3 ( open angle ) in which scleral spur is observed for the majority of the image , ( 3 ) grade 2 ( narrow but open angle ) in which the posterior tm is the most posterior structure seen for the majority of the image , ( 4 ) grade 1 ( narrow occludable angle ) in which only the anterior tm is seen for the majority of the image , and ( 5 ) grade 0 ( closed / appositional angle ) in which no angle structures are seen for most of the image . these angle grading categories matched the shaffer classification system used to grade the eyes clinically on gonioscopy . for both eyecam and gonioscopy , angle closure was defined as grade 0 in the classification scheme described . sampaolesi 's line was graded as positive if a linear pigmented deposition was observed anterior to the schlwabe 's line . abnormalities that were specifically denoted included focal peripheral anterior synechia , iris processes , patchy tm pigmentation , iris elevation , iris lesion ( mass with increased pigmentation ) , and neovascular vessels . the right eye of each patient was used for analysis unless that eye met one or more of the exclusion criteria , in which case the left eye was used ( provided it met no exclusion criteria ) . the mcnemar test was used to test the null hypothesis of marginal homogeneity ( rater agreement ) in 2 2 cross classifications of paired responses to dichotomous items ( quadrant / angle closure ) . the simple kappa ( ) statistic was used to assess the strength of agreement between dichotomized ( open / closed ) variables , while the weighted was used to measure agreement for ordinal variables ( the polychotomous grade variable used to quantify angle width ) , and to measure intra- and interrater reliability . the first - order agreement coefficient ( ac1 ) statistic was used in addition to , as the latter statistic can yield coefficients that are paradoxically low relative to overall agreement under conditions where high trait prevalence causes a marked imbalance in contingency table marginals . receiver operating characteristic curves with calculations of area under the curve ( auc ) and 95% confidence intervals ( cis ) were used as an index of performance for identification of eyes with angle closure , using gonioscopy as the reference standard . statistical analyses were performed using sas version 9.4 ( sas institute , inc . , cary , nc , usa ) . ches identified a total of 5782 eligible subjects . of these , 4582 completed both a home questionnaire and a clinical eye exam . one hundred forty - one subjects refused angle assessment , leaving 4441 who underwent both gonioscopy and eyecam imaging . of those who were assessed , 292 subjects were subsequently excluded due to previous intraocular surgery , resulting in an analytic sample of 4149 participants for analysis . individuals with incomplete , unavailable , or poor quality eyecam images in some quadrants were excluded from the appropriate quadrant - specific analyses ; no gonioscopy cases were excluded , incomplete , or unrecorded . the sample included 1523 men and 2626 women . inter- and intraobserver agreement for eyecam ( defining angle closure as 2 or more quadrants closed ) was excellent ( = 0.82 and 0.87 , respectively ) . tables 1 and 2 show the agreement between gonioscopy and eyecam for various definitions of angle closure , as well as agreement between the two modalities on angle closure on a per - quadrant basis . gonioscopy and eyecam showed moderate agreement according to the statistic , and excellent agreement according to the ac1 statistic for all quadrants and angle closure definitions . agreement was best when defining angle closure as two or more quadrants on gonioscopy ( = 0.60 , ac1 0.90 , p < 0.0001 ) . when angle closure was examined by quadrant , the superior and temporal quadrants showed the least agreement between gonioscopy and eyecam images ( = 0.52 ) compared with the other quadrants ( nasal quadrant = 0.57 , inferior quadrant = 0.54 ) . gonioscopy identified angle closure at higher rates than eyecam when angle closure was defined as closure in two or more quadrants , three or more quadrants , all four quadrants ( p < 0.0001 ) , and in the superior and temporal quadrants . there was no change in the agreement statistics when subjects with previous lpi were removed from the analysis ( data not shown ) . agreement between eyecam and gonioscopy in iridocorneal angle assessment using binary ( open / closed ) outcome measures agreement between eyecam and gonioscopy in iridocorneal angle assessment using ordinal outcome measures ( angle width in degrees , categorized using the shaffer classification system ) the auc was greater than 70% for all definitions of angle closure , as well as for quadrant - specific angle closure ( table 3 ) . when defined as two or more quadrants closed , angle closure was not diagnosed using eyecam despite being classified in gonioscopy in 9.0% ( 359/3994 eyes ) of cases , while 3.4% of cases saw angle closure diagnosed in eyecam but not gonioscopy . angles graded as open on eyecam and closed on gonioscopy were attributed to partial angle closure within the quadrant ( fig . a ) or the presence of a pigmented schwalbe 's line within the quadrant ( fig . b ) . performance of eyecam relative to gonioscopy for each quadrant and various definitions of angle closure eyecam images classified as ( a ) open versus ( b ) closed angles . this study is the first of its kind to use eyecam in a population - based setting and reports the largest number of patients studied with eyecam to date . while prior studies have used eyecam in smaller , clinic - based populations constituting less than 200 patients , our study examined over 4000 individuals in a community setting . in our study , grading of eyecam images showed moderate to excellent agreement with gonioscopic assessment , which is consistent with prior studies in smaller clinic - based populations . our study also demonstrated excellent intra- and interobserver agreement equivalent to prior studies in a clinical setting . differences exist between our results and prior studies regarding the agreement rate between gonioscopy and eyecam , which can be attributed to the different size and demographics of the populations studied , and the prevalence of angle closure within these populations . we noted a higher rate of angle closure detected by gonioscopy than by eyecam in this study . this difference can be attributed to the presence of partial angle closure within the quadrant that was not detected by eyecam but detected on gonioscopy , as well as a highly pigmented schwalbe 's line that may have led to disparate interpretations of the angle structures . eyecam is unlikely to replace gonioscopy as the reference standard , and disparities in angle assessment using the two methods must be interpreted with caution . the moderate to high diagnostic performance of eyecam in detecting angle closure and its agreement with gonioscopy make it a useful screening tool in patient populations with known high rates of angle closure , such as chinese and chinese americans . in this study the utility of eyecam in populations at high risk for angle closure includes : ( 1 ) the ability of eyecam to be operated by a trained technician rather than a physician , allowing for a wider range of angle documentation across a greater number of patients , ( 2 ) providing a view of the angle similar to that observed with gonioscopy ( apart from its inability to provide a dynamic view of the angle ) , allowing for 360 visualization of the iridocorneal angle , unlike the cross - sectional views afforded by traditional as - oct and ubm , and ( 3 ) the ability to monitor the angle over time . eyecam imaging provides an unprecedented objective method of gonioscopic angle documentation that can be followed longitudinally , in a manner similar to how optic disc stereophotographs are used to track glaucomatous disease progression . these stored eyecam images can also be used in training sessions to improve agreement of gonioscopic angle grading between ophthalmologists , which remain suboptimal and subjective . furthermore , the standardization of the eyecam image acquisition process , such that identical portions of the iridocorneal angle are obtained at each exam , provides the clinician with a baseline of the gonioscopic exam for each patient and each angle quadrant that can be referenced to evaluate disease progression and changes related to treatment . in our study , 72% and 96% of eyes identified as closed and open angle using the eyecam , respectively , were given the same diagnosis using gonioscopy . separate clinicians assessed gonioscopy and eyecam images in order to prevent bias in agreement between the two methods , but there was excellent intra- and interrater reliability . there are several disadvantages to eyecam imaging , such as the inability to perform dynamic imaging , as well as its requirement for supine patient positioning . however the eyecam is not an invasive measure , and is similar to gonioscopy in this way . obtaining eyecam imaging also takes longer than gonioscopy , approximately 5 to 10 minutes per eye , which may present a challenge in employing it as a standard screening tool for all populations . this study was limited by the fact that the there was little experience with the eyecam prior to this study ; additional experience may have resulted in better quality images with time . in summary , our study suggests that expert grading of eyecam images of the iridocorneal angle can provide moderate to excellent agreement with gonioscopic grading in a large population - based cohort . eyecam can be an effective screening tool for the identification of closed angles or narrow angles developing creeping angle closure over time in populations with a known predisposition to angle closure . usc roski eye institute , university of southern california : rohit varma , md , mph ( principal investigator ) ; roberta mckean - cowdin , phd ( co - investigator ) ; stanley p. azen , phd ( co - investigator ) ; mina torres , ms ( project director ) ; chunyi hsu , mph ( project manager ) ; david dinh , ba ( research assistant ) ; ruzhang jiang , md ( examiner ) ; jie sun , md , phd , mph ( examiner ) ; dandan wang , md ( examiner ) ; yuping wang , cot ( examiner ) ; justine wong , ba ( clinical interviewer ) ; shuang wu , ms ( statistician ) ; rucha desai , ms ( programmer ) . the battelle survey research center : lisa v. john , phd ( recruitment director ) ; michelle cheng , ms ( field supervisor ) . alfred sommer , md , mhs ( chair ) ; anne coleman , md , phd ; dennis han , md ; craig hanis , phd ; louise wideroff , phd ; and terri young , md .
abstractobjectivesnanothechnology found to be increasingly implemented in implantology sphere over the recent years and it shows encouraging effect in this field . the aim of present review is to compare , based on the recent evidence , the influence of various nanostructure surface modifications of titanium for implants , on osteoblasts proliferation.material and methodsa literature review of english articles was conducted by using medline database restricted to 2009 - 2014 and constructed according prisma guidelines . search terms included titanium implant , titanium surface with nanostructure , osteoblast . additional studies were identified in bibliographies . only in vitro and/or in vivo studies on nano structured implant surfaces plus control sample , with specific evaluation method for osteoblasts proliferation and at least one ti sample with nanostructure , were included in the review.results32 studies with 122 groups of examined samples were selected for present review . each study conducted in vitro experiment , two studies conducted additional in vivo experiments . all studies were dispensed by type of surface modification into two major groups ; direct ablative titanium implant surface nano - modifications with 19 studies and nanocomposite additive implant surface modifications with 13 studies . overall 24 studies reporting on positive effect of nanostructured surface , 2 studies found no significant advantage and 6 studies reported on negative effect compared to other structure scales.conclusionsfrom examination of selected articles we can notice marked advantage in implementation of various nanostructures onto implant surface . yet for discovering the ultimate implant surface nanostructure , further comparable investigations of ti surface nanostructures need to be done .	dental implant treatment is very widely spread and reliable treatment that provides good clinical results with high success rates over 90% [ 1 - 2 ] . titanium is commonly used as an implant material as it has high biocompatibility and bonding ability with the bone . these characteristics were found in 1952 by the swedish scientist per - ingvar brnemark . since then , the results of many studies have demonstrated that titanium has high biocompatibility . titanium has no adverse effect on the human body and bonds readily with the new bone , which penetrates into the titanium surface . implant survival rate and prognosis depends on quality of osseointegration as more direct bone - to - metal interface take place without interposition of non - bone tissue . however , differences in bonding force between the implant body and bone occur depending on the differences in surface structures of the implant . many studies have concluded that certain characteristics of the implant surface play an important role in altering the quality of osseointegration [ 7 - 9 ] . it is commonly thought that the slightly roughened implant surface allows better osseointegration compared with the smooth implant surface . moreover nanostructured materials have shown increased cell attachment over microstructured or smooth surfaces . an essential role of osseointegration processes is played by osteoblast progenitor stem cells during recruitment , adhesion , proliferation , differentiation , and mineralized matrix deposition during bone regeneration phases . nanoporous topography tend to help the proliferation processes , acting directly on the selective adhesion of osteoblastic cells on the surface , which can accelerate the healing process around implants . low osteoblasts cell number and proliferation have been closely associated with negative results when considering it to osseointegration . many studies were conducted to investigate various implant surface nanostructures and their influence on cell behaviour as proliferation , in contrast to other scopes of surface structures dimensions . however , the optimal implant surface nanostructure covering for osteoblasts proliferation is yet to be established . the aim of the present review is to compare , based on the recent available evidence , the influence of various nanostructure surface modifications of titanium for implants , on osteoblasts proliferation . protocol and registration the review is registered in international prospective register of systematic reviews prospero . the protocol can be accessed at : http://www.crd.york.ac.uk/prospero/display_record.asp?id=crd42014009436 registration number : crd42014009436 . the review included laboratory research studies , in vitro studies that using cells from human or animals and in vivo studies on animals . studies published on english language between january 2009 and june 2014 with various evaluation methods for osteoblasts proliferation and various evaluation intervals between hours and days . , titanium surface with nanostructure , osteoblast . for more recent and updated information , search included only articles that were publicated from january 2009 to june 2014 to ensure sensitivity of the review . ( figure 1 ) illustrates the flow diagram of present articles selection according to prisma guidelines . exclusion criteria inclusion criteria for the selection were : in vitro and/or in vivo studies . the search displayed 97 results from which 72 abstracts were screened ( figure 1 ) . preliminary exclusion was made by the title and its relevancy , later by abstract and its relevancy . finally , articles that did not meet the inclusion and exclusion criteria , where filtered as followed : no proliferation described ( n = 12 ) , microstructured surface analyzed ( n = 4 ) , no titanium sample included ( n = 3 ) , nanostructure and morphology has not been described ( n = 1 ) , organic coating has been used ( n = 5 ) . data collection process data was independently extracted from reports in form of variables according the aim and themes of present review as listed onwards . variables on which data were sought are as follow : type of study , indicates whether it was in vivo or in vitro or both and materials respectively . sample , describes the number of particular investigated samples in the study and its singularity ( e.g. , a - machined , b - polished , c - acid etched ) . topography , describes the nanoscale topography of the nanostructures on the surface of the sample , can be interrelated with sample description of nanostructure ( e.g. , nanograins 100 nm ) . evaluation , describes evaluation methods and duration of osteoblasts proliferation cultured on the sample ( e.g. , histology , 24 , 48 and 72 hours).result , describes the impact of surface structure on osteoblast proliferation . description of nanostructure peculiarities can vary from study to study and may be located under sample column or topography or both . risk of bias assessment risk of bias ( e.g. , lack of information or selective reports on variables of interest ) was assessed on study level . the risks were indicated as lack of precise information of interest in each individual study that can blind the reader from particular information about examined samples . the cochrane collaboration s tool for assessing risk of bias was used to assess bias across the studies that can affect cumulative evidence . relevant data of interest according stated previously variables , was collected and organized in two tables that divided according type of implant surface modification . separation of articles by their samples fabrication methods of nanostructured surfaces into two groups can provide possibility for simple comparison . numbers of samples that provide positive or negative effect on osteoblasts proliferation are assessed in each article for each modification method . in addition samples of three topography types ( e.g. , control , microstructured and nanostrucruted ) are included in each modification methods groups for better understanding of nanostructured surface superiority . quantitative and relative comparison between examined groups of samples and illustration of their relative efficiency in cells proliferation by means of diagrams . preliminary exclusion during screening stage was made by the titles and abstracts relevancy ( n = 13 ) and ( n = 4 ) respectively . during eligibility stage articles that did not meet the inclusion and exclusion criteria , where filtered as followed : no proliferation described ( n = 12 ) , microstructured surface analyzed ( n = 4 ) , no titanium sample included ( n = 3 ) , nanostructure and morphology has not been described ( n = 1 ) , organic coating has been used ( n = 5 ) . additional relevant articles were added after manual selection from references according eligibility ( n = 2 ) . finely 32 studies with 122 groups of examined samples were included in present review ( figure 1 ) . study characteristics all 32 studies finally selected for the review were in vivo and in vitro studies published in english with description of osteoblast proliferation . each study conducted in vitro experiment by culturing osteoblasts on several investigated samples and controls when three studies conducted additional in vivo experiments . all authors except four , reported on conducting the experiments at least twice to ensure statistical validity . the duration of osteoblasts proliferation evaluation varied from 2 hours to 14 days across all the studies except three articles of gittens et al . the main evaluation methods for osteoblasts proliferation across the studies were : histology , mtt assay , alamarblue assay , dna assay and wst-1 or brdu marker evaluations , additional visual evaluation by scanning electron microscope ( sem ) was described by six authors , when tet et al . uses sem as main evaluation method . all examined studies were assessed for specific variables described previously and were further divided into two groups characterized by type of implant surface modification . the division provided better understanding of nanosurface structure characteristics and contributed to sensitivity of the review . first group direct ablative titanium implant surface nano - modifications deals with titanium implant samples that were treated by various methods directly without addition of other materials to the implant surface , include 19 studies with 70 samples ( table 1 ) . nanocomposite additive implant surface modifications deals with samples that were treated by addition of variable non organic particles , includes 13 studies with 50 samples , showed in ( table 2 ) . direct ablative titanium implant surface nano - modifications nanocomposite additive implant surface modifications risk of bias within studies only 22 from 32 studies fulfilled the expected markers of validity . the risk of bias that indicated within other 10 studies presented as lack of information values that grouped as followed : nano scale topography was not indicated , evaluation methods with timing description and significance of experiments indicated in the study result ( p < 0.05 ) ( table 3 ) . assessment of the risk of bias present review focused on describing the studies , their results and qualitative synthesis rather than meta - analysis because the analyzed studies were not presented with clear quantitative results of osteoblasts proliferation , furthermore examined samples , evaluation methods and duration varied markedly . overall 24 studies with 89 examined samples from which 63 are various nanostractured patterns , reporting positive effect of 33 nanostrucure modified ti samples , thus enhance osteoblasts proliferation on nanostructured features with significant differences ( p < 0.05 ) between nanostructured surface , and microstructured or smooth control surfaces in each study . no significant difference in positive effect on proliferation of cells cultured on nanostructured samples compared to microstructured or smooth control samples , was described in 2 studies with 6 examined samples ; one study reports no significant differences between all three examined samples , another study result reveals equal proliferation on smooth sample and sample with micronanohybrid surface , whereas microstructured sample showed impaired proliferative activity . negative effect was described in 6 studies with 27 samples from which 15 are nanostructured samples , when results vary from , microstructured surface that promote osteoblasts proliferation to smooth or control samples showed better proliferation results . results by type of modification method , 19 belong to direct ablative titanium implant surface nano - modifications ( table 1 ) with 72 examined samples from which 41 are nanostructured and only 19 samples produce positive effect for cellular proliferation . 13 studies belong to nanocomposite additive implant surface modifications ( table 2 ) , with 50 examined samples from which 40 are nanostructured with 25 samples reported as having significantly positive effect on osteoblasts proliferation . results of individual studies results of individual studies are shown in table 1 and table 2 . risk of bias across studies all studies examined did nt show numerical data on the osteoblast proliferation results , what did not allow us to estimate precisely the advantage of one nanostructured sample on another . all reviewed studies except two indicated significance of their results by ( p < 0.05 ) what can be interpreted as study s quality guaranty , but as mentioned previously , absence of quantitative results and calculations prevent conformation of significance and comparison across the studies by the reviewer . in addition there are 10 studies with 41 examined samples were we could not find exact information as , nanoscale topography of the specific surface structure and/or evaluation timing . review with and without inclusion of these studies found no differences in the patterns of our review results but only leave the reader blinded by lack of specific features of investigated sample . the division of articles into two groups by surface modification type contributed to better understanding of nanosurface structure characteristics and provide possibility for comparison between two main nanostructure modification methods ( table 4 ) . additionally the examined groups of samples can be compared for their relative influence on cell proliferation by surface topography . percentage of groups of samples that promote proliferation in each surface topography , within nanostructures been the greatest illustrated in ( figure 2 ) . division of samples by modification and topography percentage of groups of samples that promote proliferation in each surface topography . overall , in most of reviewed articles 24 in number , nanostructured surfaces enhanced osteoblast proliferation compared to microstructured or smooth surfaces . dielectric barrier discharge ( dbd ) modification of ti with spherical nanoparticles of 50 - 125 nm , significantly enhance cell proliferation , adhesion and spread without negative effect on differentiation . strong evidence delivered by two different studies that acid etched microfeature and tio deposited samples with nanonodules of 100 nm , 300 nm and 500 nm increase in proliferation , with that on 300 nm being the greatest . porous ti6al4v substrate with 10 - 20 nm grains appeared to have a not only higher but also longer growth phase for cell proliferation . full contact coverage coating that was obtained by galvanotactic anodizing process in a phosphate - sulfate bath , ( fcc ) characterized by unique nanotopography of regular volcanoes with circular pores of 10 m and 700 nm shows greater amount of cell proliferation than micro and nanopore of 2 m and 150 nm . . stated that crystal structure of nanotube layer can override the chemistry effect and plays a main role in cell proliferation , when anatase / rutile ~80 nm nanotube layers showed significantly higher proliferation than smooth layer and amorphous nanotube layers . this statement confirmed with evidence found in another study where ~80 nm nanotube surface increase cell number when the same author reports on reduced cell number cultured on 130 nm nanotubes and in other study of same author , no difference was found between ~80 nm nanotubular pattern and polished sample . final conformation for nanotubes benefit in such dispute was found in in vivo and in vitro experiment . one of the most advantageous nanotube diameters for better cell proliferation appears to be ~30 nm . additional nanofeature with ~30 nm was reported to have largest cell response , including proliferation adhesion and differentiation , as ~30 nm saw tooth nanonetwork surface with 200 - 300 nm inter tooth distance was examined . interesting findings were observed in additional combined in vivo and in vitro studies when one reports on higher cell proliferation rate on nanoleaf feature with roughness of 228 nm than on 60 - 80 nm nanotubs and significant reduction in proliferation on nanoneedle feature with 940 nm roughness , whereas another latest in vivo and in vitro study controversially reports on enhanced proliferation on nanoneedle structure . concerning nanocomposite materials incorporated into the implants surface we saw niobium ( nb2o5 ) doped tio2 producing nanoplate structure that promote cell adhesion and proliferation . strong evidence brought to us in two studies that reported on strontium doped hydroxyapatite with 21.6 3.7 nm grain morphology , accelerates cell proliferation . furthermore , cell proliferation can be directly regulated by sr1-ha interrod spacing , with 71.4 nm interrod space three - dimensional patterns being the greatest . controversial results were noticed about incorporation of ha within nanotubes as two authors claim that there is an obvious positive change in the spreading and viability of osteoblasts on ha coated ~45 - 50 nm titania nanotubes layer comparing to cells on pure titanium or tio2 nanotubes and in contrast gu et al . reports on reduced proliferation on 90 nm nanotubules with ha compared to same nanotubes without ha . another report reveals higher cell numbers on ha - tio2 nanocomposite coated sample with 300 nm spherical tio , 20 - 30 nm in diameter and 50 - 100 nm in length ha nanorods . significantly improved bone cell proliferation on the biomimetic nano coatings compared to uncoated ti and nano - ha coated ti was reported , as nano - ha combined with both magnetically and non magnetically treated single walled carbon nanotubuls can achieve the highest osteoblasts proliferation density when diameter of swcnt is 1.19 and 1.52 nm respectively . structures with 80 nm selenium clusters incorporated onto ti implant surface , significantly increase healthy cell density compared to untreated ti , on which cancerous osteoblasts found to be prevailed . tic layer deposited on titanium sample by ion plating plasma assisted deposition , increase osteoblast growth rate as was claimed in an in vitro and in vivo studies . another nanocomposite material that succeeds to increase proliferation rate and vitality was tio2/casio3 which exists on the surface as casio3 nanocristals on 20 - 10 nm tio2 grains pattern . in contrast to 23 articles that describe positive effect of nanostructured surfaces , we are dealing with negative reports as follow . reporting superiority of microstructure surface with pore size of 1 - 2 m over nanostructured samples . yu et al . described lower proliferation rate on 80 nm nano - foveolae structure compared to smooth control sample . [ 24 - 26 ] in three different studies describes negative results for micro and nano - modified samples compared to smooth and control surfaces in regard to osteoblasts proliferation , possibly due to transcriptionally - restricted transition . transcriptionally - restricted transition between proliferation and differentiation is a process that forces osteoblasts to stop dividing once they start maturing [ 59 - 61 ] . zhao et al . from three articles included in this work , first reporting on significant smaller osteoblasts numbers cultured on nanotubules of 130 nm than on flat ti sample , the number become even smaller when 10 - 20 nm ag particles added to the surface . second article finds no significant difference in cell numbers between polished sample , 25 nm nanonet texture and 80 nm nanotubular texture . third article reports on slightly enhanced cell number on the ~80 nm nt acid - etched/20 v anodized surface . describe relatively equivalent proliferation level on tio2 smooth and 198.5 22.3 nm tio2 micronanohybrid . after all , the most mentioned advantageous pattern is nanotubular structure with nanotube diameter of ~30 nm . other nanostructures mentioned in our review need to be further investigated and compared for most advantageous nanoscale within each particular nanostructure . furthermore this review analysed articles that present synergic effect of ablative and additive nanocomopsite surface coating to osteoblasts proliferation . most articles that were including hydroxyapatite incorporation into ti implant nanostructures describe obvious positive effect on cells proliferation especially when doped with strontium . strontium doped ha nanorods appear to be beneficial with nanoscale of ~20 - 30 nm diameter and with interrod spacing of less than 96 nm . beside strontium , nanostructures doped with niobium , selenium and casio2 nanoparticles showed promising results , when selenium substrates suggesting a more favourable environment for healthy than cancerous osteoblasts . the main limitation of this overview is that the samples group types , the culture techniques and evaluation methods are not the same across studies and can not be compared . all studies examined did nt show numerical data on the osteoblast proliferation results , what did not allow us to estimate precisely the advantage of one nanostructured sample on another . all reviewed studies indicated significance of their results by ( p < 0.05 ) what can be interpreted as study s quality guaranty , but as mentioned previously , absence of quantitative results and calculations prevent conformation of significance and comparison across the studies by the reviewer . in addition there are 10 studies with 41 examined samples were we could not find exact information as , nanoscale topography of the specific surface structure and/or evaluation timing . review with and without inclusion of these studies found no differences in the patterns of our review results but only leave the reader blinded by lack of specific features of investigated sample . from examination of selected articles we can notice marked advantage in implementation of various nanostructures onto implant surface . in our review 24 articles reporting on positive effect of nanostructured surfaces on osteoblasts proliferation , when 33 samples with particular nanostructures markedly enhance cell proliferation . most of the examined nanostructures showed obvious positive impact on osteoblasts proliferation compared to other topography scales . yet for discovering the ultimate implant surface nanostructure , further investigations of ti nanopatterns with various nanoscales need to be done , moreover for reaching the most sensitive outcome , the experiments should be statistically compared , what can be achieved only when different studies will use the same concerted evaluation method for osteoblasts proliferation .
context : there is ample evidence to prove that medical graduates are not prescribing rationally and this can be improved by proper training.aims:to design and implement a prescription writing teaching module for second professional medical students.subjects and methods : a module of 3 h duration consisting of didactic lecture , interactive audiovisual small group session , and evaluation method was framed for every disease and implemented . completeness of the prescriptions was evaluated on a scale of 14 . appropriateness of the prescription , knowledge about the rationale behind the drugs used and adverse events related to the drugs used was judged in three categories , that is , appropriate and complete ; appropriate but insufficient ; and inappropriate.results:one thousand six hundred and seven response sheets to 24 health problems were collected . completeness score of 18% was 2 , 59% was 3% and 24% was 4.41% prescriptions were appropriate and complete , 58% appropriate but insufficient and 1% inappropriate . the rationale behind the drugs used was appropriate and complete 24% , appropriate but insufficient 68% , inappropriate 8% . documentation of adverse events was appropriate and complete 23% , appropriate but insufficient 49% , inappropriate 28% . all facilitators were satisfied with the duration , contents and conduct of the sessions.conclusions:a module is an effective tool for teaching prescription writing to undergraduate students ; modifications required in contents and strategy to emphasize the need of complete documentation .	is focused on symptoms and diagnosis , and little or no time is given to the principles of drug treatment . unfortunately , many medical schools still do not provide a structured training in pharmacotherapy but only lectures in basic pharmacology . this approach goes a long way to explaining why many medical graduates feel insufficiently prepared to assume prescribing responsibilities after graduation , and the many hospital admissions and even deaths caused by possibly avoidable medication errors . there is also ample evidence that prescription writing by medical students , interns , and fresh graduates can be improved by proper training . the study was conducted in the department of pharmacology , of a tertiary care health center , after taking permission from institutional ethics committee . a core committee consisting of faculty and senior residents of the department was formed . in consultation with faculty and after studying the feasibility ( depending on the approved sequence of topics to be covered in the semester ) 24 diseases were selected for the purpose of this study . a module consisting of didactic lectures , interactive audiovisual small group sessions with evaluation methods were framed for every disease . didactic lecture , 30 min audiovisual session on case history and records , 20 min preparation of prescriptions by the small groups based on focused group discussion and available literature , 40 min discussion on prescriptions prepared by the groups , its rationale and likely adverse events of the drugs prescribed , 15 min writing of final prescription and answer of questions by every student individually , and 15 min completing the reaction questionnaire by the facilitators . the contents of the didactic lectures and audiovisual sessions were decided using standard text books , the world health organization ( who ) guide to good prescribing , the who teachers guide and other relevant information available on internet . the resource persons for didactic lectures and facilitators were decided , and a pilot study was conducted consisting of one full session of the module for feedback and based on that the module was finalized . october 15 , 2014 , and until december 31 , 2014 , 24 prescriptions were completed . six of the prescriptions were completed with the batch 2012 and 18 with batch 2013 . a uniform question in the form of write an appropriate prescription for the given problem , write the rationale behind the drugs prescribed and write the adverse drug events ( ades ) that may arise because of the use of this prescription was given in every session . the questions put to the facilitators were : was the duration appropriate , were the contents of the session appropriate , was the conduct of the session appropriate , and are you satisfied with the session ? the completeness of the prescription was evaluated based on the four parts of the prescription , that is : ( 1 ) information about the patient . ( 2 ) information about the medicines prescribed ( type of formulation , name of the medicine , strength , and duration ) . ( 3 ) instructions about the consumption of the medicine to the pharmacist and patient and related advise and ( 4 ) information about the prescriber . according to the parts appropriateness of the prescriptions , the documentation of rationale of the medicines used and probable adverse events that may be caused because of the prescription were judged in three categories , that is , perfectly appropriate , appropriate but insufficient , and inappropriate . a total of 1607 response sheets consisting of prescription slip for the given health problem , the rationale of the medicines prescribed and likely adverse events because of the prescription were collected . three hundred and twenty - three response sheets were from batch 2012 as response to the six health problems , that is , duodenal ulcer , amoebic liver abscess , shigellosis , pulmonary tuberculosis , iron deficiency anemia and hypertension stage ii . rest of the 1284 response sheets were from batch 2013 as response to the 18 health problems , that is , belladona poisoning , acute organophosphate poisoning , chronic organophosphate poisoning , anaphylactic shock , benign prostate hypertrophy , motion sickness , narrow angle glaucoma , open angle glaucoma , pheochromocytoma , mild depression , major endogenous depression , posttraumatic stress , jet lag , chronic insomnia , sleep onset insomnia , generalized tonic - clonic seizures , absence seizures , status epilepticus . on evaluation , completeness score of 18% was 2 , 59% was 3 and 24% was 4 , that means a majority 83% of the prescriptions were almost complete with a score of three or four [ figure 1 ] . similarly , 99% of the prescriptions were appropriate ; 41% prescriptions were appropriate and complete , 58% appropriate but insufficient and 1% inappropriate . the rationale given for the prescription was appropriate in 92% of the cases ; appropriate and complete 24% , appropriate but insufficient in 68% and inappropriate in 8% . documentation of adverse events was appropriate in 72% ; appropriate and complete in 23% , appropriate but insufficient 49% and inappropriate in 28% [ figure 2 ] . completeness score of prescriptions in different categories ( n = 1607 ) appropriateness of prescriptions and documentation of rationale and adverse drug reactions ( n = 1607 ) all the facilitators were satisfied with the duration , contents and conduct of the sessions . eighty - three percent of the prescriptions were almost complete with a score of three or four . similarly , the rationale given for the prescription was either perfectly appropriate or appropriate but insufficient in 92% of the cases , and documentation of ades was either perfectly appropriate or appropriate but insufficient in 72% cases . the figure of 28% incorrect knowledge of ades is an area of concern , and necessary modifications are required in the module . another area of concern that needs to be addressed is a large number of insufficient documentation . the reason may be , they were a part of the panel who decided the contents and implementation strategy of the sessions .
the purpose of this cross - sectional study is to assess the desire , need , perceptions , and knowledge of assisted reproductive technologies ( arts ) for women living with hiv ( wlwhiv ) and determine correlates of art knowledge desire . wlwhiv of reproductive age were surveyed using the survey instrument the hiv pregnancy planning questionnaire at hiv / aids service organizations across ontario , canada . of our cohort of 500 wlwhiv , median age was 38 , 88% were previously pregnant , 78% desired more information regarding art , 59% were open to the idea of receiving art , 39% felt they could access a sperm bank , and 17% had difficulties conceiving ( self - reported ) . age , african ethnicity , and residence in an urban center were correlated with desire for more art information . of participants , 50% wanted to speak to an obstetrician / gynecologist regarding pregnancy planning , and 74% regarded physicians as a main source of fertility service information . while the majority of participants in our cohort desire access to art information , most do not perceive these services as readily accessible . healthcare practitioners were viewed as main sources of information regarding fertility services and need to provide accurate information regarding access . fertility service professionals need to be aware of the increasing demand for art among wlwhiv .	with the advent of highly active antiretroviral therapy ( haart ) to treat hiv infection over the past 15 years , dramatic reductions in hiv - related morbidity and mortality have yielded improvements in quality of life and life expectancy . the risk of vertical transmission through pregnancy has also been reduced to < 1% with timely antiretroviral therapy , continuing viral suppression , delivery by caesarean section if appropriate , and avoidance of breastfeeding . women now represent 22% of canada 's hiv - positive population , and with over 80% of hiv - positive women being of reproductive age , there is a need for safe pregnancy planning as more women living with hiv ( wlwhiv ) desire to become pregnant . while prevention of vertical transmission of hiv to the child remains a key component , the main focus of safe pregnancy planning includes prevention of horizontal transmission to an uninfected partner and treatment of infertility issues . to achieve such objectives , assisted reproductive technologies ( arts ) have become central to meeting the reproductive needs of wlwhiv in resource - rich countries . indeed , access to conception and fertility services including art has become an important component within the recent global movements to establish national guidelines or update existing recommendations on pregnancy planning and conception for people living with hiv ( plwhiv ) in canada , the united kingdom , germany , and france [ 511 ] . furthermore , as of july 2010 , the most recent american society for reproductive medicine ( asrm ) ethics committee guidelines also recommend that fertility clinics offer services to hiv - positive individuals and couples willing to use art for reduction of transmission risk . compared to conception through unprotected intercourse with timed ovulation , associated with a horizontal transmission risk of 4.3% for serodiscordant couples , art procedures such as sperm washing , intra uterine insemination ( iui ) , in vitro fertilization ( ivf ) , and intracytoplasmic sperm injection ( icsi ) have been found to offer significant reductions in horizontal hiv transmission or coinfection for couples wishing to conceive . as well , art is also recommended for the treatment of infertility , an issue of importance to wlwhiv as studies have indicated lower fertility rates among hiv - positive women [ 1422 ] . various studies have documented an increased rate of tubal factor infertility in hiv - positive women [ 7 , 14 , 15 , 23 ] . previous literature on the fertility intentions of wlwhiv has been important in eliciting trends and predictors of the decision to attempt pregnancy [ 2439 ] . as well , studies exist regarding the prevalence of fertility service centers in north america receiving requests from or providing services to plwhiv [ 6 , 12 , 31 ] . although the asrm guidelines recommend that fertility clinics offer reproductive assistance to hiv - positive patients within technical and economic feasibility , less than 3% of fertility practices registered with the american society for assisted reproductive technologies provide services to couples where both partners are hiv positive [ 12 , 31 ] . low patient volume has been cited as the most common reason for the limitation of fertility services by american service providers . few studies have investigated the demand for or desire to use art or the trends in and predictors of conception strategies amongst plwhiv . as women are the main recipients of art , it is important to assess the current intentions , knowledge , and perceptions regarding art among the current cohort of wlwhiv in north america . the purpose of this cross - sectional study was to assess the desire and need for art among hiv - positive women of reproductive age living in ontario , canada , and to determine correlates of art knowledge desire and the knowledge and perceptions of accessibility of art by the wlwhiv in this cohort . a cross - sectional study using a survey instrument was carried out with participants who met the following inclusion criteria : ( 1 ) hiv - positive , ( 2 ) biologically female , ( 3 ) of reproductive age ( ages 1852 ) , ( 4 ) living in ontario , canada , and ( 5 ) ability to read english or french . the upper age limit was chosen to reflect the cut - off for fertility clinic consultation in canada . recruitment was conducted from october 5th , 2007 to march 31st , 2009 through 28 aids service organizations ( asos ) , eight primary care and specialty hiv clinics , and two community health centers ( chcs ) across the province of ontario . an invitation email was sent out to all ontario asos listed on the canadian aids society website ( the national society for asos ) and all clinics and chcs known by the investigators that care for hiv - positive women . recruitment and study qualification determination were carried out by a single research staff member at each site following a predeveloped recruitment plan ( available upon request ) . our study determined geographic distribution of our data collection sites based on the provincial regions laid out by the provincial public health authorities with the specific regions noted in table 1 . this nonrandom sampling technique was used in an effort to obtain a representative sample of hiv - positive women of reproductive age living in ontario in the absence of a registry of hiv - positive individuals . once written consent was obtained , the survey was completed at the site or at home . an amended ethics approval was obtained for this specific research analysis . a 189-item survey instrument , the hiv pregnancy planning questionnaire , was created using the methods of fowler for instrument development and has previously been described in detail . the survey consisted of 12 domains including : ( 1 ) interest / desire to have children , ( 2 ) intent to have children in the future , ( 3 ) behavior related to the pursuit of fertility , ( 4 ) menstrual , birth control , and sexual history , ( 5 ) pregnancy and birth history , ( 6 ) perceived support for becoming pregnant , ( 7 ) satisfaction with providers , ( 8) needs assessment , ( 9 ) hiv medical history , ( 10 ) demographics , ( 11 ) anxiety and depression , and ( 12 ) hiv stigma ( full survey instrument available upon request ) . the survey was first developed in english and then translated into french using the back translation method . content validity was achieved by using items from 4 previously validated surveys [ 13 , 24 , 32 , 42 ] , by developing items based on a literature search exploring factors that determined reproductive decisions in hiv - positive women ( 939 ) and by reviewing the questions with a project advisory panel of experts including hiv specialists , obstetricians , midwives , community members , and plwhiv . face validity was achieved by initially piloting the survey with 20 hiv - positive women in 2 focus groups . the focus group participants confirmed that the survey instrument items actually appeared to be measuring the intended items related to the domains . furthermore , participants were asked to comment on each item in terms of comprehension , clarity , and relevance . further pilot testing was carried out with an initial 52 hiv - positive women that met the inclusion criteria . baseline characteristics of the study population were summarized using medians and interquartile ranges ( iqrs ) for continuous variables and frequencies and proportions for categorical variables . the geographic distribution of the study population was compared to the distribution of hiv - positive women living in ontario using the chi - square test . the primary outcome of interest for this analysis was desire for information about art ( art knowledge desire ) . the question used to represent desire for art information was i would like to learn more about fertility technologies and options for people living with hiv . the response options characterized agreement with the statement on a likert - type scale . participant responses were dichotomized into yes or no with regard to desire for art . agree were classified as yes ; participant answers of strongly disagree or the question used to represent previous need for art was if you have been pregnant before , did you ever experience problems or difficulties in trying to get pregnant ? women who had never been pregnant and women who had been pregnant but did not answer this question were excluded from the analysis . univariate logistic regression analysis was conducted to determine the unadjusted odds ratios with 95% cis for correlates of the desire for art and need for art . the final multivariate logistic regression model determined the adjusted odds ratios with 95% confidence intervals ( cis ) for correlates of the desire for art and need for art . the final multivariable logistic regression models included covariates that were a priori believed to be related to desire for art and need for art , such as age , ethnic background , marital status , history of hiv medication , and current contraceptive use . variables which were significant at p < 0.20 in the univariate analyses were candidates for inclusion in final multivariable logistic regression models for desire and need for art . when multiple covariates measured similar phenomenon ( e.g. , ethnic background and country of birth ) , the variable representing each construct with the most statistical significance was chosen . finally , the qualifying variables were entered into the multivariable model and selected by stepwise - selection method . only the correlates with a significant effect ( p < 0.05 ) remained in the final multivariable models . other outcomes of interest included perceptions and knowledge of art . the outcomes were reported using medians and iqr for continuous variables and proportions for categorical variables . statistical analyses were performed using sas version 9.2 ( sas institute , cary , nc , usa ) . five hundred and four wlwhiv in ontario , canada , were recruited from 38 sites . of these participants , four did not meet the inclusion criteria ( two were over the age of 52 and two were not living in ontario ) . of the remaining 500 participants , 10 did not answer the question used to represent desire for art . surveys from the remaining 490 participants were used for the final analysis for the primary outcome . the median age for this final study population was 38 years ( iqr 32 , 43 ) . sixty - one percent of the participants were born outside of canada and 46% were of african ethnicity . within this cohort , 55% were in sexual relationships and 46% were married , common - law , or living with a partner . thirty - three percent of the women had hiv - negative partners and 24% had hiv - positive partners ; 36% were currently using contraception ; 88% had been pregnant previously ; 74% had given birth . the demographic characteristics of the entire study population are presented in table 1 . in the final study population of 490 hiv - positive women living in ontario , canada , 78% ( n = 384 ) indicated that they would like to learn more about fertility technology options . the distribution of participant characteristics by desire for art information is shown in the logistic regression models for correlates of desire for art in table 2 . in the final multivariate model , african ethnicity ( or = 3.86 , p < 0.0001 ) and current contraceptive use ( or = 1.70 , p = 0.05 ) being married , common - law , or living with a partner ( or = 0.52 , p = 0.04 ) and history of hiv medication ( or = 0.34 , p = 0.02 ) were associated with a lack of desire for art . of the 500 participants who met the study inclusion criteria , 65 did not answer the question used to represent need for art and 10 had never been pregnant . the distribution of participant characteristics by need for art is shown in the logistic regression models for correlates of need for art in table 3 . for the study participants , 17% ( n = 72 ) indicated they had difficulties getting pregnant . in the final multivariate model , annual household income of $ 20,000 to $ 40,000 ( or = 2.36 , p = 0.02 ) greater than $ 40,000 ( or = 2.63 , p = 0.01 ) and current hiv treatment ( or = 2.53 , p = 0.01 ) were associated with difficulties conceiving and two or more lifetime pregnancies ( or = 0.41 , p < 0.01 ) were associated with no difficulties conceiving . the final analysis included surveys from the 500 participants who met the study inclusion criteria and responded to these set of questions . the distribution of participant answers to the various questions representing perceptions towards and knowledge of art is shown in table 4 . the majority of participants ( 59% ) were open to medical techniques that help with conception and 39% felt they could access a sperm bank if needed . of the women surveyed , 74% agreed they could ask a physician if they needed help finding a fertility clinic . to help with the decision to become pregnant , 50% of the participants indicated they wanted information booklets and publications on becoming pregnant , 59% wanted to speak to a health professional with fertility expertise , and 50% wanted to speak to an obstetrician / gynecologist . in this study , a large majority of wlwhiv ( 78% ) indicated a desire for information regarding reproductive technology options . correlates of desire for art , a measure of pregnancy intention , included younger age , ethnicity , residence in an urban region , and birth place outside of canada . previous publications support our findings of younger age and black / african ethnicity as significant correlates of pregnancy intention in wlwhiv [ 26 , 28 , 29 , 34 , 35 , 39 , 43 , 44 ] . although african ethnicity has not been associated with desire to have children , women of african ethnicity may be more likely to intend pregnancy due to cultural factors and traditional gender roles that stress the importance of childbearing [ 4447 ] . history of hiv medication use was also associated with a lack of desire for art in our study . however , other studies have indicated a positive influence on childbearing decisions associated with haart [ 27 , 30 , 32 , 33 , 35 ] . these other studies , conducted in vietnam , india , and south africa , may have reflected the more advanced disease of the study populations , where haart access is associated with better health and greater perceived ability to be a fit parent . in direct contrast to a previous study in british columbia ( bc ) , canada , being in a stable relationship this discrepancy can be attributed to the epidemiology of hiv infection in bc , differences in sample characteristics of the study , and the location and timing of the recruitment process . these differences highlight potential regional differences in the reproductive concerns of wlwhiv , attitudes towards those seeking art , and a need for continued research in this area . in addition to desire , our study also investigated potential need for reproductive technology options among wlwhiv by assessing correlates of self - reported conception difficulty . although not a direct measure of the true infertility rates among wlwhiv , conception difficulty does provide an estimate of the prevalence of infertility concerns within the population . infertility , defined as inability to conceive 12 months after initiating attempts , affects 1015% of the general population . various previous studies have documented the lower fertility rates of wlwhiv [ 1423 ] and the percentage of wlwhiv who reported difficulties conceiving in our study ( 17% ) was higher than that found in general population . wlwhiv with resolved primary infertility are not reflective of the population of wlwhiv and our results may underestimate the true infertility rates of wlwhiv . it appears then that the potential need for art among wlwhiv may be higher than that of the general population . greater household income and current hiv treatment were both correlates of difficulty conceiving , potentially due to the association between higher income and age . the lack of association between age , a major predictor of infertility generally , and difficulty conceiving in this study may be attributed to the relative youth of the study population . the age range of study participants was 3243 , and previous attempts at pregnancy may have occurred when the participants were younger ( < 35 years ) when age did not significantly affect fertility . current hiv treatment , while associated with increased fertility in developing countries [ 27 , 30 , 32 , 33 , 35 ] , was associated with difficulties conceiving in our study . thus far , studies on the effects of haart on pregnancy rates have not shown conclusive evidence of any impact on fertility [ 4951 ] . more research is required to further evaluate the true effects of haart on the fertility of wlwhiv . this study also assessed the perceptions and knowledge of wlwhiv of art . in our study , at least half of the participants viewed information booklets and publications on becoming pregnant , speaking with a health care professional with fertility expertise , or speaking with a obstetrician / gynecologist as important resources needed to help with the decision to become pregnant . more than one - third of participants also indicated a desire to have access to a fertility clinic as a resource needed for making pregnancy decisions . the majority of women surveyed were open to receiving art but only 39% of participants believed they had access to art services such as sperm banks . it appears that although women are interested in pursuing art services , most do not perceive these services as being accessible . since 74% of study participants indicated they regard their physician as the first source of information for art , doctors therefore have an important role in informing their patients of their full reproductive options . with the current movement towards increasing provision of art to plwhiv for prevention of horizontal transmission and treatment of subfertility , there is an increased need for physicians to provide information and initiate discussions regarding pregnancy planning and conception to their patients who are hiv positive . the assessment of art desires of a cohort of wlwhiv in ontario , canada , has important implications for healthcare providers , policy makers , and researchers . the results of this survey indicate wlwhiv of reproductive age are open to and desire access to art for the purposes of safe conception and treatment of infertility . while this desire and demand for fertility technology options is supported by asrm ethics committee guidelines for safe pregnancy planning in north america , access to fertility services although the majority of fertility clinics surveyed across canada and the us have expressed willingness to provide care to hiv - positive individuals , less than 50% have actually offered treatment [ 31 , 52 ] . even though previous restrictive state laws that have explicitly limited the availability of art services to plwhiv did contribute to the lack of access to fertility clinics within the us , low patient volume is the most common reason given by clinics for not providing services . the perceived inaccessibility of these services by plwhiv may contribute to the low number of clinics who actually treat patients from this population . there is a need for both provider and patient education to ensure the necessary provision of safe reproductive services to hiv - positive individuals . healthcare practitioners need to provide accurate information regarding reproductive technology options and access to such services , while fertility service professionals need to be aware of the increasing demand for art among plwhiv and current recommendations regarding provision of treatment . the limitations of our study include a potential for selection bias , as the survey used for this study was provided only in english and french and potentially limited to women literate in these two languages . as the survey was self - administered to ensure privacy , the lack of added clarification by an interviewer may have contributed to certain questions being unanswered . the participants of this study were also relatively healthy , with high cd4 counts and good viral suppression on haart , and may not represent the full spectrum of wlwhiv in ontario . finally , women who desire and intend pregnancy may be more motivated to complete a survey assessing reproductive intention . however , the large sample size in the study and the matching of study recruitment to the geographic distribution of hiv - positive women living in ontario allowed for generalizability of study results to the population of wlwhiv in ontario . a significant proportion of the study enrolment was also conducted using a community - based research model involving aids service organizations and wlwhiv in the recruitment coordination and process , which allowed the engagement of participants who do not usually partake in research . our findings indicate wlwhiv of reproductive age living in ontario , canada both desire and need to receive art for safe pregnancy planning and conception and view healthcare providers as the main source of information regarding reproductive options . this study continues a series of assessments intended to guide the development of canadian national guidelines on safer pregnancy planning and conception , as well as provincial and national hiv pregnancy planning and fertility programs . potential future areas of research include an exploration of healthcare provider attitudes towards fertility and pregnancy for hiv - positive individuals as well as exploration of potential regional differences in their reproductive needs . to fully assess the reproductive needs of plwhiv , similar research
bitterness - masking compounds were identified in a natural white mold cheese . the oily fraction of the cheese was extracted and further fractionated by using silica gel column chromatography . the four fractions obtained were characterized by thin - layer chromatography and nuclear magnetic resonance spectroscopy . the fatty acid - containing fraction was found to have the highest bitterness - masking activity against quinine hydrochloride . bitterness - masking activity was quantitated using a method based on subjective equivalents . at 0.5 mm , the fatty acid mixture , which had a composition similar to that of cheese , suppressed the bitterness of 0.008% quinine hydrochloride to be equivalent to that of 0.00490.0060% and 0.5 mm oleic acid to that of 0.00320.0038% solution . the binding potential between oleic acid and the bitter compounds was estimated by isothermal titration calorimetry . these results suggest that oleic acid masked bitterness by forming a complex with the bitter compounds .	the five basic tastes comprise sweet , sour , bitter , salty , and umami . of these tastes , sour and bitter are generally unfavorable and avoided by humans , because these tastes are associated with spoiled and unripe foods as well as bitter toxins . the perception of bitterness may have evolved in humans and animals to avoid the intake of toxins . the origins and structural diversity of bitter compounds are also much greater than those of other substances . metal ions , some amino acids , peptides , and the secondary metabolites produced by plants , such as alkaloids , phenols , flavonoids , isoflavones , terpenes , and glucosinolates , are bitter . these substances are perceived by taste 2 receptors ( tas2r ) , which are classified as g protein - coupled receptors . in human , bitter - tasting foods are not preferred in most cases ; a few exceptions include coffee , beer , and wine . even though humans are averse to bitter taste , pharmaceutical compounds with physiological benefits often taste bitter . therefore , the masking of bitterness is considered to be important in food processing and pharmacology . some substances with potent tastes such as salts , acids , and sugars can suppress bitterness . cyclodextrin includes some bitter substances intramolecularly , thereby inhibiting the binding of these substances to taste receptors . phosphatidic acid ( pa ) and its lipoprotein derivative , formed by interactions with -lactoglobulin , are reported to suppress the bitterness of quinine sulfate . the activation pattern of tas2rs with some bitter chemicals , including quinine hydrochloride ( qhcl ) , is now known . recently , an antagonist for several tas2r taste receptors was identified by screening a chemical compound library . this compound binds the tas2r activation pocket to inhibit ligand binding , thereby effectively suppressing bitter taste . however , these are not applicable for processed food because the safety of the antagonists is not guaranteed . to utilize these compounds for processed food therefore , it is more appropriate to screen for bitterness - masking compounds in conventional foods to meet the requirements of the food - processing industry . its unique flavors and tastes are produced by the action of microorganisms such as lactic acid bacteria and fungi . during the fermentation process , various compounds not contained in milk are produced . peptides and free amino acids are generated by the digestion of milk proteins by microbial proteinases and exopeptidases . bitter- or astringent - tasting peptides are often produced by digestion of the caseins in cheese . many kinds of tastants are present in foods ; they may interact with each other to enhance or suppress the taste . the aim of this study was to purify and identify compounds in cheese with bitterness - masking properties and to reveal the mechanism of their suppression by using isothermal titration calorimetry ( itc ) . the following cheeses were purchased from supermarkets in tokyo , japan : baraka cheese ( lincet saint - julien , trappes , france ) , gouda ( frico , wolvega , the netherlands ) , ricotta ( galbani , milan , italy ) , and brie ( bongrain sa , viroflay , france ) . chemicals were obtained from commercial sources : 9-anthryldiazomethane ( adam ) from funakoshi co. , tokyo , japan ; qhcl from nacalai tesque inc . , kyoto , japan ; fatty acids ( fas ) including myristic acid , palmitic acid , stearic acid , and oleic acid ( oa ) , glycerides including trioleoylglycerol ( tog ) , dioleoylglycerol ( dog ) , and monooleoylglycerol ( mog ) , and promethazine hydrochloride ( phcl ) from sigma - aldrich co. , tokyo , japan ; and caffeine and cdcl3 containing 1 vol % of tetramethylsilane ( tms ) from wako pure chemical industries , ltd . wakogel c-200 ( chromatography grade , particle size = 75150 m ) was purchased from wako pure chemical industries , ltd . , osaka , japan , and silica gel 60f254 ( hx953368 ; 5 25 cm ) for thin - layer chromatography ( tlc ) was from merck kgaa , darmstadt , germany . itc was performed on a microcal itc200 instrument ( ge healthcare japan corp . , a kitchen knife was used to cut 150 g of cheese into small pieces after removal of the mold - covered surface , prior to the addition of 600 ml of ethanol . the mixture was homogenized at 20000 rpm for 10 min by using a polytron homogenizer ( pt1300d , da1607/2 ; ishii laboratory works co. , ltd . , osaka , japan ) and then centrifuged at 15300 g for 10 min at room temperature . the liquid layer was separated from the debris and re - extracted twice with 400 ml of ethanol . the liquid layers collected were then concentrated to dryness by using a rotary evaporator . the dried residue was dissolved in 200 ml of ethyl acetate , dehydrated with na2so4 anhydrate , and filtered with filter paper ( advantec no . the plate was spotted with the samples , developed with a mixture of n - hexane / acetone ( 2:1 ) , and exposed by spraying with 10% h2so4 , prior to heating on a hot plate , to detect the separated samples . silica gel ( 80 g ) was packed into a glass column ( 29 300 mm ) and equilibrated with n - hexane . next , 510 g of the oily fraction was applied and eluted with n - hexane . the elution was performed with a n - hexane / acetone mixture , with the ratio of acetone increasing stepwise . the typical elution profile in sequence is 200 ml of n - hexane , 210 ml of n - hexane / acetone ( 100/5 ) , 220 ml of n - hexane / acetone ( 100/10 ) , 240 ml of n - hexane / acetone ( 100/20 ) , 260 ml of n - hexane / acetone ( 100/30 ) , and 280 ml of n - hexane / acetone ( 100/40 ) . the resulting fractions with the same tlc patterns were gathered , and the four fractions a , b , c , and d were obtained . fraction a corresponded to the n - hexane / acetone mixtures of 100/5 and 100/10 , fraction b to 100/20 , fraction c to 100/30 , and fraction d to 100/40 . the separation was performed eight times , and each pooled fraction was further purified by rechromatography , eluting using the n - hexane / acetone system . baraka , ricotta , gouda , and brie cheeses were analyzed to determine their free fa composition . the oily fraction from each cheese was prepared according to the above - described method . the free fas were then analyzed using high - performance liquid chromatography ( hplc ) using the adam method , as reported previously . panelists ( n = 9 ) were selected using the difference test with five basic tastes and the discrimination test for the differences in the concentrations of four basic tastes and were trained using the methods described below . for the discernment of bitter taste , each panelist was trained with triangular tests to distinguish bitter taste at four concentration levels near the threshold value . in addition , each panelist was trained in the discernment of difference in the concentrations of bitter taste by using a ranking test involving the qhcl solution at seven concentration levels ( common ratio 1.11.2 ) . in sensory tests with panelists ( n = 4 ) , a piece of baraka cheese was placed on the tongue after peeling off the mold - covered surface and spread over the whole tongue prior to tasting 0.0080% qhcl solution . to estimate the bitterness - masking activity , fractions a , b , c , and d were each solubilized in 0.0080% qhcl containing 1% -lactoglobulin , at a concentration of 12 mg / ml . because the oily fractions dissolved poorly in water , -lactoglobulin was added as a solubilizer . -lactoglobulin ( 1% ) alone did not possess bitterness - masking activity under these conditions ( data not shown ) . the free fas were solubilized by the addition of equimolar naoh and stirred with a magnetic stirrer . then , 1 ml of bitter - tasting solutions containing each of the four fractions was put in the mouth , and the bitter taste intensity was evaluated on a three - level scale : bitterness equal to 0.0080% qhcl ( 1 ) , slightly less than 0.0080% qhcl ( 2 ) , or significantly less bitterness ( 3 ) . panelists ( n = 7 ) performed sensory tests by using the beer . here , a piece of baraka , gouda , brie , or ricotta cheese was put in the mouth , and a sip of beer was consumed . the bitter taste was evaluated using a four - point categorical scale : strong ( 0 ) , medium ( 1 ) , weak ( 2 ) , or very weak ( 3 ) . for evaluation of scores , the steel dwass test , a nonparametric multiple - comparison method , was applied for detecting between - sample differences . two test solutions were prepared : ( a ) 0.2 mm oa , 0.2 mm palmitic acid , 0.05 mm myristic acid , 0.05 mm stearic acid , and 0.0080% qhcl in 5 mm sodium phosphate buffer ( ph 7.0 ) ; ( b ) 0.5 mm oa and 0.0080% qhcl in 5 mm sodium phosphate buffer ( ph 7.0 ) . standard solutions with seven different concentrations of qhcl , that is , 0.0026 , 0.0030 , 0.0035 , 0.0040 , 0.0046 , 0.0053 , and 0.0060% , in 5 mm sodium phosphate buffer ( ph 7.0 ) were also prepared . panelists ( n = 9 ) who could discriminate the seven standard solutions in order of concentration participated in this test . each of the standard solutions ( 5 ml ) was put in a clear plastic cup , whereas each of the test solutions ( 5 ml ) was in a white paper cup because the test solutions were slightly cloudy . first , the panelists tasted the three standard solutions , 0.0030 , 0.0040 , and 0.0053% qhcl , to remember the bitterness of each solution . then , 5 ml of the test solution was held in the mouth for 15 s prior to its being spat out . after that , the mouth was rinsed with water to remove any bitter aftertaste , and the panelist waited for 30 s before moving to the next test . an interval of 60 s was provided before and after tasting each fa solution to avoid confusing the tastes . the bitter taste intensities of test solutions a and b were estimated in comparison with seven standard solutions . panelists were allowed to repeatedly taste the standard solutions before selecting the solution that was the closest to the bitterness of the test solutions . analysis of variance ( anova ) was applied to detect the variation of judged sensory scores at significance level = 0.05 . all statistical analyses were carried out using the computer software jmp 9.0.2 ( sas institute , inc . , bitter taste intensity evaluations were performed by using the following paired difference tests : between 0.22 mm qhcl and 0.22 mm qhcl containing 0.5 mm oa ( involving 20 panelists ) , between 1.5 mm phcl and 1.5 mm phcl containing 0.5 mm oa ( involving 6 panelists ) , and between 50 mm caffeine and 50 mm caffeine containing 0.5 mm oa ( involving 10 panelists ) . statistical analysis of scores was conducted using a one - tailed binomial test ( significance level = 0.05 ) . the concentrations of oa , qhcl , and phcl used in titration were 0.5 , 2.2 , and 1.5 mm , respectively . they were dissolved in 5 mm sodium phosphate buffer ( ph 7.0 ) containing 5% ethanol . the reference cell was filled with milli - q water ( millipore corp . , billerica , ma , usa ) . each titration was carried out with initial injection ( 0.4 l ) followed by 18 main injections ( 2 l each ) at intervals of 120 s. the first titration ( 0.4 l ) was excluded for the analysis . the dilution calorie of the ligand in the buffer was subtracted from the titration data of qhcl . the titration of oa with phcl was also performed except for using at 150 s intervals . although the titration of 0.5 mm oa with 2.2 mm caffeine was performed at 150 s intervals , the titration was dispersed . therefore , 50 mm caffeine containing 50 mm sodium phosphate buffer ( ph 7.0 ) was adopted . the data were analyzed according to a model for one set of sites provided in the origin 7.0 software for microcal itc200 . the dissociation constant ( kd ) and enthalpy change of binding ( h ) were obtained from the fitted curve . the entropy change of binding ( s ) and free energy change of binding ( g ) were obtained from eq 1 ; r is the gas constant , t , the thermodynamic temperature , and k , the association constant.1 seventy - one brands of cheeses were commercially obtained and subjected to sensory tests to identify the cheese with bitterness - masking activity . in addition , panelists were asked to consider which of the 71 cheeses had the strongest bitterness - masking activity . as a result of the screening , baraka cheese was selected ( supporting information ) . the oily fraction was extracted with ethanol and then with ethyl acetate ( figure 1a ) , resulting in a yield of 56 g from 150 g of baraka cheese . it was further separated by silica gel column chromatography by using a n - hexane / acetone stepwise elution system . four fractions , a , b , c , and d , weighing 24.3 , 0.77 , 0.012 , and 0.17 g , respectively , were eluted in this order . tlc was performed with tog , dog , mog , and oa as the references ( figure 1b ) . the mobilities of tog , dog , mog , and oa are indicated by their rf values of 0.96 , 0.72 and 0.68 , 0.63 , and 0.50 , respectively . the mobilities of the fractions a d were compared with those of the standard compounds . fraction a moved to the front of the plate and accounted for 96.2% of the oily fraction . the main spot of fraction b had a mobility equal to that of the dog spots , whereas that of fraction c nearly coincided with those of dog and oa . extraction and separation of oily fraction from baraka cheese : ( a ) fractionation scheme ; ( b ) thin - layer chromatography ( tlc ) analysis . the four fractions were analyzed by tlc , developed using n - hexane / acetone ( 2:1 ) , and detected by spraying with 10% h2so4 and heating on a hot plate . the rf values for each authentic sample , applied at 1 g / lane , are as follows : trioleoylglycerol ( tog ) ( rf = 0.96 ) ; dioleoylglycerol ( dog ) ( rf = 0.72 , 0.68 ) ; oa , oleic acid ( oa ) ( rf = 0.63 ) ; monooleoylglycerol ( mog ) ( rf = 0.50 ) . to identify the substances in the fractions b , c , and d , the samples were analyzed by h and c nuclear magnetic resonance ( nmr ) spectroscopy ( varian inova 500 ) . the methyl , methylene , and olefin signals of the fas are not shown because these signals were derived by a mixture of many fa molecules . fraction b ( diacylglycerol ( dg ) mixture of 1,2-isomer ( 53% ) and 1,3-isomer ( 47% ) ) : h nmr ( 500 mhz , cdcl3 , tms ) 3.61 ( 2 , 3-ch2 of 1,2-isomer ) , 3.97 ( 1 , 2-ch of 1,3-isomer ) , 4.05 ( 4 , 1-ch2 and 3-ch2 of 1,3-isomer ) , 4.10 ( 1 , 1-ch2 of 1,2-isomer ) , 4.25 ( 1 , 1-ch2 of 1,2-isomer ) , 5.00 ( 1 , 2-ch of 1,2-isomer ) ; c nmr ( 125 mhz , cdcl3 , tms ) 60.9 ( 3-ch2 of 1,2-isomer ) , 62.1 ( 1-ch2 of 1,2-isomer ) , 64.7 ( 1-ch2 and 3-ch2 of 1,3-isomer ) , 67.7 ( 2-ch of 1,3-isomer ) , 71.8 ( 2-ch of 1,2-isomer ) , 173.2 ( 2-ch o co of 1,2-isomer ) , 173.5 ( 1-ch2o co of 1,2-isomer ) , 173.6 ( 2 , 1-ch2o co and 3-ch2o co of 1,3-isomer ) . the ratio of the 1,2- to 1,3-isomers was calculated from the integration value of the two signals corresponding to the 2-ch protons . fraction c ( dg of 1,3-isomer and fas ) : h nmr ( 500 mhz , cdcl3 , tms ) 4.1 ( 1 , 2-ch ) , 4.22 ( 4 , 1-ch2 and 3-ch2 ) ; c nmr ( 125 mhz , cdcl3 , tms ) 62.1 ( 1-ch2 and 3-ch2 ) , 68.9 ( 2-ch ) , 173.3 ( 1-ch o co and 3-ch o fraction d ( 1-monoacylglycerol ( mg ) ) : h nmr ( 500 mhz , cdcl3 , tms ) 3.60 ( 1 , 3-ch2 ) , 3.69 ( 1 , 3-ch2 ) , 3.89 ( 1 , 2-ch ) , 4.15 ( 1 , 1-ch2 ) , 4.21 ( 1 , 1-ch2 ) . fractions b and d were found from their nmr spectra to include dg and mg , respectively ( table 1 ) . fraction c was determined to mainly include 1,3-dg and free fas by tlc and nmr analyses . the concentration of free fas in this fraction was 43.6% , as determined by hplc by using the adam method . the compounds containing fractions b , c , and d were identified from h and c nmr spectra . next , the bitterness - masking activity of each fraction was analyzed by sensory tests . using the examination of the wilcoxon rank - sum test and steel dwass test , fraction c had the strongest bitterness - masking activity compared to the other three fractions with a significant level of 10% ( wilcoxon rank - sum test , p < 0.025 ; steel dwass test , fractions a , b , and d , p = 0.063 , 0.089 , and 0.089 ) ( table 2 ) . these results suggest that free fas are the bitterness - masking compounds in cheese . sensory test i : 0.5 g of baraka cheese was put on the tongue and spread over the entire tongue prior to tasting 0.0080% quinine hydrochloride ( qhcl ) . sensory test ii : each fraction was solubilized in 0.0080% qhcl and evaluated for bitterness . the bitterness was evaluated on a 3-score scale : ( 1 ) equal to that of 0.0080% qhcl , ( 2 ) slightly less bitter than 0.0080% qhcl , or ( 3 ) significantly less bitter . each score is the average of four trials . to confirm the bitterness - masking activity of free fas the total concentration of free fas in baraka cheese was 12.15 mm , with oa present at the highest concentration ( 4.29 mm , 35.3% ) followed by palmitic acid ( 3.70 mm , 30.5% ) , myristic acid ( 1.27 mm , 10.5% ) , and stearic acid ( 0.86 mm , 7.0% ) ( table 3 ) . free fatty acids ( fas ) in the cheese samples were quantitated from their oily fractions . the molar concentration and weight percentage of free fas in the whole cheeses were calculated . nd , not detected . to examine the correlation of free fa content with bitterness - masking activity , baraka and the other three cheeses with different bitterness - masking activities were analyzed . first , the bitterness - masking activities of baraka , gouda , brie , and ricotta were estimated using beer . the bitterness - masking activity of baraka cheese was found to be significantly stronger than that of the other three cheeses by using the nonparametric steel dwass test . the significant differences between baraka and the other cheeses were as follows : baraka versus gouda , p = 0.0159 ; versus brie ; p = 0.0423 ; and versus ricotta ; p = 0.0077 ( figure 2 ) . the average values of the panelists answers concerning the masking activity of cheese to the bitter taste of beer are shown ( n = 7 ) . the intensity of bitter taste was evaluated using a 4-point categorical scale , as follows : strong ( 0 ) , medium ( 1 ) , weak ( 2 ) , or very weak ( 3 ) . the steel dwass test , which uses the nonparametric multiple - comparison method , was applied for detecting the between - sample differences . the baraka cheese sample showed significantly higher bitterness - masking activity than the other three samples [ gouda , p = 0.0159 ( ) ; brie , p = 0.0423 ( ) ; ricotta , p = 0.0077 ( ) ] . next , the concentrations of free fas in the other three cheeses were analyzed . the total concentrations of free fas were 1.13 mm in ricotta , 3.86 mm in brie , and 2.25 mm in gouda . thus , the total free fas are apparently high in baraka and low in the other three cheeses . these results suggest that free fa content is correlated with the bitterness - masking activity . although the total concentrations of free fas in the four cheeses are quite different , their free fa compositions are almost identical ( table 3 ) . in all of the cheeses analyzed , four fas ( oa , palmitic acid , myristic acid , and stearic acid ) comprised up to 60% of the total free fas . a 0.5 mm solution of mixed free fas ( equivalent to the average free fa composition of cheese ) , 0.2 mm oa , 0.2 mm palmitic acid , 0.05 mm myristic acid , and 0.05 mm stearic acid were subjected to a bitterness - masking test . a 0.0080% qhcl solution was used to determine the bitterness - masking activity of the mixed free fas . a test was constructed using seven concentrations of qhcl solution to evaluate the degree of bitterness - masking activity . wilk w test ( p = 0.2123 and w = 0.932201 ) ( figure 3a ) . on the basis of these results , it was concluded that the bitterness - masking effect existed within the 95% confidence interval of the mean response . we found that the 0.5 mm mixed fa solution reduced the bitterness of qhcl from 0.0080 to 0.00490.0060% . however , there was a significant difference in the evaluation score between the panelists according to the f test of anova ( p = 0.001 ) . the mixed free fas apparently suppressed the bitterness of qhcl , but the data varied among panelists . this could be explained by the fact saturated fas , myristic acid , palmitic acid , and stearic acid were little dissolved and could not be suspended in buffer and , therefore , the recognition of bitter taste would be variable . two sample solutions were prepared : ( a ) 0.0080% quinine hydrochloride ( qhcl ) containing 0.2 mm oleic acid ( oa ) , 0.2 mm palmitic acid , 0.05 mm myristic acid , and 0.05 mm stearic acid ; ( b ) 0.0080% qhcl containing 0.5 mm oa . bitter taste intensity was evaluated by comparing the sample solutions with seven standard qhcl solutions , as follows : 1 , 0.0026% ; 2 , 0.0030% ; 3 , 0.0035% ; 4 , 0.0040% ; 5 , 0.0046% ; 6 , 0.0053% ; and 7 , 0.0060% . the right panels are box plots with the smallest observation , lower quartile , median , upper quartile , and largest observation . wilk w test for non - normality showed p = 0.2123 and w = 0.932201 in ( a ) and p = 0.1376 and w = 0.921529 in ( b ) . to eliminate the factor of low solubility of these fas , oa was used for the bitterness - masking test , because it can be suspended in buffer as a sodium salt and it was the predominant fa in baraka cheese . the normality of distribution of the sensory evaluation score , using the seven concentration levels of qhcl for quantitatively evaluating the degree of bitterness - masking activity , was confirmed by the shapiro wilk w test ( p = 0.1376 and w = 0.921529 ) ( figure 3b ) . on the basis of these results , it was estimated that the bitterness - masking effect existed within the 95% confidence interval of the mean response . our findings showed that a 0.5 mm oa solution reduced the bitterness of qhcl from 0.0080 to 0.00320.0038% . in addition , there was no significant difference in the evaluation score among the panelists , as demonstrated by the f test of anova ( p = 0.131 ) . thus , in the measurements conducted using the 0.5 mm solution of oa alone , the panelists evaluations were found to be highly consistent with one another . these results demonstrate that fas , especially oa , suppress the bitterness of qhcl . as already shown , the bitter taste of 0.0080% qhcl was suppressed by 0.5 mm oa . a further study involved the bitterness - masking activity of 0.5 mm oa against 0.22 mm qhcl , 1.5 mm phcl , and 50 mm caffeine . because of the sensory tests , the bitterness of qhcl and phcl was significantly suppressed by oa ( one - tailed binomial test : p = 0.0059 , 0.0156 ) . on the other hand , that of caffeine was not suppressed ( one - tailed binomial test : p = 0.0547 ) . to validate the bitterness - masking activity of oa , the binding potential between oa and qhcl was examined by itc , which analyzes the interaction of two compounds by measuring the change in caloric output when they are mixed . when oa was titrated with qhcl , the reaction was exothermic , and the dissociation constant was 11 1 m ( figure 4a ; table 4 ) . the interactions between oa and phcl were also examined , and the dissociation constant was 8.8 1.6 m ( figure 4b ; table 4 ) . the interactions between oa and caffeine were also examined , but were not detected under the same conditions ( figure 4c ) . isothermal titration calorimetry profiles of oleic acid binding to quinine hydrochloride ( qhcl ) and promethazine hydrochloride ( phcl ) : ( a ) 0.5 mm oleic acid ( oa ) was titrated with 2.2 mm qhcl ; ( b ) 0.5 mm oa was titrated with 1.5 mm phcl ; ( c ) 0.5 mm oa was titrated with 2.2 mm caffeine . the experiments were performed at 25 c , and the titration was repeated 19 times at ( a ) 120 s and ( b , c ) 150 s intervals . in the lower panels , the area of the peak was integrated and plotted against the molar ratio of qhcl or phcl to oa . kd and h were obtained from the fitted curve according to a model for one set of sites . qhci , quinine hydrochloride ; phci , promethazine hydrochloride ; kd , dissociation constant ; g , free energy change of binding ; h , enthalpy change of binding ; s , entropy change of binding . in this study , we have purified a bitterness - masking fraction in cheese , and free fas were identified as the compounds responsible for the bitterness of this fraction . the total concentration of free fas in baraka cheese was about 12 mm , which included 4.3 mm oa . a sensory test showed that both a 0.5 mm free fa mixture ( oa , palmitic acid , stearic acid , myristic acid ) and 0.5 mm oa alone reduced the bitterness of qhcl . the concentration of oa in baraka cheese is about 10 times larger than that of oa used for the sensory test , which is adequate for it to develop the bitterness - masking effects . several kinds of free fas other than oa were also contained in the four cheeses analyzed . although individual free fas were not examined , a mixture of these fas showed bitterness - masking activity , proving that they must possess this property . in fact , linoleic acid has been reported to mask the bitterness of caffeine . however , the concentration of linoleic acid used in the previous experiment was 70 times greater than that of the oa used in the present experiment . pa and pi exhibit a strong bitterness - masking activity against qhcl , although some other bitter substances are not similarly influenced . bitterness - masking activity can be determined by the affinity of a bitter tastant for a masking compound . on the basis of the itc analyses , the kd values between oa versus qhcl and oa versus phcl were 11 1 and 8.8 1.6 m , respectively . several studies have shown that a midmicromolar dissociation range of interaction leads to the formation of a complex between two compounds . similarly , 1-aminoanthracene interacts with horse spleen apoferritin ( hsaf ) with a dissociation constant of 100 m , which was shown to be appropriate for binding . when the results of this study are considered comprehensively , the most likely hypothesis is that oa binds to qhcl or phcl to form complexes in aqueous solution , thereby masking the bitterness of qhcl . we speculate that a complex may form because of the interaction between alkyl chains in oa or in other free fas and the hydrophobic partial structure in qhcl and phcl . the calorimetric parameters as well as the values of h and ts are in line with the assumption that the suppression is caused by hydrophobic interactions . on the other hand the interaction between oa and caffeine was not detected by itc under the present conditions . even the higher concentrations of 0.5 mm oa and 50 mm caffeine did not show interaction . these results suggest that the binding ability of oa and bitter compounds is a factor determining the bitterness - masking activity . moreover , it was suggested that the kd value was related to the strength of the bitter - masking activity . however , fermented foods contain free fas produced by the digestion of tg by the lipases secreted from microorganisms . some studies showed that short - chain fas liberated from milk lipids generate a cheese - specific flavor , although the bitterness - masking activity of free fas has not been discussed . the present study found a close relationship between the high bitterness - masking activity of baraka cheese in sensory tests and its high free fa content . to the best of our knowledge , this study is the first to report the bitterness - masking effect of foods containing free fas in general and of oa in particular . however , no convincing method for analyzing the interaction of food components at the molecular level is available . in particular , interactions among small molecules have not been detectable in the intact molecular form . in this study , we directly analyzed the interaction between a bitter tastant and its masking compound . our approach will be useful for studying the interactions concerned with tastants in foods .
highlightsthe colonic igg4-rd is rare.we report the case of a74-year - old female with igg4-rd of the ileocecal region.the patient was diagnosed asmalignant lymphoma and underwent right - hemi colectomy.postoperative pathologicalexamination revealed igg4-rd of the ileocecal region.surgical resection for igg4-rdis necessary for cases with concerns of malignancy .	immunoglobulin g4-related disease ( igg4-rd ) is a systemic disease characterized by chronic fibrosing inflammation with abundant igg4-positive plasma cells and elevated serum igg4 levels ; it tends to be mistaken for malignancy and responds well to steroids . clinical manifestations are common in the pancreas , salivary glands , hepatobiliary tract , orbit , lymph nodes , and retroperitoneum but are rare in the gastrointestinal tract . we herein report a case of igg4-rd of the ileocecal region diagnosed after surgical resection performed for a suspected malignancy . we also provide a review of the literature , with an emphasis on the treatment of this disease . a 74-year - old female presented with a two - month history of right lower abdominal pain and a slight fever . an approximately 5 cm mass in her right lower abdomen was detected by abdominal ultrasonography ( us ) , and she was admitted for further examination . colonoscopy revealed moderate stenosis caused by edematous wall thickening of the lower ascending colon , with reddening of the mucosa ( fig . since the ileocecal valve was also swollen , the scope could not be inserted into the terminal ileum ( fig . a colonic biopsy from the swollen wall showed a nonspecific inflammation with lymphoid aggregates , with no evidence of malignancy . a radiographic contrast enema indicated edematous asymmetric stenosis with erosion from the terminal ileum to lower ascending colon ( fig . 1c ) . an abdominal computed tomography ( ct ) scan indicated edematous wall thickening from the terminal ileum to the lower ascending colon ( fig . positron emission tomography with f - fluorodeoxyglucose ( fdg - pet ) revealed increased fdg uptake in the ascending colon ( suv max : 13.3 ) , terminal ileum ( suv max : 6.9 ) , and also in the spleen ( suv max : 5.9 ) and paraaortic lymph node ( suv max : 5.3 ) ( fig . blood tests revealed high levels of c - reactive protein ( 27.48 mg / dl ) , elevated ldh ( 252 u / l ) and elevated alp ( 561 u / l ) . the soluble interleukin-2 receptor ( sil-2r ) level was elevated ( 2305 u / ml , normal range : 122496 u / ml ) , but none of the other tumor marker levels were remarkable ( cea : 1.0 ng / ml , ca19 - 9 : 7.1 u / ml ) . although a pathological diagnosis was not obtained preoperatively , she was diagnosed to have malignant lymphoma based on these findings . it seemed that passage of stool would be completely obstructed by tumor growth in the near future . consequently , she underwent a right hemi - colectomy in order to obtain a pathological diagnosis , and to avoid ileus caused by tumor growth . grossly , the segmental bowel resection included 25 cm of ascending colon and 125 cm of ileum ( fig . there was irregular wall thickening with submucosal sclerosis , which was 10 cm in length from the terminal ileum to the ascending colon that accompanied the sclerosis of the mesentery of the ileum . since the sclerosing mesentery needed to be extirpated , the long ileum that received its blood supply from the mesentery was also resected . microscopically , the lesion was composed of the ileal , cecal and colonic wall with lymphoplasmacytic infiltration , lymphoid follicles and fibrosis in the subserosa and surrounding mesenteric adipose tissue ( fig . atypical lymphoid cells were not aggregated in these specimens . the elastica van gieson ( evg ) staining showed obliterative phlebitis , which is one of the characteristic histological features of igg4-rd ( fig . immunohistochemical staining revealed an increased number of igg4-positive plasma cells at 150/hpf ( 400 ) , and the igg4/igg ratio was 50% ( fig . the serum igg4 level was postoperatively found to be normal level at 102 mg / dl ( normal range : 6121 mg / dl ) . the patient 's postoperative course was uneventful , and she was discharged home on postoperative day 18 tolerating a diet by mouth . she is currently doing well 10 months after the operation . during the preoperative fdg - pet examination , increased fdg uptake had been seen not only in the resected ileocecal region , but also in the spleen and paraaortic lymph node . while igg4-rd might remain in these sites thus , we decided to perform careful follow - up without any additional treatment , such as steroid therapy . many previous reports of igg4-rd have focused on pancreatic involvement , which is almost interchangeably called autoimmune pancreatitis ( aip ) , and it has been reported that infiltration of many igg4-positive plasma cells was observed in the gastric mucosa , colonic mucosa and major duodenal papillae of some aip patients . in addition , igg4-positive plasma cell infiltration is sometimes detected in the colonic mucosa of patients with ulcerative colitis ( uc ) . on the other hand , gastrointestinal igg4-rds without other lesions have also been reported in the esophagus and stomach . colonic igg4-rds without other lesions , such as aip or uc , have also been reported , but these have been quite rare . chetty et al . reported two cases of solitary igg4-rd at the cecum and sigmoid colon . in this case report , we presented a case of igg4-rd of the ileocecal region without any evidence of aip or uc based on the postoperative pathological examinations . the lesion was located at the subserosa of the ileum , cecum , ascending colon and mesenteric adipose tissue . a likely explanation for these findings is that the igg4-rd of the present case might have originated at the mesentery and involved the gastrointestinal tract . in fact , some recent reports have presented igg4-related sclerosing mesenteritis involving the gastrointestinal tract . since gastrointestinal igg4-rds without other igg4-rds , such as aip or uc , appear to be difficult to diagnose prior to surgical resection because of their rarity and features similar to malignancy , most of the solitary gastrointestinal igg4-rds in the previous reports were diagnosed after surgical resection , like the present case . on the other hand , a few cases that were successfully diagnosed as gastrointestinal igg4-rd by the immunohistochemical staining of preoperative biopsy specimens have also been reported . in the present case , although a preoperative colonic biopsy was performed to rule out malignancy , we did not perform immunohistochemical examination for igg4-positive plasma cells . it may be difficult to diagnose this disease based on biopsy specimens if there is sclerosis and an increased number of igg4-positive plasma cells were spread predominantly in the subserosa like the present case . according to the comprehensive clinical diagnostic criteria for igg4-rd proposed in 2011 , igg4-rd is diagnosed when there is a characteristic diffuse / localized swelling or mass in a single or multiple organs with elevated serum igg4 levels , or when there are histological findings of abundant infiltration of igg4-positive plasma cells and lymphocytes , along with fibrosis . however , approximately 30% of patients have normal serum igg4 levels , despite classic histopathological and immunohistochemical findings . in our present case , although histological features of igg4-rd such as dense lymphoplasmacytic infiltration , fibrosis and obliterative phlebitis were observed , the serum igg4 level was normal . one possible explanation for this finding is that the serum igg4 level was decreased to the normal level at the point of measurement because it was measured postoperatively . although some igg4-rd patients might show false - negative serum igg4 findings , the levels should be measured prior to surgical resection in case of gastrointestinal stenosis or obstruction of uncertain cause , and where this disease might be suspected . as described above , measurement of the serum igg4 levels , immunohistochemical examinations of biopsy specimens and the use of several imaging modalities might help to diagnose the condition without the need for surgical resection . if gastrointestinal igg4-rd can be diagnosed without surgical resection , steroid therapy can be useful for the disease . however , if a malignancy can not be ruled out completely after these examinations , surgical resection as a treatment for malignancy should be performed to obtain a definite diagnosis . in addition , many patients with gastrointestinal igg4-rd may develop obstructive problems such as ileus . in such cases , surgical resection should be performed if the obstruction does not improve with conservative management . gastrointestinal igg4-rd often mimics malignancy , and a preoperative diagnosis of this disease remains difficult . the most important thing to avoid unnecessary resection is that igg4-rd in the gastrointestinal tract should be included in the differential diagnosis when marked wall thickening or a pseudotumor - like lesion is observed in the gastrointestinal region . however , surgical resection for igg4-rd may still be necessary for patients with concerns regarding malignancy or intractable gastrointestinal obstruction caused by this disease . written informed consent was obtained from the patient for publication of this case report and accompanying images . a copy of the written consent is available for review by the editor - in - chief of this journal on request . eiji oki , yoko zaitsu , koji ando , hiroshi saeki , masaru morita , hideo baba , yoshihiko maehara : writing . hidetaka yamamoto : pathologist.key learning pointsthe colonic igg4-rd is rare.we herein report the case of a 74-year - old female with igg4-rd of the ileocecal region who underwent right hemi - colectomy for suspected malignant lymphoma.the patient was diagnosed with igg4-rd by postoperative pathological examinations.the measurement of the serum igg4 levels , immunohistochemical examination of biopsy specimens and use of several imaging modalities might help us to diagnose the disease without surgical resection , and this disease can generally be treated with steroid therapy.surgical resection for igg4-rd may still be also necessary for patients with concerns regarding malignancy or with intractable gastrointestinal obstruction caused by this disease . the colonic igg4-rd is rare.we herein report the case of a 74-year - old female with igg4-rd of the ileocecal region who underwent right hemi - colectomy for suspected malignant lymphoma.the patient was diagnosed with igg4-rd by postoperative pathological examinations.the measurement of the serum igg4 levels , immunohistochemical examination of biopsy specimens and use of several imaging modalities might help us to diagnose the disease without surgical resection , and this disease can generally be treated with steroid therapy.surgical resection for igg4-rd may still be also necessary for patients with concerns regarding malignancy or with intractable gastrointestinal obstruction caused by this disease . we herein report the case of a 74-year - old female with igg4-rd of the ileocecal region who underwent right hemi - colectomy for suspected malignant lymphoma . the measurement of the serum igg4 levels , immunohistochemical examination of biopsy specimens and use of several imaging modalities might help us to diagnose the disease without surgical resection , and this disease can generally be treated with steroid therapy . surgical resection for igg4-rd may still be also necessary for patients with concerns regarding malignancy or with intractable gastrointestinal obstruction caused by this disease .
aim : the aim was to evaluate the bonefill in periodontal osseous defects with the help of guided tissue regeneration , bioresorbable membrane ( periocol ) + bone graft ( grabio glascera ) in combination and with bonegraft ( grabio glascera ) alone.materials and methods : the study involved total 30 sites in systemically healthy 19 patients . the parameters for evaluation includes plaque index sulcus bleeding index with one or more periodontal osseous defects having ( i ) probing depth ( pd ) of 5 mm ( ii ) clinical attachment loss ( cal ) of 5 mm and ( iii ) 3 mm of radiographic periodontal osseous defect ( iv ) bonefill ( v ) crestal bone loss ( vi ) defect resolution . the study involved the three wall and two wall defects which should be either located interproximally or involving the furcation area . the statistical analysis was done using statistical package for social sciences , the wilcoxon signed rank statistic w + for mann whitney u-test.results:the net gain in pd and cal after 6 months for group i ( [ periocol ] + [ grabio glascera ] ) and group ii ( grabio glascera ) was 3.94 1.81 mm , 3.57 2.21 mm and 3.94 1.81 , 3.57 2.21 mm , respectively . the results of the study for group i and group ii with regards to mean net bonefill , was 3.25 2.32 ( 58% ) mm and 5.14 3.84 ( 40.26 19.14% ) mm , crestal bone loss 0.25 0.68 mm and 0.79 1.19 mm . defect resolution 3.50 2.34 mm and 5.93 4.01 mm , respectively.conclusion:on comparing both the groups together after 6 months of therapy , the results were equally effective for combination of graft and membrane versus bone graft alone since no statistical significant difference was seen between above parameters for both the groups . thus , both the treatment modalities are comparable and equally effective .	periodontitis is a disease characterized by inflammation of the gingiva that results in periodontal pocket formation with loss of the supporting periodontal ligament and alveolar bone around teeth . various terminologies including scaling and root planing ( srp ) or open flap debridement ( ofd ) are conventional methods used for treatment of periodontitis . but the use of specific biomaterials was more effective than ofd in improving attachment levels in periodontal defects . in the past three decades , periodontal regenerative treatment has received increasing attention as an alternative to tooth extraction in patients with periodontal disease . implant insertion , although highly predictable , has been shown to be less predictable than saving a periodontally compromised tooth with regenerative therapy . with the advent of guided tissue regeneration ( gtr ) , restoration of periodontium the technique using barrier was introduced by nyman in 1982 , and the term gtr was coined by gottlow in 1986 . gtr is based upon the biological behavior of different periodontal tissues . during 1980s , researchers published information that led to the conclusion that the periodontal ligament was the most important source of periodontal progenitor regenerative cells . current concept of gtr membrane , that is , resorbable and nonresorbable membranes act as a physical barrier to avoid connective and epithelial tissue down - growth into the defect , favoring the regeneration of periodontal tissues . the bioabsorbable membranes are the second generation gtr membranes and were developed to avoid the second surgical procedure to remove the barrier . these gtr devices fall into two broad categories , natural products ( collagen membrane ) and the synthetic ( copolymer ) materials . in an attempt to further induce attachment gain for intrabony defects , regenerative therapy with bone replacement grafts did not gain acceptance as predictable therapy until the 1980 . these materials act either as osteogenic and osteoinductive or osteoconductive . among alloplastic graft materials , bioactive ceramics is a group of osteoconductive materials which include hydroxyapatite ( ha ) , fluorapatite , bioactive glass , and tricalcium phosphate . the food and drug administration approved original composition of bioactive glass designated 45s5 was 45 mol% of sio2 , 26.9 mol% of cao , 24.4 mol% of na2o , and 2.5 mol% of p2o5 . this material can bond to bone through development of a surface layer of carbonated ha in situ . the calcium phosphate is layer thought to promote adsorption and concentration of osteoblast derived protein necessary for mineralization of extracellular matrix . objectives of periodontal bonegrafts are probing depth ( pd ) reduction , clinical attachment gain , bonefill of the osseous defect and regeneration of new bone , cementum , and periodontal ligament . to evaluate regenerative potential of the combination of gtr ( periocol , eucare pharmaceuticals private limited , chennai , india ) and bonegraft ( grabio glascera , dorthom medi dents pvt ltd , coimbathore , india ) ; and bonegraft ( grabio glascera ) alone individually with respect to clinical parameter including clinical attachment level and radiographically the bonefill , change in alveolar crest ( ac ) bone and defect resolutionto compare the regenerative potential of the combination of gtr ( periocol ) and bonegraft ( grabio glascera ) ; and bonegraft ( grabio glascera ) alone between each other with respect to clinical parameter including clinical attachment level and radiographically the bonefill , change in ac bone and defect resolution . to evaluate regenerative potential of the combination of gtr ( periocol , eucare pharmaceuticals private limited , chennai , india ) and bonegraft ( grabio glascera , dorthom medi dents pvt ltd , coimbathore , india ) ; and bonegraft ( grabio glascera ) alone individually with respect to clinical parameter including clinical attachment level and radiographically the bonefill , change in alveolar crest ( ac ) bone and defect resolution to compare the regenerative potential of the combination of gtr ( periocol ) and bonegraft ( grabio glascera ) ; and bonegraft ( grabio glascera ) alone between each other with respect to clinical parameter including clinical attachment level and radiographically the bonefill , change in ac bone and defect resolution . to evaluate regenerative potential of the combination of gtr ( periocol , eucare pharmaceuticals private limited , chennai , india ) and bonegraft ( grabio glascera , dorthom medi dents pvt ltd , coimbathore , india ) ; and bonegraft ( grabio glascera ) alone individually with respect to clinical parameter including clinical attachment level and radiographically the bonefill , change in alveolar crest ( ac ) bone and defect resolutionto compare the regenerative potential of the combination of gtr ( periocol ) and bonegraft ( grabio glascera ) ; and bonegraft ( grabio glascera ) alone between each other with respect to clinical parameter including clinical attachment level and radiographically the bonefill , change in ac bone and defect resolution . to evaluate regenerative potential of the combination of gtr ( periocol , eucare pharmaceuticals private limited , chennai , india ) and bonegraft ( grabio glascera , dorthom medi dents pvt ltd , coimbathore , india ) ; and bonegraft ( grabio glascera ) alone individually with respect to clinical parameter including clinical attachment level and radiographically the bonefill , change in alveolar crest ( ac ) bone and defect resolution to compare the regenerative potential of the combination of gtr ( periocol ) and bonegraft ( grabio glascera ) ; and bonegraft ( grabio glascera ) alone between each other with respect to clinical parameter including clinical attachment level and radiographically the bonefill , change in ac bone and defect resolution . in the present study , a total of 30 sites of systemically healthy 19 patients ( 14 males and 5 females ) who referred to the department of periodontology and implantology of sardar patel postgraduate institute of dental and medical sciences lucknow with moderate to severe periodontitis having one or more periodontal osseous defects were treated during the period of 20092010 . the other inclusion criteria were ( i ) pd of 5 mm ( ii ) clinical attachment loss ( cal ) of 5 mm after phase one therapy of 46 weeks and ( iii ) 3 mm of radiographic periodontal osseous defect . each patient exhibited at least one site chosen randomly which should be either located interproximally or involving the furcation area : ( i ) medically compromised patient , ( ii ) lactating and pregnant women , ( iii ) smokers and ( iv ) teeth with hopeless prognosis were excluded from the study . the patient had completed basic periodontal therapy including scaling oral hygiene instruction and reinforcement , srp with both hand and ultrasonic instrumentation at the time of enrollment before 46 weeks of surgeries . endodontic treatment involving root canal treatment and crown rehabilitation was done in the required cases . patient who agreed to participate signed an informed consent . using randomized parallel method the defects were randomly assigned ( flip of the coin ) to the two treatment groups designated as group i and group ii . group i ( 16 sites ) was treated with bioresorbable gtr membranes ( periocol ) ( sterile collagen periodontal membrane - fish origin 25 mm 30 mm ) and bone graft in combination ( grabio glascera ) - containing 50% bioactive glass ( [ 17% silicon [ sio2 ] , 53% calcium [ cao ] , 30% phosphorous [ p2o5 ] ) and 50% synthetic hydroxyapatite ( ha ) with particle size of 0.150.50 mm ) . group ii - ( 14-sites ) was treated with bonegraft alone ( grabio glascera ) . soft tissue measurements include plaque index , ( le , 1967 ) sulcus bleeding index , ( muhlemman and son , 1971 ) , pd and clinical attachment level which were performed with university of north carolina probe ( unc-15 ) and hard tissue measurements ( bonefill , ac bone change and defect resolution ) were recorded in radiographs which were studied preoperatively and at 6 months postoperatively by single examiner . the maximum pd and clinical attachment were measured at four aspects of the tooth , that is , buccal , lingual , mesial , and distal . the radiographic measurement includes standardized reproducible intraoral periapical radiographs which were taken before surgery preoperatively ( baseline ) and 6 months postoperatively by the paralleling technique with rinn holder xcp system , dentsply [ dentsply ] hamm moor lane / addlestone / weybridge / surrey kt15 2se ( x - ray machine - sansin dig 7 with microprocessor controlled digital timer and calibrated by national physical laboratory with settings of 70 kvp , 10 ma ) . radiographic measurements were made utilizing a millimeter x - ray grid , which consisted of vertical and horizontal squares , with small squares measuring 1 mm . vertical linear distances between the cementoenamel junction ( cej ) and the most apical extension of the defect were obtained by counting the number of squares of 1 mm in a grid [ figure 1 ] . ( a ) measurement of preoperative , ( b ) six months postoperative radiograph with 1 mm grid , also showing landmarks cej , ac , and bd although the radiographic pairs were almost identical , still some amount of the vertical distortion between baseline radiographs and the 12-month radiograph was estimated . in order to get the exact values , the following measures were taken . in order to estimate this distortion , an anatomically nonvariable distance as the root length ( distance from cej to root apex was measured on both the radiographs and a correction factor was calculated as follows : in case where it was not possible to measure the root length , the crown length was assessed ( distance from incisal margin of the crown to the cej ) . the following anatomical landmarks of the intrabony defect that is , cej , ac , and bd were identified on the radiographs [ figure 1a ] based on the criteria set by bjorn et al . ac : the most coronal position of the alveolar bone crest of the intrabony defect when it touches the root surface of the adjacent tooth before treatment , thst is , the top of the crest . bd : the most apical extension of the intrabony destruction where the periodontal ligament space still retained its normal width before treatment , that is , the bottom of the defect . if the restoration was present , the apical margin of the restoration was used to replace the cej as a fixed reference point . the following linear measurements were performed with the help of grid mount : cej - bd ( cej to bottom of the defect)cej - ac ( cej to the most coronal extent of the interdental crest)ac - bd ( depth of intabony defect at baseline was obtained by subtracting above parameters [ ( cej - bd)(cej - ac ) ] ) . cej - bd ( cej to bottom of the defect ) cej - ac ( cej to the most coronal extent of the interdental crest ) ac - bd ( depth of intabony defect at baseline was obtained by subtracting above parameters [ ( cej - bd)(cej - ac ) ] ) . pre- and post - treatment radiograph pairs were analyzed in order to measure [ figures 1a , b and 2a c ] the following . ( a ) preoperative radiograph , ( b ) postoperative after 6 months with grid , ( c ) postoperative after 6 months without grid the radiographic bonefill after 6-month was calculated after applying the correction factor as follows : ( cej - bd [ baseline ] ) ( cej - bd [ 6-month ] correction factor ) = bonefill ( cej - ac [ baseline ] ) ( cej - ac [ 6-month ] correction factor ) = ac bone change if the results were negative , this meant that a process of bone resorption had occurred . the radiographic defect resolution was evaluated subtracting radiographic alveolar bone crest change from radiographic bonefill . defect resolution = bonefill alveolar bone crest change . after a preoperative radiographic assessment , prior to surgery , a presurgical rinse with chlorhexidine ( 0.2% ) ( clohex plus , 0.2% w / v chlorhexidine gluconate , dr reddys , hyderabad ) to maintain asepsis followed with local anesthesia 2% lignocaine containing adrenaline at a concentration of 1:80,000 was administered . intracrevicular incision along with vertical releasing incisions was performed if necessary for a better access . mucoperiosteal flaps were raised , granulation tissue was removed , roots were thoroughly scaled and planned , followed by conditioning with tetracycline . group i received combination of gtr ( periocol ) ( sterile collagen periodontal membrane - fish origin 25 30 mm 30 mm soaked in normal saline about half an hour prior to its placement ) and bone graft ( grabio glascera ) - containing 50% bioactive glass ( 17% silicon [ sio2 ] , 53% calcium [ cao ] , 30% phosphorous [ p2 o5 ] ) and 50% synthetic ha with particle size of 0.150.50 mm was used and mixed with normal saline 5 min prior to the placement in the defect . whereas group ii received bone graft alone . the defect was filled in both the groups with bone graft ( bioactive ceramic composite granule with particle size of 0.150.50 mm ) . the graft material was moistened in sterile saline for 5 min before placement into the defects , care was taken not to overfill the defect . following bone grafting , an aluminum foil template was trimmed and adapted over the entire defect so as to cover 23 mm of the surrounding alveolar bone , followed by a membrane of the same size and shape was trimmed and adapted over the entire defect so as to cover 23 mm of surrounding alveolar bone . sutures ( 50 vicryl resorbable ) , if needed , were placed to secure the membrane and to ensure stability of the graft material . finally , the mucoperiosteal flap was repositioned and sutured at the original level using 30 silk with an interrupted suture . the same surgical technique was used for group ii except the placement of the membrane , only bone graft was used . amoxicillin for 7 days ) and analgesic ( ibuprofen 400 mg t.d.s for 3 days ) . the postoperative care consisted of 0.2% chlorhexidine rinse twice daily for 4 weeks . for clinical and radiographic measurement purposes , the patient was recalled after 6 months [ figures 1a , b , and 2a c ] . soft tissue measurements include plaque index , ( le , 1967 ) sulcus bleeding index , ( muhlemman and son , 1971 ) , pd and clinical attachment level which were performed with university of north carolina probe ( unc-15 ) and hard tissue measurements ( bonefill , ac bone change and defect resolution ) were recorded in radiographs which were studied preoperatively and at 6 months postoperatively by single examiner . the maximum pd and clinical attachment were measured at four aspects of the tooth , that is , buccal , lingual , mesial , and distal . the radiographic measurement includes standardized reproducible intraoral periapical radiographs which were taken before surgery preoperatively ( baseline ) and 6 months postoperatively by the paralleling technique with rinn holder xcp system , dentsply [ dentsply ] hamm moor lane / addlestone / weybridge / surrey kt15 2se ( x - ray machine - sansin dig 7 with microprocessor controlled digital timer and calibrated by national physical laboratory with settings of 70 kvp , 10 ma ) . radiographic measurements were made utilizing a millimeter x - ray grid , which consisted of vertical and horizontal squares , with small squares measuring 1 mm . vertical linear distances between the cementoenamel junction ( cej ) and the most apical extension of the defect were obtained by counting the number of squares of 1 mm in a grid [ figure 1 ] . ( a ) measurement of preoperative , ( b ) six months postoperative radiograph with 1 mm grid , also showing landmarks cej , ac , and bd although the radiographic pairs were almost identical , still some amount of the vertical distortion between baseline radiographs and the 12-month radiograph was estimated . in order to get the exact values , the following measures were taken . in order to estimate this distortion , an anatomically nonvariable distance as the root length ( distance from cej to root apex was measured on both the radiographs and a correction factor was calculated as follows : in case where it was not possible to measure the root length , the crown length was assessed ( distance from incisal margin of the crown to the cej ) . the following anatomical landmarks of the intrabony defect that is , cej , ac , and bd were identified on the radiographs [ figure 1a ] based on the criteria set by bjorn et al . ac : the most coronal position of the alveolar bone crest of the intrabony defect when it touches the root surface of the adjacent tooth before treatment , thst is , the top of the crest . bd : the most apical extension of the intrabony destruction where the periodontal ligament space still retained its normal width before treatment , that is , the bottom of the defect . if the restoration was present , the apical margin of the restoration was used to replace the cej as a fixed reference point . the following linear measurements were performed with the help of grid mount : cej - bd ( cej to bottom of the defect)cej - ac ( cej to the most coronal extent of the interdental crest)ac - bd ( depth of intabony defect at baseline was obtained by subtracting above parameters [ ( cej - bd)(cej - ac ) ] ) . cej - bd ( cej to bottom of the defect ) cej - ac ( cej to the most coronal extent of the interdental crest ) ac - bd ( depth of intabony defect at baseline was obtained by subtracting above parameters [ ( cej - bd)(cej - ac ) ] ) . although the radiographic pairs were almost identical , still some amount of the vertical distortion between baseline radiographs and the 12-month radiograph was estimated . in order to get the exact values , the following measures were taken . in order to estimate this distortion , an anatomically nonvariable distance as the root length ( distance from cej to root apex was measured on both the radiographs and a correction factor was calculated as follows : in case where it was not possible to measure the root length , the crown length was assessed ( distance from incisal margin of the crown to the cej ) . the following anatomical landmarks of the intrabony defect that is , cej , ac , and bd were identified on the radiographs [ figure 1a ] based on the criteria set by bjorn et al . ac : the most coronal position of the alveolar bone crest of the intrabony defect when it touches the root surface of the adjacent tooth before treatment , thst is , the top of the crest . bd : the most apical extension of the intrabony destruction where the periodontal ligament space still retained its normal width before treatment , that is , the bottom of the defect . if the restoration was present , the apical margin of the restoration was used to replace the cej as a fixed reference point . the following linear measurements were performed with the help of grid mount : cej - bd ( cej to bottom of the defect)cej - ac ( cej to the most coronal extent of the interdental crest)ac - bd ( depth of intabony defect at baseline was obtained by subtracting above parameters [ ( cej - bd)(cej - ac ) ] ) . cej - bd ( cej to bottom of the defect ) cej - ac ( cej to the most coronal extent of the interdental crest ) ac - bd ( depth of intabony defect at baseline was obtained by subtracting above parameters [ ( cej - bd)(cej - ac ) ] ) . pre- and post - treatment radiograph pairs were analyzed in order to measure [ figures 1a , b and 2a c ] the following . ( a ) preoperative radiograph , ( b ) postoperative after 6 months with grid , ( c ) postoperative after 6 months without grid the radiographic bonefill after 6-month was calculated after applying the correction factor as follows : ( cej - bd [ baseline ] ) ( cej - bd [ 6-month ] correction factor ) = bonefill ( cej - ac [ baseline ] ) ( cej - ac [ 6-month ] correction factor ) = ac bone change if the results were negative , this meant that a process of bone resorption had occurred . the radiographic defect resolution was evaluated subtracting radiographic alveolar bone crest change from radiographic bonefill . the radiographic bonefill after 6-month was calculated after applying the correction factor as follows : ( cej - bd [ baseline ] ) ( cej - bd [ 6-month ] correction factor ) = bonefill ( cej - ac [ baseline ] ) ( cej - ac [ 6-month ] correction factor ) = ac bone change if the results were negative , this meant that a process of bone resorption had occurred . the radiographic defect resolution was evaluated subtracting radiographic alveolar bone crest change from radiographic bonefill . after a preoperative radiographic assessment , surgical therapy was planned . prior to surgery , a presurgical rinse with chlorhexidine ( 0.2% ) ( clohex plus , 0.2% w / v chlorhexidine gluconate , dr reddys , hyderabad ) to maintain asepsis followed with local anesthesia 2% lignocaine containing adrenaline at a concentration of 1:80,000 was administered . intracrevicular incision along with vertical releasing incisions was performed if necessary for a better access . mucoperiosteal flaps were raised , granulation tissue was removed , roots were thoroughly scaled and planned , followed by conditioning with tetracycline . group i received combination of gtr ( periocol ) ( sterile collagen periodontal membrane - fish origin 25 30 mm 30 mm soaked in normal saline about half an hour prior to its placement ) and bone graft ( grabio glascera ) - containing 50% bioactive glass ( 17% silicon [ sio2 ] , 53% calcium [ cao ] , 30% phosphorous [ p2 o5 ] ) and 50% synthetic ha with particle size of 0.150.50 mm was used and mixed with normal saline 5 min prior to the placement in the defect . whereas group ii received bone graft alone . the defect was filled in both the groups with bone graft ( bioactive ceramic composite granule with particle size of 0.150.50 mm ) . the graft material was moistened in sterile saline for 5 min before placement into the defects , care was taken not to overfill the defect . following bone grafting , an aluminum foil template was trimmed and adapted over the entire defect so as to cover 23 mm of the surrounding alveolar bone , followed by a membrane of the same size and shape was trimmed and adapted over the entire defect so as to cover 23 mm of surrounding alveolar bone . sutures ( 50 vicryl resorbable ) , if needed , were placed to secure the membrane and to ensure stability of the graft material . finally , the mucoperiosteal flap was repositioned and sutured at the original level using 30 silk with an interrupted suture . the same surgical technique was used for group ii except the placement of the membrane , only bone graft was used . amoxicillin for 7 days ) and analgesic ( ibuprofen 400 mg t.d.s for 3 days ) . the postoperative care consisted of 0.2% chlorhexidine rinse twice daily for 4 weeks . for clinical and radiographic measurement purposes , the patient was recalled after 6 months [ figures 1a , b , and 2a c ] . regeneration of lost structures has become the primary therapeutic goal in periodontics over the past several decades . in the present study , the material used was bioresorbable collagen membrane of fish origin . the purpose to use this was to avoid a second surgery . before its placement , a priority was given to synthetic graft that is , bioactive glass . among the various comparative clinical trials involving the use of synthetic graft and various other types of graft , better results were shown in favor of synthetic bone graft . though autogenous bone is still the gold standard in bone augmentation procedures but due to its low availability and donor site morbidity necessitates the development of alternative products for it and with allogenic graft there is a possibility of disease transfer , e.g. , with freeze - dried bone allografts . clinically and statistically , a significant improvement was seen pd , cal , bonefill , crestal bone loss , and defect resolution from baseline till an observation duration of 6 months individually in both the groups . the change in pd and cal for both the groups shows a statistically significant difference between preoperative and postoperative values , that is , both the treatment modalities are effective [ tables 1 and 2 ] [ graphs 1 and 2 ] but on comparing the net gain between group i and group ii , group i , that is , combination therapy shows slightly higher values than group ii , yet there was no statistically significant difference ( p = 0.790 ) between the two groups . the net gain in pd and cal for combination and bonegraft were 3.94 1.81 mm , 3.57 2.21 mm and 3.94 1.81 and 3.57 2.21 mm , respectively [ table 3 and graph 3 ] . a similar type of study done by prathap et al . , also shows slightly higher values in combination therapy than the bone graft alone and concluded that statistically no significant difference was found between both the treatment modalities . comparison of pre- and post - operative periodontal health status in group i comparison of pre- and post - operative periodontal health status in group ii comparison of pre- and post - operative periodontal health status in group i comparison of pre- and post - operative periodontal health status in group ii comparison of net pd change , net cal change , net bone fill , net alveolar bone crest change and net defect resolution in two study groups comparison of net probing depth change , net clinical attachment loss change , net bone fill , net alveolar bone crest change , and net defect resolution in two study groups the results of the study with respect to mean net bonefill for group i and group ii in terms of percentage were 33.23 15.58% and 40.26 19.14% [ table 4 and graph 4 ] , respectively . though the value was higher in group ii than in group i statistically , there was no significant difference found between the two groups . in accordance , the present study , some other similar type of clinical trials also shows no statistically significant difference between combination and bone graft alone type of therapy . tsao et al . , showed the vertical bone fill of 1.9 1.4 mm in mineralized cancellous bone allograft ( mba ) and 0.7 0.9 mm in combination , while keles et al . used autogenous cortical bone grafting ( acb ) and atrisorb as an absorbable polylactide membrane , and the values regarding the gain in radiographic alveolar bone height were 5.50 2.24 mm in the acb graft with gtr - treated group and 5.92 1.83 mm in the acb graft - treated group . both the studies show slightly higher values for the bonegraft group with no statistically significant difference between the two treatment modalities as in the case of the present study . these studies were in agreement with our study which records no additional benefit of gtr membrane together with bone graft alone . comparison of proportional change ( in percentage ) in net pd change , net cal change , net bone fill , net alveolar bone crest change and net defect resolution in two study groups comparison of proportional change ( in percentage ) in net probing depth change , net clinical attachment loss change , net bone fill , net alveolar bone crest change , and net defect resolution in two study groups however , there are certain studies which favor the combination therapy with respect to bonefill . , used autogenous bone graft + gtr and autogenous bonegraft alone and recorded probing bone level gain as 3.9 0.8 versus 1.3 0.9 mm , p = 0.034 , respectively . contrary to the above study designs , there are few studies which use only bonegraft , though of different types , without the use of any membrane and showed significant improvement in defect fill . meffert et al . used hydroxylapatite as an alloplastic material for a period of 9 months and showed a defect fill of 66.89% . yukna used hard tissue replacement polymer for a period of 6-month and showed 60.8% defect fill respectively when compared with control groups . grover et al . , used bioactive glass and showed a mean radiographic defect fill of 64.76% ( 2.49 0.5 mm ) after 6-month observation . all the above studies used synthetic bone graft and showed the positive results thus again favoring the use of synthetic bone grafts as a regenerative material . the net alveolar crestal bone change value was negative which showed that a crestal bone resorption had occurred in both the groups , [ tables 3 and 4 ] [ graphs 3 and 4 ] yet there was no statistically significant difference found between both the groups ( p > 0.05 ) . this was a significant finding as many of the synthetic bone graft reported bone fill still resulted in some amount of crestal bone loss . the net value was higher in group ii which showed that more crestal resorption occurred in group ii than in group i , but there was no statistically significant difference present between both the groups ( p > 0.05 ) tsao et al . showed the 1.3 1.1 mm crestal bone resorption in mba alone and 0.7 0.9 mm in combination with gtr . the study of luepke et al . showed that when combination therapy was used , that is , bioabsorbable alone and its combination with decalcified freeze - dried bone allograft ( dfdba ) , the crestal bone resorption seen was 0.10 1.11 mm and 0.27 1.15 mm . the result of the study was almost comparable to combination group as seen in the present study . in terms of defect resolution , the mean net defect resolution in group i and group ii [ table 3 and graph 3 ] in terms of percentage 62.86 26.49 and 80.58 24.09 , [ table 4 and graph 4 ] respectively , the above observation shows that the net value is higher in group ii than in group i yet there was no significant difference between both the groups . the defect resolution value was obtained after taking into consideration of the net crestal bone resorption and net bone fill . if the net crestal bone resorption would have not taken into consideration , then the values of defect resolution would have been different as compared to the actual value . thus , this error was rectified initially and after rectifying it only the net mean defect resolution was calculated . evaluated the combination of expanded polytetrafluoroethylene and dfdba versus dfdba alone but showed no statistically significant difference regarding defect resolution ( 81% vs. 64% ) thus supporting to our study . used a combination of deproteinized bovine mineral and a collagen membrane in the treatment of intrabony defects and compared with papilla preservation flap alone and showed the radiographic resolution of approximately 60% which was almost comparable to the present study with respect to combination therapy . meffert et al . , showed the radiographic resolution of 53.57 4.79% with hydroxylapatite as an alloplastic graft and 19.49 4.52% with the control group , that is , without graft . richardson et al . , compared the graft , that is , bio - oss ( wolhusen ) and dfdba and showed the defect resolution of 77.6% for bio - oss and 59.4% for dfdba . in the present study , the results were evaluated up to the end of 6-month following surgeries . had the time been permitted longer for the observation the results might have been better . the radiographic outcome after the two treatment modalities should be interpreted with caution as these grafts in the radiograph are hardly distinguishable from the host bone and the graft appearing on radiographs may not necessarily be incorporated in bone . this could introduce some bias in the radiographic examination process and overestimation of radiographic bone level in both the groups . the calculation of a correction factor assessing the level of distortion between preoperative and postoperative radiograph helped to minimize errors . the correction factor for both the groups was close to one which means that the projection of pre- and post - operative radiographs was similar in both the groups . all the clinical measurements were done using unc-15 probe . since in most of the studies the measurements such as bonefill , crestal resorption , and defect resolution were done with re - entry procedure which is traumatic to the patient and on other ethical issues . within the limitations of present study , it can be concluded that the combination of gtr + bone graft and bone graft alone resulted in a significant reduction in both the hard and soft tissue parameters when compared individually from baseline data to 6-month duration . on comparing both the groups together after 6 months of therapy , the results were equally effective with combination of graft + membrane versus bone graft alone since no significant difference was seen between any of the parameters between both the groups . thus , both the treatment modalities are comparable and effective . taking combination of membrane and bone graft into consideration , it can be concluded that using gtr membrane with bonegraft does not have any additional benefit as compared to bone graft alone .
freely available automated mr image analysis techniques are being increasingly used to investigate neuroanatomical abnormalities in patients with neurological disorders . it is important to assess the specificity and validity of automated measurements of structure volumes with respect to reliable manual methods that rely on human anatomical expertise . the thalamus is widely investigated in many neurological and neuropsychiatric disorders using mri , but thalamic volumes are notoriously difficult to quantify given the poor between - tissue contrast at the thalamic gray - white matter interface . in the present study we investigated the reliability of automatically determined thalamic volume measurements obtained using freesurfer software with respect to a manual stereological technique on 3d t1-weighted mr images obtained from a 3 t mr system . further to demonstrating impressive consistency between stereological and freesurfer volume estimates of the thalamus in healthy subjects and neurological patients , we demonstrate that the extent of agreeability between stereology and freesurfer is equal to the agreeability between two human anatomists estimating thalamic volume using stereological methods . using patients with juvenile myoclonic epilepsy as a model for thalamic atrophy , we also show that both automated and manual methods provide very similar ratios of thalamic volume loss in patients . this work promotes the use of freesurfer for reliable estimation of global volume in healthy and diseased thalami .	the thalamus is of central interest in many disorders of the nervous system ( andreasen 1997 ; dom , et al . 1976 ; lee and marsden 1994 ; meador - woodruff , et al . 2003 ; speedie and heilman 1983 ; williams 1965 ; xuereb , et al . 1991 ) . the functioning of the thalamus is crucial to many sensory , motor and cognitive systems , and therefore has also been subject to a great deal of investigation in the cognitive neurosciences ( basso , et al . 2005 ; engelborghs , et al . it is in these capacities that analysis of thalamic structure and function is a continually researched theme in neuroimaging investigations , particularly using magnetic resonance imaging ( mri ) . analysis of volume or shape using mri techniques may provide important information with respect to the involvement of the thalamus in neurological and neuropsychiatric disorders , including generalized ( du , et al . 2011 ; pulsipher , et al . 2009 ) and partial ( gong , et al . 2008 ; pulsipher , et al . 2007 ) epilepsy , schizophrenia ( adriano , et al . 2010 ) , huntington s disease ( douaud , et al . 2006 2008 ) , and alzheimer s disease ( de jong , et al . 2008 ) . reliable measurement of thalamic structure is , however , notoriously difficult to achieve , particularly given the typically poor between - tissue mr contrast of the thalamic nuclei and adjacent white matter ( amini , et al . it is therefore important to develop new and improve and validate existing methodologies that provide thalamic metrics . like for other subcortical brain structures , there are several approaches freely available to estimate thalamic volume from mr images . at either end of the mr image analysis spectrum , there are manual and automated approaches ; manual approaches are user - dependent , time consuming but are considered to be the gold standard of mr image analysis techniques ( bonilha , et al . 2004 ; collins and pruessner 2010 ; crum , et al . 2001 ; pruessner , et al . automated approaches remove the need of an expert anatomist , are dependent on computer algorithms , and are time efficient , but require a great deal of validation against manual methods to determine the specificity and validity of measurements ( chupin , et al . the primary goal of the present study was to evaluate the validity of thalamic volume measurements obtained from a frequently used automated approach with respect to a reputable manual approach widely used in the imaging , anatomical and histological sciences . the fully automated approach investigated in the present study was the subcortical segmentation and volume estimation techniques ( fischl , et al . 2002 ) incorporated into freesurfer software ( http://surfer.nmr.mgh.harvard.edu/ ) , which provide observer - independent volumes for individual subcortical nuclei from conventional mr images . similarly to other methods that automatically segment and estimate subcortical volume such as first ( patenaude , et al . 2011 ) incorporated into fsl software ( http://www.fmrib.ox.ac.uk/fsl/first/index.html ) , there has been a recent proliferation of studies using freesurfer methods for volumetric studies , some of which have included comparison with manual methods , most notably for the hippocampus ( cherbuin , et al . to our knowledge , there has been no independent comparison between manual methods and freesurfer methods for volume estimation of the thalamus . the manual approach used to evaluated freesurfer - based thalamic volumes in the present study was the cavalieri method of design - based stereology in conjunction with point counting ( gundersen and jensen 1987 ; gundersen , et al . 2000 ) , which is a 100 % investigator interactive technique that requires manual determination of sampling density for a given brain structure ( i.e. the stereological parameters necessary to produce a reliable volume estimate ) and investigator decisions on whether or not sampling probes ( i.e. points ) intersect the brain region - of - interest ( roi ) . stereology requires the use of a human anatomist with expert knowledge of anatomical boundaries that divide legitimate ( i.e. thalamic ) and illegitimate ( i.e. non - thalamic ) brain tissue . manual approaches such as stereology are considered gold standard because it is assumed that human knowledge and perception is superior to computer algorithms that determine regional brain boundaries . firstly , we compared thalamic volume estimates in a sample of neurologically and psychiatrically healthy subjects . secondly , we compared the methods in their sensitivity in detecting thalamic atrophy in patients with juvenile myoclonic epilepsy ( jme ) . jme is an electro - clinical syndrome that by definition is non - lesional and without abnormality on conventional magnetic resonance imaging ( mri ) ( berg , et al . 2010 ; ilae 1989 ) , but has previously been shown to be associated with thalamic structural alterations ( deppe , et al . 2009 ) , and is generally considered to be intimately associated with thalamic dysfunction ( holmes , et al . we studied a neurologically and psychiatrically healthy control group that was composed of 62 adult volunteers ( 32 females , mean age 27.9 4.3 sd , range 2143 ) , all of whom had normal neurological examination and normal mri ( t1- , t2-weighted , and flair ) . we also studied ten patients ( 6 females , mean age 28.6 8.8 sd , range 1942 ) with jme . 2011a ) . there was no statistical difference in age between patients and controls ( t = 0.38 , p = 0.70 ) . all participants underwent high resolution mri t1-weighted , t2-weighted and flair imaging at 3 t ( philips intera t30 , t / r head coil ) . all mri modalities were used to exclude the possibility of brain lesions in patients and controls . for volumetric analysis , we acquired t1-weighted structural mris using a high - resolution 3d turbo - field - echo sequence ( matrix 256 256 160 over a field of view of 25.6 25.6 16 cm reconstructed after zero filling to 512 512 320 cubic voxels with an edge length of 0.5 mm ) . prior to morphometric analyses , all mr images were intensity inhomogeneity corrected and resampled to isotropic voxels of 1 1 1 mm ( 256 256 160 slices ) using in - house software ( eval 3.0 ) . to confirm that there was no systematic difference in head size between patient and control groups , we automatically obtained relative brain size , vscale ( global tissue volumes normalized for head size ) and csf volume estimates from the 3d t1-weighted images of all subjects using the sienax protocol ( smith , et al . 2002 ) integrated into fsl software ( http://www.fmrib.ox.ac.uk/fsl/siena/index.html ) . there were no inter - group differences in relative brain size ( p = 0.40 ) , vscale ( p = 0.96 ) or csf volume ( p = 0.95).stereology the cavalieri method of design - based stereology in conjunction with point counting ( gundersen and jensen 1987 ; gundersen , et al . 2000 ) was used as an unbiased estimator of the volume of the left and right thalamus in all subjects . by using the cavalieri method , volume is directly estimated from equidistant and parallel mr images of the brain with a uniform random starting position . a second level of sampling is required to estimate the section area from each image by applying point counting within the roi . the mathematical justification and implementation of the methodology is simple and it can be applied to structures of arbitrary shape ( garcia - finana , et al . this technique has been frequently applied to reliably estimate brain volume and surface area on mr images ( acer , et al . 2008 ; mackay , et al . 1998 ; mackay , et al . 2000 ; ronan , et al . 2006 1996 ) , and more widely applied to study other aspects of anatomy with and without the use of mri . stereology has been shown to be at least as precise as tracing and thresholding volumetry techniques and substantially more time efficient , with validation relative to post - mortem measurements ( garcia - finana , et al . windows - compatible easymeasure software ( keller , et al . 2007 ; puddephat 1999 ) was used for point counting on mr images . given that the borders of different thalamic nuclei are almost indistinguishable on conventional mri , the thalamus was sampled as an entire complex , including the anterior thalamic nucleus , mediodorsal thalamic nucleus , lateral dorsal thalamic nucleus , ventral lateral thalamic nucleus , ventral postero - lateral / medial thalamic nuclei , and pulvinar ( duvernoy 1999 ) . the lateral and medial geniculate nuclei were not included in measurements , similar to previous studies ( natsume , et al . on axial sections , point counting began on the dorsal most section , where the area of the lateral dorsal thalamic nucleus was demarcated laterally by the corona radiata and medially by the lateral ventricles . as measurements progressed ventrally , the posterior limb of the internal capsule bordered the thalamus laterally . on ventral sections , care was taken to delineate the thalamic nuclei from the neighbouring pars medialis of the globus pallidus , and at the most ventral sections , to segregate the pulvinar and area of the ventral posterolateral nucleus from the emerging hypothalamus anteriorly , superior colliculus posteromedially , and the hippocampus posteriorly / posterolaterally . figure 1 shows point counting within the thalamic roi relative to the automated freesurfer approach described below . more information on the mri - based anatomy of the thalamus with respect to post - mortem sections can be found at http://www.psychiatry.uiowa.edu/mhcrc/pdf/papers/thalamus.pdf.fig . 1stereological and freesurfer methods used to estimate thalamic volume shown at approximately the same axial sections . both methods are shown for the same hemisphere ( the freesurfer axial sections are flipped to show maximal correspondence between techniques ) of the same randomly selected subject . the sagittal mr section illustrates the approximate levels of the axial sections shown . abbreviations : di , ventral diencephalon ( peach ) ; pa , pallidum ( violet ) ; put , putamen ( lilac ) ; th , thalamus ( dark green ) ; thp , pulvinar of the thalamus ( dark green ) stereological and freesurfer methods used to estimate thalamic volume shown at approximately the same axial sections . both methods are shown for the same hemisphere ( the freesurfer axial sections are flipped to show maximal correspondence between techniques ) of the same randomly selected subject . the sagittal mr section illustrates the approximate levels of the axial sections shown . abbreviations : di , ventral diencephalon ( peach ) ; pa , pallidum ( violet ) ; put , putamen ( lilac ) ; th , thalamus ( dark green ) ; thp , pulvinar of the thalamus ( dark green ) the sampling density ( i.e. size of points , distance between sections ) were optimized to achieve a coefficient of error ( ce ) of less that 5 % ( roberts , et al . 2000 ) , an approach that has been adopted in our stereological analysis of the hippocampus ( keller , et al . 2009a ) , planum temporale ( keller , et al . 2007 ; keller , et al . the ce is essentially a statistical estimate of how accurate the stereological volume estimation is for each structure . separation between test points on the square grid used for point counting was 0.312 cm ( i.e. , 4 pixels ) and slice interval was 1 mm ( singular axial mr sections ) . thalamic transect area was obtained by multiplying the total number of points recorded by the area corresponding to each test point . an estimate of thalamic volume was obtained as the sum of the estimated areas of the structure transects on consecutive systematic sections multiplied by the distance between sections . between approximately 400550 points were recorded on approximately 20 systematic random sections.freesurfer freesurfer software ( http://surfer.nmr.mgh.harvard.edu/ ) was used to obtain thalamic volumes for all subjects using an observer - independent approach , which could be contrasted with the manual stereological measurements of the thalamus . thalamic segmentations are based on the assignment of neuroanatomical labels to each voxel in an mr image based on the probabilistic information automatically estimated from a manually labelled training set . the methods of the automated volumetric approach have been described in detail previously ( fischl , et al . 2002 ) , and the accuracy of automated labelling and volumetry of subcortical structures have been independently validated with respect to gold standard manual volumetric techniques , predominantly for the hippocampus ( cherbuin , et al . , there has been no independent comparison of the automated thalamic volumetry offered by freesurfer and a manual volumetric method ( although thalamic tracings were compared with the performance of freesurfer in the original methods paper by fischl et al . figure 1 shows the comparison of automated labelling of the thalamus ( and extra - thalamic structures ) in an individual control subject using freesurfer relative to stereological volume estimation of the thalamus in the same subject . freesurfer analyses were performed on a mac pro ( version os x 10.6.6 , 32 gb , 2 2.93 ghz 6-core intel xeon ( ht ) ) , which permitted the freesurfer recon - all function ( for cortical reconstruction and brain segmentation ; http://surfer.nmr.mgh.harvard.edu/fswiki/recon-all ) to complete 23 participants in less than 20 h. after the recon - all function , the neuroanatomical labels were inspected for accuracy in all patients and controls . despite that freesurfer permits manual editing to improve subcortical segmentation , no obvious errors in the automatic labelling were observed for any subject , and so all data obtained from freesurfer analyses were 100 % automated and not influenced by manual intervention . two - way mixed intra - class correlation coefficients for absolute agreement ( shrout and fleiss 1979 ) were used to determine inter - rater agreement between two human raters using manual stereology and freesurfer for volume estimation of the thalamus in ten randomly selected controls using the statistics software spss ( version 18 , www.spss.com ) . intra - class correlations were subsequently performed between stereological volumes obtained by one human rater and freesurfer volumes for the entire sample of patients and controls ( n = 72 ) . univariate anovas were used to investigate patient - control differences in volumes , and corrected for multiple comparisons using statistica version 9.1 , ( stat soft . we studied a neurologically and psychiatrically healthy control group that was composed of 62 adult volunteers ( 32 females , mean age 27.9 4.3 sd , range 2143 ) , all of whom had normal neurological examination and normal mri ( t1- , t2-weighted , and flair ) . we also studied ten patients ( 6 females , mean age 28.6 8.8 sd , range 1942 ) with jme . 2011a ) . there was no statistical difference in age between patients and controls ( t = 0.38 , p = 0.70 ) . all participants underwent high resolution mri t1-weighted , t2-weighted and flair imaging at 3 t ( philips intera t30 , t / r head coil ) . all mri modalities were used to exclude the possibility of brain lesions in patients and controls . for volumetric analysis , we acquired t1-weighted structural mris using a high - resolution 3d turbo - field - echo sequence ( matrix 256 256 160 over a field of view of 25.6 25.6 16 cm reconstructed after zero filling to 512 512 320 cubic voxels with an edge length of 0.5 mm ) . prior to morphometric analyses , all mr images were intensity inhomogeneity corrected and resampled to isotropic voxels of 1 1 1 mm ( 256 256 160 slices ) using in - house software ( eval 3.0 ) . to confirm that there was no systematic difference in head size between patient and control groups , we automatically obtained relative brain size , vscale ( global tissue volumes normalized for head size ) and csf volume estimates from the 3d t1-weighted images of all subjects using the sienax protocol ( smith , et al . 2002 ) integrated into fsl software ( http://www.fmrib.ox.ac.uk/fsl/siena/index.html ) . there were no inter - group differences in relative brain size ( p = 0.40 ) , vscale ( p = 0.96 ) or csf volume ( p = 0.95).stereology the cavalieri method of design - based stereology in conjunction with point counting ( gundersen and jensen 1987 ; gundersen , et al . 2000 ) was used as an unbiased estimator of the volume of the left and right thalamus in all subjects . by using the cavalieri method , volume is directly estimated from equidistant and parallel mr images of the brain with a uniform random starting position . a second level of sampling is required to estimate the section area from each image by applying point counting within the roi . the mathematical justification and implementation of the methodology is simple and it can be applied to structures of arbitrary shape ( garcia - finana , et al . this technique has been frequently applied to reliably estimate brain volume and surface area on mr images ( acer , et al . 2011 ; howard , et al . 2003 ; jelsing , et al . 2005 ; keller , et al . 2008 ; mackay , et al . 1998 ; mackay , et al . 2000 ; ronan , et al . 2006 ; salmenpera , et al . 2005 ; sheline , et al . 1996 ) , and more widely applied to study other aspects of anatomy with and without the use of mri . stereology has been shown to be at least as precise as tracing and thresholding volumetry techniques and substantially more time efficient , with validation relative to post - mortem measurements ( garcia - finana , et al . 2003 ; garcia - finana , et al . 2009 ; keller and roberts 2009 ; keshavan , et al . windows - compatible easymeasure software ( keller , et al . 2007 ; puddephat 1999 ) was used for point counting on mr images . given that the borders of different thalamic nuclei are almost indistinguishable on conventional mri , the thalamus was sampled as an entire complex , including the anterior thalamic nucleus , mediodorsal thalamic nucleus , lateral dorsal thalamic nucleus , ventral lateral thalamic nucleus , ventral postero - lateral / medial thalamic nuclei , and pulvinar ( duvernoy 1999 ) . the lateral and medial geniculate nuclei were not included in measurements , similar to previous studies ( natsume , et al . point counting began on the dorsal most section , where the area of the lateral dorsal thalamic nucleus was demarcated laterally by the corona radiata and medially by the lateral ventricles . as measurements progressed ventrally , the posterior limb of the internal capsule bordered the thalamus laterally . on ventral sections , care was taken to delineate the thalamic nuclei from the neighbouring pars medialis of the globus pallidus , and at the most ventral sections , to segregate the pulvinar and area of the ventral posterolateral nucleus from the emerging hypothalamus anteriorly , superior colliculus posteromedially , and the hippocampus posteriorly / posterolaterally . figure 1 shows point counting within the thalamic roi relative to the automated freesurfer approach described below . more information on the mri - based anatomy of the thalamus with respect to post - mortem sections can be found at http://www.psychiatry.uiowa.edu/mhcrc/pdf/papers/thalamus.pdf.fig . 1stereological and freesurfer methods used to estimate thalamic volume shown at approximately the same axial sections . both methods are shown for the same hemisphere ( the freesurfer axial sections are flipped to show maximal correspondence between techniques ) of the same randomly selected subject . the sagittal mr section illustrates the approximate levels of the axial sections shown . abbreviations : di , ventral diencephalon ( peach ) ; pa , pallidum ( violet ) ; put , putamen ( lilac ) ; th , thalamus ( dark green ) ; thp , pulvinar of the thalamus ( dark green ) stereological and freesurfer methods used to estimate thalamic volume shown at approximately the same axial sections . both methods are shown for the same hemisphere ( the freesurfer axial sections are flipped to show maximal correspondence between techniques ) of the same randomly selected subject . the sagittal mr section illustrates the approximate levels of the axial sections shown . abbreviations : di , ventral diencephalon ( peach ) ; pa , pallidum ( violet ) ; put , putamen ( lilac ) ; th , thalamus ( dark green ) ; thp , pulvinar of the thalamus ( dark green ) the sampling density ( i.e. size of points , distance between sections ) were optimized to achieve a coefficient of error ( ce ) of less that 5 % ( roberts , et al . 2000 ) , an approach that has been adopted in our stereological analysis of the hippocampus ( keller , et al . 2002a 2009a ) , planum temporale ( keller , et al . 2007 ; keller , et al . ; keller , et al . 2009b ) and insula ( keller , et al . the ce is essentially a statistical estimate of how accurate the stereological volume estimation is for each structure . separation between test points on the square grid used for point counting was 0.312 cm ( i.e. , 4 pixels ) and slice interval was 1 mm ( singular axial mr sections ) . thalamic transect area was obtained by multiplying the total number of points recorded by the area corresponding to each test point . an estimate of thalamic volume was obtained as the sum of the estimated areas of the structure transects on consecutive systematic sections multiplied by the distance between sections . between approximately 400550 points were recorded on approximately 20 systematic random sections.freesurfer freesurfer software ( http://surfer.nmr.mgh.harvard.edu/ ) was used to obtain thalamic volumes for all subjects using an observer - independent approach , which could be contrasted with the manual stereological measurements of the thalamus . thalamic segmentations are based on the assignment of neuroanatomical labels to each voxel in an mr image based on the probabilistic information automatically estimated from a manually labelled training set . the methods of the automated volumetric approach have been described in detail previously ( fischl , et al . 2002 ) , and the accuracy of automated labelling and volumetry of subcortical structures have been independently validated with respect to gold standard manual volumetric techniques , predominantly for the hippocampus ( cherbuin , et al . to our knowledge , there has been no independent comparison of the automated thalamic volumetry offered by freesurfer and a manual volumetric method ( although thalamic tracings were compared with the performance of freesurfer in the original methods paper by fischl et al . figure 1 shows the comparison of automated labelling of the thalamus ( and extra - thalamic structures ) in an individual control subject using freesurfer relative to stereological volume estimation of the thalamus in the same subject . freesurfer analyses were performed on a mac pro ( version os x 10.6.6 , 32 gb , 2 2.93 ghz 6-core intel xeon ( ht ) ) , which permitted the freesurfer recon - all function ( for cortical reconstruction and brain segmentation ; http://surfer.nmr.mgh.harvard.edu/fswiki/recon-all ) to complete 23 participants in less than 20 h. after the recon - all function , the neuroanatomical labels were inspected for accuracy in all patients and controls . despite that freesurfer permits manual editing to improve subcortical segmentation , no obvious errors in the automatic labelling were observed for any subject , and so all data obtained from freesurfer analyses were 100 % automated and not influenced by manual intervention . two - way mixed intra - class correlation coefficients for absolute agreement ( shrout and fleiss 1979 ) were used to determine inter - rater agreement between two human raters using manual stereology and freesurfer for volume estimation of the thalamus in ten randomly selected controls using the statistics software spss ( version 18 , www.spss.com ) . intra - class correlations were subsequently performed between stereological volumes obtained by one human rater and freesurfer volumes for the entire sample of patients and controls ( n = 72 ) . univariate anovas were used to investigate patient - control differences in volumes , and corrected for multiple comparisons using statistica version 9.1 , ( stat soft . stereology vs freesurfer : consistency between volumetric measures stereology vs freesurfer : consistency between volumetric measures figure 2 shows the comparison of the three approaches ( rater one ( r1 ) for stereology , rater two ( r2 ) for stereology and freesurfer ) to estimate thalamic volume in the randomly selected ten subjects . this inter - rater / between - technique analysis indicates consistency across measures , and most notably , that freesurfer performed at least as consistent as r2 relative to r1 . mean ( sd ) left and right thalamic volume was 7339.6 mm ( 567.3 ) and 7339.2 mm ( 489.3 ) for r1 , 7456.3 mm ( 597.3 ) and 7444.3 mm ( 590.6 ) for r2 , and 7365.0 mm ( 641.3 ) and 7317.6 mm ( 610.0 ) for freesurfer , respectively . measurements between r1 and r2 , and between manual raters and freesurfer , achieve high intra - class correlations ( all > 0.9).fig . 2inter - rater consistency in stereological volume estimation of the left ( blue ) and right ( red ) thalamus , and relation to volume estimates obtained from freesurfer . stereological volume estimates are reproducible , and freesurfer is entirely consistent with the volumes obtained with both rater one and rater two . error bars indicate the 95 % confidence intervalstable 1inter - rater intra - class coefficients for volumetric measures rater 1rater 2fsleft thalamus rater 10.9770.969 rater 20.9770.925 fs0.9690.925 right thalamus rater 10.9520.923 rater 20.9520.916 fs0.9230.916 inter - rater consistency in stereological volume estimation of the left ( blue ) and right ( red ) thalamus , and relation to volume estimates obtained from freesurfer . stereological volume estimates are reproducible , and freesurfer is entirely consistent with the volumes obtained with both rater one and rater two . error bars indicate the 95 % confidence intervals inter - rater intra - class coefficients for volumetric measures figure 3 presents the relationship between stereological ( r1 ) and freesurfer estimates of left and right thalamus volume across the entire sample of 72 subjects investigated in the present study . intra - class correlations revealed a slightly reduced level of consistency between human and freesurfer analysis of the entire sample relative to the sub - sample of ten subjects ( left thalamus = 0.812 , right thalamus = 0.881 ) . mean ( sd ) left and right thalamic volume was 7422.4 mm ( 824.3 ) and 7390.1 mm ( 805.8 ) for stereology and 7343.6 mm ( 824.3 ) and 7388.3 mm ( 844.0 ) for freesurfer . there was no difference between the volume of the left and right thalamus across all 72 subjects using stereology ( f = 0.09 , p = 0.82 ) or freesurfer ( f = 0.10 , p = 0.75 ) ( and no differences when patients and controls were separated , p > 0.80 ) . although there were occasional differences in the direction of inter - hemispheric thalamic volume asymmetry in individual cases between stereology and freesurfer ( fig . 3 , right panel ) , this did not represent a statistically significant group effect ( f = 1.46 , p = 0.23).fig . 3relationship between stereological and freesurfer volume estimates of the left a and right b thalamus in the whole study sample . the relationship between left - right asymmetries determined for each individual participant by each technique is shown in c. although stereology and freesurfer determined left - right asymmetries of the thalamus in the same direction for the vast majority of subjects ( lower left and upper right quandrants ) , there were some disassociations ( top left quadrant)stereology vs freesurfer : identification of thalamic atrophy in jme relationship between stereological and freesurfer volume estimates of the left a and right b thalamus in the whole study sample . the relationship between left - right asymmetries determined for each individual participant by each technique is shown in c. although stereology and freesurfer determined left - right asymmetries of the thalamus in the same direction for the vast majority of subjects ( lower left and upper right quandrants ) , there were some disassociations ( top left quadrant ) stereology vs freesurfer : identification of thalamic atrophy in jme both techniques were equally sensitive in detecting bilateral thalamic volume atrophy in patients with jme relative to controls ( fig . 4 ) . using stereology , mean ( sd ) left and right thalamic volume was 6843.2 mm ( 746.6 ) and 6763.3 mm ( 824.0 ) in patients with jme , and 7507.8 mm ( 805.6 ) and 7482.6 mm ( 767.2 ) in controls , respectively . volume reduction in patients was found to be statistically significant for the left ( f(1,70 ) = 5.43 , p = 0.02 ) and right ( f(1,70 ) = 6.77 , p = 0.01 ) thalamus compared to controls . using freesurfer , mean ( sd ) left and right thalamic volume was 6803.4 mm ( 732.8 ) and 6750.0 mm ( 714.3 ) in patients with jme , and 7430.7 mm ( 809.9 ) and 7491.3 mm ( 822.3 ) in controls , respectively . freesurfer thalamic volumes were similarly smaller in patients relative to controls in the left ( f(1,70 ) = 4.76 , p = 0.03 ) and right ( f(1,70 ) = 7.36 , p = 0.008 ) hemispheres.fig . 4volume reduction of the left ( blue circles ) and right ( red squares ) thalamus in patients with jme relative to healthy controls using stereology a and freesurfer b. error bars indicate the 95 % confidence intervals volume reduction of the left ( blue circles ) and right ( red squares ) thalamus in patients with jme relative to healthy controls using stereology a and freesurfer b. error bars indicate the 95 % confidence intervals the volume of the thalamus is a notoriously difficult metric to estimate reliably given the low contrast between thalamic gray matter and adjacent white matter on t1-weighted mr images , which is a particular challenge for automated mr image analysis methods ( amini , et al . only by comparing such automated methods with manual investigator - intensive methods can we establish the reliability of volume estimates . the present study provides important data indicating the specificity and validity of automated thalamic volume estimation using freesurfer software . in particular , further to demonstrating consistency between stereological and freesurfer volume estimates of the thalamus in healthy subjects and neurological patients , we demonstrate that the extent of agreeability between stereology and freesurfer is equal to the agreeability between two human anatomists estimating thalamic volume using stereological methods . freesurfer software is now a frequently used tool for the estimation of subcortical structure volume . at the time of writing , a pubmed search using freesurfer and volume yields 87 articles ( october 2011 ) . the vast majority of these articles are application studies , particularly in neurological disorders , and only a few have sought to evaluate the validity of volume measurements . various levels of consistency between freesurfer and manual roi methods have been reported for the hippocampus ( cherbuin , et al . ; morey , et al . 2009 ) and striatum ( dewey , et al . dewey et al . ( 2010 ) performed a series of comparisons between the fully automated techniques of freesurfer and individual brain atlases using statistical parametric mapping ( ibaspm ) with auto - assisted manual tracings of the hippocampus , amygdala , putamen and caudate . the authors report that freesurfer segmentations exhibited significantly higher mean spatial overlap with auto - assisted tracings in all structures compared to ibaspm using dice coefficients . we were not in a position to perform spatial overlap analyses of the thalamus given that stereology and freesurfer are two inherently distinct mr image analysis approaches . however , this is one of the primary strengths of the data presented here , insomuch that a reliable volume estimate obtained using a gold - standard ( non - voxel labelling ) manual approach on mr images without automated spatial transformations ( i.e. in native space ) is comparable to a fully automated approach that requires spatial transformations in order to label an roi and obtain a volume . our interest was with respect to the reliability of the volume estimate of the thalamus . to our knowledge , the present study is the first to independently provide data validating the application of freesurfer to obtain automated volumes of the left and right thalamus . based on the congruence between the data obtained from freesurfer and manual stereology the latter of which is considered to represent the gold standard approach due to the requirement of an expert anatomist we recommend the use of freesurfer software for accurate volumetric quantification of the thalamus using high - resolution t1-weighted mri . the removal of an expert anatomist for volumetric analyses is cost effective and time efficient , particularly in large - scale volumetric studies . importantly , we demonstrate that the automated technique is as sensitive in detecting pathological alterations of the thalamus relative to stereology , which promotes the use of freesurfer in neurological contexts . there are two additional issues that should be highlighted . the thalamus is composed of lamellae that segregate multiple nuclei with distinct connections and functions , which are likely to be differentially affected in various neurological and neuropsychiatric disorders . for example , in disorders where the thalamus is implicated in patients also exhibiting deficits in frontal lobe functioning such as in jme ( pulsipher , et al . 2009)it would be expected that anterior thalamic nuclei that project to the frontal lobe would be preferentially affected ( deppe , et al . in such circumstances it will be interesting to investigate structural alterations of differential thalamic subregions , which are measures that the techniques applied in the present study can not provide . there are other techniques that may provide the basis for quantitative measurements of thalamic subregions based on dti and quantitative t1 and t2 imaging ( behrens , et al . secondly , the global estimates of thalamic volume using freesurfer in the present study was obtained on a philips intera t30 3 t mri system , requiring no additional manual edits for ( obvious ) incorrect labelling of the thalamic roi after the application of our in - house image inhomogeneity and resampling algorithm . different mri systems and head coils may have different image contrast characteristics that can potentially affect the performance of automated mr image analysis techniques . however , reproducibility of freesurfer estimated thalamic volume from serially acquired mr images on the same mr system is high ( jovicich , et al . 2010 ) , and mr system manufacturer has been shown to have little effect on volume estimates ( jovicich , et al . 2009 ) . in summary , this study provides convincing evidence for the reliability of global thalamic measurements using freesurfer in healthy and damaged thalami . the use of this software is cost effective and particularly advantageous in large - scale cross - sectional studies and longitudinal investigations in neurological settings . freesurfer software is publicly and freely available from the freesurferwiki resource ( http://surfer.nmr.mgh.harvard.edu/fswiki/freesurferwiki ) , which is developed and maintained at the martinos center for biomedical imaging ( http://www.nmr.mgh.harvard.edu/martinos/noflashhome.php ) . easymeasure software for volume estimation using stereology is freely available from the authors of this manuscript upon request .
case summarylumbosacral agenesis is a rare congenital condition reported in children . we report a 17-week - old female domestic shorthair cat with lumbosacral agenesis on whole - body radiographs . the cat was euthanized shortly thereafter presentation . a necropsy was not permitted.relevance and novel informationthis is the first reported feline case of lumbosacral agenesis .	we report a 17-week - old female domestic shorthair cat with lumbosacral agenesis on whole - body radiographs . lumbosacral agenesis ( lsa ) is a rare congenital condition found in people , where segments of the lumbar spine are missing with total or partial absence of the sacrum . the exact etiology is unknown , although several theories exist , including a potential association with maternal insulin - dependent diabetes , inherited genetic mutations and failure of certain mechanisms of embryonic differentiation of parts of the vertebral column . the purpose of this case report is to describe a clinical feline case of lsa . a 17-week - old female intact domestic shorthair cat presented on emergency for acute onset of lethargy and open - mouth breathing . the owner had adopted the cat at approximately 2 weeks of age from the local shelter . the cat was paralyzed in the hind end with severe muscle wasting in both hindlimbs and ankylotic joints . the owner reported that other than hindlimb paralysis the cat was bright , alert and responsive , and appeared otherwise normal . the cat ambulated quite well on the forelimbs , urinated and defecated regularly , and always ate well . approximately 2 days prior to presentation , the cat became lethargic and lost its appetite . the cat was taken to its regular veterinarian , who identified a left - sided inguinal hernia and the cat was given 20 ml / kg of a balanced crystalloid solution subcutaneously ( sc ) , maropitant 1 mg / kg sc , a warm soapy enema and was scheduled for surgical repair of the hernia . the cat declined rapidly at home and presented on emergency for lethargy and open - mouth breathing . physical examination on presentation revealed dull mentation , open - mouth breathing and cyanotic mucous membranes . the cat s heart rate was 200 beats per minute , with no identifiable heart murmur and the lungs were clear . the hindlimbs were noted to be ankylotic and have severe muscle wastage ; they were crossed behind the body with no attempts at ambulation . the visible external genitalia appeared normal , but there was poor anal tone and no movement of the tail . there was visible malformation of ribs 12 and 13 , as well as of the first and second lumbar vertebrae . there was a large colon distended with feces with a left inguinal hernia and possible bowel entrapment . shortly after radiographs were performed the cat became agonal and had an episode of respiratory arrest . there is visible malformation of ribs 12 and 13 , as well as of the first and second lumbar vertebrae . there is no evidence of any other lumbar or sacral vertebrae as indicated by the arrows . there is a large colon distended with feces with a left inguinal hernia and possible bowel entrapment . lsa is a rare malformation occurring in approximately 1 of 25,000 live births in people . it is part of a group of disorders characterized by the absence of different portions of the caudal spine , otherwise known as caudal regression syndrome . newborn children exhibit similar physical examination findings to those found in this case , with severe hindlimb muscle atrophy most common . concurrent malformations in children include hydrocephalus , myelomeningocele , kidney malformations , inguinal hernia , atresi ani , rectovaginal fistulas and congenital heart defects . concurrent orthopedic abnormalities include cervical vertebral fusion particularly at c2c3 , malformed ribs , hemivertebrae and kyphosis . the cat in the case reported here had a left inguinal hernia with possible bowel entrapment , no visible kidneys radiographically , and malformed ribs 12 and 13 . to the owner , the cat appeared to be neurologically normal in the cranial body but an occult neurological defect can not be completely ruled out . other intra - abdominal abnormalities may have been present but , owing to the absence of a necropsy , not identified . type ii involves partial sacral agenesis with a partial but bilaterally symmetrical defect and a stable articulation between the ilia and a normal or hypoplastic first sacral vertebra . type iii is variable lumbar and total sacral agenesis with the ilia articulating with the sides of the lowest vertebra . finally , type iv is variable lumbar and a total sacral agenesis . considering this human classification as there is no veterinary classification available , the cat presented in this report would be classified as type iv . treatment in children involves either amputation of the lower extremities and long - term prothesis or spinal pelvic fusion . many children will still have some proprioception in their lower extremities so correction of limb deformities is also pursued in some . although lsa has never been reported in any veterinary species , the malformed cat shared several important lesions with perosomus elumbis ( pe ) , which has been documented in various species . in animals with pe the hindlimbs have severe muscle wastage and the joints are ankylotic owing to the absence of nervous innervation from the absence of the coccygeal , sacral and portions of the lumbar vertebrae . this is in contrast to lsa , where patients only have agenesis of the lumbar and sacral portions of the spine . abdominal abnormalities identified in both pe and lsa include atresia ani , cryptorchidism , renal abnormalities and hernias . however , pe can lead to altered pelvic bone structure , with the pubic bone sometimes being absent , leading to pelvic narrowing ; this does not occur in people with lsa . the long - term prognosis of caudal spinal malformations is usually poor as most animals are stillborn or die shortly after birth . the cat reported herein lived longer than similar reported cases in the veterinary literature ; the previous reported longest survival was 12 days in a holstein calf with pe . it is unclear what caused the sudden demise of the cat but , as in pe , it appears the identification of lsa in a cat warrants a poor prognosis . the clinical presentation in this cat is similar to pe reported in other species and , as in other species , the prognosis for caudal spinal vertebral malformations appears poor .
the ability of the cellular prion protein ( prpc ) to bind copper in vivo points to a physiological role for prpc in copper transport . six copper binding sites have been identified in the nonstructured n - terminal region of human prpc . among these sites , the his111 site is unique in that it contains a mkhm motif that would confer interesting cui and cuii binding properties . we have evaluated cui coordination to the prp(106115 ) fragment of the human prp protein , using nmr and x - ray absorption spectroscopies and electronic structure calculations . we find that met109 and met112 play an important role in anchoring this metal ion . cui coordination to his111 is ph - dependent : at ph > 8 , 2n1o1s species are formed with one met ligand ; in the range of ph 58 , both methionine ( met ) residues bind to cui , forming a 1n1o2s species , where n is from his111 and o is from a backbone carbonyl or a water molecule ; at ph < 5 , only the two met residues remain coordinated . thus , even upon drastic changes in the chemical environment , such as those occurring during endocytosis of prpc ( decreased ph and a reducing potential ) , the two met residues in the mkhm motif enable prpc to maintain the bound cui ions , consistent with a copper transport function for this protein . we also find that the physiologically relevant cui-1n1o2s species activates dioxygen via an inner - sphere mechanism , likely involving the formation of a copper(ii ) superoxide complex . in this process , the met residues are partially oxidized to sulfoxide ; this ability to scavenge superoxide may play a role in the proposed antioxidant properties of prpc . this study provides further insight into the cui coordination properties of his111 in human prpc and the molecular mechanism of oxygen activation by this site .	transmissible spongiform encephalopathies are a group of neurodegenerative disorders that include scrapie in sheep , bovine spongiform encephalopathy , chronic wasting disease in elk and deer , and creutzfeldt jakob disease in humans . the infectious agent is an abnormally folded isoform of cellular prion protein ( prp ) , designated as prp . this misfolded protein is rich in -sheet structure , insoluble , and protease - resistant and possesses a tendency to polymerize into amyloid aggregates . these fibrils accumulate as plaques in the nervous system and have been associated with the induction of neuronal death . prp is a cell surface glycoprotein expressed throughout the body but mainly found in the central nervous system of all mammals and avian species . the n - terminal region of prp is largely unstructured , while the c - terminal region has a globular structure with three -helices and two short -sheets . the physiological role of prp has not been determined ; however , it has been proposed to participate in the allosteric function , signal transduction , cell the ability of prp to bind cu in vivo has led to the proposals that it may play a role in copper sensing , buffering , and/or transport . furthermore , cellular studies show that copper or zinc binding to prp induces its endocytosis . it has been established that prp can bind up to six cu ions in its flexible n - terminal region . cu coordination to the n - terminal region of prp as well as related synthetic peptides have been extensively studied . because these copper binding sites are unstructured , the study of peptide fragments has been a successful approach to studying copper binding to prp . in the octarepeat region ( or ) , a domain comprised of four tandem repeats of eight amino acids with the sequence phgggwgq , cu sites are populated in response to the ph and cu concentration . outside the or region , two sites have been identified ; these are associated with his96 and his111 . the his96 cu site can adopt a 3n1o or 4n equatorial coordination mode , with a pka value of 7.8 for conversion between these species . regarding cu binding to his111 , several proposals of coordination modes have been suggested . in our previous study , cu - prp(106115 ) complexes were characterized using different spectroscopic techniques [ absorption , circular dichroism ( cd ) , and electron paramagnetic resonance ( epr ) ] in combination with electronic structure calculations . this study shows a ph - dependent cu - prp(106115 ) coordination mode with a pka value of 7.5 ( structures given in scheme 1 ) . electrochemical measurements of copper bound to the or region have concluded that the low - occupancy copper binding mode [ e1/2(cu - or / cu - or ) = 0.323 v vs nhe ] can not reduce dioxygen to hydrogen peroxide [ e1/2(o2/h2o2 ) = 0.296 v vs nhe ] , while the high - occupancy copper binding mode [ e1/2(cu4-or / cu4-or ) = 0.172 v vs nhe ] can . with respect to the n - terminal his111 site , spin - trapping and nbt / formazan experiments with the prp(106126 ) and prp(106114 ) fragments suggested that these peptides are also capable of hydrogen peroxide or superoxide production in the presence of cu , ascorbic acid , and dioxygen . while the nature of the cu coordination properties of the n - terminal region of the prp protein has been widely explored , studies of its cu binding properties have been limited . unlike the other sites , the his111 binding site contains two adjacent methionine ( met ) residues , which could provide good ligands for cu and promote interesting redox behavior for this site . in fact , extended x - ray absorption fine structure ( exafs ) studies have demonstrated cu s interactions in the cu complexes of prp(106114 ) and prp(91126 ) fragments at ph 7.4 . however , at this ph , the exafs spectra would have contributions from different protonation states of the complex ( vide infra ) , obscuring the coordination mode assignments . in this study , we used different spectroscopic techniques ( x - ray absorption spectroscopy and nmr ) in combination with electronic structure calculations to elucidate the coordination modes involved in cu binding to his111 in the prp(106115 ) fragment . a detailed ph study of the cu binding properties of this fragment has allowed us to identify species that would be physiologically relevant . we have also evaluated the oxygen reactivity of these cu species , identifying specific sites for copper - catalyzed oxidation . the roles of met109 and met112 in cu and cu coordination , as well as oxygen activation have been evaluated . peptides ktnmkhmaga [ prp(106115 ) ] , ktnakhmaga [ prp(106115)m109a ] , ktnmkhaaga [ prp(106115)m112a ] , and ktnakhaaga [ prp(106115)m109&m112a ] were prepared by solid - phase fluorenylmethoxycarbonyl ( fmoc ) methods , using a fmoc - rink amide mbha resin , as previously described . all peptides were acetylated at the amino terminus and amidated in the carboxyl terminal . peptides were purified by semipreparative reversed - phase high - performance liquid chromatography ( hplc ) . the final purity was determined by analytical hplc and was found to be > 95% . copper(ii ) peptide complexes were prepared either at a 2 mm peptide concentration with 0.5 equiv of cu ( at ph 6.5 ) or 1 mm peptide with 0.8 equiv of cu at ph 8.5 , in a mixture of 20 mm 2-(n - morpholino)ethanesulfonic acid ( mes ) buffer with 20 mm n - ethylmorpholine ( nem ) and 50% glycerol ; the ph was adjusted by adding small volumes of naoh or hcl . the nature of the copper(ii ) peptide complexes was probed by electron paramagnetic resonance ( epr ) spectroscopy , yielding g and a values that are consistent with those previously reported . the copper(i ) peptide complexes were obtained upon reduction of the copper(ii ) peptide complexes with 100 equiv of ascorbic acid ( adjusted to the corresponding ph ) , under anaerobic conditions . the reduced complexes were characterized by epr , and in all cases , they were found to contain less than 3% of the residual cu species ( figure s1 in the supporting information , si ) . the cu k - edge xas data were collected at the ssrl on the unfocused 20-pole , 2.0-t wiggler beamline 7 - 3 under storage ring parameters of 3 gev and 300350 or 500 ma . a rhodium - coated premonochromator flat bent mirror was used for harmonic rejection and vertical collimation . the samples were loaded into 2 mm lucite xas cells with 38 m kapton windows and maintained at a constant temperature of 10 k during data collection using an oxford instruments cf 1208 continuous - flow liquid - helium cryostat . a canberra ge 30-element solid - state array detector was used to collect cu k fluorescence signals , using a soller slit and a zinc filter inserted between the sample and detector . internal energy calibration was accomplished by simultaneous measurement of the absorption of a copper foil placed between two ionization chambers located after the sample . the first inflection point of the foil spectrum was assigned to 8980.3 ev . extended x - ray absorption fine structure ( exafs ) data are reported to k = 12.8 in order to avoid interference from the zn k - edge . no photodamage was observed to cu samples , and thus all of the scans were used in the final average . each data set includes an average of 727 scans . data from cu samples were collected on four physically separate spots on the sample cells with 24 scans / spot to minimize the effect of photoreduction . the energy - calibrated and averaged data were processed by fitting a second - order polynomial to the preedge region and subtracting this from the entire spectrum as a background . a three - region polynomial spline of orders 2 , 3 , and 3 was used to model the smoothly decaying postedge region . the data were normalized by scaling the spline function to an edge jump of 1.0 at 9000 ev . the least - squares fitting program opt in exafspak(41 ) was used to fit the data . initial ab initio theoretical phase and amplitude functions were generated in feff 7.0(42 ) using calculated structures of cu models as the starting structures . atomic coordinates were adjusted as necessary as fits were improved . during the fitting process , the bond distance ( r ) and the mean - square deviation or bond variance in r arising from thermal and static disorder ( ) were varied for all components . the threshold energy ( e0 ) was also allowed to vary for each fit but was constrained to the same value for all components in a given fit . coordination numbers ( n ) were systematically varied to provide the best chemically viable agreement to the exafs data and their fourier transform ( ft ) but were fixed within a given fit . the fits were evaluated based on a comparison of the normalized error ( fn ) of each fit along with inspection of individual fits to the data and the agreement of the fts and of individual wave components . cu - prp(106115 ) complexes were prepared as described above but at a lower peptide concentration ( 0.3 mm ) and without glycerol , and the ph was varied from 3.4 to 9.2 . h total correlated spectroscopy ( tocsy ) , and h c heteronuclear single quantum coherence ( hsqc ) experiments were performed at ph 6.5 and 8.5 . h tocsy and h c hsqc cross peaks affected by cu ( 0.8 equiv ) were identified by comparing their chemical shift values in the absence and presence of the metal ion . all spectra were acquired at 288 k in nmr tubes sealed under a n2 atmosphere , using a bruker avance ii 600 mhz spectrometer with a triple - resonance probe equipped with z - axis self - shielded gradient coils . acquisition , processing , and visualization of the nmr spectra were performed using topspin 2.1 ( bruker ) and sparky . in all calculations , the structure of the prp(106113 ) peptide with sequence ktnmkhma was used , with an acetylated n - terminus and amidated c - terminus . the structures had a total of 141161 atoms , depending on the protonation state of the backbone amides and the number of explicit water molecules included in each model . each initial copper(i ) peptide complex model was built in gaussview 4.1.2 , starting from the previously reported cu models . using a restricted kohn sham ( rks ) approach , all copper(i ) peptide structures with a spin multiplicity of 1 ( singlet ) were fully optimized without geometry constraints , and the stationary points of selected cu structures were characterized by a harmonic analysis ( table s1 in the si ) . all geometry optimizations were performed using the demon2k code with the functional opbe ( which combines handy and cohen s optx exchange functional with the pbe correlation functional ) . it should be noted that calculations at the lda level and with the nonempirical pbe functional were also performed for all models ; however , only the results obtained with opbe are presented here because this functional provides the best description and distances for cu s bonds . the orbital and auxiliary basis sets used were tzvp and gen - a2 , respectively . solvent effects on selected optimized structures were evaluated in the demon2k code , including six explicit water molecules in the gas phase and reoptimizing with opbe and tzvp ; in these cases , after reoptimization , the water molecules remained outside the coordination sphere ( figure s2 in the si ) . implicit solvation of selected models was also calculated using the model cosmo , as implemented in the orca(52 ) program . because explicit solvation with six water molecules provides a geometric description very similar to that of implicit solvation ( table s2 in the si ) , herewith we report the results corresponding to explicit solvation with six water molecules for all of the structures . the inner - sphere reorganization energy for the electron - transfer step was computed using the following expression , which depends on the energies of the oxidized ( ox ) and reduced ( red ) geometries at the selected structures that best reproduce the copper complex at ph 6.5 and 8.5:where eox(redgeom ) is the energy of the oxidized state in the reduced structure and ered(oxgeom ) is the energy of the reduced state in the oxidized structure . kinetic studies of the reduction of the copper(ii ) peptide complexes by ascorbate were carried out on a sx20 stopped - flow system operated by the pro - data software ( applied photophysics ) with a 150 w xenon light source and equipped with a photodiode array for multiwavelength analysis . all experiments were performed in a single - mixing mode of the instrument , with a 1:1 ( v / v ) mixing ratio in a 1 cm optical path . the temperature was maintained at 25.0 0.1 c using a water bath and monitored via the internal sensor of the mixing unit . cu - prp(106115 ) complexes were degassed in a vacuum line and loaded into the driving syringes anaerobically . all flow lines of the instrument were extensively washed with degassed sodium dithionite and 20 mm nem / mes buffer , before charging the driving syringes with reactant solutions . the final concentration of the copper(ii ) peptide complexes in the cell was 0.15 mm . the reduction was followed under pseudo - first - order conditions with a 20-fold excess of reductant , and the loss of absorbance was monitored at 570 nm ( ph 8.5 ) or 600 nm ( ph 6.5 ) as a function of time . the reported rate constants represent the average values and standard deviations of three independent runs . kinetic studies of the reoxidation of the copper(i ) peptide complexes by oxygen were carried out on an agilent 8453 diode - array spectrometer . all experiments were performed in a screw - cap quartz cuvette with a 1 cm optical path . the cu - prp(106115 ) complexes at ph 6.5 were prepared by adding 1 equiv of ascorbate to a degassed solution of the copper(ii ) peptide complex ( 0.8 mm peptide with 0.5 equiv of copper ) in 20 mm nem / mes ; full reduction under these conditions was confirmed by uv vis absorption . reoxidation of the cu complexes was followed by the appearance of a characteristic d d band at 600 nm after the addition of an air - saturated buffer ( 0.5 equiv of dioxygen ) . the reported rate constants represent average values and standard deviations of three independent runs . the cu - prp(106115 ) complexes have been spectroscopically characterized previously . the coordination mode of the cu - prp(106115 ) complex is ph - dependent with a pka of 7.5 . at low ph , the equatorial coordination is the 3n1o mode , while at high ph , a 4n coordination mode is stabilized with a third deprotonated amide of the backbone ( scheme 1 ) . here , we use xas to evaluate these models and the role of met109 and met112 in copper coordination . the cu k - edge spectra of the cu - prp(106115 ) complexes at ph 6.5 and 8.5 are presented in figure 1 . the extremely weak signal around 8979 ev is the 1s 3d transition , associated with the cu d ion . figure 1 shows that the spectra are not affected by the replacement of met109 and met112 by ala . thus , at both ph values , the thioether groups of the met residues are not involved in the coordination of cu . exafs fits for the data shown in figure 1 are consistent with 4n / o tetracoordinated complexes with cu ligand distances between 1.96 and 1.98 ( table 1 and figure s3 in the si ) . these results are in a good agreement with the previously proposed coordination models ( scheme 1 ) . the effect of the ph on the copper coordination is not reflected in xas because of the similar light - atom ( n / o ) ligands at both ph values . however , the difference between the two coordination modes ( 3n1o and 4n ) is clearly observed by cd and epr . the reoptimized structures [ from density functional theory ( dft ) calculations ] of the 3n1o and 4n models reported in ref ( 33 ) were used to simulate the exafs spectra at ph 6.5 and 8.5 , respectively , without any fits against the experimental data . the simulated exafs spectra show reasonable frequency agreement with the experimental data ( figure s4 in the si ) , thus validating the proposed models for the cu - prp(106115 ) complexes in scheme 1 . xanes ( a and d ) , exafs ( b and e ) , and ft ( c and f ) spectra of cu - prp(106115 ) ( black line ) , cu - prp(106115)m09a ( green line ) , cu - prp(106115)m112a ( red line ) , and cu - prp(106115)m109&m112a ( blue line , at ph 6.5 ( a c ) and 8.5 ( d f ) . the parameters obtained for the best fits for these data ( figure s3 in the si ) are listed in table 1 . the goodness - of - fit fn ranges between 0.19 and 0.31 depending on the signal - to - noise ratio of the data set . the cu k - edge x - ray absorption near - edge structure ( xanes ) spectra of the cu - prp(106115 ) complexes show an intense signal assigned to the electric dipole - allowed cu 1s 4p transition at 8984 ev , characteristic of cu complexes ( figures 2a , d ) . both xanes and exafs spectra ( figure 2 ) of the cu - prp(106115 ) complexes change with the ph ( figure s5 in the si ) , while the most drastic changes are observed upon substitution of the met residues by ala . regardless of the ph , the xanes spectra of the cu - prp(106115 ) complex ( black line ) and the m109a and m112a complexes ( red and green lines ) show a characteristic signal of four - coordinate complexes ( figures 2a , d ) , while the cu complex without met residues ( cu - prp(106115)m109&m112a , blue line ) displays a spectrum indicative of a two - coordinate complex . xanes ( a and d ) , exafs ( b and e ) , and ft ( c and f ) spectra of cu - prp(106115 ) ( black line ) , cu - prp(106115)m09a ( green line ) , cu - prp(106115)m112a ( red line ) , and cu - prp(106115)m109&m112a ( blue line ) at ph 6.5 ( a c ) and 8.5 ( d f ) . at ph 6.5 , the exafs spectra ( figure 2b ) of the cu - prp(106115 ) complex change significantly upon each met - to - ala substitution , suggesting that both met109 and met112 participate in cu coordination . consistently , the fts of the exafs spectra ( figure 2c ) show a drastic loss in intensity in the met - to - ala variants compared to those of the cu - prp(106115 ) complex ; the low intensity of these signals is best modeled by a lower number of sulfur atoms in the coordination sphere ( see below ) . these changes are most dramatic for the cu - prp(106115)m109&m112a complex , consistent with the xanes results . in contrast , at ph 8.5 , the exafs spectra of the cu - prp(106115 ) complex ( black line ) and the single met - to - ala variants ( red and green lines ) are practically identical ( figure 2e ) , while only the spectrum for the cu - prp(106115)m109&m112a ( blue line ) , where there are no sulfur atoms available for coordination , differs from the original complex . consistently , the fts of the exafs spectra of cu - prp(106115 ) ( black line ) and the m109a ( green line ) and m112a ( red line ) variants ( figure 2f ) are also very similar in intensity , indicating that they have the same sulfur content in the first coordination sphere , while the spectrum of the cu - prp(106115)m109&m112a complex ( blue line ) shows essentially no intensity at the r value of a cu overall , the exafs results indicate that at ph 8.5 only one met residue participates in cu coordination , while at ph 6.5 , both met residues play an important role . consistently , a comparison of the spectra of the cu - prp(106115 ) complexes at ph 6.5 and 8.5 in figure s5 in the si shows differences that can be attributed to a larger contribution of sulfur atoms in the first coordination sphere at low ph . the best fits of the cu - prp(106115 ) complexes at ph 6.5 and 8.5 are shown in figure 3 , and the corresponding parameters are listed in table 2 . at ph 6.5 , the best fit is achieved with two sulfur atoms at 2.37 and two nitrogen or oxygen ligands at 2.17 . the four - coordinate nature of the complex is supported by a bond valence sum ( bvs ) analysis : the bvs value is in better agreement with a four - coordinate structure ( 0.98 ) compared to a three - coordinate fit ( bvss = 0.82 ) . at ph 8.5 , the best fit of the exafs data involves only one sulfur ligand at 2.35 and three nitrogen or oxygen ligands displaying one short cu n / o bond at 1.97 and two longer cu2n / o bonds at 2.15 ( table 2 ) . again , bvs analysis is in better agreement with a four - coordinate structure ( bvss = 1.10 ) relative to a three - coordinate complex ( bvss = 0.74 ) . exafs spectra ( a and c ) and their fts ( b and d ) of cu - prp(106115 ) at ph 6.5 ( a and b ) and 8.5 ( c and d ) . experimental data are shown as solid black lines , and their best fits , using the parameters listed in table 2 , are shown as dashed red lines . the reported goodness - of - fit fn is the normalized error given bywhere the summation is over the fitted k range , k is the photoelectron wave vector , and is the experimental or calculated data point . in order to further explore the nature of the ligands in the cu - prp(106115 ) complexes , 1d ( h ) and 2d h h tocsy and h c hsqc nmr spectra of prp(106115)m109a , prp(106115)m112a , and prp(106115 ) in the presence and absence of 0.8 equiv of cu at ph 6.5 and 8.5 were used to assign the signals of the h of met109 and met112 , the h and h of his111 , and other residues ( figures s6s8 in the si ) . h tocsy and h c hsqc nmr spectra shows that the most affected amino acids in the presence of cu are met109 , met112 , his111 , and lys110 ( figures s6s8 in the si ) . figure 4 shows the aromatic ( 6.98.1 h ppm ) and aliphatic ( 1.92.4 h ppm ) regions of the h nmr spectra of the free peptide prp(106115 ) ( black line ) and the cu complex ( red line ) at ph 6.5 ( figure 4a ) and ph 8.5 ( figure 4b ) . at ph 6.5 , the chemical shifts associated with the h of met109 and met112 are clearly affected ( figure 4a , aliphatic region ) having a broadening and downfield shift ( 0.15 ppm of h met112 and 0.23 ppm of h met109 ) in the presence of cu . in agreement with xas data , nmr data indicate that cu is coordinated by both met residues at low ph . the aromatic region of the spectrum displays a broadening and an upfield shift in the h ( 0.12 ppm ) and h ( 0.04 ppm ) signals of his111 in the presence of cu ( figure 4a ) . this result indicates that at ph 6.5 the his111 imidazole nitrogen participates in cu binding . h nmr spectra of prp(106115 ) ( black line ) and cu - prp(106115 ) ( red line ) at ph 6.5 ( a ) and 8.5 ( b ) . black lines indicate the chemical shifts for the h protons of met residues and the h and h protons of histidine in the peptide without copper . gray arrows show the shifts of these signals in the presence of cu . at ph 8.5 , in the aliphatic region of the h nmr spectrum , the chemical shifts of h of met109 and met112 undergo a broadening and a downfield shift ( 0.12 ppm of h m112 and 0.21 ppm of h met109 ) upon cu binding ( figure 4b ) . in the aromatic region , signals of the h and h of his111 also show broadening and downfield shift . the effect of cu on the signals of the h and h of his111 suggests that at ph 8.5 the his111 imidazole nitrogen participates in cu binding . however , the line broadening observed at ph 8.5 in both regions is much more dramatic than that observed at ph 6.5 . such large signal broadening may be related to the presence of two species at ph 8.5 , one with the sulfur atom from met109 coordinating cu and another complex with the sulfur atom from met112 . this interpretation is consistent with the behavior of the met - to - ala variants observed in xas , where the only sulfur available in each variant is found to coordinate cu . in summary , our nmr data corroborate the involvement of sulfur atoms from met109 and met112 of the prp(106115 ) fragment in the coordination of cu , while they clearly indicate that the his111 imidazole nitrogen also binds to the metal ion . to further explore the role that met109 , met112 , and his111 play on the coordination with cu , we tested both the prp(106115 ) peptide and the cu - prp(106115 ) complex by h nmr spectroscopy at several ph values ranging from 3.5 to 9.5 . figure 5a shows the behavior of h ( dashed black line ) and h ( solid black line ) of his111 in the free peptide and in the cu - prp(106115 ) complex ( dashed and solid red lines ) . the pka associated with the free histidine is 6.2 , while in the presence of cu , the pka of histidine shifts to 5 because of the coordination of his111 to cu in the cu - prp(106115 ) complex . this shift is consistent with that observed in cu complexes in blue copper proteins . the signals associated with the met residues in the cu - prp(106115 ) complex also change drastically with the ph . figure 5b shows in green ( h met109 ) and red ( h met112 ) dots chemical shifts of h of met109 and met112 in the presence of cu at several ph values ( 3.59.5 ) . the chemical shift of these protons is 2.10 ppm in the free peptide ( figure 4 ) , and they do not change with the ph ( data not shown ) . however , in the presence of cu , even at a ph as low as 3.5 , the signals associated with h of met109 and met112 are both shifted to 2.29 ppm , indicating coordination to cu . also , at higher ph values ( above 8.5 ) , both signals show a tendency to return to the chemical shift associated with the free peptide ( 2.10 ppm ) . this result indicates that , at higher ph values , both met residues leave the coordination sphere of cu , leading to a species without thioether ligands ; such species would not be physiologically relevant and were not further characterized . ( a ) chemical shifts of the h and h protons of his111 in the free peptide ( dashed and solid black lines ) and in the cu - prp(106115 ) complex ( dashed and solid red lines ) as a function of the ph . a shift in the pka of his111 is observed in the presence of cu . ( b ) chemical shifts of the h protons of met109 ( green dots ) and met112 ( red dots ) in the cu - prp(106115 ) complex as a function of the ph . the ph - independent chemical shift of these protons is 2.10 ppm in the free peptide . these nmr data provide further insight into the role of met109 , met112 , and his111 in the coordination of cu to the prp(106115 ) fragment as a function of the ph . at very low ph ( 3.5 ) , cu is coordinated by both met109 and met112 . with a pka of 5 , the nitrogen imidazole from his111 enters the coordination sphere , while the two met residues remain coordinated . as the ph is further increased , the met residues play a less important role in cu coordination , and only one met residue ( met109 or met112 ) coordinates cu at ph > 8 ( scheme 2 ) . at ph < 5 , cu is anchored by both met residues , while at a ph > 5 , the his111 imidazole also participates in cu coordination . at ph > 8 , the box in scheme 2 shows the structures for the cu - prp(106115 ) complexes under extracellular conditions , derived from the exafs and nmr data . in order to gain further insight into the nature of the unidentified n / o ligands in these cu complexes , several models were built and rks calculations were performed , using the cu models previously described as starting points . the copper ion was reduced and three groups of ligands were evaluated ( table s3 in the si ) . group 1 included four - coordinate cu-1n2s1x models for the complex formed at ph 6.5 , with two sulfur atoms from both met residues ( met109 and met112 ) , a his111 imidazole nitrogen , and a fourth ligand x , where x could be an oxygen atom from a backbone carbonyl group , a solvent h2o molecule , or a nitrogen atom from a backbone amide in a protonated ( n ) or deprotonated form ( n ) ( including those of the most affected amino acid residues in the 2d tocsy and hsqc experiments ; figures s6s8 in the si ) . because at ph 8.5 the exafs results indicate that one met is coordinated while nmr data show that both met residues are affected , group 2 included four - coordinate cu-1n1s2x models , where the sulfur atom is provided by either met109 ( group 2a ) or met112 ( group 2b ) , his111 imidazole provides a nitrogen ligand , and 2x could be any of the following combinations : 2n , 1n1n , 2n , 1n1o , 1n1o , or 2o ( described above ) . it should be noted that , because the xanes data indicate that cu complexes are four - coordinate , only the structures that optimized as four - coordinate complexes were analyzed in each group . five structures optimized as four - coordinate complexes in group 1 ( table s3 in the si ) ; these are shown in figure 6 , and relevant geometric parameters with and without solvent effects are listed in table s4 in the si . the group 1 models are divided into two subgroups , according to the total number of atoms ( 160 or 161 ) , which depends on the protonation state of the backbone amides . in this group , when x is a deprotonated amide ( 2n2sa and 2n2sb ) , the average cu s bond distances ( 2.365 and 2.34 ) are in good agreement with the experimental values ; however , the two cu n bonds are quite short ( 2.092.10 ) . when x is a backbone carbonyl ( 1n1o2sa and 1n1o2sb ) or a water molecule ( 1n1o2sc1 ) , the average cu s bond lengths ( 2.35 , 2.34 , and 2.36 ) and cu n / o distances are in good agreement with the experimental data ( 2.17 , 2.145 , and 2.16 ; table s4 in the si ) . however , the 1n1o2sb model is discarded because it is the structure that has the highest energy in this subgroup , 13.32 kcal / mol above 1n1o2sa . energy diagram of optimized four - coordinate cu-1n2s1x models for the cu - prp(106115 ) complex at ph 6.5 ( group 1 ) , with explicit solvent . most side chains are not shown for clarity ; however , the geometry optimizations were done with the complete cu - prp(106113 ) complex . thus , the best candidates for the ph 6.5 cu structures ( group 1 ) are the 1n1o2sa and 1n1o2sc1 models ( figure 6 ) because they display metal ligand distances that best reproduce the experimental data in trends and values . furthermore , using 1n1o2sc1 to simulate an exafs spectrum using the exafspak program yielded a spectrum that was in good intensity agreement ( but indicating an overall longer distance ) with the experimental data for cu - prp(106115 ) at ph 6.5 ( figure s9 in the si ) . therefore , at ph 6.5 the best model to represent the cu - prp(106115 ) complex has two sulfur atoms from met109 and met112 , a his111 imidazole nitrogen , and an oxygen atom as the fourth ligand , which could be the carbonyl oxygen atom from his111 ( as in model 1n1o2sa ) or a water molecule ( as in model 1n1o2sc1 ) . in these models , the sulfur atom can be provided by met109 ( group 2a ) or met112 ( group 2b ) . the group 2 models are divided into two subgroups , according to the total number of atoms ( 159 or 160 ) , which depends on the protonation state of the backbone amides . five structures optimized as four - coordinate complexes in group 2a and only four in group 2b ( table s3 in the si ) . the structures are shown in figure 7 , and their geometric parameters with and without solvent effects are listed in tables s5 and s6 in the si . energy diagram of optimized four - coordinate cu-1n1s2x models for the cu - prp(106115 ) complexes at ph 8.5 , with explicit solvent and either met109 ( group 2a ) or met112 ( group 2b ) as the ligand . most side chains are not shown for clarity ; however , the geometry optimizations were done with the complete cu - prp(106113 ) complex . for the models with met109 , when 2x is a combination of two deprotonated amides ( 3n1sm109a and 3n1sm109c ) , only the 3n1sm109c model yields cu ligand distances that are in agreement with the experimental trend , one short cu n and two long cu n bonds ( 2.04 and 2.13 , respectively ) and a cu s bond distance at 2.36 ( table s5 in the si ) , although it is the highest - energy structure of the group ( figure 7 ) . in contrast , in the models where 2x is the combination of a deprotonated amide and a backbone carbonyl ( 2n1o1sm109a , 2n1o1sm109b , and 2n1o1sm109c ) , the cu n / o bonds are reproduced in the 2n1o1sm109a model ( 2.32 , 2.02 , and 2.15 , respectively ; table s5 in the si ) , which is the lowest - energy structure in this subgroup . thus , we propose that the best model for the cu - prp(106115 ) complex at ph 8.5 with a sulfur atom from met109 is 2n1o1sm109a , with a nitrogen atom from the deprotonated amide of lys110 and an oxygen atom from the carbonyl of his111 . the cu-1n1s2x models with the met112 ligand ( figure 7 and table s6 in the si ) , having 2x as a combination of two deprotonated amides ( 3n1sm112a and 3n1sm112c ) , yield a cu s bond at 2.282.30 and three similar cu n bonds ( 2.032.04 ) , which is not consistent with the experimental trend . on the other hand , when 2x is a combination of a deprotonated amide and a backbone carbonyl ( 2n1o1sm112a and 2n1o1sm112d ) , the 2n1o1sm112d model leads to a three - coordinate structure , while the cu ligand distances in the 2n1o1sm112a model show excellent agreement with the experimental data : a short cu n / o bonds ( 2.17 ) , and a cu s bond at 2.34 ( table s6 in the si ) . therefore , the best model for the cu - prp(106115 ) complex with met112 at ph 8.5 is the 2n1o1sm112a structure . overall , the best models for the cu - prp(106115 ) complex at ph 8.5 are 2n1o1s structures with a nitrogen from the deprotonated backbone amide of lys110 and an oxygen from the backbone carbonyl of his111 , regardless of which met participates in the coordination shell . figure 8 summarizes the results from dft modeling , showing the best models for the cu and cu complexes with prp(106115 ) at ph 6.5 and 8.5 . it is clear that the cu complexes ( cu-3n1o and cu-4n ) have very different geometries and coordination spheres than their reduced counterparts ( cu-1n1o2s and cu-2n1o1s ) . we calculated the inner - sphere reorganization energies associated with the reduction of the cu species to cu , at ph 6.5 and 8.5 . for the low - ph structures cu-3n1o and cu-1n1o2s , the calculated i is 1.79 ev when the oxygen ligand is the backbone carbonyl of his111 and 2.15 ev when it is a water molecule . at ph 8.5 , the cu-4n and cu-2n1o1s structures yielded a reorganization energy of 1.60 ev when met109 is bound and 1.66 ev for the met112 structure ( table s7 in the si ) . these are necessary for evaluating the reduction and o2 reactivity in the next section . dft - derived models for cu and cu complexes with the prp(106115 ) peptide at different ph values . for the cu complex at ph 6.5 , the best model has a 1n1o2s coordination sphere , where both met residues are bound to copper and the oxygen atom can be the carbonyl oxygen from his111 ( as shown here ) or a water molecule . at ph 8.5 , the best models are 2n1o1s structures with a deprotonated amide of lys110 , an oxygen atom from the backbone carbonyl of his111 , and either met109 or met112 as the sulfur ligand . once the geometries and electronic structures of the cu - prp(106115 ) complexes had been elucidated , we evaluated the role of met109 and met112 on the reactivity of these complexes . first , the kinetics of reduction of the cu - prp(106115 ) complexes with ascorbate was studied , followed by reactivity studies of the resultant cu - prp(106115 ) complexes with dioxygen . the reduction of both complexes cu-3n1o at ph 6.5 and cu-4n at ph 8.5 by ascorbate was examined by stopped - flow absorption spectroscopy at 25 c . electronic absorption at the wavelength for the maximum intensity of the ligand - field transitions ( i.e. , 600 nm at ph 6.5 and 570 nm at ph 8.5 ) was followed over time after rapid mixing of the cu complexes with the reductant . the reduction was followed under pseudo - first - order conditions , using at least a 20-fold excess of the reductant over the copper complex concentration . plots of the absorption intensity at 600 and 570 nm as a function of the reaction time are shown in figure 9 , while representative data for the absorption spectra are shown in the insets ; fits to the kinetic traces are summarized in table 3 . the reduction of the cu-3n1o complex at ph 6.5 can be fitted to a single - exponential equation to obtain kobs = 0.287 0.010 s ( figure 9a ) . in contrast , the reduction of cu-4n complex at ph 8.5 exhibited a slow biphasic behavior ( figure 9b ) . the kinetic trace for cu-4n can be fitted to a double - exponential equation , yielding rates of 0.134 0.023 and 0.010 0.004 s. thus , even the fast phase of reduction of the cu-4n complex at ph 8.5 is 2-fold slower than reduction of the cu-3n1o complex at ph 6.5 ( table 3 ) . considering the reorganization energies calculated for these two complexes , this could be due to a difference in the reduction potentials of the cu-3n1o and cu-4n species , which would result in different driving forces for the two reactions . using anaerobic reductive titrations of the cu complexes at ph 6.5 and 8.5 with ascorbate , we estimate a reduction potential of 152 mv for the cu-3n1o complex and 75 mv for the cu-4n complex . thus , a significant difference in g for their reduction by ascorbate is expected { g = [ g(3n1o ) g(4n ) ] = 3.52 kcal / mol}. using the semiclassical marcus equation for the rate of intermolecular electron transfer and considering the differences in the reorganization energies and reduction potentials , the ratio of the reduction rates for cu-3n1o and cu-4n species is calculated to be k3n1o / k4n = 2.3 ( see the si ) , which is consistent with the experimental values . thus , the difference in the reduction potentials for these two complexes results in a faster reduction for the cu-3n1o complex compared to that for the cu-4n species . stopped - flow absorption data for the reduction of the cu - prp(106115 ) complex at ph 6.5 ( 3n1o complex ) ( a ) and at ph 8.5 ( 4n complex ) ( b ) with 20 equiv of ascorbate . d transitions as a function of time , are shown in each inset . in order to evaluate the effect of met109 and met112 on the rate of reduction of the cu-3n1o complex , the reduction of the cu - prp(106115)m109a , cu - prp(106115)m112a , and cu - prp(106115)m109&m112a complexes by ascorbate at ph 6.5 was also studied ; representative kinetic traces are shown in figure s10 in the si , and the reduction rates are summarized in table 3 . within experimental error , cu - prp(106115)m109a , cu - prp(106115)m112a , and cu - prp(106115)m10&m112a exhibit a reduction rate constant that is the same as that of the cu - prp(106115 ) complex . these results suggest that the met residues are not involved in the rate - limiting step of the reduction of the cu - prp(106115 ) complex at ph 6.5 . thus , it is likely that this involves electron transfer , followed by a slower rearrangement of the peptide , to provide the met ligands to form the ph 6.5 cu-1n1o2s complex . we next investigated the first step of dioxygen activation by the cu - prp(106115 ) complexes . reduced complexes of prp(106115 ) , prp(106115)m109a , prp(106115)m112a , and prp(106115)m10&m112a were mixed with an oxygenated buffer , and the reaction was followed by uv vis absorption spectroscopy . d transition , indicating cu oxidation , while the reoxidation rate for each copper peptide is listed in table 4 . the end products of the reaction of cu - prp(106115 ) complexes with oxygen were spectroscopically characterized by cd and epr ; in all cases , at least 50% of the original cu complexes were recovered ( figure s11 in the si ) . also , analysis of the samples by matrix - assisted laser desorption / ionization time of flight / time of flight ( maldi - tof / tof ) showed metal - catalyzed oxidation for the prp(106115 ) , prp(106115)m109a , and prp(106115)m112a peptides ( figure s12 in the si ) . in particular , for the prp(106115 ) peptide , two new signals with m / z peaks corresponding to the mass of the peptide , + 16 and + 32 da , were observed , while single - variant peptides [ prp(106115)m109a and prp(106115)m112a ] only show one + 16 da peak . in contrast , the double - variant prp(106115)m109&m112a peptide , which contains no met residues , suffered no modification . these results indicate that redox cycling of these copper peptide complexes results in oxidation of the met residues into sulfoxides , while no evidence was found for their oxidation to sulfone . kinetic traces for the reoxidation by dioxygen of the cu - prp(106115 ) complex ( black ) and the variants cu - prp(106115)m109a ( green ) , cu - prp(106115)m112a ( red ) , and cu - prp(106115)m109&m112a ( blue ) at ph 6.5 , followed by uv vis absorption spectroscopy at 600 nm . inset : representative absorption data showing the increase in the intensity of the ligand - field transitions for the cu complex as a function of time . the reoxidation of cu - prp(106115 ) by dioxygen may follow inner- or outer - sphere electron - transfer pathways . in an outer - sphere mechanism , the cu complex would be oxidized by dioxygen in a single step to regenerate the cu complex , while an inner - sphere reaction would involve binding of dioxygen to the cu complex and formation of a potential copper(ii ) superoxide intermediate , followed by the release of superoxide to regenerate the cu complex . the possibility of outer - sphere reoxidation of the cu - prp(106115 ) complexes was evaluated using the marcus equation to analyze the rates in table 4 . using an estimated reduction potential for the copper peptide complex of 150 mv , the potential of one - electron reduction of dioxygen to o2 ( 165 mv vs nhe ) , and the calculated reorganization energy of the site , the outer - sphere electron - transfer rate from the cu complex at ph 6.5 to the oxygen molecule is estimated to be at most 2.81 10 s ( see the si ) , which is 1010 times slower than the experimental rate of cu reoxidation from the kinetic data ( 3.6 10 ) . thus , single - electron transfer from cu - prp(106115 ) to dioxygen likely proceeds via an inner - sphere pathway that would involve formation of a copper(ii ) superoxide species . this is consistent with a previous study that showed the formation of superoxide species upon redox cycling of the cu - prp(106114 ) complex and with our finding that the met residues are oxidized to sulfoxides upon redox cycling of the cu - prp(106115 ) complex . it is important to note that the reoxidation rate shows an inverse correlation to the number of met residues in the initial cu complex , following the trend cu - prp(106115 ) < cu - prp(106115)m109a cu - prp(106115)m112a < cu - prp(106115)m109&m112a ( table 4 ) . the reoxidation rate of the cu - prp(106115)m109&m112a complex is 6 times faster than those of the single variants and 10 times faster than that of the cu - prp(106115 ) complex containing both met residues . these results suggest that the presence of met ligands at the cu complex affects the rate - limiting step in the reoxidation process , which likely involves formation of the copper(ii ) superoxide species . xanes data clearly show that , in the absence of met residues , the cu - prp(106115)m109&m112a complex is two - coordinate , while the cu - prp(106115 ) complex is four - coordinate ( vide supra ) . thus , in an associative mechanism for formation of the copper(ii ) superoxide species , a faster reaction would be expected for the cu - prp(106115)m109&m112a complex because of its coordination unsaturation , as observed . the nature of the mechanism ( associative vs dissociative ) for the inner - sphere oxygen reaction of the four - coordinate cu - prp(106115 ) complex requires further computational evaluation . cu - prp(106115 ) complexes at ph 6.5 and 8.5 were studied by xas , and the role of met109 and met112 in cu coordination was evaluated using peptides with met - to - ala variants . our xas results show that cu coordination to his111 occurs through nitrogen and oxygen atoms at low ph , and all nitrogen ligation at high ph ( pka = 7.5 ) , which is consistent with the previously proposed coordination models , as shown in scheme 1 , while the possibility of met residues acting as ligands for cu can be discarded . xas and nmr spectroscopy , in combination with electronic structure calculations , show that cu coordination to prp(106115 ) is also ph - dependent ( scheme 2 ) . at ph < 5 , the thioether groups of met109 and met112 bind cu ; with a pka of 5 , the nitrogen imidazole from his111 enters the coordination sphere , while both met residues remain coordinated , adopting a 1n1o2s coordination mode . at ph 8.5 , two cu species with 2n1o1s ( 2nosm109 and 2nosm112 ) coordination modes are formed , with a single met residue bound to cu ( scheme 2 ) . although this study has used a short fragment of prp that only includes his111 , the 2s and 1n1o2s species described here would form in longer prp protein chains . at low ph ( below 5 ) , all his residues would be protonated and met109 and met112 would dominate cu coordination to form the 2s species . at higher ph , other n - terminal his residues , such as his96 , could participate in cu binding ; however , a recent study of cu and ag coordination to the 91127 fragments demonstrated that a helical structure at the hydrophobic region of the 91127 fragments causes his96 to fall out of the coordination sphere , leaving his111 as the main copper anchoring site ; thus , the 1n1o2s complex described herein would be the dominant species . prp is a synaptic glycolipid - anchored membrane protein predominantly expressed on presynaptic membranes , where it is exposed to cu ions in a range of concentrations : from 15 m at the synaptic cleft during synaptic vesicle release up to 100300 m during neuronal depolarization . thus , prp can interact with cu ions in the extracellular space , and at ph 7.4 , there is a mixture of two species 3n1o and 4n ( scheme 1 and figure 11a ) . also , biological reductants , such as ascorbic acid , are found in concentrations of 150 m in the cerebrospinal fluid . thus , cu - prp complexes anchored at his111 in the synaptic cleft may be reduced to cu by available reducing agents . our results indicate that , in the presence of ascorbate , the cu-3n1o complex would reduce faster to generate a cu-1n1o2s complex . therefore , at physiological ph , 3n1o and 4n complexes are the relevant cu species , while the 1n1o2s complex is the relevant cu coordination mode at the his111 site in prp ( figure 11b ) . schematic representation of the main cu - prp complexes under different physiological conditions . ( a ) at low cu concentration ( nm ) , cu would be anchored at the or ( cu - or ) and at his96 and his111 sites ( cu-3n1o and cu-4n ) . ( b ) cu complexes in the presence of reducing agents would be reduced . at the his111 site , ( c ) high copper concentrations ( 100500 m ) can stimulate endocytosis ; in the endosomes , only the his111 site would coordinate cu through met109 and met112 . ( d ) in the extracellular space , oxygen activation by copper bound to his111 could generate superoxide , causing the subsequent oxidation of met residues . footnotes : a , ref ( 27 ) ; b , ref ( 28 ) ; c , ref ( 33 ) ; d , this work ; e , ref ( 34 ) ; f , ref ( 35 ) . unlike all other copper binding sites in the human prp protein , the his111 binding site contains the mkhm motif , which provides it with unique coordination and redox properties . mx2mx2 m motifs have been identified in copper - transport proteins ( e.g. , yeast yctr1 ) that bind cu selectively via met residues with affinities in each motif on the order of 2.5 10 m at ph 4.5 . on the other hand , several proteins involved in copper transport that utilize his in combination with met residues for cu binding have emerged , such as pcoc with a cu-(his)(met)2 coordination mode , copc [ cu-(his)(met)3 ; kd 10 m ] , and cusf [ cu-(his)(met)2(o ) ] . in these cases , the presence of the his residue in the coordination sphere plays two roles : ( i ) to increase the affinity for cu and ( ii ) to enable the coordination of cu ions . furthermore , it has been demonstrated that thioether ligands can be particularly effective at chelating cu when they are separated by no more than two amino acids . the mkhm motif in the human prp(106115 ) sequence fulfills these conditions , such that at physiological ph cu can be anchored via the his residue , while upon reduction , the cu ion is bound by his and met residues . high cu concentrations stimulate endocytosis of the prion protein , taking the protein from the extracellular space at ph 7.4 to the endosomal space , where there is a high concentration of protons ( ph 45 ) and biological reductants , such as glutathione or nicotinamide adenine dinucleotide ( nadh ) , that are found in concentrations of 13 mm and 200 m , respectively . under these conditions , his111 would be protonated and cu would be anchored only by met residues , as illustrated in figure 11c . thus , this site is optimized to chelate both cu and cu ions and is likely to be involved in cu transport into the cell . cu and cu bound to prp(106115 ) form complexes with very different geometries and coordination spheres : cu-3n1o versus cu-1n1o2s at ph 6.5 and cu-4n versus cu-2n1o1s at ph 8.5 , resulting in large reorganization energies of the site upon reduction of 1.79 and 1.6 ev , respectively . in spite of its higher reorganization energy , reduction of the cu-3n1o complex is faster than that of the cu-4n species because of its higher reduction potential . considering that , at physiological ph , cu-3n1o and cu-4n complexes are present , our results indicate that , under reducing conditions , the cu-3n1o complex would reduce faster , shifting the equilibrium between 3n1o and 4n toward the cu-3n1o complex and favoring production of the cu-1n1o2s complex . overall , the reorganization energies for the cu - prp(106115 ) complexes are large compared to those of copper sites that are optimized to support electron transfer ( blue copper t1 and cua sites ) , which range from 0.5 to 0.82 ev . despite its large reorganization energy , the cu - prp(106115 ) complex still reacts with dioxygen . several studies regarding the redox activity and functional implications of cu - prp complexes have been put forward . some studies have reported the generation of reactive oxygen species ( h2o2 or superoxide ) by cu - prp complexes ; however , little is known about the mechanism of dioxygen activation by the different cu - prp complexes . this study investigated the kinetics of the first step of dioxygen activation by the cu-1n1o2s complex at ph 6.5 . the estimate for the rate of outer - sphere reoxidation is 10 times slower than the experimental rate of cu reoxidation from the kinetic data . thus , we conclude that the cu-1n1o2s complex at the his111 site reacts with dioxygen in an inner - sphere electron - transfer pathway that involves binding of dioxygen to the cu complex and formation of a copper(ii ) superoxide intermediate , followed by the release of superoxide and partial regeneration of the cu complex . our study also shows that the met residues can be selectively oxidized to sulfoxide by the copper - catalyzed reaction ( figure 11d ) . it has been proposed that copper - catalyzed oxidation of met residues may trigger a structural transition , leading to aggregation of prp protein , or it may interfere with conversion of the prion protein into the fibrillar proteinase k - resistant conformation . beyond that , met residues in prp may act as an innate antioxidant defense in the protein by their ability to scavenge the produced superoxide and undergo oxidation to form methionine sulfoxide . without the met residues , the enzymatic peptide methionine sulfoxide reductase ( msra ) reverses methionine sulfoxide back to met , which once again is able to scavenge oxidants . in fact , in the alzheimer s disease brain , a decrease in the activity of msra compared to control subjects was observed , while the induction of msra activity protects neuronal cells from amyloid toxicity . it is plausible that a reversible oxidation / reduction of met residues at the n - terminal region of the prp protein could be acting as an ros sink . this would be consistent with the notion that prp plays a role in cellular antioxidant defense . in summary , our study shows that cu binding to his111 is highly ph - dependent and that the presence of one his and two met residues in the mkhm motif of the human prp(106115 ) fragment confers this site with unique cu binding properties . even upon drastic changes in the chemical environment , in particular those occurring during endocytosis , the two met residues in the mkhm motif allow prp to keep cu ions anchored , consistent with a copper - transport function for this protein . on the other hand , in the extracellular space , in the presence of reducing agents , the most populated cu - his111 complex would be a cu-1n1o2s species , which is capable of activating dioxygen . our study provides further insight into the molecular mechanism of oxygen activation by this site . the cu-1n1o2s complex reacts with dioxygen through an inner - sphere mechanism , likely involving the formation of a copper(ii ) superoxide intermediate , followed by the release of superoxide and partial regeneration of the cu complex . the met residues are oxidized to sulfoxide in this process , and their ability to scavenge superoxide may play a role in the proposed antioxidant properties of prp .
we describe a case of acute myeloid leukemia m5 showing a balanced t(2;10 ) ( q31;p12 ) translocation . this has never been described before as the sole cytogenetic abnormality in a bone marrow cell clone at onset . using fluorescence in situ hybridization with properly designed bacterial artificial chromosome probes , we mapped the breakpoint regions on both derivative chromosomes 2 and 10 : der(2 ) and der(10 ) , respectively . the mpp7 gene , disrupted by the breakpoint on chromosome 10 , was juxtaposed upstream of both hnrna3 and nfe2l2 genes on chromosome 2 , without the formation of any fusion gene . using real - time quantitative polymerase chain reaction , we tested the possible disregulation of any of the breakpoint - associated genes as a consequence of the translocation , but we found no statistically significant alteration . considering the potential role of this clonal cytogenetic abnormality in leukemogenesis , we speculate that this translocation could have an impact on additional genes mapping outside the breakpoint regions . however , the limited amount of rna material available prevented us from testing this hypothesis in this present case .	the majority of chromosomal rearrangements in acute myeloid leukemia ( aml ) result in fusion genes or position effects . indeed , according to the world health organization classification of hematopoietic tumors , a consistent group of aberrations is associated with specific aml subtypes , with both diagnostic and prognostic significance . in this report , we describe a case of aml ( fab m5 subtype ) in which the karyotype displayed a novel t(2;10)(q31;p12 ) balanced translocation as the sole cytogenetic abnormality in a bone marrow cell clone at onset . in november 2007 , a 42-year old man was admitted to the department of hematology and oncological sciences of the sergnoli institute , bologna , northern italy . peripheral blood count showed white blood cells 81.710/l with 9% neutrophils and 84% blast cells , hemoglobin 12 g / dl , and platelets 8910/l . bone marrow aspirate was hypercellular and showed a wide population of medium and large sized cells characterized by a high nuclear / cytoplasmatic ratio and basophil cytoplasm . immunophenotyping identified a cd13 , cd15 , cd33 , cd117 , mpo7 population with aberrant expression of cd19 . bone marrow biopsy confirmed a widespread infiltrate with cd68 ( pgm1 ) positive blast cells . mutational analysis was negative for flt3-itd , flt3-tkd and npm1 . in december 2007 , induction chemotherapy with fludarabine ( 50 mg / die for 5 days ) , cytosine arabinoside ( ara - c ) ( 4 g / die for 5 days ) , idarubicin ( 20 mg / die on days 1 , 3 and 5 ) followed by gemtuzumab - ozogamicin infusion ( 5 mg on day 6 ) was started and a morphological and cytogenetic remission was achieved . the patient later received a first consolidation cycle with idarubicin and ara - c , and a second consolidation cycle with high - dose ara - c . in may 2008 , bone marrow examination revealed an initial relapse , confirmed by the cytogenetic analysis showing karyotype 47,xy,+8/46 , xy . reinduction chemotherapy was started but the patient showed resistance to conventional chemotherapy . in june 2008 , he started therapy with tipifarnib and bortezomib , but he experienced disease progression with peripheral blastosis , anemia and thrombocytopenia . fluorescence in situ hybridization analysis mapped the chromosome 2 breakpoint within rp11 - 25l17 ( chr2:177,938,840178,096,310 ) , precisely within the non - overlapping region ( approx . 70 kb ) of this clone with rp11 - 28m17 ( chr2:178,009,023178,198,838 ) ( figure 1a ) . the breakpoint was then located upstream from the heterogeneous nuclear ribonucleoprotein a3 ( hnrnpa3 ) gene , encoding a protein involved in alternative splicing , and the nuclear factor erythroid 2-like 2 ( nfe2l2 ) gene , encoding a basic leucine zipper ( bzip ) transcription factor . interestingly , both genes had been previously described as deregulated in some tumor types . on chromosome 10 , the breakpoint was identified by the splitting signals of rp11 - 49d12 ( chr10:28,369,83728,539,930 ) on both der(2 ) and der(10 ) ( figure 1a ) . it encompassed the palmitoylated membrane protein 7 ( mpp7 ) gene , encoding a member of the p55 subfamily of maguk proteins . figure 1a ) left : fluorescence in situ hybridization ( fish ) results obtained with bacterial artificial chromosome clones delimiting the breakpoint regions on der(2 ) and der(10 ) . right : maps of the breakpoint regions in chromosome bands 2q31.2 ( top ) and 10p11.23 ( bottom ) , according to the latest release of the ucsc human genome browser ( grch37/hg19 ) ( february 2009 ) . the reported clones have the same color code as the fish image on the left . b ) expression analyses of exons 6 and 14 of mpp7 ( red bars ) , exon 5 of hnrnpa3 ( green bar ) , and exon 4 of nfel2l2 ( yellow bar ) evaluated by real - time quantitative polymerase chain reaction , in the present case [ t(2;10 ) ] , 4 control akute myeloid leukemia ( aml ) m5 samples , the mean ct value of the controls ( mean value ) , and normal bone marrow . the results showed comparable genes transcriptional levels in the patient with t(2;10 ) compared with the mean ct value of the 4 m5 aml controls . a ) left : fluorescence in situ hybridization ( fish ) results obtained with bacterial artificial chromosome clones delimiting the breakpoint regions on der(2 ) and der(10 ) . right : maps of the breakpoint regions in chromosome bands 2q31.2 ( top ) and 10p11.23 ( bottom ) , according to the latest release of the ucsc human genome browser ( grch37/hg19 ) ( february 2009 ) . genes are indicated by yellow bars . the reported clones have the same color code as the fish image on the left . b ) expression analyses of exons 6 and 14 of mpp7 ( red bars ) , exon 5 of hnrnpa3 ( green bar ) , and exon 4 of nfel2l2 ( yellow bar ) evaluated by real - time quantitative polymerase chain reaction , in the present case [ t(2;10 ) ] , 4 control akute myeloid leukemia ( aml ) m5 samples , the mean ct value of the controls ( mean value ) , and normal bone marrow . the results showed comparable genes transcriptional levels in the patient with t(2;10 ) compared with the mean ct value of the 4 m5 aml controls . neither of these two breakpoint regions has ever been described as being involved in tumor - associated chromosome rearrangements . to assess the possible impact of this translocation on the hnrnpa3 , nfe2l2 , and mpp7 genes , real - time quantitative polymerase chain reaction assays were performed on the patient 's bone marrow ( bm ) rna , and compared to 4 aml m5 control cases ( without the translocation ) , as well as to normal bm . we used three reference genes ( hprt1 , ywhaz , and sdha ) and the mean expression value of the control amls as calibrator . the results obtained showed that there was no statistically significant change in the expression of any of the genes investigated ( figure 1b ) . to summarize , we describe for the first time a novel , non - recurrent t(2;10)(q31;p12 ) translocation in aml which did not lead to any gene fusion or position effects . remarkably , the molecular consequences of this translocation are to be found outside the breakpoint regions ' gene domains . we might speculate that the chromatin relocation due to the t(2;10)(q31;p12 ) rearrangement might have influenced the expression pattern of additional genes , mapping along both derivative chromosomes 2 and 10 . however , we were not able to evaluate this because of the limited amount of rna material available . it is not possible to draw clear conclusions about the possible clinical impact of this translocation , also because the rearrangement was not present at relapse . however , on the other hand , the t(2;10)(q31;p12 ) was the only chromosomal aberration observed in the karyotype of a cell clone in the patient 's bone marrow at onset , suggesting it might have an impact on leukemogenesis . notably , the patient was negative for flt3 and npm1 mutations , excluding the possibility that this translocation might be a secondary event to this type of alteration . the study of further aml cases with t(2;10)(q31;p12 ) would allow us to gain a better understanding of the clinical and molecular impact of this translocation on patient outcome .
crisscross heart ( cch ) is a rare cardiac malformation characterized by crossing of the inflow streams of the two ventricles due to an apparent twisting of the heart about its long axis . the developmental mechanisms and causes of cch are remaining unknown . neonates mainly presents with cyanosis and a systolic murmur . we herein present a case of cch with concordant atrioventriculo connections with double outlet right ventricle ( dorv ) which was diagnosed by echocardiography .	criss - cross heart ( cch ) , or superoinferior ventricles , is a complex congenital rotational abnormality in which the systemic and pulmonary venous streams cross at the atrioventricular ( av ) level without mixing . its frequency is less than 8/1000000 and accounts for < 0.1% of congenital heart defects ( chd ) . in the normal heart , av structures are parallel to each other when viewed from the front , whereas in cch the av structures are not parallel but angulated by as much as 90 degrees.14 the atrium connects with the contralateral ventricle and the ventricular chambers are arranged in a superoinferior fashion , with the right ventricle ( rv ) superiorly and the left ventricle ( lv ) inferiorly located , regardless of whether the av connection is concordant or discordant . the diagnosis is made by using 2-dimensional and color doppler echocardiography.35 here , we report a rare case of cch with concordant atrioventricular connections with dorv which was diagnosed by echocardiography . a 2 month old female infant , weighing 3.7 kg presented with history of breathlessness , feeding difficulty and cyanosis . she was a second product of non - consanguineous marriage and born by normal vaginal delivery . respectively . blood pressure was recorded at 75/35 mmhg and oxygen saturation 85% in room air . cardiac auscultation revealed normal first and second heart sound along with a grade 3/6 ejection systolic murmur audible best at the left upper parasternal area . the two - dimensional echocardiography , subcostal coronal views demonstrated the connection of the left - sided left atrium and the right - sided left ventricle through the mitral valve and the right - sided right atrium to be connected to the left - sided right ventricle through the tricuspid valve by anterior angulation of the transducer . it also shows the two great arteries arising from the right ventricle ( figure 1 ) . subcostal coronal views shows the connection of the right - sided ra to be connected to the left - sided rv through the tricuspid valve , inlet vsd and the two great arteries arising from the rv . short - axis views shows the right ventricle was superior and left ventricle was inferior with horizontal position of the ventricular septum ( figure 2 ) . short - axis views shows the superior ( rv ) and inferior ( lv ) ventricle with horizontal position of the ventricular septum . the standard 4-chamber view was not showing simultaneously all four chambers and both atrioventricular valves ( figure 3 ) . a large size inlet type of ventricular septal defect ( bidirectional shunt ) and a moderate size ostium secundum atrial septal defect ( bidirectional shunt ) were present . cch is a complex congenital anomaly produced by the rotation of ventricular mass along its long axis . this positional anomaly can coexist with a horizontal displacement of the ventricular mass along the horizontal plane of long axis , which produces superior - inferior ventricles . in 1961 , lev and rowlatt described unusual arrangement of the cardiac inlets that is ventricular chambers arranged in a superoinferior fashion , with the rv superiorly and the lv inferiorly.2 in 1974 , anderson et al used the term cch for cardiac anomaly producing the illusion of crossing of the systemic and pulmonary venous stream without mixing at the av level.3 in the later years , other cases was described with situs solitus or situs inversus or isomerism , mostly with av concordance.45 the physiology is determined by the concordant or discordant atrioventricular ( av ) and ventriculoarterial ( va ) connection and the associated cardiac defects.67 cch may be seen in three forms , that is complete transposition , corrected transposition , and normal hearts . in our case , there was av and va concordance with malposed great arteries . a review of the medical literature does not reveal any isolated presentation of cch cases . most patients with cch has other anomalies such as vsd , transposition of great vessels , right ventricular hypoplasia , subpulmonary stenosis , straddling av and others.78 the diagnosis should be suspected by echocardiography when the parallel arrangement of the av valves and ventricular inlets can not be achieved , and the two valves are not easily visualized simultaneously on apical 4 chamber view . color flow mapping can help in assessing the av connection , visualization of the direction of intracardiac blood flows and recognition of the crossover of the inflow streams.910 yang yl et al11 reported that the failure to obtain a characteristic 4-chamber view was diagnostic for recognition of the cch . the echocardiographic features suggested by yang yl et al11 were present in our case . surgical options vary according to the exact sequential segmental analysis and associated abnormalities . in conclusion , cch is a rare cardiac anomaly than can be diagnosed by a transthoracic echocardiography by an alert echocardiographer to determine the relationships of the cardiac chambers and associated cardiac anomalies .
we report a case of small bowel ischemia secondary to sickle cell disease . acute bowel ischemia is an uncommon presentation of patients with sickle cell disease . historically , only a handful of cases have been reported . we also provide a summary of the literature relevant to sickle cell patients with acute bowel ischemia .	while abdominal pain is a common presentation in patients with sickle cell disease , its progression to an acute surgical abdomen is relatively rare . only a few case reports describing ischemic bowel secondary to sickle cell crisis have been published . ischemic bowel secondary to sickle cell disease has been recognized with increasing frequency [ 1 , 2 , 3 ] . most patients with ischemic bowel are elderly and often have a past medical history significant for atherosclerotic disease manifesting with congestive heart failure or cardiac dysrhythmias . in contrast , patients with sickle cell disease are considerably younger , often with no history of cardiovascular disease . a 42-year - old african - american man with a past medical history of hypertension , end - stage renal disease , and mechanical heart valve was admitted to the hospital in sickle cell crisis with complaints of chest and bilateral upper extremity digit pain . cardiac work - up showed an ekg with st depression in the left lateral wall leads . cardiac catheterization was performed , but demonstrated no significant coronary artery lesions and required no further cardiac intervention . the patient had an elevated amylase and lipase of 456 and 334 u / l , respectively . electrolytes were within normal limits except for a total bilirubin of 2.1 mg / dl . as such , the patient was managed conservatively and eventually started on oral intake . once the patient was started on a regular diet , he developed acute generalized abdominal pain which was similar in his estimation to previously experienced pain associated with sickle cell crisis . upon examination , nevertheless , his examination revealed a tense , distended abdomen which revealed diffuse rebound tenderness and guarding . complete blood cell count demonstrated a white blood cell count of 16,200 with 80% neutrophils . an upright abdominal film demonstrated pneumoperitoneum . the patient was immediately given intravenous piperacillin / tazobactam , hydrated aggressively , and taken to the operating room for exploration . intraoperatively , evaluation of the small bowel demonstrated 3 lesions that were grossly infarcted and leaking bile . the ischemic regions and infarcted / perforated portions of duodenum and jejunum were resected with primary anastomosis . the patient 's operative course was uneventful and he was transferred to the intensive care unit for recovery . histologic sections of the duodenal and jejunal specimens of infarcted bowel demonstrated transmural infarction with necrotizing acute inflammatory cell exudates along with submucosal edema and necrotizing acute serositis consistent with ischemic bowel ( fig . 1 , fig . its etiology has been attributed to a myriad of sources including mesenteric and retroperitoneal adenopathy , infarction in vertebral bodies , hepatobiliary disease and splenic infarction . while the above may at some point in the natural history of the disease contribute to abdominal pain , it is more likely that intermittent ischemia , stemming from transient low - flow states due to red blood cell deformity , is the primary source of the abdominal pain . at the capillary level , red blood cell deformability is the major determinant of viscosity with the capillary diameter at which red blood cell deformation inhibits normal blood flow being < 10 m . increased membrane rigidity resulting in decreased deformability may be the primary contributor in transient vaso- occlusion in capillaries that often range in diameter from 3060 m . one classic study by boley et al . reproduced ischemic colitis pathology in dogs after injection of microspheres of 30100 m into the mesenteric circulation [ 6 , 7 ] . we believe that the bowel injury found in our particular patient likely represents an extreme manifestation of what is likely to be a spectrum of pathology that ranges from minor abdominal pain secondary to transient ischemia to full - blown transmural bowel infarction . most case reports and reviews emphasize that the majority of patients with abdominal pain will not have clinically significant bowel injury and thus can be managed conservatively with bowel rest , aggressive hydration , and intravenous analgesics . however , our case highlights the fact that the far end of the clinical spectrum , i.e. transmural bowel injury with pneumoperitoneum requiring operative intervention , does indeed occur and should be kept in mind when managing and following sickle cell patients with abdominal pain .
we investigated the androgenic effects of 17- methyltestosterone ( mt ) on gonadal sex reversal in juvenile red spotted grouper epinephelus akaara . the fish were immersed in 17-mt at 1 and 5 mg / l . treatment method of 17-mt was once weekly for 4 and 8 weeks . fish were sampled at 12 months after end of the treatment period in order to histological analysis . at the initiation of an experiment ( 70 day after hatching ) , juvenile red spotted grouper have the paired primordial gonads with somatic cells bellow kidney in the posterior portion of the body cavity . formation of ovarian cavity indicates that the ovarian differentiation beginning at 70 dah in red spotted grouper . at 12 months after end of the treatment period , control group , 17-mt 1 mg / l treatment group for 4 and 8 weeks , and 17-mt 5 mg / l treatment group for 4 weeks were all female . however , sex - changed males without ovarian cavity were observed in the 17-mt 5 mg / l treatment group for 8 weeks . in grouper , we firstly reported that the red spotted grouper be able to induce the primary males by hormone treatment prior to gonadal sex differentiation .	the red spotted grouper , epinephelus akaara is one of the most important grouper species for aquaculture in korea , china , japan and southeast asian countries . in particular , groupers are ideal candidate species for intensive aquaculture in asia pacific region because of high consumer demand , desirable taste , efficient feed conversion , and rapid growth ( kohno et al . , 1993 ; commercial importance culture species is still in infancy with adult management , natural spawning and seed production because most groupers require culture periods of several years until first maturation and spawning is achieved ( song et al . , 2005 ) . the problems of grouper aquaculture development has been hindered by the difficulty of mature male grouper security , which is due to the lack of a standardized method of sex change and the unavailability of mature male broodstock . most groupers including the red spotted grouper are protogynous hermaphrodites , first become sexually mature as females and later change sex to become males . natural sex change in grouper species occurs between 3 and 11 years of age depending on the species ( tan & tan , 1974 ; chauvet , 1988 ) . the steroid hormones play important role of sex change in the grouper species ( li et al . many study have been reported on different grouper species to develop standardized methods for sex reversal by treatment with androgens or aromatase inhibitors ( bhandari et al . , 2005 ) . but sex - changed male grouper had testis with ovarian cavity and sperm production is very low . thus , in order to have a stable supply of sperm , it is necessary for male groupers without ovarian cavity using hormone treatment prior to ovarian cavity formation . in this study , we examined that the effect of 17-methyltestosterone ( mt ) to induce the primary male of juvenile red spotted grouper e. akaara . the experiments were carried out in marine science institute , jeju national university . at each experimental group , 200 fish were reared in 300 l circle tank with flowthrough sea water , under natural water temperature and photoperiod . at 70 days after hatching ( total length , 3.33 0.38 cm ; body weight , 0.470.14 g ) , fish were immersed in 17-mt at 1 and 5 fish were sampled at 12 months after end of the treatment period in order to histological analysis . the gonads were fixed in bouin 's solution , embedded in paraffin , cross - sectioned , and stained with haematoxylin and eosin using the standard methods for light microscopy . at the initiation of an experiment ( 70 day after hatching ) , juvenile red spotted grouper have the paired primordial gonads with somatic cells bellow kidney in the posterior portion of the body cavity . each lobe of the primordial gonads began elongation to form an ovarian cavity ( fig . gonad development during sexual differentiation is a little studied but similar patterns as early characteristic of female differentiation were reported ( murata et al . , 2009 ; liu & sadovy , 2009 ; tsai et al . , 2011 ; sao et al . , 2012 ) . in e. malabaricus , the initial ovarian cavity was observed at 47 day after hatching by elongations of somatic cells in the gonads on the sides facing the gonad lateral walls , an early event of initial ovarian cavity formation ( murata et al . , 2009 ) . in e. coioides and e. bruneus , the initial ovarian phase was observed at 4 month after hatching and 60 day after hatching , respectively ( tsai et al . , 2011 ; sao et al . , 2012 ) . in this study , early morphological change to form the initial ovarian cavity in red spotted grouper were observed by around 70 day after hatching . sex pattern of protogynous hermaphroditic fish can be divided into two types , mondary and diandry , according to the male development pathway ( reinboth , 1967 ) . monandric species follow a single male developmental pathway ; all males in a population are secondary males derived exclusively from functional females via sex change . diandric species follow two male developmental pathways , i.e. , primary males develop from juveniles through sexual differentiation and secondary males develop via sex change . in halichoeres most grouper is mondary type with secondary male via sex change from functional female ( lee et al . , 2006 ) . at 12 months after end of the treatment period , control group , 17-mt 1 mg / l treatment group for 4 and 8 weeks , and 17-mt 5 mg / l treatment group for 4 weeks were all female , which is contained oogonia and perinucleolus oocytes in ovary . however , sex - changed males were observed in the 17-mt 5 mg / l treatment group for 8 weeks . the gonad of sex changing individuals was consisted of numerous testicular lobules and filled with sperm , but can not observed the ovarian cavity ( fig . successful induction of artificial sex change has been reported mostly in small sized females ( tukashima & kitajima , 1983 : chao & chow , 1990 ; tsuchihashi et al . , 2003 ) . androgen administration including 11-ketotestosterone ( 11-kt ) and the synthetic 17-methyl - testosterone ( mt ) widely used for sex reversal induction in grouper , but various dose concentrations and methods of hormone administration ( chao & lim , 1991 ; tsuchihashi et al . , 2003 ; however , these methods of hormone administration have problem such as incomplete sex inversion and reverted back to being females during the next reproductive period ( marino et al . therefore , the most effective treatment time for artificial sex change induction by steroid hormone is during gonadal sex differentiation . in this study , sex - changed males without ovarian cavity were observed when juvenile red spotted grouper ( 70 dah ) were immersed in 17-mt 5 mg these results suggested that the red spotted grouper be able to induce the primary males by hormone treatment prior to gonadal sex differentiation .
the incidence of syphilis has consistently increased from 2000 to 2002 . we report a case of acquired syphilis with symptoms of tullio phenomenon in a patient concurrently diagnosed with hiv infection . the resurgence of syphilis in hiv - positive groups at high risk has public health implications for prevention of both diseases .	a 35-year - old man with an unremarkable medical history sought treatment for headaches , hearing loss , and night sweats . they came on as the day progressed and were neither positional nor associated with nausea , vomiting , or visual changes . three weeks before arriving at the hospital , he noted a sense of " fullness " in his ears ; he said that spoken voices sounded muffled , and he had difficulty hearing telephone conversations . when someone at the restaurant where he worked dropped a dish , he heard that sound clearly and reported that it was almost painful , causing him to become dizzy . his appetite was good , and he had not lost weight . although he reported no fever , he did report night sweats for several weeks . he had no rash , diarrhea , abdominal pain , chest pain , shortness of breath , joint complaints , or dysuria . he also said that he was exposed to dust at his workplace because of remodeling . he lived in new hampshire , had no pets , and worked as part owner of a restaurant . exposures included multiple male and female sexual partners with inconsistent condom use , and he had acquired several tattoos 8 months earlier while traveling in spain and italy . he had no known tuberculosis exposure . temperature was 36.7c , blood pressure 128/84 mm hg , pulse 80 , respirations 16/minute . sclerae were anicteric , and his pupils reacted to direct and consensual testing and responded normally to accommodation . bedside testing showed that his hearing was diminished to low volume sounds , but he was able to hear loud sounds , which he found painful . when the examiner clapped his hands loudly a few feet from the patient 's ear , the patient exhibited nystagmus . the patient 's neck was supple ; a 1.5-cm , nontender lymph node was palpated in the left upper anterior cervical chain . the remainder of the examination was unremarkable , with negative romberg test ; normal gait ; and normal motor , sensory , and reflex performance . rapid plasma reagin ( rpr ) was positive at a titer of 1:128 and was confirmed by a fluorescent treponemal antibody test . subsequent studies showed a cd4 receptor positive t - cell ( cd4 ) count of 899/ml and an hiv viral load of 878 copies / ml . a lumbar puncture showed protein 33 mg / dl , glucose 78 mg / dl , 9 erythrocytes/l , and 8 leukocytes/l ( 100% lymphocytes ) . cerebrospinal fluid venereal disease research laboratory test ( csf - vdrl ) results were negative . csf - vdrl , cerebrospinal fluid venereal disease research laboratory test ; elisa , enzyme - linked immunosorbent assay ; wb , western blot ; rt - pcr , reverse transcription polymerase chain reaction . the patient was started on 24 mu of iv penicillin per day for 14 days . at the end of this period , he reported notable improvement in his headaches , hearing loss , and vertiginous symptoms . one month after completing treatment , his rpr titer was 1:32 ; 3 months after he completed treatment , it was nonreactive . the course of syphilis in an hiv - positive patient may be altered from the natural history of the disease in hiv - negative patients . an increased frequency of ocular disease , multiple and slower resolving primary chancres , and a higher titer rpr have been reported ( 16 ) . in addition , delay or failure of titer decline after treatment , predilection for developing the jarisch - herxheimer reaction , and clinical relapse have also been described in hiv infection ( 1,4,7,8 ) . treponema pallidum is thought to invade the central nervous system in 25% of patients with syphilis , irrespective of hiv status ( 9 ) . most of these persons successfully clear the infection ( 10 ) , but other patients harbor the spirochete and remain at risk for sequelae of neurosyphilis . syphilitic meningitis , meningovascular disease ( often occurring with stroke ) , labyrinthitis , or cranial nerve palsies , as seen in this case , can be early findings . loud sounds or even routine acoustic stimuli can result in vertigo , nystagmus , or nausea and vomiting . the physiologic underpinnings of the tullio phenomenon were first described in 1929 , when tullio noted that experimentally induced fenestrations in the bony capsule of the lateral semicircular canals of pigeons caused the canals to be sound - responsive , inducing vestibular activation ( 11,12 ) . shortly thereafter , benjamins described a tullio reaction in a human patient with fistulizing cholesteatoma ( 13 ) . today , the term has been generalized to include vestibular activation in response to stimulation by sound of any part of the vestibular apparatus ( 12 ) . the tullio phenomenon is seen in a range of clinical contexts , including congenital deafness , meniere disease , suppurative middle ear disease , and spirochetal infections , such as syphilis or lyme disease . nields et al . describe a woman who had nystagmus and vertigo with routine sounds ; running tap water caused her to fall to the floor or retch in pain ( 14 ) . describe a series of patients with oscillopsia induced by pencil tapping , telephone ringing , or the sound of cutlery falling on the floor . patients often report a vague sense of ear blockage and an unpleasant awareness of their own voice vibrating in their ear ( 12 ) . symptomatic cranial nerve viii involvement in this patient prompted treatment for neurosyphilis , despite equivocal ( but typical ) csf findings . interpreting csf findings in hiv - positive patients with syphilis is challenging because commonly encountered mild lymphocytic pleocytosis may be attributable to hiv . csf - vdrl is often negative , with a 20%70% false - negative rate ( 15 ) . optimally managing patients with hiv and syphilis has been debated ; to date routine recommendations are the same as for hiv - negative persons ( 16 ) . although any patient in whom syphilis is diagnosed and who has neurologic symptoms should undergo lumbar puncture , csf evaluation should also be considered in asymptomatic patients in whom syphilis is diagnosed and who have an rpr titer > 1:32 or a cd4 + count < 350 , as these markers have been associated with increased risk for neurosyphilis ( 10 ) . because of the high rate of relapse that has been reported in some series ( 4,7,8 ) , close clinical and serologic follow - up is essential . a study of 59 patients with neurosyphilis showed that hiv - positive study participants were 2.5 times less likely to normalize csf - vdrl reactivity than hiv - negative patients . this effect was even more pronounced in patients with cd4 + counts < 200 ( 17 ) . the findings suggest that more intensive therapy for neurosyphilis in immunocompromised patients merits further study . after an all - time low case - rate of syphilis in 2000 , reports of rising trends , specifically in groups at risk , such as men who have sex with men , were reported in 2001 ( 18 ) . the outbreaks marked a 9.1% overall increased rate of primary and secondary syphilis ( 19 ) and were characterized by a high rate of hiv infection . parallel increases in hiv infection rates were a concern because of the common mechanism of transmission and the increased efficiency of hiv transmission with coexistent genital ulcers ( 20 ) . in 2001 , increases in hiv rates were reported in several states with large urban populations ; men who have sex with men accounted for the largest known subgroup at risk in incident adult or adolescent hiv cases ( 32% ) and aids cases ( 44% ) ( 21 ) . new hiv diagnoses increased in 29 states with mandatory reporting from 1999 to 2002 , notably by 17% among gay and bisexual men ( 22 ) . syphilis incidence also continued to rise in the united states in 2002 , with a 12.4% increase since 2001 ( 19 ) , and the highest trends were estimated to be among men who have sex with men . in san francisco , primary and secondary syphilis rates increased by > 1,000% from 1998 to 2002 among men who have sex with men ( 23 ) . high - risk behaviors have been documented in this group ( 24 ) and factor prominently in syphilis reemergence in the early 21st century . this case report describes a patient who had tullio phenomenon as the index symptom of neurosyphilis with previously undiagnosed hiv infection . his rpr titer was 1:128 , his cd4 count was preserved , and he responded well clinically and serologically to standard therapy . to our knowledge , this case is the first report of syphilis occurring as tullio phenomenon in an hiv - positive patient . we suggest that syphilis be considered in patients who have cranial nerve viii symptoms and an appropriate risk - factor profile . eradicating treponemes may be limited from sanctuary sites such as the cns . because control of syphilis likely depends not only on antimicrobial effect but also on host immune response , awareness of unusual symptoms of a relatively common disease will benefit not only the patient but also public health efforts in managing both syphilis and hiv infection . preventive and educational efforts focused on men who have sex with men may prove particularly important in modifying behaviors that foster the growth of both epidemics .
[ purpose ] the aim of this study was to analyze stabilometry in athletes during an indoor season in order to determine whether injured athletes show different stabilometric values before injury than non - injured athletes in two different training periods ( volume and pre - competition periods ) . [ subjects ] the subjects were 51 athletes from unicaja athletic club who trained regularly . [ methods ] at the end of the preseason and volume periods , athletes were subjected to bipodal and monopodal stabilometry . in addition , all injuries happening in the periods after performing stabilometry ( volume and pre - competition periods ) were tracked . [ results ] variance analysis of bipodal stabilometric measurements taken at the end of the preseason period showed that athletes with higher values for the center - of - pressure spread variables suffered injuries during the volume period . the right - leg monopodal stabilometric measurements taken at the end of the volume period showed that athletes with higher values in the center - of - pressure position variables suffered injuries during the pre - competition period . [ conclusion ] athletes showing the worst values for center - of - pressure spread variables are more prone to sports injuries in the subsequent training period . in monopodal measurements , athletes with poorer mediolateral stability were more prone to injuries in the subsequent training period .	postural stability is deeply linked with sports injuries , and copious evidence exists indicating that postural stability is greatly reduced by sports injuries . back in 1965 , freeman first described stabilometric alterations in patients suffering from ankle sprain and correlated lateral instability of the ankle with a lack of postural control1 . after freeman , several authors analyzed the effects of sports injuries on postural stability of athletes and concluded that its deterioration may cause re - injury or even new injuries2 , 3 . in addition to previous injuries , postural stability may be affected by changes in the level of activity , as well as by its type , intensity , and volume4,5,6 , which constitute a risk of injury for athletes7 . some authors have carried out prospective assessments of athletes in order to determine how stabilometric changes can be a causal factor for injuries8 . assessed the first two weeks of the season for basketball players , and correctly described balance as a predictor of injuries , showing that those with higher postural sway values at the start of the season were the ones most injured in the long term9 . similar results were found by wang et al . , who established a correlation between poor mediolateral stability and suffering from ankle injuries later on10 . likewise , trojian and mckeag showed in 2006 that the ability to maintain single - leg balance of athletes at the beginning of the preseason was a predictor of ankle sprains for the autumn season11 . also , soderman et al . correlated all lower extremity injuries with increases in postural sway in female soccer players12 . from a physiological point of view , murphy et al . affirmed in 2003 that the main cause of deteriorated postural stability as a risk factor is an alteration in the neuromuscular control strategy , which increases intersegmental joint forces and consequently increases the development of forces involving ligaments , tendons , and muscles13 . in spite of the findings reported by these authors , whether postural stability is hopper et al . analyzed single - leg stability by assessing the duration for which female netball athletes could maintain a unilateral posture ; however , they did not find any correlation between postural stability and an increase of injuries14 . similar results were reported by beynnon et al . in soccer , field hockey , and lacrosse athletes15 . based on the controversy existing regarding postural stability as an injury predictor and the evidence of effects of training on postural stability and considering that to date , no studies have taken into account the type of training as a cause of postural stability deterioration in their prospective investigations , the aim of the present study was to analyze the stabilometry in athletes during their training stage in order to determine whether injured athletes scored differently in stabilometric tests than those who were injury - free in two different training periods . this was a five - month descriptive prospective study ( september january ) that was divided into two parts according to the different training periods . the first part consisted of bipodal and monopodal stabilometry tests at the end of preseason ( in september , mainly general exercises ) and a record of injuries suffered by the athletes during the subsequent period of training , which was volume period ( in october and november , with training based on alternating general and specific high - volume , low - intensity exercises ) . the second part consisted of bipodal and monopodal stabilometry tests at the end of volume period and a record of injuries suffered by the athletes during the subsequent period of training , which was pre - competition period ( in december and january , with an majority of specific low - volume , high intensity exercises ) . in order to avoid interference from previous injuries , a total of 51 track and field athletes who were from an athletic club in the city , were 17 to 35 years old and had at least three years of experience took part in the first part of the present study , after excluding those who did not train regularly or who had been injured during the preseason period . in the second part of the study , athletes who had been injured previously were excluded in order to avoid interferences in the results . therefore , 39 athletes between the ages of 17 and 35 participed in this part of investigation . anthropometric and demographic data are shown in table 1table 1 . statistical description of the samplefirst part of the study n=51second part of the study n=39injured in volume period n=12non - injured n=39injured in pre - competition period n=6non - injured n=33age ( years)23.28.022.37.526.59.021.67.0height ( m)1.740.071.740.081.750.051.740.09weight ( kg)61.011.062.911.761.57.963.212.4bmi ( kg / m)20.12.420.72.520.11.520.82.7gendermen866.7%2769.2%583.3%2266.7%women4 33.3%12 30.8%1 16.7%11 33.3%experience ( years)7.84.77.24.610.04.86.74.4quantitative variables are shown as means and sd . p values are from the student s t - test and test , respectively . p values are from the student s t - test and test , respectively . bmi : body mass index before the start of the study , all athletes were briefed on how they would be tested , and written informed consent was obtained from each subject or from their legal guardians in the case of underage athletes according to the standards of the declaration of helsinki ( 2008 revision)16 . weight and height measurements were performed with a tefal ( france ) digital precision scale ( 100 g300 kg ) and an asimed t201-t4 ( spain ) measuring rod , respectively . for stabilometric measurements , a modular electronic baropodometer was used comprising a 120 160 cm sensormdica ( spain ) platform with 19200 active sensors . athletes were subject to a bipodal and a monopodal stabilometric measurements once at the end of each period . for the bipodal test , athletes were instructed to remain as still as possible on the baropodometric platform for 52 seconds , with a between - heels separation of 5 cm and their feet forming a 30 angle . for the monopodal test , athletes stood on each of their lower limbs for 15 seconds ( left leg first ) at the center of the platform ( fig . the following parameters were recorded for the bipodal test as well as for the left - leg and right - leg monopodal tests : length ( length ) and area ( area ) of the path described by the center of pressure , the speed of center of pressure movement ( speed ) , and the position of the center of pressure in the mediolateral ( xmean ) and anteroposterior ( ymean ) planes . r to indicate whether they belong to the left or right leg , respectively . tests were carried out before training started , in order to avoid any interference . also , athletes were instructed not to engage in any sports activity on the day of testing . bipodal , left monopodal and right monopodal stabilometry tests in order to track injuries , athletes were instructed in advance and then interviewed weekly by a sports physical therapist during the volume and pre - competition periods in order to record any musculoskeletal injury on the lower limbs they might have sustained during these periods . injury was defined as physical damage that resulted in missing or modifying one or more training sessions or competitions ( kolt et al . a description of data included means and standard deviations for the categorical variables ( table 1 ) . differences in social and demographic variables between the injured and non - injured athletes each period ( volume and pre - competition ) were analyzed by means of the student s t - test for independent samples in the case of the continuous variables and a test for the categorical variables . for comparison between stabilometric variables between groups ( injured and non - injured ) , the student s t - test for independent samples was used . a significance level of p0.05 was set for all statistical procedures , and the spss v. 19 software was used . table 1 shows social and demographic statistics for the injured and non - injured subjects during the volume and pre - competition periods . there was a similar number of injured athletes in both periods . no differences were apparent in the social and demographic characteristics of the injured and non - injured athletes ( p>0.05 ) . mean values of stabilometric variables in bipodal and monopodal support taken at the end of the pre - season periodtotal injuredin volume period n=12non - injuredn=39preseason measuresmeansdmeansdlength ( mm)366.5183.9340.2104.0area ( mm)36.527.147.549.9speed ( mm / sec)7.43.96.72.0xmean ( mm)3.94.74.43.2ymean ( mm)4.54.16.05.1lengthl ( mm)272.0112.5267.865.5areal ( mm)419.1493.7408.7350.8speedl ( mm / sec)24.411.424.76.8xmeanl ( mm)4.93.66.013.5ymeanl ( mm)6.65.1410.28.2lengthr ( mm)255.6119.9275.582.8arear ( mm)340.2282.9378.2298.8speedr ( mm / sec)23.112.024.77.9xmeanr ( mm)3.42.93.02.8ymeanr ( mm)7.06.58.65.2results are split for athletes injured during the volume period.length= length of the path described by the center of pressure . area = area of the path described by the center of pressure . speed = speed of the center of pressure . the first section is for bipodal support , the second section is for the left foot ( indicated by addition of l to each variable ) , and the third section is for the right foot ( indicated by addition of r to each variable ) . * p<0.05 shows mean values for the center - of - pressure spread and position variables in both bipodal and monopodal support at the end of the preseason period for the athletes who were injured and non - injured during the volume period . bipodal stabilometry tests showed that those injured during the volume period had scored significantly higher in length and speed than those who were not injured ( p=0.009 and p=0.003 , respectively ) . the first section is for bipodal support , the second section is for the left foot ( indicated by addition of l to each variable ) , and the third section is for the right foot ( indicated by addition of r to each variable ) . mean values of stabilometric variables in both bipodal and monopodal support at the end of the volume periodtotal injuredin pre - competition n=6non - injuredn=33volume measuresmean sdmean sdlength ( mm)310.1131.8307.7143.6area ( mm)128.5103.198.8109.6speed ( mm / sec)6.22.813.542.0xmean ( mm)4.51.85.75.0ymean ( mm)8.72.712.49.4lengthl ( mm)335.395.9316.3127.4areal ( mm)615.0512.0489.6506.8speedl ( mm / sec)31.19.635.340.2xmeanl ( mm)3.52.55.812.3ymeanl ( mm)10.87.016.311.6lengthr ( mm)349.089.3322.7132.8arear ( mm)631.1328.3608.1624.0speedr ( mm / sec)31.99.436.740.2xmeanr ( mm)15.230.34.33.6ymeanr ( mm)12.06.815.39.6results are split for athletes injured during the pre - competition period.length= length of the path described by the center of pressure . area = area of the path described by the center of pressure . speed = speed of the center of pressure . the first section is for bipodal support , the second section is for the left foot ( indicated by addition of l to each variable ) , and the third section is for the right foot ( indicated by addition of r to each variable ) . p<0.05 displays the mean values for the center - of pressure spread and position variables in both bipodal and monopodal support at the end of the volume period for the athletes who were injured and non - injured during the pre - competition period . right - leg monopodal stabilometry showed that injured subjects scored significantly higher with respect to xmeanr ( p=0.041 ) . left - leg monopodal and bipodal stabilometry did not show any statistically significant difference ( p>0.05 ) . the first section is for bipodal support , the second section is for the left foot ( indicated by addition of l to each variable ) , and the third section is for the right foot ( indicated by addition of r to each variable ) . the aim of the present study was to analyze stabilometric values of athletes through their training in order to determine their value as a predictor for injuries in each of the training periods . to this end , athletes were subjected to a bipodal and monopodal stabilometry at the end of the preseason period and a tracking of their injuries in the subsequent volume period ( first part ) . in addition , bipodal and monopodal stabilometry was carried out at the end of volume period , and tracking of their injuries was carried out in the subsequent pre - competition period ( second part ) . the results obtained from stabilometry at the end of the preseason period show that athletes with poorer values for center - of - pressure length and speed in bipodal support at the end of the preseason were the ones who were injured in the two subsequent months , which comprised the volume period . although these findings agree with previous studies in which high variation of postural sway correlated or predicted posterior sports injuries9 , 10 , 12 , our results did not show any significant difference in monopodal stability , which differ from most of the previous studies . mcguine et al . observed in 2000 that basketball players with poorer preseason unilateral balance values were seven times more prone to ankle sprains during the season9 , and soderman et al . reported similar findings for female soccer players , with the ones with lower scores for unilateral postural balance being more prone to leg - related injuries12 . similar results were found for unilateral stability of athletes by watson et al . , who assessed the monopodal postural sway by making soccer players maintain unilateral balance for 15 seconds , and those who were not able to perform it without touching down were classified as having abnormal postural sway . this group was the most affected by posterior ankle sprains19 . also , our findings contrast with the study of trojian and mckeag in 2006 , in which they found a positive association between monopodal support stability and ankle sprains11 . in addition , the results of stabilometric tests carried out in the volume period show that athletes with worse center - of - pressure values in the mediolateral plane while performing during right - leg monopodal support were more commonly injured during the two subsequent months , which comprised the pre - competition period . in spite of the fact that these results were found in monopodal stabilometry instead of bipodal stabilometry , these findings show the same trend ilustrated by the previous values of center - of - pressure spread found during the first part of the present study , in which injured athletes exhibited stability deterioration . besides , these results support the research by mcguine et al . and soderman et al . , who reported in basketball players and soccer players , respectively , poorer unilateral stabilometric center - of - pressure values in subjects who were later found to be injured more frequently9 , 12 . these results are also in accord ance with those of trojian and mckeag , who associated worse monopodal stabilometry with later ankle sprains in athletes11 , and agree with those of wang et al . , who established in 2006 a correlation between poorer center - of - pressure mediolateral position and later ankle injuries10 . on the other hand , in 2001 and 1995 , respectively , beynnon et al . and hopper et al . did not find data to support the value of postural balance as a predictor of ankle sprains14 , 15 , which differs from the rest of the studies and the present investigation . the same procedure was used by willems et al . in 2005 , who did not find significant differences in postural stability variables ; however , these authors discovered that subjects with weaker control over their center of gravity were more prone to later suffering ankle sprains20 . similar trends were seen for the results of both forms of stabilometric testing performed in the preseason period and the number of athletes injured in the volume period compared with the testing performed in the volume period and the number of athletes injured during the pre - competition period . both sets of data point to athletes with higher values ( and therefore poorer scores ) for stabilometric variables being more prone to injuries in the subsequent periods . however , higher stabilometric variables were found for the bipodal stance in the first part , whereas higher stabilometric variables were found for the monopodal stance in the second part . based on the difference between the results of the parts of the present study , it is important to take into account that the injuries that occurred during the pre - competition period could have been influenced by other risk factors such as higher intensity of trainings or explosive actions , which are thought to be actions involving more damage21 and characteristic of this training period . also , it is important to indicate that the present investigation is the only study to date in which all lower extremity injuries were correlated with increases in postural sway . in addition , certain limits of our study may have played a part in determining the lack of more significant differences . the low number of injuries recorded as a consequence of the small size of the sample might conceivably have contributed to this discrepancy in results . indeed , the difference in number of athletes participating in both parts of the study could have affected the results due to the small size of the sample . further research with a larger sample size is recommended , as well as inclusion of other age groups . in conclusion , athletes who show higher center - of - pressure spread during bipodal stabilometry at the end of the preseason are more prone to injuries during the subsequent training period . also , athletes who exhibit poorer stability in the mediolateral plane when performing right - leg monopodal support at the end of the volume period are more prone to injuries in the subsequent training period . as a practical application , inclusion of specific proprioceptive training in the training routines of athletes is recommended in order to improve stabilometric parameters and decrease and/or eliminate their role as risk factors .
summary introduction : in the dentistry practice , dentists are exposed to harmful effects caused by several factors , such as the noise produced by their work instruments . in 1959 , the american dental association recommended periodical hearing assessments and the use of ear protectors . aquiring more information regarding dentists ' , dental nurses ' , and prosthodontists ' hearing abilities is necessary to propose prevention measures and early treatment strategies . objective : to investigate the auditory thresholds of dentists , dental nurses , and prosthodontists . method : in this clinical and experimental study , 44 dentists ( group i ; gi ) , 36 dental nurses ( group ii ; gii ) , and 28 prosthodontists ( group iii ; giii ) were included , , with a total of 108 professionals . the procedures that were performed included a specific interview , ear canal inspection , conventional and high - frequency threshold audiometry , a speech reception threshold test , and an acoustic impedance test . results : in the 3 groups that were tested , the comparison between the mean hearing thresholds provided evidence of worsened hearing ability relative to the increase in frequency . for the tritonal mean at 500 to 2,000 hz and 3,000 to 6,000 hz , giii presented the worst thresholds . for the mean of the high frequencies ( 9,000 and 16,000 hz ) , gii presented the worst thresholds . conclusion : the conventional hearing threshold evaluation did not demonstrate alterations in the 3 groups that were tested ; however , the complementary tests such as high - frequency audiometry provided greater efficacy in the early detection of hearing problems , since this population 's hearing loss impaired hearing ability at frequencies that are not tested by the conventional tests . therefore , we emphasize the need of utilizing high - frequency threshold audiometry in the hearing assessment routine in combination with other audiological tests .	noise is one of the most harmful agents for health , primarily for hearing , and it is often unavoidable at workplaces and entertainment venues . noise - induced hearing loss ( nihl ) , which is hearing loss that is induced by high levels of sound pressure , is one of the most common occupational diseases . currently , noise is a constant part of people 's daily activities , as it is present in traffic , leisure , and work ; therefore , nihl may become one of the main chronic diseases in the future ( fiorini , 2000 ) . nihl is defined as sensorineural hearing loss that occurs due to systematic occupational exposure to high levels of sound pressure , thus causing damage to the hair cells of the organ of corti . generally , this hearing loss is bilateral and symmetrical , insidious and irreversible , and is directly related to the period of exposure and individual susceptibility ( brazilian national noise and hearing preservation committee , 1999 ; rabinowitz , 2000 ) . this hearing disturbance is initially manifested at the frequencies of 6,000 hz , 4,000 hz , and 3,000 hz , and broadens progressively to the frequencies of 8,000 hz , 2,000 hz , 1,000 hz , 500 hz , and 250 hz . noise rarely leads to profound hearing loss , which generally does not exceed 75 db for high frequencies and 40 db for low frequencies , and reaching its upper limit in the first 10 to 15 years of exposure ( luxon , 1998 ; hanger , barbosa - branco , 2004 ; gatto et al . , generally , professionals only notice a hearing difficulty when the lesion is at an advanced stage , as the hearing symptoms are insidious and manifest late ( sava , 2005 ) . continuous exposure to high levels of sounds may not only result in hearing damage , but also in a few secondary alterations , such as tinnitus , stress , physiological alterations to the heart rhythm and to blood pressure , as well as difficulty in discriminating speech sounds , especially in noisy environments . noise causes physical exhaustion , chemical , metabolic , and mechanic disturbances to the sensory hearing organ , thus resulting in partial or total hearing loss of the organ of corti , which is located in the internal part of the ear . ( otoni a , boger me , barbosa - branco a , shimizu he , maftum ma , 2008 ) . dentists are typically exposed to 2 types of noise : external noise from the work environment and noise from their own work equipment , such as the noise from high and low rotation motors , compressors , air conditioners , amalgamators , aspirators , and others ( hinze , deleon , and mitchel , 1999 ) . in addition to noise , they are exposed to other factors , including chemicals ( manipulated substances , especially mercury ) , biological agents ( the oral cavity is full of microorganisms and the risk of contracting diseases such as hepatitis and aquired immune deficiency syndrome ( aids ) is high ) , mechanical agents ( body lesions caused by to the instruments that are used ) , social stresses ( having occupation that involves tension and requires mastery of the situation to facilitate the patient / professional relationship ) , and ergonomic challenges ( due to the body position while working , the professional is subjected to back and arm problems and varicose veins ) ( souza , 1997 ) . according to presta et al . ( 2004 ) dentists have frequently presented with discomforts that are related to the nature of their profession , which may progress to repetitive strain injuries or work - related musculoskeletal disorders . in 1959 , the american dental association ( ada ) had already recommended periodic hearing assessments for dentists , due to their prolonged exposure to high - frequency sounds , which were caused by instruments like high speed drills , ejection systems , ultrasound equipment , model clippers , and instruments with high suction and vibration speeds , which can lead to hearing loss . studies ( altinoz et al . , 2001 ; fernandes et al . , 2004 ) that measured the noise levels in dental practices observed noise levels that were higher than 80 db sound pressure level ( spl ) . in brazil , law number 6.514 of the brazilian labor code ( consolidao da leis do trabalho ; clt ) , relates the parameters that provide acoustic comfort for practitioners with the norms of the brazilian association of technical norms ( abnt ) . the brazilian employment legislation considers the maximum tolerable level of noise to be 85 db spl in an 8-hour shift ( segurana e medicina do trabalho , 1991 ) ; the brazilian standard ( nbr ) 10.152 indicates that this level should be between 35 and 45 db ( a ) for a dental office . paraguay ( 1999 ) verified that dentists with 5 or more years of practice presented hearing disturbances by tonal threshold audiometry . leggat ( 2000 ) stated that the reason that more experienced dentists suffer from hearing alterations may be due to previous exposure to older equipment . however , this possibility was not considered in this study , as the study population was relatively young . previous studies ( oliveira et al . , 2007 ; torrs et al . , 2007 ; melo et al . , 2008 ) have shown that dental professionals should be aware of occupational noise , as well as of the harmful consequences that it can have on their health . this understanding should begin early , during the undergraduate course , when the professionals are being educated , so that , aware of the risks that they may be exposed to , they will be able to prevent them , instead of attempting to lessen or treat the problems caused by them . in brazil , the prevention of occupational diseases and occupational accidents began with the clt in 1943 , and ever since , the attention to hearing in noisy work environments has intensified . with the promulgation of ordinance number 3.214/78 there was an important improvement in the scope of hearing conservation efforts , by means of the control program occupational health medical - pcmso ( nr-7 ) , which made the tonal threshold audiometry mandatory . the interest in early diagnosis has increased , and considering the progress of the technology that is aimed at the diagnosis of hearing loss , as well as the prevalence of hearing alterations even in the absence of complaints , which are commonly found by tonal threshold audiometry , other methods have been used to identify hearing alterations at an early stage . according to previous studies , high - frequency ( between 9,000 and 18,000 hz ) tonal audiometry is an instrument that is effective for the early diagnosis of hearing problems due to exposure to noise ( porto et al . ( 2004 ) that investigated hearing at conventional frequencies and high frequencies in dental practices have shown a tendency toward lower thresholds , suggesting that significant hearing problems accumulate with time . as the frequency , time of exposure , and age increases these studies also observed a greater incidence of hearing loss in the frequencies of 6,000 hz and 14,000 hz . lopes and godoy ( 2006 ) compiled a literature review regarding the importance and contribution of high - frequency audiometry for the early identification of nihl . they demonstrated that conventional tonal threshold audiometry alone may not be effective in the identification or prevention of nihl , and also described the methodological variables for its proper execution . the authors also suggested this method should be added to the routine that is indicated by the occupational hearing loss prevention program . therefore , the purpose of this study was to investigate the hearing thresholds at conventional and high frequencies , thus enabling early prevention of hearing loss and and improving the overall hearing health of the population as a whole . this study began after the approval of the ethics committee of the university of so paulo under process number 043/2007 . this research study was financed by the so paulo research foundation ( fapesp ) funding agency under process number 2007/01074 - 7 . this was a cross - sectional study with 108 volunteer participants the bauru community , which were divided into 3 experimental groups . group i ( gi ) consisted of 44 dentists ( 16 males and 28 females ) aged between 23 and 57 years ( average of 34 years of age ) . group ii ( gii ) consisted of 36 female dental nurses , aged between 21 and 59 years ( average of 38 years of age ) . lastly , group iii ( giii ) consisted of 28 prosthodontists ( 17 males and 11 females ) aged between 17 and 53 years ( average of 35 years of age ) . professionals from private dentistry offices or laboratories , universities in bauru , so paulo , and hospitals that employ dentists on their staff were invited to participate . the participants initially received clarifications regarding the purpose of this study , which only began after the agreement to participate and the signature of the informed consent were obtained . for the inclusion and exclusion criteria , only professionals in the dentistry field with at least 2 years of experience , who did not present any pre - existing diseases such as mumps , high blood pressure , diabetes , meningitis , human immunodeficiency virus ( hiv ) , syphilis , and other conditions that can compromise hearing and/or pre - existing hearing impairment were considered . all the participants were underwent : a specific interview and middle ear inspection : these procedures were performed to investigate the individual features such as age , time in the profession , noisy entertainment habits , exposure to chemical products , as well as health conditions and other diseases that can aggravate the effect of environmental risks . conventional tonal threshold audiometry ( 250 to 8,000 hz ) , high - frequency tonal threshold audiometry ( 9,000 to 16,000 hz ) , and speech reception threshold tests were performed using a siemens sd 50 audiometer . for the tests of the tonal thresholds the warble tone was used , which was presented using aural headphones ( hda 200 ) . the hearing threshold was established when there was a 50% positive answer for sound detection ( lopes and godoy , 2006 ) . acoustic impedance test : the acoustic immitance measures and the ipsilateral and contralateral acoustic reflex tests of the stapedius muscle were performed with gsi tymp star the data analysis from the specific interview revealed that 65 participants were bothered by noise at work , 50 participants reported difficulties in speech comprehension , 8 had served in the army , 11 had acoustic trauma , 32 were exposed to chemical products , and 35 referred to being exposed to noise during recreational activities . graph 1 shows the mean hearing thresholds for all of the frequencies that were tested in the 3 groups . hearing thresholds of the right ear in all of the tested groups . hearing thresholds of the left ear in all of the tested groups . the comparison between the mean hearing thresholds of each frequency that was tested in the 3 groups was performed using the kruskall - wallis test , and considered significant by the dunn test . we obtained a statistically significant difference in the right ear for the frequencies of 2,000 hz ( p = .0446 ) , 8,000 hz ( p = .0492 ) , and 16,000 hz ( p = .0441 ) when the mean of group i ( mean threshold at 2,000 hz = 5.91 db , at 8,000 hz = 11.59 db , and at 16,000 hz = 21.59 db ) was compared to the mean of group ii ( mean threshold at 2,000 hz = 10.69 db , at 8,000 hz = 18.61 db , and at 16,000 hz = 32.78 db ) ; hence , we verified that gii presented worse thresholds at the frequencies of 2,000 hz , 8,000 hz , and 16,000 hz in the right ear compared to gi . it can observed that the right and left ears presented similar configurations in conventional and high - frequency audiometry , when the mean hearing thresholds for all of the groups is considered ; however , the right ear presented worse hearing thresholds than the left ear . for the left ear , the frequencies of 4,000 hz ( p = .0238 ) and 6,000 hz ( p = .0310 ) presented statistically significant differences between gi ( mean threshold at 4,000 hz = 8.41 db and at 6,000 hz = 14.32 db ) and gii ( threshold mean at 4,000 hz = 14.03 db and at 6,000 hz = 20.69 db ) and between gi and giii ( mean threshold at 4,000 hz = 15.36 db and at 6,000 hz = 22.32 db ) . at the frequency of 9,000 hz in the left ear , there was a statistically significant difference ( p= .0397 ) between gi ( mean theshold at 9,000 hz = 10.91 ) and gii ( mean threshold at 9,000 hz = 20.28 db ) . the comparison of the mean hearing thresholds at the frequencies of 500 hz to 2,000 hz , 3,000 hz to 6,000 hz , 9,000 hz to 16,000 hz , and 12,000 hz to 16,000 hz was performed using the kruskall - wallis test , and the statistical significance was determined by the dunn test . for these comparisons , a statistically significant difference ( p = .0147 ) was obtained only when the mean thresholds between the frequencies of 3,000 hz to 6,000 hz in the left ear between gi ( 10 db ) and gii ( 15.93 db ) and those between gi ( 10 db ) and giii ( 16.25 db ) were compared . the speech audiometry , which was performed using the speech recognition threshold , confirmed the results of the conventional audiometry in 100% of the participants , as did the speech recognition index , which was compatible with the hearing thresholds found in 100% of the participants . for the acoustic immitance and evaluation of the ipsilateral and contralateral stapedius muscle reflexes , normal tympanograms were obtained bilaterally in 100% of the participants , thus indicating that the middle ear did not interfere with the results that were obtained . this study focused on a sample of dentists , dental nurses , and prosthodontists with more than 2 years of experience in the dentistry field and with an average age of 35 years among the 3 groups . in this population , hearing loss can occur due to prolonged exposure to high - frequency sounds that are produced by the instruments that are used in their daily practice , which results in subsequent handicaps to their communication and quality of life ( hinze , deleon , and mitchel , 1999 ) . this study showed that only 60% of the participants were bothered by noise at work ( souza , 1997 ; trres et al . , 2007 ; melo et al . , 2008 ) , that 43.3% of the participants reported difficulties in speech comprehension , and that 32.4% mentioned that they were exposed to noise during recreational activities . this study revealed that the mean hearing thresholds of all of the groups worsened according to increases in frequency ( matthews et al . , 1997 ; beltrami , 1999 ; fernandes and mota 2001 ; mota , 2002 ; porto et al . , 2004 ; silva and feitosa , 2006 ; lopes et al . , 2006 ; lopes and godoy , 2006 ; lopes , almeida , zanconato , and mondelli , 2007 ; carvalho , koga , carvalho , and ishida , 2007 ) . for all of the groups , both conventional and high - frequency audiometry of each ear showed similar configurations ; however , by comparing both ears in the 3 groups , we observed that the right ear showed worse mean thresholds than the left ear . these results are in agreement with zubick , tolentino , and boffa ( 1980 ) , and are in disagreement with gijbels et al . ( 2006 ) , who gathered data on the effects of occupational health among dentists and observed that hearing loss was greater for the left side for right - handed professionals , which can be explained by the short distance between the left ear and the circular motion / vibration equipment for right - handed professionals . the tritonal means of 500 to 2,000 hz and 3,000 to 6,000 hz revealed that prosthodontists ( gii ) had the worst hearing thresholds . for the high - frequency mean ( 9,000 to 16,000 hz ) the dental nurses ( gii ) had the worst hearing thresholds . these results highlight the importance of using complementary tests in the hearing evaluation ( which in this case was high - frequency tonal threshold audiometry ) , and that if these tests are used as a routine procedure in the evaluation of these professionals it would assist in the early detection and prevention of hearing problems . in this study , we concluded that the conventional hearing assessment did not identify hearing problems in the 3 groups that were tested . however , the assessments of the high - frequency thresholds indicated disturbances to the peripheral auditory system , specifically on the external hair cells , thereby demonstrating greater sensitivity for the early detection of hearing problems and favoring the use of complementary tests as prevention tools .
visceral leishmaniasis , a vector - borne tropical disease that is threatening about 350 million people worldwide , is caused by the protozoan parasite leishmania donovani . metalloids like arsenic and antimony have been used to treat diseases like leishmaniasis caused by the kinetoplastid parasites . arsenic ( iii ) at a relatively higher concentration ( 30 g / ml ) has been shown to have antileishmanial activity , but this concentration is reported to be toxic in several experimental mammalian systems . nanosized metal ( 0 ) particles have been shown to be more effective than their higher oxidation state forms . there is no information so far regarding arsenic nanoparticles ( as - nps ) as an antileishmanial agent . we have tested the antileishmanial properties of the as - nps , developed for the first time in our laboratory . as - nps inhibited the in vitro growth , oxygen consumption , infectivity , and intramacrophage proliferation of l. donovani parasites at a concentration which is about several fold lower than that of as ( iii ) . moreover , this antileishmanial activity has comparatively less cytotoxic effect on the mouse macrophage cell line . it is evident from our findings that as - nps have more potential than as ( iii ) to be used as an antileishmanial agent .	visceral leishmaniasis or kala - azar , an infective disease encountered in tropical and subtropical regions of the world including indian subcontinent , is caused by protozoan parasite , leishmania donovani . l. donovani has two morphological forms in its life cycle : the motile flagellated slender promastigotes that stay inside the midgut of sandfly vector and the immotile nonflagellated oval amastigote present in the phagolysosomal complex of mammalian host macrophages . the survival and proliferation of the amastigotes followed by the reinfection of new macrophages by evading the host immune machinery ensure the progression of the disease in the mammalian host . overall annual prevalence of the disease is approximately 12 million people and the size of the population at risk is approximately 350 million . it is estimated that about 500,000 cases of visceral leishmaniasis occur worldwide each year [ 14 ] . two closely related metalloids like arsenic and antimony are the drugs of choice to treat diseases caused by the kinetoplastid parasites . antimony and arsenic are members of the same group , xv , of the periodic table and are transported into cells by the same channels , carriers , and pumps . interestingly , arsenic - resistant leishmania species show cross resistance to antimony [ 7 , 8 ] . previously , it has been shown that 30 g / ml of sodium arsenite has antileishmanial activity . however , this concentration of arsenic is reported to be toxic in several systems [ 10 , 11 ] . the selectivity of these arsenical drugs towards trypanosomes rather than the host is apparently conferred by the aromatic ring structure [ 12 , 13 ] . nowadays , nanomedicine is rapidly evolving as an extensively studied potent therapeutic tool [ 15 , 16 ] . various exciting new works were done throughout the world in the interdisciplinary field of therapy for leishmaniasis using nanomedicine [ 1720 ] . so far , only a few reports are known describing the usage of metal nanoparticles as antileishmanial agents [ 2123 ] . despite usages of arsenic ( iii ) form as antileishmanial agent , use of arsenic nanoparticle ( as - nps ) is yet to be reported . in our laboratory , for the first time , stable yellowish - brown colored spherical as - nps have been synthesized . the aim of this study is to investigate whether the antileishmanial efficacy of as is enhanced in its nanoparticulate form . here , we have tested the effect of as - nps on the growth , oxygen consumption , infectivity , and intracellular proliferation of this parasite at a concentration which is manyfold less than that of as ( iii ) . unless otherwise mentioned , all biochemicals such as fcs , antibiotics , and g418 were of highest quality and were purchased from sigma chemical co. ( usa ) . promastigotes of l. donovani ( mhom / in/83/ag83 ) were cultured at 23c in rpmi 1640 media containing 10% heat inactivated fcs ( fetal calf serum ) , 25 mm hepes , 2 mm l - glutamine , 50 u / ml penicillin , and 50 g / ml streptomycin . parasites were inoculated at a density of 1 10 cells / ml and grown for 3 days to obtain exponentially growing log phase cells , which were used for subculture of the parasites . elemental grey arsenic ( 0 ) , in the bulk stage ( as4 ) , is solid and insoluble in aqueous solution . under ordinary condition thus , the effect of elemental arsenic ( 0 ) is difficult to evaluate in an aqueous media ; that is , why arsenic ( 0 ) can not be applied in the cell culture medium . arsenic ( 0 ) is made water soluble only by making it a nanoparticle . as - nps used in this work were prepared using a simple wet - chemical procedure and characterized by sem and tem as published earlier . briefly , in a typical procedure , 1.0 ml of naaso2 ( loba chemicals ) having concentration 1.0 10 m was mixed with 12 ml of distilled water , and , to this solution , 0.6 ml of ice - cold nabh4 solution was added having concentration 1.0 10 m . the mixture was allowed to stand at room temperature for 2 hours when yellowish - brown colored as ( 0 ) solution appeared . the solution was further heated to ~60c for 15 minutes and cooled to room temperature . sem , tem , and dls ( malvern ) measurements of the as ( 0 ) solution indicated that the particles were of spherical shape , and the calculated average particle size was 76 nm ( see supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2014/187640 ) . the dls measurement was carried out in a malvern nano zs instrument equipped with a 4 mw he - ne laser ( = 632.8 nm ) according to manufacturer 's protocol . the zeta potential of the solution has been measured and found to be 18.1 mv , indicating that the particles are negatively charged . parasites were inoculated at a density of 0.5 10 cells / ml and incubated at 23c in 5 ml rpmi-1640 media for 24 hours . following incubation , 0.6 10 cells / ml were incubated further without and with different concentrations of as - nps . promastigote numbers were counted at an interval of 24 hours for 2 consecutive days in a neubauer improved counting chamber ( haemocytometer ) using a phase contrast microscope . ic50 value was determined by plotting log of cell numbers after 24 hours of incubation with as - nps versus concentrations of as - nps used . briefly , log phase leishmania cells ( 1 10 cells per each well ) in 96-well plate were incubated without or with indicated concentrations of as - nps or as ( iii ) solution in growth media at 23c for defined time intervals . after the treatment , 10 l mtt solution ( cck-8 , sigma ) was added to each well , following which , the readings were collected at an interval of one hour up to three hours with a microplate reader ( biorad ; model no . 5000 raw 264.7 cells were seeded in each well of a 96-well plate and incubated for 24 hours at 37c . in the following day , these cells were treated for 24 and 48 hours with indicated concentrations of as - nps in fresh media at 37c . after the treatment , 100 l fresh media was added to each well followed by 10 l of mtt reagent ( cck-8 , sigma ) , following which , the readings were collected at an interval of one hour up to four hours with a microplate reader ( biorad ; model no . the quantitation of the viable cells in control and sets treated with different concentrations of as - nps was obtained from these average values ( o.d./o.d . a standard curve was generated to determine the equivalence of the cell numbers and o.d . p values were determined using paired t - test between untreated sets and sets treated with as - nps . transformations of empty egfp ( enhanced green fluorescence protein ) plasmid into l. donovani promastigotes were performed by electroporation with a bio - rad gene pulsar apparatus using 450 v and 550 f capacitance . briefly , late log - phase promastigotes ( 0.51.0 10 ) were harvested at 1200 g ( 4c ) for 10 min and washed twice in electroporation buffer ( 21 mm hepes , 137 mm nacl , 0.7 mm nah2po4 , and 6 mm glucose , ph 7.4 ) . cells were finally suspended at a density of 1 10/ml and 0.40 ml was taken into a 0.2 mm ice - chilled electroporation cuvette . thirty micrograms of plasmid dna dissolved in 40 l of electroporation buffer was then added to the cuvette and incubated on ice for 10 min . the cells were incubated for another 10 min on ice and added to 10 ml of drug - free growth medium ( rpmi-1640 ) . after 24 hours of revival , 20 g / ml g418 was added and the cells were kept at 26c for another 10 days with mild shaking . the presence of transfected cells was monitored visually by microscope ( supplementary figure 2 ) and the drug concentration was increased gradually with each passage . finally , all the transfected cells were maintained in 300 g / ml of g418 . consumption of oxygen by parasites was measured by gilson oxygraph ( model : gilson oxygraph 56 ) according to the procedure published earlier . parasites inoculated at a density of 1 10 cells / ml were incubated at 37c for 24 hours . from there , 1 10 cells were further incubated without or with indicated concentrations of as - nps or as ( iii ) for defined time periods . following incubation , 1 10 parasite cells either treated or untreated were taken in pbs and transferred in the cell of the oxygraph to record the oxygen consumption . total oxygen dissolved in pbs inside the oxygraph cell was measured by the addition of sodium metabisulphite and is expressed as 100% . averages of three independent measurements of percent of oxygen consumed by the parasite cells were presented graphically . raw 264.7 cell line ( atcc tib-71 ) that was used for the purpose of infection studies with leishmania donovani promastigotes is a transformed murine macrophage cell line routinely used by several researchers in this field . parasites stably expressing egfp ( parasite / macrophage = 20 : 1 ) . after 4 hours of incubation for internalization at 37c , the coverslips were washed twice with media to remove nonphagocytosed promastigotes . the plate containing coverslips were then incubated without or with indicated concentrations of as - nps or as ( iii ) at 37c for indicated period of time . infected cells and the parasite positive dots within the infected cells were visualized in gfp and uv filters ( for dapi ) in the nikon inverted fluorescence microscope ( nikon eclipse ti - u ) . pictures were taken in tiff image format and were processed and merged in adobe photoshop cs5 . from the images , total and infected macrophage cell numbers were counted . the numbers of gfp positive punctate dots representing internalized parasites ( amastigotes ) were also determined . the numbers of dots per 100 infected cells thus obtained were plotted against corresponding concentrations of as - nps and the ic50 value was determined accordingly from the graph . to investigate the attachment after addition of the parasites to the macrophages , subsequent processing was similar to that of the infected sets . to see the curing effect of these nps , as - nps ( 2 m ) were added to the 24-hour postinfected macrophages and were further incubated for 24 and 48 hours . numbers of total cells and average numbers of parasite positive ( + ) dots ( from three independent experiments ) within each infected cell were computed and represented graphically . statistical significance of the results obtained was determined using the following analysis : standard deviation , paired t - test , unpaired t - test , and fisher exact test . the effect of different concentrations of as - nps and as ( iii ) on growth of l. donovani promastigotes was quantitatively determined by haemocytometer under a phase contrast microscope . the ic50 value of the as - np that was found to be 2.37 m was determined using the parasite cell numbers obtained from the 24-hour sample ( methods and figure 1(a ) ) . to further confirm our result , the cytotoxicity of as - nps and as ( iii ) on the promastigotes was assayed by mtt . our results clearly demonstrated that treatment with 2 m as - nps for 72 hours has affected the growth of promastigotes most prominently in comparison with other lower concentrations of as - nps ( 0.1 and 1.0 m ) ( figure 1(b)(i ) ) . whereas as ( iii ) at the same range of concentration imparted minor effect on the survival of the parasites ( red , blue , and green lines in figure 1(b)(ii ) ) , similar killing effect was only observed by treatment with 1020 m of as ( iii ) which is much higher than that of as - nps ( figure 1(b)(ii ) ) . the results of the mtt assay correlated with that of the cell counting by haemocytometer . having shown growth inhibitory activity on the promastigotes , both as - nps and as ( iii ) were tested for their effect on the parasite respiration . the respiratory activity of l. donovani parasites was directly assessed by measuring the percent of oxygen consumed using an oxygraph as described in materials and methods . oxygen consumption by the parasites decreased gradually upon treatment with 2 m as - nps for 24 , 48 , and 72 hours as compared to that by the cells left untreated ( figure 2(a ) ) . however , as expected , the parasites treated with 2 m as ( iii ) showed almost no effect in their oxygen consumption ( figure 2(b ) inset ) . even 10-fold higher concentration of as ( iii ) was shown to have much less effect on the respiration of promastigotes ( figure 2(b ) ) . the effects of as - nps and as ( iii ) on the oxygen consumption by the parasites were found to be statistically significant ( tables 1(a ) and 1(b ) ) . the inhibitory effects of as - nps on the growth and metabolic activity of the parasites led us to investigate their effect on the infectivity of leishmania parasites in vitro . we have also compared the effect of as - nps and as ( iii ) on the infectivity of promastigote . the macrophages that were infected with promastigotes in presence of as - nps had modest number of gfp positive puncta ( figure 3(a ) ; panels ( f ) and ( h ) ) as compared to the set without as - nps indicating reduction of parasite burden in the macrophages ( figure 3(a ) ; panels ( b ) and ( d ) and table 2(a ) ) . however , in presence of as ( iii ) , the numbers of internalized parasites ( amastigotes ) were very similar to those found in control sets ( figure 3(a ) ; compare panels ( j ) , ( l ) and ( b ) , ( d ) with ( f ) , ( h ) ) . ic50 values that were determined using range of concentrations of as - nps in the infection studies ( 37c for 24 hours of incubation ) were found to be 1.5 m ( figure 3(b ) ) . moreover , the number of fluorescent parasites attached on macrophage surface was also reduced by the treatment with the nanoparticles indicating an inhibition in the attachment of the parasites to the macrophages prior to internalization ( figure 3(c ) ; compare panels ( b ) and ( d ) and table 2(b ) ) . these experiments were repeated several times and resulted in very similar observation . in the preceding section , we have demonstrated the suppressive effect of as - nps on the attachment and infectivity of the parasite . furthermore , we asked whether the nanoparticles can exert effect on the postinfection proliferation of the parasites inside the macrophages . to address that , 24-hour postinfected macrophages were treated with as - nps for another 24 and 48 hours ( figure 4(a ) ; panels ( g ) , ( h ) and ( i ) , ( j ) , resp . ) . our results clearly showed that as - nps can also block parasite proliferation inside the macrophage significantly ( figure 4(a ) : compare panels ( d ) , ( f ) with ( h ) , ( j ) and figure 4(b ) ) . while the parasite positive fluorescent puncta in as - np untreated cells increased significantly over time ( figure 4(a ) ; panels ( b ) , ( d ) , and ( f ) ) , the parasite positive dots in the infected cells that were treated remained static ( figure 4(a ) : compare panels ( b ) , ( d ) , and ( f ) with ( h ) and ( j ) and figure 4(b ) : in treated samples : parasite positive dots ) . repeated addition of fresh as - nps after 24 hours of the first addition did not yield much effect ( data not shown ) . the result of this experiment is satisfactorily reproducible and the p values for this experiment that were determined by fisher exact test are statistically significant ( figure 4(b ) ) . here , to explore the possibility whether the as - nps treatment had any effect on the macrophage itself , several microscopic images of nanoparticle treated macrophages were captured . figure 5(a ) shows representative of such images which clearly indicate that there is no change in the morphology ( phase and dapi ) of macrophages treated with as - nps . additionally , we have checked the viability of the macrophages treated with the nanoparticles by mtt assay . figure 5(b ) shows that as - nps treatment has resulted in 25% loss in viable cells as compared to the untreated control . it is to be noted that the viable cell number in the control set ( ~4000 ) showed a reduction from the initially seeded cell number ( 5000 ) due to loss during the processing required for this experiment . it exists in nature mostly as two biologically relevant oxidation states : arsenate [ as ( v ) ] and arsenite [ as ( iii ) ] . in proteins , as ( iii ) binds to both cysteine and histidine residues and causes depletion of intracellular glutathione . in the pentavalent form , arsenic is known to compete with phosphate , particularly in the citric acid cycle to alter the production of atp . several studies on the antibacterial , antiviral , and antifungal activities of arsenic have been carried out . according to a previous report , 30 g / ml of as ( iii ) ( equivalent to about 230 m ) is shown to be antileishmanial . this concentration of as is very toxic in mammalian system [ 10 , 11 ] . the present study is the first to demonstrate that the novel as - nps possess better antileishmanial efficacy than that of as ( iii ) . it is noteworthy that the concentration at which the as - nps imparted significant inhibitory effect on the viability , oxygen consumption , and infectivity of the parasite is too low for as ( iii ) to show any significant inhibitory effect on the parasites ( figures 14 ) . moreover , the antileishmanial effect comparable to that of as - nps is only achieved by at least 10-fold concentrated as ( iii ) . ic50 value determination shows that the amastigotes are more sensitive to the as - nps than the promastigotes . this data indicates that the as - nps will be equally effective as an antileishmanial agent in vivo . furthermore , the negatively charged nanoparticles as indicated by the zeta potential value would possibly face no problem to reach the phagolysosomes of macrophages in the infected internal organs , namely , liver and spleen . while the cellular and nuclear morphologies of the host macrophage cells treated with as - nps remain unchanged ( figure 5(a ) ) , the viability of the treated macrophages was reduced only by 25% ( figure 5(b ) ) . it is noteworthy that 1 m as ( iii ) was shown to have severe toxicity in vitro and in vivo [ 10 , 11 ] . moreover , it is not intuitive to predict the extent of its toxicity in vivo from the in vitro observation . there are reports where it was shown that the levels of toxicity between these two systems vary significantly [ 10 , 11 ] . taken together , our findings suggest that the as - nps have very good potential as compared to the as ( iii ) to be developed as an effective antileishmanial agent with much less effect on the host macrophages . it is evident that the pathogenesis depends on the proliferative capacity of the parasite inside the macrophages . if , somehow , this proliferation can be controlled , the pathogen can be rendered ineffective . the main problem of the conventional antileishmanial drugs for leishmaniasis is their toxicity and high prices . so , the development of new drug(s ) which would be cheaper with lesser side effects is a long lasting requirement . arsenic ( iii ) at a relatively higher concentration ( 30 g / ml ) has been shown to have antileishmanial activity ; but this concentration is reported to be toxic in several experimental mammalian systems . we have shown that the as - nps , developed for the first time in our laboratory , significantly inhibited the oxygen consumption and intracellular proliferation of the parasitic pathogen . the concentration of as - nps is about several fold lower than that of as ( iii ) . moreover , this antileishmanial activity has comparatively less cytotoxic effect on the mouse macrophage cell line . thus , it is evident from our findings that as - nps have more potential than as ( iii ) to be developed as a cheap and less toxic antileishmanial agent .
[ purpose ] the purpose of this study was to examine the effects of extracorporeal shock wave therapy on pain , disability , and depression of chronic low back pain patients . [ subjects ] in this study , 30 chronic low back pain patients were divided into an extracorporeal shock wave therapy group ( eswtg , n=15 ) and a conservative physical therapy group ( cptg , n=15 ) . [ methods ] the eswtg received extracorporeal shock wave therapy and the cptg received general conservative physical therapy two times per week for six weeks . pain was measured using a visual analog scale ( vas ) , the degree of disability of the patients was assessed using the oswestry disability index ( odi ) , and their degree of depression was measured using the beck depression index ( bdi ) . [ results ] in intra - group comparisons , eswtg and cptg showed significant decreases in vas , odi , and bdi scores . intergroup comparisons revealed that these decreases in vas , odi , and bdi scores were significantly larger in eswtg than in cptg . [ conclusion ] extracorporeal shock wave therapy is an effective intervention for the treatment of pain , disability , and depression in chronic low back pain patients .	low back pain is a disease that frequently occurs in daily life . apart from headache , it is the most common pain experienced by humans . approximately 80% of people in the entire population experience low back pain at least once during their lives . this pain is one of the factors that greatly affect the quality of life of patients because it leads to socioeconomic problems associated with increases in the costs of treatment , together with problems in daily living activities1 . simple low back pain can often be prevented by forming the habit of sitting with a proper posture and avoiding maintaining a sitting posture for prolonged periods . however , many types of low back pain are treated with therapeutic methods that are as diverse as the causes of low back pain . in most cases , therapeutic methods such as bed rest , assistive devices , traction treatment , hyperthermia , electric stimulation , and manual therapy are the first choice treatments2 . when these fail , invasive therapeutic methods such as selective nerve root blocks and epidural injections are used , and surgery is performed if no response to the other treatments is seen , or if the condition of the disease is serious3 . recently , new conservative treatments have been adopted , including extracorporeal shock wave therapy ( eswt)4 . eswt is a therapeutic method that applies shock waves to lesions from the outside of the body to promote revascularization and to stimulate or reactivate the curing process of connective tissues including tendons and bones , thereby relieving pain and improving function5 . eswt can be used for pain relief as well as for muscle strength improvement through appropriate motor stimulation of muscles and tendons by the shock waves6 . currently , eswt is administered for musculoskeletal system diseases , but studies of the effects of eswt on chronic low back pain are rare , and few studies have examined its effects on pain , disability , and depression . the purpose of this study was to examine the effects of extracorporeal shock wave therapy on pain , disability , and depression of chronic low back pain patients . this study was conducted with 30 patients ( male : 9 , female : 21 ) who had been diagnosed as having chronic low back pain by specialists in orthopedics , who had low back pain that had persisted for at least three months , and who had visited s orthopedics hospital located in daegu , korea . the subjects were divided into a conservative physical therapy group ( cptg ; n=15 ; mean age 46.08.9 years , mean height 163.79.1 cm , and mean weight 61.914.3 kg ) and an extracorporeal shock wave therapy group ( eswtg ; n=15 ; mean age 49.78.3 years , mean height 161.35.9 cm , and mean weight 62.29.3 kg ) . ethical approval for the study was granted by the youngdong university institutional review board . the study subjects had not received any surgery to the lumbar vertebrae region ; had no vertebral compression fractures , spinal tumors , or intervertebral disc infections ; were free of inflammatory diseases such as rheumatism ; and had no heart disease or structural abnormalities . the intention of this study and the experimental procedures were sufficiently explained to the study subjects and their voluntary agreement to participation was obtained before conducting the experiment . a vitera ( comed , korea ) was used to conduct extracorporeal shock wave therapy for eswtg . each patient assumed a prone position , and1,000 shock waves ( 7 times per sec ) were applied at 2.5 hz at low energy flux densities of 0.010.16 mj / mm using a 17 mm head . the treatment was conducted at the quadratus lumborum muscle and the sacroiliac joint , where the patients complained of pain . cptg was treated with hyperthermia using hot packs ( 20 minutes ) , ultrasound ( 5 minutes ) , and electrotherapy using tens ( 15 minutes ) . this scale has 10 items which are scored on a scale of 05 points based on functional performance , with higher scores indicating more severe disabilities . in this study , nine items were assessed , excepting the item for sex life considering subjects without spouses . the scores measured for individual items were summed and the results were divided by the maximum score of 45 points . depression was assessed using the beck depression index ( bdi ) developed by beck et al7 . scores from 09 indicate conditions free of depression , from 1015 indicate slightly depressed conditions , from 1623 indicate quite depressed conditions , and from 2463 indicate seriously depressed conditions . for statistical processing , the degrees of the chronic low back pain patients pain , disability , and depression were examined using the paired sample t - test to analyze intra - group differences and the independent sample t - test for analysis of intergroup differences . spss 12.0 for windows was used for statistical processing and the significance level , , was chosen as 0.05 . intra - group comparisons revealed significant decreases in vas , odi , and bdi scores of eswtg and the cptg at the end of the experimental period . intergroup comparisons showed that the decreases in vas , odi , and bdi scores were larger in eswtg than in cptg ( table 1table 1.intra- and inter - group comparisons of vas , odi , and bdi scoresgroupprepostvas ( point)cptg6.61.54.91.3eswtg7.00.763.61.1odi ( % ) cptg30.411.725.011.4eswtg30.112.417.58.1bdi ( point)cptg18.76.416.34.5eswtg19.66.712.55.7vas : visual analog scale , odi : oswestry disability index , bdi : beck depression index , cptg : conservative physical therapy group , eswtg : extracorporeal shock wave therapy group , * : paired t - test , : independent sample t - tests : p<0.05 , : p<0.01 ) . vas : visual analog scale , odi : oswestry disability index , bdi : beck depression index , cptg : conservative physical therapy group , eswtg : extracorporeal shock wave therapy group , * : paired t - test , : independent sample t - tests : p<0.05 , : p<0.01 chronic low back pain that brings about discomfort and inconvenience in daily life tends not to respond to conservative therapy in many cases , which has prompted a search for other therapeutic methods . rompe et al.8 reported that when one - off wave therapy was implemented for patients with calcific tendinitis of the shoulder , 60% of the patients recovered to their normal states and 72% of the patients recovered to the extent that they felt only intermittent discomfort as assessed by the constant and murley scale . lee sb5 reported that when eswt was applied to patients with lateral epicondylitis of the elbow joint , the patients pain was relieved , their simple elbow test scores improved significantly , and 83% of the patients showed satisfactory outcomes . cho et al.9 reported that when patients with lateral epicondylitis of the elbow joint were treated with eswt , the patients pain significantly decreased and their muscle strength increased significantly . na jy3 found that when eswt was administered to chronic low back pain patients , the patients pain significantly decreased . in addition , some researchers have suggested that eswt is an effective and non - invasive new therapeutic method for the treatment of lateral epicondylitis of the elbow joint that does not respond to conservative therapy . lee et al.10 reported that when eswt was implemented for chronic low back pain patients , exercise programs used together with extracorporeal shock wave therapy relieved pain and improved dynamic balance ability to a greater extent than was seen with a combination of exercise programs and conservative physical therapy . the intra - group comparisons in the present study revealed that there were significant decreases in the vas , odi , and vas scores of eswtg and cptg at the end of the intervention . the intergroup comparison revealed that these decreases were larger in eswtg than in the cptg . rompe et al.8 advised that minute and repetitive stimuli applied through shock wave therapy were effective at relieving pain . hammer et al.11 recognized that breaking calculi and gallstones with extracorporeal shock waves increases the blood flow rate and rate of revascularization , and used this effect to stimulate and activate the curing processes of tendons , surrounding tissues , and bones to remove and stabilize inflammations in the tendons and ligaments , which resulted in pain relief . the results of the present study also indicate that decreases in vas scores were occurring through mechanisms described by presented in previous studies . in addition , eswt appeared to relieve chronic low back pain , thereby improving the patients physical functions and consequently their ability to perform daily living activities , leading to significant decreases in low back pain disability indexes . holmes12 argued that the limitations or disability in patients daily living would cause psychosocial problems that would further impair the quality of their lives . depression is closely related to physical disability and chronic pain in chronic disease patients , and it is an important element that interferes with pain relief by delaying recovery or aggravating diseases13 . in the present study , eswt relieved pain and reduced disability indexes , thereby improving the patients physical functions and ability to perform daily living activities , which in turn had positive effects on the patients psychological , emotional , and cognitive states , decreasing the degree of their depression . decreased pain in chronic low back pain patients is a more influential factor than disability and depression . first , the number of chronic low back pain patient subjects was limited , so the results can not be generalized . second , the lumbar lesion sites were limited and the subjects daily lives could not be completely controlled . third , no previous studies have measured the effects of eswt on depression , so our present results could not be compared with those of other studies .
while ultrasonography is widely performed prior to biopsy , colour doppler examination is often used only to discover post - biopsy complications . aim of this paper was to evaluate the usefulness of colour doppler examination in planning the optimal site of puncture for renal biopsy . present analysis includes 561 consecutive percutaneous renal biopsies performed from the same operator . until august 2000 332 biopsies were performed after a preliminary ultrasonography ( group a ) . from september 2000 , 229 patients underwent even a preliminary colour doppler study ( group b ) . postbioptic bleeding were categorized as minor ( gross hematuria or subcapsular perinephric hematoma < 4 cmq of greater diameter ) or major ( hematoma > 4 cmq of greater diameter ; requiring blood transfusion or invasive procedures ; leading to acute renal failure , urine tract obstruction , septicaemia , or death ) . major complications were seen in 2.1% in group a while in group b only one case was reported ( 0.43% ) . minor clinically significant complications occur in 7.8% in group a and in 3.4% of cases of group b. colour doppler reduced drastically the incidence of complications observed before the introduction of routine colour doppler examination prior to biopsy . in our opinion , these data support the use of preliminary colour doppler study when a biopsy is planned .	percutaneous renal biopsy has become an essential tool in the diagnosis and treatment of patients with renal disease , and although less invasive , it is fraught with potential complications . introduction of realtime ultrasound and automated biopsy needles increased quality of specimens and dramatically reduced the incidence of biopsy - related accidents [ 14 ] . nonetheless , the potential for serious complications still remains . on average , clinically significant complications occur in 7.4% of cases , but also complications rates as high as 19.5% have been reported . many variability in complication incidence attains certainly to operator experience , but it is our opinion that a part of this heterogeneity is linked to the attention paid in planning correctly the optimal site of puncture , in order to avoid undesired puncture of anomalous vessels . while preprocedural ultrasonography is in fact widely used to assess the anatomical landmarks of the kidney and to exclude anatomical conditions considered as absolute contraindications , to date , power and colour doppler ( cd ) examination are used less frequently in this setting , if not only to discover postprocedural complications . in our experience , anomalous perirenal vessels were present in many biopsies complicated by renal bleeding . in our department , until august 2000 , we performed a total of 332 biopsies , using b - mode ultrasonography , only occasionally integrated by cd study , in planning procedures . from september 2000 if this systematic approach reduced or not complication incidence after procedure , we designed a retrospective analysis of observed complications before and after the systematic introduction of cd study in the preparation of renal biopsy . present analysis includes 561 consecutive ultrasound - guided percutaneous renal biopsies performed in our department from august 1995 to december 2009 from the same operator . until august 2000 , a total of 332 biopsies were performed , after a b - mode ultrasonographic study of kidneys . from september 2000 all patients ( total 229 ) underwent a preliminary systematic cd study prior to biopsy . in these patients , vessels in the region of the biopsy low pulse repetition frequency were used , between 750 and 1000 hz . 332 patients biopsied before september 2000 were named group a. the group of 229 patients systematically cd - studied was named group b. demographical data are listed in table 1 . postbiopsy complications were categorized as either minor ( gross hematuria and/or subcapsular perinephric hematoma < 4 cmq of greater diameter ) or major ( > 4 cmq of greater diameter ; requiring transfusion of blood products or invasive procedures ; leading to acute renal failure , urine tract obstruction , septicaemia , or death ) [ 5 , 6 ] . to minimize the risk of bleeding , prothrombin time , partial thromboplastin time , bleeding time ( ivy ) , and salicilate , ticlopidine , and nsaids were withdrawn for at least 7 days before biopsy . use of heparin was prohibited two days before biopsy . in case of arterial hypertension under treatment , biopsy was performed only when normotensive range was achieved ( less then 140/90 mm / hg ) . patients with renal cortex width < 10 mm and/or < 90 mm of kidney longitudinal diameter were not considered suitable for renal biopsy . all biopsies were performed using realtime ultrasound - fixed guidance ( 3.5 mhz convex probe , technos mp ; esaote , italy ) , and 16-gauge automated spring - loaded gun . in all cases the lower pole of kidney was used for biopsy , mainly at left side . only in cases in which cysts , small cortex , peri - renal vessels were detected in this location , the right kidney was biopsied . in group b , the presence of medium - size arteries / veins at lower pole of left kidney was an indication to perform biopsy on right kidney . biopsy was performed in prone position with patients lying with the abdomen on a firm pillow . the most appropriate needle - skin angle was selected by adjusting the puncturing adaptor ( usually 20 ; see figure 1 ) . after disinfection of the skin , lidocaine ( 1% ) was applied locally under ultrasound control along the needle insertion tract . a small incision of the skin was made to facilitate the introduction of the biopsy needle . under us imaging guidance the needle was advanced at an angle of 20until the capsule of the lower kidney pole had been reached . patients were asked to hold their breath , the biopsy device was unlocked and tissue obtained . the sampling time was < 1 s. length of the obtained biopsy specimens was usually 1820 mm and at least two samples were taken . following the biopsy , patients lay in bed on their backs for a 24-hour observation time . during this time , both clinical ( gross hematuria , flank pain , and hypotension ) and imaging test ( 2-d realtime sonography , cd sonography , and power doppler sonography ) were performed in order to evaluate the presence and sizes of potential bleedings . statistical analysis was performed through statistica 5.0 ( statsoft inc . ) , using fisher 's exact test . histological analysis , including conventional light and immunofluorescence microscopy , could be performed by the pathologist in 100% of cases . all complications registered during the study are reported in table 2 . in group a an arteriovenous fistula was observed in one patient , and spontaneously resolved 18 months after procedure . in group one of this two requested an angiographic procedure to stop bleeding , through gianturco method . three major hematomas ( > 4 cmq ) were observed in group a , while only one in group b. in none of these cases surgical procedures were indicated after urological consulting . minor clinically significant complications occur in 7.8% of cases in group a and in 3.4% of cases of group b. microhematuria was highly frequent but it was not intended as a complication . prebioptic cd evaluation showed anomalous vessels at the lower pole of the right kidney in 15 patients and at the lower pole of left kidney in 19 patients , respectively , as shown in table 3 . in 15 of 19 patients with anomalous vessels at the left kidney , it was impossible to identify an alternative trajectory for the needle ( figures 1 and 2 ) and biopsy was carried out on the right kidney . in the other 4 cases was sufficient to accurately study the trajectory . only one patient in group a underwent right kidney biopsy , showing a large inferior pole simple cyst . anomalous interlobar vessels were observed in 9/229 pts while inferior pole arteries in 6 patients . cumulatively 23 pts ( about 10% of whole group b ) showed vascular abnormalities potentially at risk of postprocedural bleeding . several authors [ 710 ] reported a drastic decrease in the incidence of complications after renal biopsy when procedure is guided by ultrasonography . systematic use of colour doppler ultrasonography in monitoring patients after a kidney biopsy , is crucial for an early diagnosis of postbiopsy av fistula . perhaps if the importance of colour doppler ultrasonography in postprocedural evaluation [ 1113 ] is clear and commonly accepted , its usefulness in preparing a biopsy is yet to be appreciated . this study , for the first time , showed a systematic , preparatory use of cd reduced postbiopsy complications incidence , in the same unit and from the same operator . it is to be underlined that during the time course of this study , the operator experience in biopsy practice grew , probably influencing in some part the results of this study . perhaps it is obvious that this study has all limitations of a retrospective analysis . on the other hand it appears acceptable that preliminary cd study helped operators to identify the safest needle trajectory to the kidney , in some cases indicating to prefer right organ for biopsy , even in absence of other ultrasonographic indications to avoid left kidney puncture . data registered in our clinic before the default use of cd study showed an incidence of major and minor complications absolutely in line with those reported in the literature [ 48 ] . cd module is largely available in most of ultrasonographers in use in biopsy - providing units . it does not require the exposition to ionizing radiations and does not expose patient to any kind of risk . perhaps it is an inexpensive tool , in term of money , and its systematic use requests little effort to the nephrologist in term of time . its systematic use can reduce complications , and then costs , frequently as high as dramatic from an ethical point of view . in other words it is an easy , useful hand and sure tool in percutaneous renal biopsy . on the other hand cd technique is highly operator - dependent , requesting experience and skill to warrant affordable reliefs . it is then to be underlined that cd module has to be used when the nephrologist plans a biopsy , to design ideal needle trajectory , and it is turned off when needle starts its descent to the kidney . in this phase , obviously , cd could be only a confounding factor for the operator , often used to vibrate needle to make it visible on ultrasonographic screen . this study opens a window on a new aspect in planning kidney biopsies : the presence of anomalous vessels potentially on the trajectory of biopsy needle . the systematic study of kidneys showed in fact anomalous vessels in about 10% of patients , giving a potential explanation to the observed reduction in complications rate in cd - studied patients . it is our opinion that this aspect could have more consideration in planning the better strategy for kidney biopsy . our data show clearly as the same operator , in the same setting , could obtain better results and drastically reduce complications only enriching preliminary study with cd . it is obvious to remind to the reader as vascular structures , particularly in case of small ones , can slip from the attention even of a well - skilled operator using only b - mode ultrasonography . cd can help him to better clarify their nature and to identify potential sources of risk in his strategy to biopsy . on the basis of our data , clinically significant postbiopsy haematomas experienced nephrologist , planning a site of puncture for biopsy , has to take care of them and design a strategy to reach renal cortex without undesired experiences .
in drosophila , widely - used mitotic recombination - based strategies generate mosaic flies with positive readout for only one daughter cell after division . to differentially label both daughter cells , we developed the twin spot generator technique ( tsg ) and demonstrate that through mitotic recombination , tsg generates green and red twin spots in internal fly tissues , visible even as single cells . we discuss the wide applications of tsg to lineage and genetic mosaic studies .
background : subacute granulomatous thyroiditis ( sgt ) is an inflammatory disease that presents with different clinical and cytological characteristics . although the diagnosis is generally made clinically , imaging methods and fine - needle aspiration ( fna ) may provide assistance , particularly in atypical cases . the objective of this study is to reveal the ultrasonographic ( usg ) and cytological characteristics of sgt.materials and methods : the clinical , usg and cytological findings of 21 cases diagnosed with sgt were reviewed.results:ultrasonographic data was available in 20 cases . a hypoechoic thyroid nodule with irregular margins was detected in 12 of the 20 total cases . of these , 9 cases complained about pain in the thyroid lodge and generally had unilateral lesions , heterogeneous and hypoechoic areas with indistinct margins , rather than nodular lesions , which were seen in 7 cases . cytologically , the multinuclear giant cells ( mngcs ) found in all cases were accompanied by a dirty background containing varying numbers of granulomatous structures , including isolated epithelioid histiocytes , proliferated / regenerated follicle epithelium cells and inflammatory cells and colloid.conclusion:though hypoechoic and heterogeneous areas with irregular margins are strongly associated with thyroiditis , sgt may also appear as painful or painless hypoechoic , solid nodules and generate challenges in differential diagnosis . although the most remarkable characteristic observed in fna cytology was the presence of multiple mngcs with cytoplasm , a dirty background accompanied by mild - moderate cellularity , degenerated - proliferated follicular epithelium cells , rare epithelioid granulomas and mixed type inflammatory cells are characteristic for sgt . the assessment of these radiological and cytological findings in conjunction with clinical findings will assist in the achievement of an accurate diagnosis .	subacute granulomatous thyroiditis ( sgt ) , also known as de quervain 's thyroiditis , is a self - limiting , inflammatory disease of the thyroid that is believed to be caused by a systemic viral infection . it was first diagnosed in 1825 , and 18 cases were reported as thyroiditis acuta simplex until 1895 . it typically occurs in the area of the gland in mid - aged hyperthyroid women complaining of pain , tenderness , fatigue and mild fever . it is usually clinically diagnosed without the need for fine - needle aspiration ( fna ) . therefore , there are few studies regarding the cytological features of sgt in the literature . however , it is important to exclude other diseases of the thyroid because sgt may be confused with them , particularly with malignancies . the objective of this study is to define the ultrasonographic ( usg ) and cytopathologic characteristics of sgt and highlight its differential diagnosis from the other lesions of the thyroid . a total of 21 cases diagnosed with sgt between 2001 and 2014 were included in our study . clinical data , such as the patients ages , clinical findings and clinical preliminary diagnoses and the usg findings , were obtained retrospectively from the pathology reports . cytological preparations were re - evaluated in terms of cellularity , multinuclear giant cells ( mngcs ) , granulomatous structures , histiocytes , colloids , follicular epithelial cells , fibrous fragments and inflammatory cells . fine - needle aspirations were performed by a cytopathologist ( np ) , who was accompanied by a radiologist ( ky ) , and with ultrasonography guidance , by using a 22-gauge needle , 10 cc syringe and aspirator . on average , the procedure was concluded upon the detection of a sufficient number of air - dried preparations stained with the diff - quik dye at the bedside . the clinical and cytological characteristics of the 21 cases included into the study have been summarized in table 1 . clinical and cytopathological features of subacute granulomatous thyroiditis all , except two cases , were women ( 90% ) and the mean age was 38.19 years ( 1850 years ) . seven cases presented with a pain complaint in the area of the thyroid lasting a few weeks , whereas 2 cases had pain and previous upper respiratory tract infection and 1 case had a history of sgt that had occurred 3 months previously . the preliminary clinical diagnosis was sgt in 2 cases , thyroiditis in 6 cases , multinodular goiter ( mng ) in 4 cases , thyroid carcinoma in 4 cases and lymphoma in 1 case . the usg findings of the other 20 cases have been summarized in table 2 . hypoechoic thyroid nodules with irregular margins were observed in 12 cases at usg , whereas heterogeneous , hypoechoic areas ( lava flow ) with indistinct margins were observed in 7 cases [ figures 1a and 2a ] . in 1 case ( case 16 ) , one of the bilateral and multiple lesions , which located on the right side , appeared with a hypoechoic area , and the others presented as nodular lesions . in the 13 cases that presented as nodular lesions , the nodules were generally unilateral and single , with the largest mean size being 22.19 mm ( range : 740 mm ) , whereas the largest mean size of the hypoechoic - heterogeneous areas was 27.52 mm ( range : 1075 mm ) . ultrasonographic findings of subacute granulomatous thyroiditis a subacute granulomatous thyroiditis case sonographically characterized by an ill - defined hypoechoic nodule ( case no . the cytologic features of the case included the following : ( b ) multi - nucleated giant cell ( papanicolau stain , 400 ) , ( c ) an epithelioid histiocytic granuloma ( papanicolau stain , 400 ) , ( d ) a group of epithelioid histiocytes adjacent to a giant cell ( papanicolau stain , 400 ) and ( e ) a collection of proliferated follicular cells ( papanicolau stain , 200 ) a subacute granulomatous thyroiditis case with a sonographic image of a poorly defined hypoechoic area . the cytologic features of the case included the following : ( b ) multinucleated giant cell ( diff quik , 400 ) , ( c ) an aggregate of epitheliod histiocytes consistent with granuloma ( diff quik , 400 ) , ( d ) a cluster of epithelioid histiocytes ( papanicolau stain 400 ) and ( e ) a group of proliferated follicular cells ( diff quik , 400 ) cytologically , the diffusions were generally normocellular or moderately cellular , and there was no highly dense ( tumoral ) cellularity . all cases presented with follicular epithelial cells , although this was rare in some such cases . the cells were generally shaped as honeycomb fragments , with a partially degenerated and proliferated appearance , and it was noted that reactive atypia accompanied 1 case ( case 15 ) . however , no papillary or microfollicular structuring , plasmacytoid appearance or intranuclear inclusion or groove was observed in any sample . the mngcs were of variable sizes and forms , contained a cytoplasmic content and were generally large . phagocyted colloid and neutrophils appeared in the cytoplasm of some mngcs . in rare cases , there were also some smaller , round , vacuole - cytoplasm mngcs that contained less nucleus . though there were epitheloid histiocytes and clusters of epitheloid histiocytes , which were believed to be granulomas , in 10 cases , the epitheloid cells were isolated or formed loose cohesive clusters in 3 cases . the granulomas that were assessed appeared similar to those in other granulomatous diseases , specifically , to be derived from bare - foot or carrot - shaped nucleus cells constituting tight syncytial groups of variable sizes [ figures 1 and 2b e ] . this colloid generally appeared in thick , small and large masses inside epithelioid histiocytes , some of which occurred in a phagocytosed manner . all cases had a dirty background that was composed of cellular debris , blood elements and pink amorphous acellular material , and this was accompanied by neutrophil leukocytes in 2 cases and mixed types of inflammatory cells with dominant lymphocytes in the remaining cases . the inflammatory diseases of the thyroid gland are classified into three groups , that is , acute , subacute and chronic . de quervain 's thyroiditis , also named giant - cell or granulomatous thyroiditis , is the subacute inflammation of the thyroid and constitutes nearly 36% of all thyroid diseases . it is a nonspecific inflammation that generally appears 2 weeks after a viral upper respiratory tract infection and regresses spontaneously within 23 months . although its etiology remains unknown , it is believed have a viral origin , similar to the mumps virus , hepatitis b and c viruses , cytomegalovirus enterovirus , and type a and b coxsackie viruses . thyroiditis predominantly affects adult females , and the ratio of females to males is 2:16:1 . clinically , patients present with localized anterior neck pain associated with glandular tenderness and diffuse pain in the ears and the jaw , which is accompanied by fatigue , weight loss , low - grade fever , elevated erythrocyte sedimentation rate , suppression in the thyroid stimulating hormone level and occasionally dysphagia . the thyroid is sensitive at palpation , and there is a mild - moderate growth in the thyroid . depending on the stage of the disease , hyperthyroidism , hypothyroidism or euthyroidism may be seen in patients . the patients recover spontaneously after weeks or months , and recovery may be supported by symptomatic treatment with nonsteroid anti - inflammatory drugs or , occasionally , cortisone . generally , an increase in the size of the thyroid and heterogeneous , diffuse , hypoechoic and confluent areas with negative margins , characteristically defined as lava flow , are observed on usg . however , these findings are nonspecific because they may also be observed in lymphocytic thyroiditis , mng and graves diseases , and the clinical findings may assist in the differential diagnosis . sgt may also present with nonsensitive solid nodules . unlike the literature findings related to this topic , the number of cases appearing as nodules in our study ( 13 cases ) was relatively higher than the number of cases appearing as hypoechoic areas ( 7 cases ) . additionally , most of our cases were unilateral . due to the risk of infiltrative malignancies , such as generalized parenchymal abnormalities , which can be interpreted as diffuse nonneoplastic thyroid disease , including sgt , repeated usg and even fna should be performed to exclude other malignancies in sensitive or nonsensitive hypoechoic and solid nodules . due to the rare however , the frequency of cytopathological use has increased with the development of usg approaches . fine - needle aspiration is beneficial in the diagnosis of thyroiditis and may provide very accurate results . fna plays an important role for accurate diagnosis , particularly when clinicians do not suspect sgt or in atypical cases . the fact that among all of our cases , there is no distinct clinical finding that may help us , except for pain in the area of the thyroid in 7 cases , pain and previous upper respiratory tract infection in 2 cases , and the presence of a history of sgt in 1 case as well as the absence of malignancies among clinical differential diagnoses in 5 cases ; this reveals the importance of fna . the key cytological characteristics for sgt are as follows : ( i ) a high number of mngcs , ( ii ) epithelioid cells with a tendency for clustering , ( iii ) epithelioid cell granulomas , ( iv ) lymphocytes , macrophages and neutrophils , ( v ) frequently degenerated follicular epithelial cells displaying mild - moderate cellularity , and ( vi ) a dirty background that is composed of cellular debris , naked , degenerated nuclei and thick colloids . the absence of one or a few of these does not exclude sgt but instead increases the number of thyroid diseases that are included in the scope of the differential diagnosis . the characteristic cytological feature of sgt is the presence of mngcs , which may comprise hundreds of large nuclei and contain colloidal residues in their cytoplasm[figure 3 ] . mngcs may be seen inflammatory conditions of the thyroid ( granulomatous diseases , hashimoto 's thyroiditis , graves disease and palpation thyroiditis ) , hyperplastic conditions ( mng displaying degenerative changes ) and neoplastic conditions ( papillary carcinoma , anaplastic carcinoma ) . ( a ) multinucleated giant cell secondary to a benign cystic nodule ( our archival case ) ( diff quik , 400 ) . ( b ) bizarre giant cells seen in a case papillary thyroid carcinoma ( our archival case ) ( papanicolau stain , 400 ) . ( c ) ( d ) multinucleated giant cell with an intracytoplasmic colloid like material ( arrow ) seen in one of our subacute granulomatous thyroiditis cases ( case no . 21 ) ( papanicolau stain , 400 ) two morphological types mngcs can be observed in fna samples : those with foamlike cytoplasm and those with dense cytoplasm . mngcs with foamlike cytoplasm are round cells with less diagnostic importance and are generally seen in cystic degeneration . they are usually composed of multinuclear forms of histiocytes , flat cytoplasmic contours , foamlike , vacuole and hemosiderin cytoplasms [ figure 3a ] . further , mngcs with dense cytoplasm have angular cytoplasm and irregular shapes , are larger and contain a higher number of nuclei . primarily , the three following conditions should come to mind in diffusions containing mngcs : ( i ) sgt , ( ii ) granulomatous diseases , such as sarcoidosis , tuberculosis and fungal infections , and ( iii ) papillary carcinoma of the thyroid ( pc ) . the presence of tumor cellularity and pc nuclear characteristics ( powdery chromatin , nuclear groove and intranuclear inclusions ) , overlapping syncytial tissue fragments , real papillary fragments , if any , and psammoma bodies are characteristics in favor of pc . the mngcs that they contain are smaller in number and size compared with sgt and granulomatous diseases . these cells are called bizarre giant cells [ figure 3b ] . however , in some pc cases , mngcs of sgt can be seen in addition to the bizarre giant cells [ figure 3c ] . dirty background with clinical , well - formed or poor - formed epithelioid granulomas and acute - chronic inflammation is included in the differential diagnosis for sgt . the mngcs that are present have a tendency to be more frequent and larger in size and to contain more nuclei . the main cells in the granulomatous diseases of the thyroid are epithelioid histiocytes , and the granulomas observed are generally well formed , comprise fewer lymphocytes and acute inflammatory components and do not contain a dirty background . however , it should also be highlighted that mngcs may not always be seen in sgt aspirates , may sometimes be too few and scattered , or may very rarely be seen in sgt as intensely as they appear in other thyroid diseases . granulomas , which appear as epithelioid histiocyte clusters , are another key characteristic for sgt but are rare or may not be seen . different rates have been reported with regard to the presence of granulomas in sgt in different studies in literature , and it was suggested that these different rates were due to the association with the stage of the disease or a sampling error . epithelioid granulomas were defined in many cases , such as tuberculosis , sarcoidosis and fungal infections , postsurgically in the neck area , auto - palpation habit , hemorrhage in nodular goiter , proximity to histiocytic reaction , hashimoto 's thyroiditis and graves diseases . the presence of a high number of mngcs accompanied by acute onset pain , which is also a characteristic clinical symptom for sgt , will be helpful for differential diagnosis . garca solano et al . reported the presence of follicular cells with intravacuolar granules and/or plump transformed follicular cells , though this was not a marked characteristic in our cases . in contrast , the follicular epithelial cells in our cases were generally degenerated / proliferated , and regenerative atypia findings were observed from an aspirate taken with the suspicion of malignancy . however , the patient was diagnosed as sgt due to the absence of cellular and structural malignant characteristics , despite the presence of a high number of mngcs and epithelioid cells accompanied by a dirty background . if the patient has the cytological characteristics specified above when he / she comes to the clinic with a clinical diagnosis of sgt , the cytological diagnosis of sgt may not be too difficult . however , the following factors may rise to challenges in the diagnosis : ( i ) lack of clinical data , ( ii ) failure of the needle to reach the target , ( iii ) absence of the typical cytological characteristics ( i.e. mngcs , granulomas ) as in acute or advanced stage of the disease , and ( iv ) presence of atypical follicular epithelium cells without other diagnostic elements . according to ofner et al . although pain and tenderness in the thyroid gland is a very important finding for the diagnosis of sgt , it is not specific and may be seen in suppurative thyroiditis , trauma , radiation , nodular goiter , hemorrhagic and infarct areas , metastatic diseases and rarely , in hashimoto 's thyroiditis . generally , the correct diagnosis can be made by combining detailed clinical and physical examination and using serological and cytological characteristics in combination . however , clinical follow - up and fna may be very beneficial in such cases . subacute granulomatous thyroiditis is a dynamic process , and usg and cytological findings may vary according to the stage of the disease . although there is no special radiological finding of thyroiditis , hypoechoic and heterogeneous areas with irregular margins in the thyroid are strongly associated with thyroiditis . however , sgt may also appear as a painful or painless hypoechoic and solid nodule , as occurs in most of our cases . they may be confused with malignancies , particularly in such cases , and give rise to difficulties in differential diagnosis . the characteristic cytological features in our series in fna were a high number of mngcs with dense cytoplasm , mild - moderate cellularity , degenerated - proliferated follicular epithelial cells and dirty background accompanied with epithelioid granulomas and rare , mixed type inflammatory cells . although the presence of neck pain in the clinical history , the detection of mngcs at fna , their morphological characteristics and their number play a key role in the diagnosis . further , utilizing tbc - fungal cultures and assessing the accompanying cell population in the background may help in the differential diagnosis . all authors of this article declare that we qualify for authorship as defined by icmje . each author has participated sufficiently in the work and takes public responsibility for appropriate portions of the content of this article.cv conceived of the study and drafted the manuscript . np participated in its design and coordination and helped to scientific revision of the manuscript . this study was conducted with the approval from all the institutions associated with this study as applicable . sgt = subacute granulomatous thyroiditis fna = fine - needle aspiration usg = ultrasonographic mngcs = multinuclear giant cells mng = multinodular goiter pc = papillary carcinoma to ensure the integrity and highest quality of cytojournal publications , the review process of this manuscript was conducted under a double blind model ( authors are blinded for reviewers and vice versa ) through automatic online system .
indel mutations play key roles in genome and protein evolution , yet we lack a comprehensive understanding of how indels impact evolutionary processes . genome - wide analyses enabled by next - generation sequencing can clarify the context and effect of indels , thereby integrating a more detailed consideration of indels with our knowledge of nucleotide substitutions . to this end , we sequenced blochmannia chromaiodes , an obligate bacterial endosymbiont of carpenter ants , and compared it with the close relative , b. pennsylvanicus . the genetic distance between these species is small enough for accurate whole genome alignment but large enough to provide a meaningful spectrum of indel mutations . we found that indels are subjected to purifying selection in coding regions and even intergenic regions , which show a reduced rate of indel base pairs per kilobase compared with nonfunctional pseudogenes . indels occur almost exclusively in repeat regions composed of homopolymers and multimeric simple sequence repeats , demonstrating the importance of sequence context for indel mutations . despite purifying selection , some indels occur in protein - coding genes . most are multiples of three , indicating selective pressure to maintain the reading frame . the deleterious effect of frameshift - inducing indels is minimized by either compensation from a nearby indel to restore reading frame or the indel s location near the 3'-end of the gene . we observed amino acid divergence exceeding nucleotide divergence in regions affected by frameshift - inducing indels , suggesting that these indels may either drive adaptive protein evolution or initiate gene degradation . our results shed light on how indel mutations impact processes of molecular evolution underlying endosymbiont genome evolution .	. a single indel may alter gene length by disrupting the reading frame , increase substitution rate in flanking regions and catalyze adaptive protein evolution ( tian et al . indels complicate multiple sequence alignments and may skew subsequent evolutionary analyses such as tests of positive selection ( fletcher and yang 2010 ; jordan and goldman 2012 ; westesson et al . 2012 ) . integrating our knowledge of nucleotide substitutions with more detailed consideration of indel mutations is necessary to fully understand processes of molecular evolution . to this end , advances in high - throughput sequencing enable genome - wide analyses of indels at different divergence levels , thereby elucidating the context and effect of indels . indels impact ongoing genome reduction in obligate bacterial endosymbionts of insects , which have the smallest genomes reported for cellular life ( moran et al . 2008 ) . genome degradation in established endosymbionts , including buchnera of aphids and blochmannia of carpenter ants , is hypothesized to occur primarily through small deletions punctuated by occasional large deletions in nonfunctional sequence ( gomez - valero et al . the spectrum of indel mutations in bacterial endosymbionts is affected by a combination of factors , including loss of dna repair mechanisms and relaxed purifying selection resulting from population bottlenecks during vertical transmission of bacteria ( moran et al . 2008 ) . the high at content of most bacterial endosymbionts results in increased density of homopolymers , providing hotspots for indels as an important first step in gene degradation ( gomez - valero et al . other repetitive regions , such as multimeric simple sequence repeats ( mssrs ) , may also serve as hotspots for indels ( williams and wernegreen 2012 ) . to investigate the roles of indels in genome evolution of bacterial endosymbionts , we assembled and annotated the genome of blochmannia chromaiodes ( nc_020075 ) , an endosymbiont of the carpenter ant camponotus chromaiodes . blochmannia chromaiodes is closely related to b. pennsylvanicus ( degnan et al . 2004 ; wernegreen et al . 2009 ) , which was previously sequenced ( degnan et al . 2005 ) . the genetic distance between these two species is close enough for accurate whole genome alignment but divergent enough that a meaningful number of indels have accumulated , thereby affording valuable opportunities to clarify the context and effect of indel mutations in endosymbiont genome evolution . camponotus chromaiodes ants were collected from two colonies in the same location less than 1 year apart . genomic dna from the two colonies was prepped separately and sequenced with either 454 or illumina technology . we used 454 reads for de novo assembly of a draft sequence , which we then corrected with illumina reads to ensure homopolymer accuracy in the final genome sequence . for 454 sequencing , we isolated endosymbiont cells from 12.9 g of ants using a percoll density gradient centrifugation protocol described previously ( wernegreen et al . chloroform extraction as described previously ( williams and wernegreen 2010 ) . for illumina sequencing , we prepared genomic dna from three gasters using the qiagen dneasy blood and tissue kit . 454 sequencing generated 295,496 single - end reads , which we assembled into a single draft contig using the gs assembler and gs mapper in newbler version 2.3 . we closed the single gap with sanger sequencing . because 454 sequencing is known for homopolymer inaccuracy , we used illumina reads to correct the draft and obtain the final genome sequence . illumina sequencing generated 28,516,837 single - end 76 bp reads , which we aligned to the 454 draft genome using mosaik ( http://bioinformatics.bc.edu/marthlab/mosaik , last accessed march 15 , 2013 ) . we corrected the draft genome and then confirmed the final genome sequence by aligning illumina reads with bwa ( li and durbin 2009 ) and the genome analysis toolkit indelrealigner tool ( mckenna et al . , we also assembled a large contig of the mitochondrial genome ( jx966368 ) , which aligns with 100% identity to a c. chromaiodes mitochondrial sequence in genbank . we used an annotation engine hosted by the institute for genome sciences at the university of maryland school of medicine to obtain an automated annotation of b. chromaiodes , which we then manually curated . protein - coding genes predicted by the annotation engine were removed from the annotation if they lacked a blast extend repraze ( ber ) alignment score less than 10 to a non - blochmannia protein . we used the locus and gene names suggested by swissprot for the homologous gene in e. coli to provide consistency with other proteobacterial genome annotations . conserved hypothetical proteins or proteins with similarity to a protein family but not a specific family member were given a locus name reflecting the gene number ( for example , bchro_042 ) . we curated the start site for each protein - coding gene using the ber alignments to non - blochmannia species . we identified two uncalled protein - coding genes ( cyod and a hypothetical protein homologous to bpen_539 ) , three rna - coding genes ( ffs , rnpb , and tmrna ) , and three pseudogenes ( rpmd , uvrd , and yqic ) by searching intergenic regions with blastx , blastn , and rfam ( altschul et al . 2000 ) and dnadiff from mummer ( kurtz et al . 2004 ) , both with default parameters , to obtain whole genome alignments of b. chromaiodes and b. pennsylvanicus . when the two alignments disagreed , we applied a set of criteria to decide the final alignment . in priority order , the criteria are as follows : 1 ) preserve reading frame in protein - coding genes , 2 ) select the most parsimonious proposal ( i.e. , the proposal with the fewest substitutions and indels ) , 3 ) when both proposals have the same total number of events , select the proposal with fewer indels , and 4 ) when both proposals have the same number of substitutions , select the proposal with fewer transversions . for 12 cases , these criteria could not differentiate between the two proposals , and we selected the emboss stretcher proposal by default . we classified substitutions and indels according to the b. chromaiodes and b. pennsylvanicus annotations using variantclassifier ( li and stockwell 2010 ) . to estimate the neutral substitution rate , we generated amino acid - based alignments of protein - coding genes with translatorx ( abascal et al . 2010 ) and mafft ( katoh et al . 2005 ) , omitting 23 genes with nontriplet indels . using codeml in paml with runmode 2 for pairwise comparisons ( yang 1997 ) , we estimated the number of synonymous changes and synonymous sites for each gene and then calculated the average number of synonymous changes per 1,000 synonymous sites across the genome . we did separate searches for homopolymers and mssrs in b. chromaiodes and b. pennsylvanicus using phobos ( http://www.ruhr-uni-bochum.de/ecoevo/cm/cm_phobos.htm , last accessed march 15 , 2013 ) . we then identified overlap between indels and repeats using intersectbed from bedtools ( quinlan and hall 2010 ) . to determine whether compensatory indels are associated with protein divergence , we used the reciprocal blastp algorithm to identify orthologs of b. pennsylvanicus genes in e. coli ( nc_000913 ) . ortholog detection and calculation of amino acid distances followed methods described previously ( wernegreen 2011 ) . this strategy invokes paml to calculate amino acid distances based on an empirical amino acid substitution rate matrix and accounts for variation in evolutionary rates among sites . the gene content of b. chromaiodes is identical to that of b. pennsylvanicus ( table 1 ) . both genomes have evidence of three pseudogenes ( rpmd , uvrd , and yqic ) , which differ slightly in length between the two species ( supplementary table s1 , supplementary material online ) . s1 , supplementary material online ) , the two nucleotide sequences are 98.0% identical . we detected 13,389 substitutions and 1,051 indels between the two sequences ( table 2 ) . substitution rates in pseudogenes and intergenic regions are very similar ( 26.8 and 29.3 substitutions / kb , respectively ) . these rates are higher than those of protein - coding and rna - coding genes ( 13.5 and 5.2 substitutions / kb , respectively ) , which is likely the result of purifying selection acting on coding regions . however , substitution rates in pseudogenes and intergenic regions are significantly lower than our estimate of the neutral substitution rate using synonymous substitutions across the genome ( 49.2 synonymous changes/1,000 synonymous sites ) ( binomial test , p < 0.001 ) . table 1gene content in blochmannia chromaiodes and b. pennsylvanicusspeciessize ( bp)gc content ( % ) total genesprotein - coding genestrnarrnaother rnapseudogenesframeshifted genesb . pennsylvanicus791,65429.6658609403334 table 2substitutions and indels between blochmannia chromaiodes and b. pennsylvanicussubstitutionssubstitutions / kbindelsindels / kbindel bpindel bp / kbprotein - coding8,16613.5630.11750.3rna - coding455.260.7212.4pseudogene6326.8187.711247.6intergenic5,09429.39605.52,00411.5ambiguous21na4na9natotal13,38916.91,0511.32,3212.9note . na , not applicable.to account for differences in the amount of each sequence type in the two genomes , rates were calculated using the b. chromaiodes and b. pennsylvanicus annotations and then averaged.ambiguous refers to positions annotated differently in the two genomes ( i.e. , protein - coding in b. chromaiodes and intergenic in b. pennsylvanicus ) . gene content in blochmannia chromaiodes and b. pennsylvanicus substitutions and indels between blochmannia chromaiodes and b. pennsylvanicus note.na , not applicable . to account for differences in the amount of each sequence type in the two genomes , rates were calculated using the b. chromaiodes and b. pennsylvanicus annotations and then averaged . ambiguous refers to positions annotated differently in the two genomes ( i.e. , protein - coding in b. chromaiodes and intergenic in b. pennsylvanicus ) . rates of indel events in pseudogenes and intergenic regions are similar ( 7.7 and 5.5 indels / kb , respectively ) , and these are higher than those of protein - coding and rna - coding genes ( 0.1 and 0.7 indels / kb , respectively ) , further demonstrating the role of purifying selection . when we consider the number of nucleotides involved in indels , protein - coding and rna - coding genes still have relatively low rates ( 0.3 and 2.4 indel bp / kb , respectively ) compared with pseudogenes and intergenic regions . however , we observed a higher rate for pseudogenes ( 47.6 indel bp / kb ) compared with intergenic regions ( 11.5 indel bp / kb ) . conservation of intergenic spacer length was observed in previous comparisons of blochmannia and buchnera endosymbionts ( degnan et al . 2005 , 2011 ) . blochmannia intergenic regions may contain regulatory sequences or other elements under constraint , as recently noted for buchnera ( degnan et al . it is also possible that truncated products from the transcription and translation of pseudogenes are detrimental to the cell and increase selective pressure for deletions . we did not detect any large ( > 60 bp ) indels between b. chromaiodes and b. pennsylvanicus . almost all of the 1,051 indels are small ; 671 ( 63.8% ) involve a single base , and 1,010 ( 96.1% ) are 6 bp . previous intraspecific analyses of blochmannia and buchnera emphasized the importance of repeat regions as indel hotspots in the at - rich genomes of these endosymbionts ( gomez - valero et al . 2008 ; moran et al . 2009 ; williams and wernegreen 2012 ) . to explore this in our interspecific comparison , we determined the repeat context of indels , focusing on two repeat types : homopolymers and mssrs . we define homopolymers as tracts of 2 bp of a single nucleotide and mssrs as 2 contiguous occurrences of a 26 bp repeat unit . for our purposes , we did not consider these repeat types mutually exclusive when identifying repeat regions . in other words , a particular region may contain an mssr , which itself includes a homopolymer , such as caaatcaaat . to ensure that we are not overlooking the contribution of either repeat type of 1,051 indels , 1,034 ( 98.4% ) occur in a repeat region ( either homopolymer or mssr ) . regarding repeat types , 932 indels ( 88.7% ) occur in homopolymers , and 611 indels ( 58.1% ) occur in mssrs . to further assess the impact of repeat type on indels , we determined whether indels comprise 1 complete repeat unit . we found that 737 of 1,034 indels occurring in repeat regions ( 71.3% ) comprise 1 repeat unit , with 579 of these comprising 1 homopolymer unit ( 78.6% ) and 158 of these comprising 1 mssr unit ( 21.4% ) . the remaining 297 indels occurring in repeat regions ( 28.7% ) either are not part of the repeat unit ( e.g. , an indel of g where the b. pennsylvanicus sequence is tattat - tat , and the b. chromaiodes sequence is tattatgtat ) or include partial repeat units ( e.g. , an indel of ttcat where the b. pennsylvanicus sequence is cattcatt , and the b. chromaiodes sequence is ca-----t ) . these data provide strong support for the importance of repeat regions as indel hotspots in at - rich endosymbionts . our analysis not only reflects the recognized and significant role of homopolymers in indel mutations but also emphasizes the role of mssrs . we initially noted the possible contribution of mssrs in an intraspecific comparison of b. vafer ( williams and wernegreen 2012 ) . the larger pool of indels afforded by our interspecific comparison here reinforces the contribution of mssrs to indel mutation in this bacterial endosymbiont . the low rate of indels in protein - coding genes ( 0.1 indels / kb ) reflects strong effects of purifying selection . of 63 indels in protein - coding genes ( table 2 ) , 41 have lengths equal to a multiple of three and do not disrupt the reading frame . of the remaining 22 nontriplet , frameshift - inducing indels , 21 fall into one of two categories : compensatory indels or indels near the 3-end of genes . the deleterious effect of these indels may be limited , thereby enabling them to escape purging by purifying selection and possibly act as drivers of protein divergence . compensatory indels involve two or more indels that combine to preserve the reading frame . for our purposes , we define compensatory indels with three criteria : 1 ) indels occur within 20 bp , 2 ) length of each individual indel is not divisible by three , and 3 ) combined length of indels is either three or zero . using these criteria , we identified three instances of compensatory indels between b. chromaiodes and b. pennsylvanicus , each involving two single - base indels ( fig . 1 ) . these six indels comprise 27.2% of the 22 frameshift - inducing indels . studies of mammalian genomes show increased frequency of compensatory indels in exons compared with introns , suggesting selection for compensation ( hu and ng 2012 ) . our data indicate that this mechanism may also operate in bacterial genomes . in at - rich endosymbiont genomes , high densities of homopolymers prone to polymerase slippage may afford more opportunities for compensatory indels to occur and correct frameshifts . fig . 1.compensatory indels detected in a whole genome alignment of blochmannia chromaiodes and b. pennsylvanicus . for each , the alternative alignment hypothesis with only nucleotide substitutions is also shown . compensatory indels detected in a whole genome alignment of blochmannia chromaiodes and b. pennsylvanicus . for each , the alternative alignment hypothesis with only nucleotide substitutions is also shown . we explored whether compensatory indels are associated with levels of protein divergence by calculating pairwise amino acid distances between b. pennsylvanicus genes and their orthologs in escherichia coli , a free - living relative . pennsylvanicus comparison are among the most divergent proteins in the comparison with e. coli ( supplementary fig . this pattern is consistent with the expected effects of purifying selection on frameshift - inducing indels . at highly conserved genes , a frameshift - inducing indel is likely to be removed by strong purifying selection . by contrast , a frameshift - inducing indel might persist longer in more divergent genes under relaxed selection , and compensation via a subsequent indel may act as a mechanism to prevent or reduce the accumulation of deleterious mutations in these genes . comparisons of yeast species suggest that compensatory indels may be a driver of rapid protein evolution ( kellis et al . even though out - of - frame sequence between compensatory indels is short ( < 6 bp ) in our b. chromaiodes b . pennsylvanicus comparison , these indels result in one or two amino acid substitutions ( fig . 1 ) , suggesting that compensatory indels may also impact protein divergence in endosymbiont genomes . interestingly , the nature of compensatory indels may hamper analyses of protein evolution , such as tests of positive selection , in more divergent species . others have explored the impact of indel - associated alignment error on tests of positive selection ( fletcher and yang 2010 ; jordan and goldman 2012 ) ; however , these analyses used codon - based alignments constraining indels to frame - preserving triplets , thereby excluding the possibility of compensatory indels . when we similarly constrain alignments of the regions with compensatory indels in b. chromaiodes and b. pennsylvanicus , this results in three or five nucleotide substitutions over < 6 bp ( fig . sixteen frameshift - inducing indels do not have compensatory indels nearby to restore the reading frame . all but one of these occur less than 60 bp from the 3'-end of the gene ( table 3 ) . although these indels disrupt the reading frame , their location minimizes the resulting impact on protein sequence . in fact , two indels involve a complete mssr repeat unit at the exact 3'-end of the gene and therefore have no effect on gene sequence or length . thirteen indels result in differences in protein length ranging from 1 to 13 aa . although we can not distinguish insertions from deletions in this comparison , previous analyses of buchnera endosymbionts of aphids showed higher prevalence of deletions in the 3'-end of genes ( charles et al . downstream of these indels , the nucleotide sequence is highly conserved , whereas the amino acid sequence shows more changes ( on average , 3.5 aa substitutions per nt substitution ) , suggesting that these indels are drivers of rapid protein divergence . previous work showed that transcription of genes with frameshifts in homopolymers generates a mixed transcript pool , including full - length transcripts in which the frameshift was corrected by polymerase slippage ( tamas et al . this may enable some frameshift - inducing indels to persist despite purifying selection , thereby catalyzing adaptive protein evolution or initiating the process of gene degradation . table 3indels occurring in the 3'-end of blochmannia chromaiodes and b. pennsylvanicus genesgeneindel size ( bp)repeat context of indeleffect of indeldifference in protein length ( aa)amino acid substitutionsnucleotide substitutionsyral / rsmi12 g hp510psd18 a hp312ftsq12 a hp130def25 at mssr000ahpc42 taag mssr000ribe14 t hp131ubig17 a hp410nuoa18 a hp1330aroc16 a hp510ureg2none333ispg14 c hp161rplj17 a hp210rplk12 t hp100gpsa16 a hp230meta14 a hp421considering the region from the indel to the end of the longest gene sequence.hp refers to homopolymers , and mssr refers to multimeric simple sequence repeats.because this indel comprises a complete repeat unit at the exact end of the gene , there is no detectable effect on the gene or protein sequence . indels occurring in the 3'-end of blochmannia chromaiodes and b. pennsylvanicus genes considering the region from the indel to the end of the longest gene sequence . because this indel comprises a complete repeat unit at the exact end of the gene , there is no detectable effect on the gene or protein sequence . the genome - wide comparison of b. chromaiodes and b. pennsylvanicus presented here provides an important view of the spectrum of indel mutations impacting endosymbiont genome evolution . the role of homopolymers and mssrs as indel hotspots , which was previously observed in intraspecific comparisons , is strongly evident in this interspecific analysis . although purifying selection limits indel mutations in coding and intergenic regions , we detected frameshift - inducing indels in protein - coding genes that may act as important drivers of protein divergence and contribute to ongoing genome reduction . supplementary figures s1 and s2 and table s1 are available at genome biology and evolution online ( http://www.gbe.oxfordjournals.org/ ) .
olanzapine is an atypical antipsychotic which is efficacious in the treatment of schizophrenia . the adverse effect profile for olanzapine is benign except for higher rates of metabolic events . orthostatic hypotension is less commonly reported with olanzapine as compared to first - generation and few atypical antipsychotics . we report a case where olanzapine , in a dose dependent fashion , caused transient postural hypotension .	olanzapine is an atypical antipsychotic with efficacy in schizophrenia slightly higher than others.1 ) the extrapyramidal adverse effects are much lower than typical antipsychotics , though it has a higher metabolic adverse effect profile.2 ) orthostatic hypotension is common with first - generation antipsychotics with rates up to 77% compared to 15% with placebo.3 ) among atypical antipsychotics , clozapine , risperidone and quetiapine have higher rates of orthostatic hypotension.3 ) we report a case where olanzapine , in a dose dependent fashion , caused transient postural hypotension . a 22-year - old female was diagnosed with paranoid schizophrenia 16 months back and was started on olanzapine , which was increased up to 15 mg / day over two weeks . she responded well to it , resuming her higher studies as well as a part - time job of a school teacher . however , after two months , she had sudden falls on five occasions over one week , seriously injuring herself with bruises and lacerations . vitals were checked on all the occasions which showed pulse rate ranging from 7090 beats per minute ( bpm ) and blood pressure ranging from 110130/ 7080 mmhg . however , in all the occasions the examinations could be done after the patient had recovered and gained consciousness . investigations including complete blood count , fasting and post prandial plasma glucose , lipid profile and liver function test were normal . her body weight has been maintained at 52 kg over the past one year , her menstrual cycles were regular and abdominal ultrasonography revealed no abnormalities . however , on current assessment both cortisol ( 9.31 g / dl ) and adrenocorticotropic hormone ( 14.5 pg / ml ) levels were within normal range with respect to age , sex and time ( morning / evening ) of sampling . she was advised rest at home , dose of olanzapine was reduced to 10 mg / day and a round - the - clock nurse was appointed for observation and monitoring . thereafter , the frequency of fall reduced , and there was only one such event when she was dizzy and about to fall but prevented by the attending nurse . her pulse rate was 140 bpm and blood pressure was 76/40 mmhg in supine position , and three minutes later pulse rate was 80 bpm and blood pressure was 110/70 mmhg in standing position , which was suggestive of orthostatic hypotension . she recovered from her dizziness and was fully oriented within five minutes of the event . subsequently , olanzapine was cross - tapered with aripiprazole up to 15 mg / day , on which she is maintaining well without further recurrence of adverse effects during follow up at two months . our patient developed orthostatic hypotension with olanzapine 15 mg per day that subsided following discontinuation of medication . there are few reports of hypotension associated with olanzapine,4,5 ) one in an elderly individual , and the other with intramuscular administration . the summary of product characteristics for olanzapine reports 5% subjects develop orthostatic hypotension as compared to 2% in placebo,6 ) which is much less as compared to the rates observed with first generation antipsychotics.3 ) nevertheless , hypotension and bradycardia has been reported with olanzapine as low as 5 mg per day.7 ) most commonly orthostatic hypotension is an incidental finding during routine examinations , and symptomatic hypotension is not usually observed except in the elderly patients . in contrast , our patient , a young female , developed symptomatic orthostatic hypotension with olanzapine that resulted in several episodes of falls , which required treatment discontinuation . interestingly , nourian et al.8 ) had shown through animal experiments that olanzapine , along with ziprasidone and aripiprazole has the least propensity to cause postural hypotension . several other putative mechanisms such as calcium blockade , inhibition of centrally mediated pressor reflexes , and negative inotropic effects , have also been proposed for this adverse effect.9 ) sedative effect of olanzapine may additionally contribute to the falls , though such mechanism appeared unlikely in our case . although the rates of orthostatic hypotension appear lower with olanzapine , an exercise of caution is definitely warranted in the light of the current case .
lymphocytic choriomeningitis virus ( lcmv ) was detected in 2 patients with acute meningitis in southern spain within a 3-year period . although the prevalence of lcmv infection was low ( 2 [ 1.3% ] of 159 meningitis patients ) , it represents 2.9% of all pathogens detected . lcmv is a noteworthy agent of neurologic illness in immunocompetent persons .	the population included patients at hospital virgen de las nieves ( granada , spain ) who had suspected ame . routine virologic investigation included reverse transcription pcr ( rt - pcr ) of cerebrospinal fluid ( csf ) samples for detection of hev ( xpert ev system , cepheid , sunnyvale , ca , usa ) , tosv ( 7 ) , and mumps virus ( 8) , and pcr of herpes simplex virus ( hsv ) and varicella - zoster virus ( lc vhs 1/2 qual and lc vzv systems , respectively ; roche diagnostics , mannheim , germany ) . negative samples were subsequently tested for epstein - barr virus , cytomegalovirus , human herpes 6 virus ( 9 ) , flavivirus ( 10 ) , arenavirus ( 2 ) , adenovirus ( 11 ) , and phlebovirus ( 12 ) . specific lcmv rt - pcr was conducted by using a previously described protocol ( 2 ) . finally , csf specimens from pcr - negative case - patients were inoculated in vero and mrc-5 cell lines for viral culture . csf and acute - phase serum samples were also tested for igg and/or igm against tosv ( eia enzywell toscana virus igg / igm , diesse , siena , italy ) , west nile virus ( elisa igg and igm - capture elisa ; focus diagnostics , cypress , ca , usa ) , and lcmv by indirect fluorescent assay ( ifa ) with further confirmation by western blot ( 2 ) . we studied 159 csf samples by using pcrs for the presence of hev , hsv , varicella - zoster virus , tosv , and mumps virus , yielding 68 positive cases . a viral agent was detected in 70 ( 44% ) cases : hev accounted for 44 ( 63% ) of positive cases , followed by varicella - zoster virus in 11 ( 16% ) , hsv-1 in 8 ( 11% ) , tosv in 4 ( 6% ) , lcmv in 2 ( 3% ) , and hsv-2 in 1 ( 1% ) . case - patient 1 , a 21-year - old woman , came to the hospital s emergency unit in april 2008 exhibiting headache , chills , fever ( 38.9c ) , confusion , nausea , vomiting , and slight nuchal rigidity . laboratory results were 415 leukocytes / mm ( 100% mononuclear cells ) , 43 mg/100 dl glucose level ( reference 3565 mg / dl ) , and 128 mg / dl protein level ( reference 1545 mg / dl ) in the csf . igg and igm titers of 640 and 128 against lcmv were detected in the serum sample by ifa and titers of 400 and 200 by western blot assay , respectively . viral culture of the csf in vero cells revealed no cytopathic effect after 1 month of incubation . viral isolate ( eeb-7 ) was used for genetic characterization . to sequence the genome , degenerate and specific primers were designed on the basis of an alignment of all complete lcmv large ( l ) , glycoprotein complex and nucleocapsid protein sequences ( table).terminal sequences were generated by using a universal arenavirus primer , targeting the conserved viral termini ( 5-cgc acm gdg gat cct agg c-3 ) , combined with 4 specific primers positioned near the ends of each segment . amplification products were size - fractionated by electrophoresis in 1% agarose gels , purified ( minelute , qiagen , valencia , ca , usa ) , and sequenced in both directions on an abi prism 3700 dna analyzer ( pe applied biosystems , foster city , ca , usa ) . to determine the evolutionary history of isolate eeb-7 , we performed bayesian phylogenetic analysis of all available complete genome sequences of lcmv using beast , beauti , and tracer analysis software packages ( 13 ) . clinical diagnosis was acute meningitis and the patient was discharged from hospital on day 9 . * lcmv , lymphocytic choriomeningitis virus ; gpc , glycoprotein comlex ; np , nucleocapsid protein . the name of the strain is followed by genbank accession number , country , and year of detection . phylogenetic tree showing genetic lymphocytic choriomeningitis virus sequences relationship within the small segment coding for the glycoprotein complex ( a ) and nucleocapsid proteins ( b ) . the name of the strain is followed by genbank accession number , country , and year of detection . case - patient 2 , a 39-year - old man , sought treatment in may 2010 with headache , nausea , vomiting , increased perspiration , and a temperature of 37.5c . csf analysis demonstrated 1,715 leukocytes / mm ( 95% mononuclear cells ) , normal glucose level ( 68 mg / dl ) , and elevated protein levels ( 240 mg / dl ) . further virologic investigation detected lcmv rna in the csf and an igg titer of 640 by ifa and igm antibodies against lcmv in the serum sample . the sequence of a 194-nt pcr product ( nucleocapsid protein gene ) obtained was most closely related to sequences of the lineage i. sequence homology among the lcmv amplicon from case - patient 2 and lineage i strains was > 87% versus 77%79% sequence homology among case - patient 2 and strains from lineages ii iv . pcr has become the reference standard for identifying common viruses involved in ame ( 6 ) . however , no commercial tests are available for lcmv ; and in - house pcrs have to be optimized according to the natural genetic diversity of the virus ( 14 ) . neurologic lcmv infection in spain has been diagnosed primarily by serologic tests ( 5,6 ) . the diagnosis in case - patients 1 and 2 in the current study was achieved by serologic testing as well . nonetheless , pcr was useful because it allowed genetic characterization of the lcmv strain from case - patient 2 . furthermore , the viral isolate of case - patient 1 was evident only by detection of lcmv rna in the cell culture supernatant because no cytopathic effect was observed . isolates from both lcmv case - patients belonged to the classical lineage i , which has been detected elsewhere in europe . lineage i is usually associated with human disease ( as are lineages ii and iii ) and is linked to the common house mouse as its reservoir ( 14 ) . lineage iv viruses were previously detected in spanish wood mice ( 2 ) and have not been associated with human disease . although no further epidemiologic studies could be conducted to search for infected reservoirs , the common genetic lineage and the fact that both case - patients resided within the same community might suggest transmission of lcmv by a common vector . previous seroprevalence studies have associated human lcmv infection with high exposure to the common house mouse ( 15 ) . human lcmv infections might be underdiagnosed because the clinical characteristics of lcmv meningitis are similar to those of other viral meningitis ; no commercial tests are available for serologic or molecular diagnostic assays , and usually no clear epidemiologic clue is available at the moment of diagnosis . thus , epidemiologic and virologic surveillance might ascertain that the true incidence of lcmv ame is more frequent than reported .
background and aims : unique post - partum endocrine hormone oxytocin secreted during breastfeeding ( bf ) has amnestic , sedative properties and down - regulates stress responses . this study was done to assess the effect of bf on consumption of propofol , sevoflurane and haemodynamic stability in women.methods:study was conducted on 120 women aged 2030 years of american society of anesthesiologists i and ii physical status scheduled for tubectomy under general anaesthesia who were randomly allocated to three groups 40 of each ; bf , withhold feeding ( wf ) , and non - feeding ( nf ) groups . all received standard premedication . heart rate ( hr ) , mean arterial pressure ( map ) and state entropy ( se ) values were recorded at regular intervals . all patients were induced with intravenous propofol until the se levels dropped to 45 , and dose of propofol recorded . airway was secured with laryngeal mask airway and anaesthesia was maintained with sevoflurane in 60% n2o and o2 . sevoflurane concentration was adjusted to maintain se between 40 and 60 . end tidal concentration of sevoflurane and consumption of sevoflurane ( ml ) was recorded by ge datex - ohmeda s/5 system . results were analysed by analysis of variance and chi - square test.results:demographic parameters were comparable . dose of propofol and sevoflurane consumption in group bf was significantly reduced by 20% and 35% , respectively ( p < 0.05 ) compared to group nf . intra - operative hr and map were persistently low in group bf and elevated in group wf ( p < 0.05).conclusion : bf before induction of anaesthesia decreases the consumption of propofol , sevoflurane and maintains the intra - operative haemodynamic stability , whereas withholding bf increases propofol and sevoflurane consumption with intra - operative higher hr and map , compared to control group .	oxytocin ( ot ) is a maternal hormone with effect on uterine contraction and milk ejection . more recently , ot has been shown to influence a wide spectrum of behavioural , physiological and endocrine functions mediated through receptors within the brain . ot induces several anti - stress - like effects ; decrease in heart rate ( hr ) , blood pressure and levels of stress hormones . administration of low dose ot can induce anxiolytic - like effect and in higher doses sedative effect . however , very few studies have explored the role of ot on anaesthetic and analgesic consumption . this study was conducted to determine the effect of ot secreted during breastfeeding ( bf ) on consumption of propofol and sevoflurane and assessment of its effect on intra - operative haemodynamics as the secondary outcome . before commencing this single blind prospective study , approval was obtained from the institutional review board . about 120 women with american society of anesthesiologists physical status i and ii , aged 2030 years , who had uncomplicated pregnancy , delivered full - term , healthy infants by the vaginal route and scheduled for tubectomy were enrolled for the study between july 2014 and december 2014 . parturients with a history of cerebrovascular disease , hypertension , diabetes mellitus and other endocrine disorders , treatment with psychoactive medication , anticipated difficult airway , parturients on sedatives or analgesics and weighing < 70% or more than 130% of ideal body weight were excluded from the study . thorough pre - anaesthetic check - up of all patients including routine investigations was carried out . the parturients were allocated to three groups of 40 each depending on their lactation status . lactating women were randomly allocated to either bf group or withhold feeding ( wf ) group based on computer - generated randomisation technique ( www.random.org ) , whereas non - lactating women were considered for the control group ( non - feeding [ nf ] ) . the bf group ( n = 40 ) consisted of lactating women who breastfed just before induction of anaesthesia . wf group ( n = 40 ) included lactating women who withheld bf for 4 h before induction of anaesthesia . nf group ( control ) ( n = 40 ) included non - lactating women ( coming for interval tubectomy ) who had delivered more than a year back and stopped bf on their own for a minimum of 4 weeks before surgery . the details of randomisation were kept with the principal investigator and not revealed to other investigators until the end of the study . the anaesthesiologist monitoring the patient intra - operatively and post - operatively was not aware of the lactation status of the women or their group allocation ; none of the cases were primigravida . on arrival in the operating room , patients were monitored for hr , blood pressure and oxygen saturation by continuous electrocardiogram , non - invasive blood pressure and pulse oximetry , respectively , and basal parameters were recorded . depth of anaesthesia was monitored by entropy analysis ( entropy module , datex - ohmeda s/5 avance workstation , ge healthcare ) . patients were started on intravenous ( iv ) infusion of ringer 's lactate solution solution premedicated with injection midazolam 1 mg iv , injection glycopyrrolate 0.2 mg iv , injection fentanyl 2 g / kg iv and preoxygenated with 100% oxygen for 3 min . anaesthesia was induced with iv propofol 10 mg increments injected over 5 s , at 10 s intervals until the state entropy ( se ) levels dropped to 45 , total dose of propofol required to achieve se 45 was recorded , and airway secured with laryngeal mask airway - classic ( lma ) ( as per manufacturer 's recommendations ) , and patients were maintained on assisted spontaneous ventilation . if more than 2 attempts were required for lma insertion , cases were excluded from the study and were managed as per standard institutional protocol . additional doses of propofol 10 mg were administered as necessary when se values increased to more than 60 during lma insertion , and the amount of supplemental propofol used was noted . anaesthesia was maintained with sevoflurane in 60% n2o and o2 , with ventilation adjusted to maintain end - tidal co2 between 35 and 40 mmhg . sevoflurane concentration was set at 2% initially and adjusted subsequently to maintain se levels between 40 and 60 , and the difference between response entropy ( re ) and se < 10 . if the difference between re and se exceeded 10 , additional doses of fentanyl 1 g / kg was administered . fresh gas flow rate was set at 4 l / min for initial 5 min and then reduced to 2 l / min . sevoflurane and n2o were discontinued at the end of closure of abdomen and lma removed after complete recovery . patients were monitored in the post - operative period for 6 h. hr , mean arterial pressure ( map ) , etco2 , se and re were recorded at baseline , at induction and every 5 min after lma insertion until 15 min into post - operative period . end - tidal concentration of sevoflurane was recorded at 5 min intervals from the time of lma insertion until discontinuation of anaesthetics . parameters recorded were total duration of surgery , duration of anaesthesia , duration of sevoflurane use , total dose of propofol consumed and total volume of sevoflurane consumed ( obtained by the anaesthesia gas module of ge datex - ohmeda s/5 anaesthetic delivery unit system ) . sample size was calculated based on findings of a pilot study conducted in our institute involving ten lactating women . the average end - tidal sevoflurane concentration was 1.45 0.32 . keeping the power of study at 80% and confidence limits at 95% , to detect a minimum of 15% difference in sevoflurane consumption between groups , assuming normal distribution of values in both groups , the minimum sample size required was thirty in each group , we included forty patients in each group for better validation of results . analysis of variance has been used to find the significance of study parameters between three or more groups of patients , student 's t - test ( two - tailed , independent ) has been used to find the significance of study parameters on continuous scale between two groups ( inter - group analysis ) on metric parameters . chi - square or fisher exact test has been used to find the significance of study parameters on a categorical scale between two or more groups . the statistical software , namely sas 9.2 , spss 15.0 , stata 10.1 , medcalc 9.0.1 , systat 12.0 and r environment version 2.11.1 ( ibm , usa ) were used for the analysis of the data and microsoft word and excel were used to generate graphs , tables , etc . a total of 120 patients were included in the study with 40 in each group . the mean duration of the surgery , mean duration of anaesthesia and mean duration of sevoflurane use were comparable between the three groups [ table 1 ] . post - partum day distribution [ table 2 ] in lactating women were comparable among the bf group and wf group ( p = 1.0 ) . post - partum week distribution in lactating women ( breastfeeding , withhold feeding groups ) baseline hr [ figure 1 ] was comparable in all the three groups ( p-0.4 ) ; intraoperatively , a significant increase in hr was observed in wf group compared to other groups at all - time points until post - operative period ( p < 0.001 ) , bf group had lower hr throughout the procedure compared to other groups ( p < 0.001 ) . changes in heart rate ( mean standard deviation ) : baseline ( pre - induction ) ; at induction ( state entropy 45 ) ; after intubation ; at 5 , 10 , 25 , 30 and 15 min post - extubation baseline map [ figure 2 ] was comparable in all the three groups ( p-0.1 ) , map was persistently higher in wf group compared to other groups ( p < 0.001 ) and bf group had lower and stable map throughout the procedure compared to other groups ( p < 0.001 ) . comparison of mean arterial pressure ( mm hg ) in three groups of patients studied changes in mean arterial pressure ( mean standard deviation ) ; baseline ( pre - induction ) ; at induction ( state entropy 45 ) ; after intubation ; at 5 , 10 , 25 , 30 and 15 min post - extubation ; ( se 45 - state entropy 45 , map - mean arterial pressure ) se [ figure 3 ] values were comparable at baseline and throughout the procedure among all the groups . state entropy : baseline ( pre - induction ) ; at induction ( state entropy 45 ) ; after intubation ; at 5 , 10 , 25 and 30 ; ( se 45 - state entropy 45 ) the difference in requirement of propofol [ table 3 ] to reach se 45 was statistically significant in bf and wf groups when compared to control group , and requirement was highest ( 102.88 5.30 mg ) in wf group and least ( 74.13 6.97 mg ) in bf group . comparison of propofol and sevoflurane usage the difference in requirement of sevoflurane [ table 3 ] to maintain anaesthesia was statistically significant in bf and wf groups when compared to control group , requirement was highest ( 6.43 0.75 ml ) in wf group and least ( 3.80 0.76 ml ) in bf group . the average end - tidal concentration of sevoflurane was significantly lower in bf group and higher in wf group compared to group nf ( p < 0.001 ) . post - hoc analysis between groups with regards to propofol and sevoflurane consumption showed statistically significant difference between groups nf , bf and wf , respectively . in the current study , the effect of ot which is secreted during bf on haemodynamics and anaesthetic drug consumption was determined . ot is mainly produced and synthesised in magnocellular neurons of the hypothalamic paraventricular ( pvn ) and supraoptic nuclei and secreted into the periphery via the posterior neurohypophysis . ot is also produced in parvocellular neurons within the pvn , and these neurons project to many areas within the brain such as other hypothalamic nuclei , the median eminence , amygdala , hippocampus , locus coeruleus , striatum , raphe nuclei , the dorsal motor nucleus of the vagus nerve ( dmx ) and nucleus tractus solitarii ( nts ) . in addition , oxytocinergic fibres project down to the spinal cord , where they terminate on the presynaptic neurons of the sympathetic chain in the intermediolateral cell column and also in the dorsal horn in the area where pain modulation takes place . the central actions of the ot are mediated via ot receptors ( otr ) which are distributed widely in the central nervous system ( cns ) . until date , only one otr is identified which is a member of class 1 g protein - coupled receptor family . during physiological and psychological stress responses , oxytocinergic neurons are activated , particularly in pvn of the hypothalamus and secreted into the circulating blood . ot induces several anti - stress - like effects such as decrease in hr , blood pressure and the levels of stress hormones , . these effects of ot are probably mediated through the hypothalamus and the vagal nuclei ( dmx and nts ) . increase in ot release from the hypothalamus inhibits hypothalamic - pituitary - adrenal ( hpa ) axis by acting at three different levels . legros et al . demonstrated that human males treated with exogenous ot followed by synthetic adrenocorticotropic hormone ( acth ) showed a blunted response in cortisol secretion , suggesting that exogenous ot inhibits corticosteroid synthesis in the adrenal gland . second , neumann et al . demonstrated that peripheral ot inhibits acth release from the pituitary gland . they injected exogenous corticotrophin - releasing factor ( crf ) into lactating rats and measured acth responses , finding a reduction in acth response . the earlier study conducted on humans showed that bf women had significantly lower hormonal stress responses ( as evident by lower cortisol and acth ) during exercise stress than non - bf mothers and women without children . several follow - up studies have detected lower cardiovascular markers of stress ( as evidenced by lower basal systolic blood pressures , higher levels of cardiac parasympathetic control and modulation of hr reactivity ) during the task in breastfeeders as compared with non - bf mothers and women without children . light et al . found similar cardiovascular patterns for bf mothers during the anticipation of the public - speaking stressor ; it is possible that any stress - buffering effects of bf are more potent during the immediate bf period . have shown that intranasal administration of ot , which passed directly into the brain , suppressed cortisol response to psychological stress , as well as attenuated emotional functions after stress episodes , indicating in humans , the inhibitory effect of ot on hpa activity mediated through cns in addition to peripheral effect . however , short - term iv administration of ot to women to enhance uterine contractions or decrease blood loss during labour or caesarean delivery confirms its effect in decreasing blood pressure . this hypotensive response to ot is due to decreases in total vascular resistance despite compensatory increases in hr , stroke volume and cardiac output . grewen et al . tested 28 early post - partum mothers , obtaining multiple blood samples for ot , the sympathetic marker , norepinephrine ( ne ) and the lactation hormone , prolactin while monitoring their cardiovascular responses to two stressors . they observed that greater overall ot level was related to lower plasma ne and higher prolactin levels ; in contrast , higher ne was linked to increases in hr and decreases in stroke volume . these data support a cardioprotective role for ot , which may influence the magnitude and haemodynamic determinants of cardiovascular stress responses . similar findings were observed in our study , with women receiving anaesthesia immediately after bf showing a better haemodynamic profile compared to those who withheld bf and nf women . the anxiolytic - like effect seems to be mediated within the amygdala , which is richly provided with otrs , during bf , there may also be increased cns gamma - amino butyric acid , a major inhibitory neurotransmitter , which may inhibit the affective state and behaviour of the lactating animal . it was observed that administration of ot can induce both anxiolytic - like effects and in higher doses , sedative effects . the results suggested there was temporary impairment although there was no correlation between ot levels and cognitive performance . the evidence that ot impairs some memory - related tasks has led to suggestions that it has a role in the forgetting of delivery pain in mothers . analgesic effect has been linked to the periaqueductal grey and the dorsal horn of the spinal cord . ot increases nociceptive thresholds through an enhancement of endogenous opioids . classical studies have demonstrated that chronic ot treatment induced not only anti - stress effects but also analgesia ; this analgesic effect was blocked by the opioid antagonist naloxone but not by an ot antagonist , indicating that ot increases endogenous opioid transmission . the above - described physiological properties of ot may have contributed to decreased requirement of induction dose of propofol and maintenance dose of sevoflurane in this study in women who breastfed just before induction of anaesthesia . plasma levels of ot normally vary between 10 and 100 pg / ml . ot secretion peaks during child birth and during bf , levels returning to normal in between bf . blunting of plasma stress hormone levels were seen up to 30 min after bf , and bf women did not have attenuated physiological or subjective anxiety responses to a laboratory psychosocial stress administered 1 h after their last episode of lactation . this may be the reason for the higher requirement of sevoflurane and propofol in women in whom bf was withheld . the possible rebound increase in noradrenaline levels following ot - induced temporary suppression may explain higher hr and map in these women , practice implication of this concept include the possibility that the withhold feeding mother might be physiologically more fragile and more susceptible to post - partum illness , further research is required to provide sufficient evidence for this effect . when ot levels are measured in the cerebrospinal fluid , levels 510 fold higher than in plasma are normally found , but the concentration of ot at its receptor sites is not known . ot does not readily cross into the brain and needless to say , blood levels often do not correlate well with brain levels , so measuring peripheral ot may not correlate levels at otr within the cns . in humans , minimum alveolar concentration ( mac ) is reduced by 30% during early gestation , and in post - partum women mac returns to that of the non - pregnant state within 2 days of delivery , possibly due to sedative effect of progesterone which returns to baseline within 2448 h after delivery . post - partum , gastrointestinal tract related and mechanical factors are relieved immediately after delivery , gastric emptying time returns to normal as early as 18 h post - partum and gastric volume and ph are comparable with non - pregnant women . all cases in our study were elective and were fasting overnight and received acid aspiration prophylaxis ; no signs of acid aspiration ( gastric contents / stain on lma at extubation time ) were seen in our cases , but it is recommended to use lma - proseal in post - partum period . the present findings in lactating women were restricted to after 3 days and within 1 week after giving birth , and we could not compare with exclusive formula feeding mothers to confirm if the same associations occur in these groups . as these data are suggestive rather than conclusive , further research is required to provide sufficient evidence on effects of ot parse on the requirement of anaesthetic and non - anaesthetic drugs . the results of this study indicate that bf before induction of anaesthesia attenuates the stress response to surgery and maintains haemodynamic stability , and reduces the consumption of both propofol and sevoflurane , and with - holding of bf increases the stress response with increased consumption of both propofol and sevoflurane .
the maximum stress induced plays vital role in fatigue life improvement of leaf springs . to reduce this maximum stress , leaves with different unassembled cambers are assembled by pulling against each other and a common curvature is established . this causes stress concentration or sets assembly stress in the assembled leaf springs which is subtractive from load stress in master leaf while it is additive to load stress for short leaves . by suitable combination of assembly stresses and stepping , it is possible to distribute the stress and improve the fatigue life of the leaf spring . the effect of assembly stresses on fatigue life of the leaf spring of a light commercial vehicle ( lcv ) has been studied . a proper combination of stepping and camber has been proposed by taking the design parameters into consideration , so that the stress in the leaves does not exceed maximum design stress . the theoretical fatigue life of the leaf springs with and without considering the assembly stresses is determined and compared with experimental life . the numbers of specimens are manufactured with proposed parameters and tested for load rate , fatigue life on a full scale leaf springs testing machine . the effect of stress range , maximum stress , and initial stress is also discussed .	leaf springs like all other springs serve to absorb , store , and release the energy . in heavy vehicles , leaves are stacked one upon another to ensure rigidity and strength . during its operation , the leaf spring requires that the load rate should vary within limit ( 7% ) and the maximum stress induced should be lower than the maximum design stress . for a given stress range the leaf spring should have maximum fatigue life or as specified . the stress range and the maximum stress induced in the leaf spring play a vital role in deciding the load rate and fatigue life of the leaf spring . the maximum stress induced can be reduced by assembling the leaves with different radii of curvature and establishing a common curvature under no load . the proper distribution of the stress between the leaves can enhance the fatigue life of the leaf springs . evaluated the axial fatigue strength of en45a spring steel specimen experimentally as a function of shot peening in the conditions used . s / n curves of the specimens were correlated with leaf springs curve in vehicles . concluded that the influence of high contact pressure and temperatures resulted in microweld between the two leaf surfaces . the fatigue strength of the leaf springs was studied as a function of shot peening parameters . an experimental leaf springs model was verified by using a leaf springs test rig that could measure vertical static deflection of leaf springs under static loading condition . the results showed a nonlinear relationship between the applied load and the leaf springs deflection for both directions of loading , in form of a hysteresis loop . . studied the origin of premature failure analysis procedures , including examining the leaf spring history . the visual inspection of fractured specimens and simulation tests on real components were also performed . it was concluded that fracture occurred by a mechanism of mechanical fatigue initiated at the region of the central hole , which suffered the highest tensile stress levels . kumar and vijayarangan described static and fatigue analysis of steel leaf springs and composite multileaf springs made up of glass fibre reinforced polymer using life data analysis . the dimensions of an existing conventional steel leaf spring of a light commercial vehicle were taken and verified by design calculations . static analysis of 2d model of conventional leaf springs was also performed using ansys 7.1 , and the results obtained were compared with experimental results . patunkar and dolas worked on nonlinear force displacement of each leaf spring as well as the spring characteristics of a pack consisting of two to four leaves using ansys . the results from ansys were compared with those from the test , which showed a fairly good agreement with each other . the objective of the present work is to determine the effect of assembly stresses on fatigue life reliability of leaf springs . it is available in open literature that with proper combination of stepping and individual leaf camber the stress distribution can be uniform along the leaf . the 65si7 leaf springs design parameter of a light commercial vehicle is taken into consideration for this work . this paper is divided into two parts . in part one , a design procedure for determination of total moment of inertia , number of leaves , and stepping and individual leaf camber is established . the stepping and individual leaf camber is proposed with a view to lower stresses . using sae approach fatigue life of the leaf spring , three leaf springs specimens lots ( four leaf spring assemblies in each lot ) have been manufactured . all the specimens are tested on a full scale leaf spring testing machine for fatigue life of the leaf spring . the theoretical and experimental fatigue life results ( with and without assembly stresses ) are compared for validation . the material is heat - treated at 880c and oil - quench - hardened and it is tempered at 410c for 90 minutes to get tempered martensite structure . a leaf spring is considered as a beam of uniform strength composed of leaves of equal thickness where the fiber stress is the same throughout the length of the beam . this approximation is justified for most of the springs within the accuracy necessary for layout work and with certain correction factors for estimate of required length , overhang , camber , width , thickness , and number of leaves . the calculation of the leaf spring parameters involves certain number of steps . as per sae spring design manual approach , the steps involved in leaf springs design are as follows . consider ( 1)i = kl332esf=34351.7 mm4 , where k = load rate , l = spring span , e = young 's modulus , and sf = stiffening factor . consider ( 2)tmax=8ismaxlpmax=8.20 mm . consider ( 3)i1=n1i1 , where n1 = number of leaves with thickness t1 , i1 = moment of inertia for section t1 = 8 mm , and width b = 70 mm and ( 4)i1=b1t1312 + 3.1428t1464=2846.5 mm4 , where b1 = b t1 = 62 mm and n1 = 11 . consider ( 5)i1=112846.13=31311 mm4i2=n2i2 , where n2 = number of leaves with thickness t2 , i2 = moment of inertia for section t2 = 7 mm , and width b = 70 mm and ( 6)i2=b2t2312 + 3.1428t2464=1918.6 mm4 , where n2 = 1 and b2 = b t2 = 63 mm and ( 7)i2=11918.6=1918.6 mm4itotal=i1+i2=33230 mm4 . n / mm% age variation of k=3.271% ( hence acceptable ) . the seat clamp is taken into consideration for stress calculation between the leaves . the inactive length in the seat for a spring without liners is estimated as distance between the outside edges of the clamp bolts which is 100 mm for this spring . the active length for both front and rear cantilever will be 50 mm less than the distance from centre of the eye to centre of an axle seat . the stress at 50 mm distance from the centre of axle seat can be calculated as the following : ( 10)front cantilever stress , sfa = palat2itrear cantilever stress , srb = pblbt2it . front cantilever ( a ) length = l = l/2 = 575 mm and rear cantilever ( b ) length = l = l/2 = 575 mm . cantilever ratio y = a / b = 1 ( symmetric spring ) and seat length sl = 100 mm . active length of front cantilever la = ( l sl/2 ) = 525 mm . active length of rear cantilever lb = ( l sl/2 ) = 525 mm . design load on front cantilever pa = p/2 = 6479.5 n. design load on rear cantilever pb = p/2 = 6479.5 n. maximum load on front cantilever pamax = pmax/2 = 14005 n. maximum load on rear cantilever pbmax = pmax/2 = 14005 n. as the leaf spring is symmetric cantilever ratio = y = a / b = 1 ; therefore , la = lb , pa = pb ( at design load ) and pamax = pbmax ( at maximum load ) . also sfa = srb = sd ( at design load ) and sfa = srb = sm ( at maximum load ) . front cantilever ( a ) length = l = l/2 = 575 mm and rear cantilever ( b ) length = l = l/2 = 575 mm . cantilever ratio y = a / b = 1 ( symmetric spring ) and seat length sl = 100 mm . active length of front cantilever la = ( l sl/2 ) = 525 mm . active length of rear cantilever lb = ( l sl/2 ) = 525 mm . design load on front cantilever pa = p/2 = 6479.5 n. design load on rear cantilever pb = p/2 = 6479.5 n. maximum load on front cantilever pamax = pmax/2 = 14005 n. maximum load on rear cantilever pbmax = pmax/2 = 14005 n. as the leaf spring is symmetric cantilever ratio = y = a / b = 1 ; therefore , la = lb , pa = pb ( at design load ) and pamax = pbmax ( at maximum load ) . also sfa = srb = sd ( at design load ) and sfa = srb = sm ( at maximum load ) . the total stress induced in the leaf spring is the summation of load stress and assembly stress : ( 11)s = sl+sa . if all the leaves are fitted with the common unassembled curvature , then the assembly stress is zero ; but the leaves are fitted in such a manner that the radius of curvature goes on increasing from the master leaf to the last leaf . with the use of different individual leaf camber , the assembly stress can be added to or deducted from the load stress of the assembled spring to obtain desirable stress pattern in the leaf spring . the assembly stress is arbitrarily chosen except they must be selected in increasing order from the main leaf to shorter leaf such that sat = 0 . a negative assembly stress in the main leaf is required to reduce the maximum stress induced in the master leaf to safe limits as the longitudinal and lateral forces are more in the master leaf . a negative assembly stress is provided in the main leaf so as to reduce the maximum stress to about 866 mpa . the longitudinal and lateral forces imposed on the main leaf and also its greater stress range are the reasons for reducing its bending stress . in view of the condition sa t = 0 , several selections of deducted and added stresses for the individual leaves are analysed before deciding on the best arrangement as shown in table 4 . in almost all leaf springs the unassembled curvatures qn are different in the unassembled leaves . in assembly , a common ( unloaded ) curvature qo is established which is variable along the spring even if the leaves are made of circular arcs . the individual leaf curvature is calculated from common curvature as the folowing : ( 12)qn = qosaey . curvature is called positive in the direction of increasing load and camber is conventionally positive in the opposite direction . camber can be converted into curvature as ( 13)qo = curvature=8camberlength2=0.000574669 mm1 . the relationship between free curvature , assembly curvature , and loaded curvature is given by ( 14)qfree+q = qloaded . for flat leaf , the curvature is zero and it increases in the direction of load application ; and for most of the springs the free curvature is negative . the no load camber requirement for this leaf spring assembly is 95 4 mm . the deflection can be determined as force per unit load rate . for different values of the load the corresponding values of deflection the stresses induced at different loads can be determined by substituting the values of the load in the stress induced formula . the analytical results for load , deflection , and bending stress are shown in table 6 . fatigue life is expressed by the number of deflection cycles a spring will withstand without failure or permanent set . a leaf spring used in a suspension will undergo a large number of cycles of small amplitude near the design load position without failure . under the greater amplitude the number of cycles without failure will be reduced since the maximum stresses as well as the stress range are increased and both are determining factors in fatigue life of the spring . as per sae spring design manual , this criteria is frequently used for determination of approximate fatigue life of the spring ; initial stress ( horizontal scale ) and maximum stress ( vertical scale ) are intersected to estimate the number of cycles the spring will withstand for given loading condition . the fatigue test stroke for leaf spring without considering assembly stress is determined as the following : deflection to design load = 12959/153.1 = 84.6 mm , maximum load = 28 kn , metal - to - metal clearance ( compression stroke ) = 94.6 mm , total deflection to maximum load = 182.9 mm , stress at metal - to - metal position = 969 mpa , stress rate = 969/182.9 = 5.29 mpa / mm , release stroke = 0.5 94.6 = 47.3 mm , fatigue test stoke = 47.3 + 94.6 = 141.9 mm , initial stress = 969 ( 141.9 5.29 ) = 218.3 mpa . deflection to design load = 12959/153.1 = 84.6 mm , maximum load = 28 kn , metal - to - metal clearance ( compression stroke ) = 94.6 mm , total deflection to maximum load = 182.9 mm , stress at metal - to - metal position = 969 mpa , stress rate = 969/182.9 = 5.29 mpa / mm , release stroke = 0.5 94.6 = 47.3 mm , fatigue test stoke = 47.3 + 94.6 = 141.9 mm , initial stress = 969 ( 141.9 5.29 ) = 218.3 mpa . the fatigue test stroke for leaf spring by considering assembly stress is determined as the following : deflection to design load = 12959/153.1 = 84.6 mm , maximum load = 28 kn , metal - to - metal clearance ( compression stroke ) = 94.6 mm , total deflection to maximum load = 182.9 mm , stress at metal - to - metal position = 885 mpa , stress rate = 885/182.9 = 4.83 mpa / mm , release stroke = 0.5 94.6 = 47.3 mm , fatigue test stoke = 47.3 + 94.6 = 141.9 mm , initial stress = 885 ( 141.9 4.83 ) = 199.6 mpa . deflection to design load = 12959/153.1 = 84.6 mm , maximum load = 28 kn , metal - to - metal clearance ( compression stroke ) = 94.6 mm , total deflection to maximum load = 182.9 mm , stress at metal - to - metal position = 885 mpa , stress rate = 885/182.9 = 4.83 mpa / mm , release stroke = 0.5 94.6 = 47.3 mm , fatigue test stoke = 47.3 + 94.6 = 141.9 mm , initial stress = 885 ( 141.9 4.83 ) = 199.6 mpa . for the production of high strength leaf springs , the process is comprised of shearing , punching , heat treatment , hot cambering , shot peening , scragging , and testing for load rate and durability . the processing of the raw material plays a vital role in achieving the required load rate and fatigue life . after punching and shearing , the raw material the structure of the raw material is partial austenite , and after quenching the structure is martensite , but after the tempering process the structure should be tempered martensite . the material is heated in the furnace in the temperature range of 880910c depending upon the cross section thickness and width . the spring steel is having the thickness of 8 mm and width of 70 mm which is heated at 880c to achieve full austenite structure . hot cambering of the spring is done in this state by passing through finger cambering tools followed by quenching in oil at temperature of 80c . tempering is done at a temperature of 410c for 90 min slow cooling till the tempered martensite structure is achieved . the final assembly is done by pulling all the leaf with a centre nut and bolt . the 65si7 leaf springs assembly consists of two full length leaves and ten graduated leaves , four rebound clips of mild steel , four shim pipes with four nut and bolts , four rivets , centre nut and bolt , and bush of bronze . the full scale testing of leaf springs was carried out in an electrohydraulic static component testing system . the laminated leaf springs were placed in a fixture simulating the conditions of a vehicle . the setup consists of a hydraulic power pack to give a hydraulic pressure of 20.6 mpa with a flow rate of 210 liters per minute ( lpm ) , which was sent to a hydraulic actuator to operate at a frequency of 0.3 hz with the displacement specified by the alternating load . this involves applying the axial load on the leaf springs and measuring the deflection and bending stress . the conventional leaf spring was tested under static load condition by using hydraulic static load ram for load application . mounting of the leaf spring was done by keeping it in inverted manner on the test bed . two eye ends were held in the clamping devices and load was applied from the top , at the center of leaf springs . to measure the load dial indicator was used , which was located beside the full scale testing machine and deflection was measured by strain gauges located at the clamping of the test rig . the springs were loaded from unladen load ( i.e. , 7.6 kn ) to maximum load ( i.e. , 28 kn ) . the vertical deflection of the springs at the unladen load , design load , flat load , rubber touching load , and metal - to - metal contact or maximum load was recorded , respectively , as per the standard operating procedure prescribed . the leaf springs were tested on a full scale testing machine under the unladen load , rated load , flat load , rubber touching load , and metal - to - metal load , and the corresponding deflection and stress values observed are shown in table 7 . table 7 depicts the observed values of deflection and stress corresponding to the loads applied on the shorter leaf by a static hydraulic ram . as per the is1135 typically this can be between 0.5 times the rated load and twice the rated load unless otherwise specified by vehicle manufacturer . consider ( 15)oa = ob(ocob)2 , where oa = load / deflection corresponding to rated load . consider ob = load / deflection corresponding to maximum load experienced under actual vehicle conditions typically 2 g , where g is the load shared by springs under the laden condition of the vehicle . for determination of experimental fatigue life , four specimens ( s-1 , s-2 , s-3 , and s-4 per batch ) for three stress ranges ( i.e. , 269896 mpa , 218969 mpa , and 200885 mpa ) are manufactured with proposed parameters . the stress range was considered for first lot of the specimens to be 627 mpa , 1.3 0.7 g. all the four specimens were tested under same stress range and fatigue life was determined . the stress range for second lot of the specimen was 751 mpa based on sae spring design manual approach , without considering assembly stresses ; that is , all the leaves with common curvature were assembled . the stress range for third lot of the specimen was 685 mpa based on sae spring design manual approach , by considering assembly stresses . the spring was clamped in the centre to simulate its installation in the vehicle as shown in figure 3 . as per the requirement specified by the vehicle manufacturer , the leaf springs are to be tested on full scale testing machine as per 1.3 0.7 g. the maximum load will be 2 g and the minimum load will be 0.6 g. here g represents the design load . the material processing for all the twelve specimens is the same , that is , normal rolling , quenching at 880c , tempering at 410c for 90 mins , shot peening at 18 a intensity , bhn 380432 , and scragging at 0.9% of yield stress . for the first lot of the specimens the maximum stress is 896 mpa and minimum stress is 269 mpa as specified by the vehicle manufacturer . the fatigue life for the four specimens is 84212 , 81961 , 82226 , and 85656 number of cycles . the second lot of specimen is assembled by considering common curvature , that is , without assembly stresses . the fatigue life for the four specimens is 66796 , 69320 , 70119 , and 69956 number of cycles . the third lot of specimens is assembled by considering the assembly stresses and leaves with proposed individual leaf camber assembled by pulling against each other to establish common curvature . the fatigue life for the four specimens is 74827 , 76658 , 79010 , and 77229 number of cycles . it is observed that the stress range for first lot , second lot , and third lot is 627 mpa , 751 mpa , and 685 mpa , respectively . the maximum stress for first lot , second lot , and third lot is 896 mpa , 969 mpa , and 885 mpa , respectively . it is observed that the lower the stress range is the higher the fatigue life will be . but for almost same stress range , the fatigue life decreases by increasing the maximum stress . it is also observed that the experimental fatigue life increases by 11.41% by considering assembly stresses . other factors like shape , size , temperature , surface , and so forth also affect the fatigue life of the leaf springs which can be considered for estimation of fatigue life . it is observed from figure 4 that the individual leaf camber is 99 mm , which is decreasing from the main leaf . the individual leaf camber is 84 mm for the second leaf with military wrapper . hence , the individual leaf camber decreases from the main leaf to the last leaf ( almost flat ) . from figure 5 it is observed that the maximum stress induced in the leaf springs without considering the assembly stresses is 969 mpa and the initial stress value is 218 mpa . the intersection of 969 mpa and 218 mpa lies in the zone of 30000 to 50000 cycles . as the point of intersection it is observed that the maximum stress induced in the leaf springs by considering the assembly stresses is 885 mpa and the initial stress value is 200 mpa . the intersection of 885 mpa and 200 mpa lies in the zone of 50000 to 75000 cycles . as the point of intersection therefore , fatigue life of leaf springs without and with considering assembly stresses would be approximately ( 460001.2 ) 55200 cycles and ( 580001.2 ) 69600 cycles , respectively . table 9 shows the fatigue life comparison between the sae spring design manual and experimental testing , by considering and not considering the assembly stresses . it is observed from table 9 that as per sae approach the fatigue life without considering the assembly stresses is 55200 cycles and by considering the assembly stress the fatigue life is 69600 cycles . as per sae the fatigue life is 69047 and 76931 cycles without and by considering the assembly stresses , respectively , in the experimental testing . in experimental testing it is observed that the fatigue life increases by 11.41% due to the reduction in stress range , and maximum stress has reduced from 969 mpa to 885 mpa by considering assembly stress . the theoretical and experimental fatigue life of a light commercial vehicle leaf spring is determined by considering assembly stresses and without considering assembly stresses , and following conclusions are made.the maximum stress induced in the leaf spring reduces and uniform stress distribution is achieved by considering the assembly stresses . the fatigue life increases due to negative assembly stresses , which reduces the maximum stress.it is also concluded that for the same stress range , higher maximum stress reduces the fatigue life of the leaf spring and higher initial stress improves the fatigue life of the leaf springs . the maximum stress induced in the leaf spring reduces and uniform stress distribution is achieved by considering the assembly stresses . the fatigue life increases due to negative assembly stresses , which reduces the maximum stress . it is also concluded that for the same stress range , higher maximum stress reduces the fatigue life of the leaf spring and higher initial stress improves the fatigue life of the leaf springs .
backgrounddespite growing interest in the therapeutic use of cannabis to manage chronic pain , only limited data that address these issues are available . in recent years , a number of nations have introduced specific laws to allow patients to use cannabis preparations to treat a variety of medical conditions . in 2015 , the italian government authorized the use of cannabis to treat several diseases , including chronic pain generally , spasticity in multiple sclerosis , cachexia and anorexia among aids and cancer patients , glaucoma , tourette syndrome , and certain types of epilepsy . we present the first snapshot of the italian experience with cannabis use for chronic pain over the initial year of its use.methodsthis is a retrospective case series analysis of all chronic pain patients treated with oral or vaporized cannabis in six hubs during the initial year following the approval of the new italian law ( december 2015 to november 2016 ) . we evaluated routes of administration , types of cannabis products utilized , dosing , and effectiveness and safety of the treatment.resultsas only one of the six centers has extensively used cannabinoids for intractable chronic pain ( 614 patients of 659 ) , only the population from azienda ospedaliero universitaria pisana ( pisa ) was considered . cannabis tea was the primary mode of delivery , and in almost all cases , it was used in association with all the other pain treatments . initial and follow - up cannabinoid concentrations were found to vary considerably . at initial follow - up , 76.2% of patients continued the treatment , and < 15% stopped the treatment due to side effects ( none of which were severe).conclusionwe present the first analysis of italian clinical practice of the use of cannabinoids for a large variety of chronic pain syndromes . from this initial snapshot , we determined that the treatment seems to be effective and safe , although more data and subsequent trials are needed to better investigate its ideal clinical indication .	the past years have witnessed a growing interest in the therapeutic use of cannabis and its constituents to manage chronic pain.1 irrespective of the number of new trials examining the use of cannabinoids for chronic pain , the evidence of its effectiveness and its safety remains limited.2 the pharmacology of cannabis sativa ( the strain generally used to treat chronic pain ) is quite complex , as it contains ~100 distinct cannabinoids,3 the relative levels of which largely determine their therapeutic effect.4 of the numerous cannabinoids , 9-tetrahydrocannabinol ( 9-thc ) is considered the most psychoactive.5,6 the current definition of cannabinoids includes all endogenous and exogenous compounds that act on cannabinoid receptors.7 thc is not the only cannabinoid with therapeutic effects8 , as cannabidiol ( cbd ) is used in the treatment of several different conditions . cbd is important not only for its therapeutic effects but also for its ability to mitigate euphoria and other side effects caused by thc,9 thereby increasing the potential therapeutic applications of cannabis . a lack of standardized dosing and uncertainty regarding the ideal ratio between thc and cbd continue to limit the medicinal usage of marijuana.10 a review by koppel et al11 analyzed six different neurological disease states and symptoms and a variety of thc / cbd ratios and methods of administration ( e.g. , oral , oromucosal , and inhaled ) without obtaining clear evidence for specific indications . as there are numerous products containing myriad thc / cbd ratios , it remains extremely challenging to draw reproducible data regarding their effectiveness in the treatment of pain.10 there are several methods of administration of cannabinoids including smoking , orally ( by infusion or by extraction in oil , as well as through edible products ) , vaporizing , and transdermally . while smoking remains the most common form of administration , a recent study found that in jurisdictions in which medical marijuana is legal , smoking is not necessarily the preferred route of administration.12 however , variance in routes of administration makes comparison of results of different studies questionable . as a number of different compounds and methods of administration exist , thorough education of the physician ( as well as the patient ) is imperative prior to prescribing or recommending these compounds.13 the most comprehensive meta - analysis of inhaled cannabis for chronic pain to date14 has recently supported its short - term effectiveness in treating neuropathic pain . the authors also concluded that more studies are needed in order to evaluate long - term effectiveness and safety . these conclusions are consistent with those from schatman s 2015 comprehensive review.10 regarding safety , aggarwal15 opined that although little data are available on the risks associated with long - term medical use in published clinical trials , it can still be used to treat complex chronic pain conditions . more recently , ware et al16 published a 1-year follow - up trial in order to better investigate the long - term efficacy and safety . the authors did not find differences in serious adverse events between chronic pain patients treated with or without cannabis . nevertheless , a higher incidence of mild adverse effects was registered among patients treated with cannabis . even though the evidence is still weak and more studies are needed , there is significantly increasing availability of cannabis to treat chronic pain in several different countries , especially in the us and canada.17 understanding of complex policy and public health issues is imperative in order to fully understand the distinction between medical vs. recreational utilization of cannabis . savage et al recently concluded that there is a need not only for additional empirical investigation but also for increased research funding to help us develop a better understanding of how to make cannabinoids more effective and safer.17 questions have also been raised regarding the legitimacy of dispensaries clientele.10 it has been demonstrated that most dispensary customers had initiated marijuana use in adolescence , with one - half presenting with indications of risky alcohol use and 20% presenting with recent histories of prescription medication or illicit drug abuse.15 hence , many concerns are arising regarding the distinct possibility that in the us and canada , some of the same problems that these nations have had with prescription opioid abuse will potentially develop in relationship to medical marijuana . currently , the us is aggressively fighting its opioid crisis while simultaneously liberalizing access to cannabis for both medical and recreational utilization.18 an italian law approved in 201519 authorizes the use of cannabis to treat chronic pain . the law allows for the utilization of cannabis not only for neuropathic pain but also for all chronic pain conditions , as well as for spasticity , cachexia , and anorexia among aids and cancer patients , ocular hypertension in glaucoma , the alleviation of spasms in tourette syndrome , and some types of epilepsy , reiterating that cannabis - based drugs should be prescribed only when other available medications have proven to be ineffective or inadequate to the therapeutic needs of the patient . in order to reduce the costs of cannabinoid products , the italian government in 2014 committed its military chemical - pharmaceutical factory to cultivate cannabis to be distributed to all pharmacies across the country ( initial production is scheduled for early in 2017 ) . in the meantime , physicians can legally prescribe different cannabis products ( such as bedrocan , bediol , and bedrolite ) , with different thc and cbd concentrations ; these drugs can be administered orally ( e.g. , through infusions in olive oil ) or via inhalation . bedrocan s constituents are 22% thc and < 1% cbd , while bediol contains 6.5% thc and 8% cbd and bedrolite contains 9% cbd and 0.4% thc and is accordingly considered non - psychoactive.20 all patients treated with cannabinoids have to be registered through a ministry of health database . in recent months , the society that includes all second - level ( hubs ) pain centers ( pinhub , www.pinhub.it ) has received a directive to evaluate all of these data among its centers . this paper presents a retrospective analysis of a case series of all chronic pain patients treated in one pinhub center over the past year in order to provide a snapshot of the initial italian experience with legalized cannabis use for chronic pain . the end point of this study has been the evaluation and identification of clinical indications and dosages currently used in second - level center of pain therapy . although our intention was to include data from all six of the centers initially involved in the study , a paucity of data from five of the six centers precluded doing so . following the approval of the italian law sanctioning the medical use of cannabis ( 2015 ) , all patients who initiated the use of medical cannabis have had to be registered , with their consent , in a database to evaluate specific data ( figure 1 ) . a retrospective group analysis of a case series of all chronic pain patients treated in one of the second - level pain clinics affiliated with the pinhub group in the first year following the approval of the italian law ( december 2015 to november 2016 ) was performed . the clinical centers intended to be involved in this study were the pain therapy services of the following hospitals : ss antonio e biagio hospital ( alessandria ) , ss annunziata hospital ( chieti ) , monaldi hospital ( naples ) , verona university hospital ( verona ) , siena university hospital ( siena ) , and azienda ospedaliera universitaria pisana ( pisa ) . patients gave their permission to use their data when the physician filled out the case report form ( crf ) . this research did not require approval by the institutional review boards of the aforementioned clinical centers as we used only raw data that was completely de - identified and anonymous . the data were presented in accordance with the strengthening the reporting of observational studies in epidemiology ( strobe ) guidelines.21 this study evaluated all patients registered as they had initiated treatment with cannabinoids for chronic pain based upon the judgment of their pain therapists . in accordance with the italian law , all patients treated with cannabinoids have had to be 18 years of age and determined to suffer from refractory chronic pain . according to italian law , cannabis could be prescribed with only two routes of administration : orally ( infusion or extraction in olive oil ) or through inhalation . smoking the cannabis is not permitted , so vaporization was the technique used for inhalation . currently , there are no national guidelines to be followed by physicians . furthermore , comorbidities and their severity that might preclude the treatment are not commonly predefined among the centers that are using this treatment . the primary end point of this study was to provide insight into how the italian second - level pain centers are utilizing medical cannabinoids in terms of routes of administration . secondary end points included the determination of the types of cannabis products most commonly utilized , as well as dosing . participating physicians were able to choose among infusion or inhalation via vaporization of the following types of cannabinoid products : pure bediol , bediol combined with bedrocan , pure bedrocan and bedrolite , and oil infused with bedrocan . as ~92% of the patients in the study used the high - thc , low - cbd bedrocan , it was unable to assess differences in efficacy and adverse events between the strains utilized in this preliminary investigation ( figure 1 ) . furthermore , the study evaluated effectiveness and safety through the analysis of patients who had at least one follow - up subsequent to initial prescription based on the data provided by the crfs defined by the italian health ministry . through the data requested by the italian health ministry for follow - up purposes , the study evaluated the dosages utilized , whether the therapy has been continued , clinical efficacy ( determined by patient - reported levels of pain severity ) , and the reason for discontinuing treatment ( pain worsened , pain not clinically improved , or presence of intolerable side effects ) . although assessing other outcomes such as functionality would have been useful , the italian health ministry has requested the provision only of data pertaining to pain relief . data have been obtained by the crf requested by the italian health ministry that has to be filled whenever patients initiated the therapy or had subsequent contact with the pain center at which they were being treated . the sample size was not calculated a priori as this study was an explorative retrospective analysis of all patients treated with cannabinoids for a year in second - level centers in italy after the approval of the law that legalized their use for chronic pain . all data are presented with percentages or means , as well as standard deviations . as this initial study is a case series , it is not surprising that the data are quite heterogeneous . however , the authors chose not to formally evaluate any statistical differences between the different treatments , as there were insufficient number of subjects using any product besides bedrocan . the sample size was not calculated a priori as this study was an explorative retrospective analysis of all patients treated with cannabinoids for a year in second - level centers in italy after the approval of the law that legalized their use for chronic pain . all data are presented with percentages or means , as well as standard deviations . as this initial study is a case series , it is not surprising that the data are quite heterogeneous . however , the authors chose not to formally evaluate any statistical differences between the different treatments , as there were insufficient number of subjects using any product besides bedrocan . of the 659 patients who initiated treatment with oral cannabis at all of the facilities originally intending to participate in the study , the study included only the subjects from the azienda ospedaliero universitaria pisana ( pisa ) for the analysis of data . as only 6% of the patients came from the other five facilities , a comparison of inter - facility data would not have been possible . on a positive note , utilizing only the data from the azienda ospedaliero universitaria pisana also allowed for a more homogeneous evaluation of current treatment . of all the subjects , 181 were male and 422 were female , and data on gender were missing for 11 subjects . of 614 patients whose data were used , 341 ( 55.5% ) had at least one follow - up at 98.42 ( 144.66 ) days . figure 3 indicates the specific symptoms for which physicians initiated cannabis treatment . in 89.2% of patients , follow - up , 76.2% patients continued the cannabinoid therapy ( of whom 64.7% reported an improvement associated with the therapy , while 34.1% reported neither an improvement nor a worsening ) , and 23.8% discontinued treatment ( 3.7% due to a worsening of their pain , 61.7% due to side effects , 29.6% due to an unsatisfactory change of their clinical condition ; for 4.9% of patients , the data were missing ) . there were no complaints of severe side effects , even though this may not have been directly assessed at follow - up visits . figure 4 illustrates the dosages ( mg / day ) of bedrocan and bediol at the initiation of the study and at initial follow - up , respectively . over the past year , a dramatic increase in the use of cannabis to treat chronic intractable pain has been witnessed.11 nevertheless , specific guidelines are still missing , and there is considerable heterogeneity in the use of this drug throughout the world . in italy , cannabis was approved legally 18 months ago for its use for several indications , including treatment of intractable pain ( not specifically neuropathic pain ) . all patients who initiated treatment with cannabis had been required to be registered in a specific national database in order to evaluate the clinical effectiveness and safety of cannabis in the 2 years immediately following its legalization . hence , a retrospective analysis of a case series of patients treated in a second - level pain clinic that is a part of the pinhub society was performed . this analysis attempted to provide an initial snapshot of how cannabinoids are used in italy in order to understand how cannabis is currently used for chronic pain treatments and its implications for clinical practices . the initial data that were obtained are those only from one of the six centers that have extensively used cannabinoids for intractable chronic pain . it was observed that cannabinoid treatment is not yet common , even though the new law has been approved . this situation could be compared to a similar situation observed in italy several years ago with regard to opioids ; despite the passage of legislation intended to facilitate opioid prescription , several years passed prior to the initiation of an appropriate increase in opioid prescription . the population in this investigation that was treated was similar to that generally observed in pain clinics ( average age of 61 years , and majority being female ) . regarding the type of cannabinoid and modality of administration , almost all patients received infusions of bedrocan , while administration of the extract of cannabinoids in olive oil or vaporization was quite uncommon . hence , as these data are quite discrepant from those from canada and the us , it is extremely difficult to compare the effectiveness and safety of the treatment to those data from studies performed in other nations . this heterogeneity currently limits the possibility of developing guidelines available and useful on an international basis . although the method of administration in this study was relatively homogeneous , a large variety ( as demonstrated through a large standard deviation ) of concentrations ( especially for bediol ) used both at the initiation of treatment and ( even more so ) at the first follow - up was observed . furthermore , in italy , a formidable variety of indications for the prescription of cannabinoids . several types of chronic pain syndromes have been treated . in order to obtain more reproducible data , in the near future , the authors intend to better focalize indications for specific pain syndromes , such as intractable neuropathic pain . that a low rate ( 22% ) of discontinuation of cannabinoids ( certainly compared to discontinuation rates of opioid analgesics ) was observed is clearly encouraging , particularly given that the iatrogeneses of cannabinoids in pain treatment are likely less substantial than those of opioids . first , it is a retrospective analysis of only one center , and unfortunately , we are compelled to acknowledge that this somewhat limits our understanding of the efficacy of medical cannabinoids for chronic pain . accordingly , we will continue to collect data from the five hospitals other than the azienda ospedaliero universitaria pisana and intend to publish data that will address inter - facility variance in outcomes . second , future investigation will analyze the impact of concomitant treatments , which will require a sufficient number of subjects for the performance of analyses of covariance . thus , we expect to move toward a better understanding of whether cannabinoid products are more effective as a monotherapeutic approach to chronic pain treatment as opposed to a component of multimodal care . finally , as the vast majority of patients enrolled in the study used the high - thc bedrocan , we were unable to assess the difference between cannabinoid treatments with different ratios of thc and cbd . future investigation will certainly look at this issue , as questions regarding the medical benefits and safety of high - thc , low - cbd cannabis have arisen.10 an initial analysis of the italian clinical practice of the use of cannabinoids for chronic pain syndromes in a reasonably large population is presented . even with the heterogeneity of the sample size and the limited data available , it can be stated that the treatment seems to be effective and safe in the majority of patients , even though the safety and effectiveness data should be confirmed in a trial better designed to assess them . nevertheless , additional data from a variety of types of trials are needed in order to better understand the benefits of cannabinoids to chronic pain sufferers . it is important for the italian and other european pain societies to more thoroughly investigate this topic in order to provide clearer and more useful guidelines , which will more adequately guide physicians in the use of this drug in the treatment of chronic pain .
we evaluated the feasibility and efficacy of intermediate - supine percutaneous nephrolithotomy ( pcnl ) in patients with renal calculi . fifteen patients were included in this study . the intermediate - supine operative position was modified by using a 1-l saline bag below the ipsilateral upper flank . a nephrostomy and stone extraction were performed as usual . after completion of the stone removal , a nephrostomy tube was used when necessary according to the surgeon 's decision . if there was no significant bleeding or renal pelvic injury , tubeless pcnl was performed . the mean stone size was 5.485.69 cm2 , the mean operative time was 78.9338.72 minutes , and the mean hospital stay was 2.601.29 days . tubeless pcnl was performed in 13 cases ( 86.7% ) , and retrograde procedures were simultaneously performed without a change of position in 2 patients ( ureteroscopic ureterolithotomy in one patient and transurethral placement of an occlusion catheter in one patient ) . there were two complications according to the clavien - dindo classification ( grade i in one patient and grade ii in one patient ) . the success rate was 80.0% and the complete stone - free rate was 73.3% . three patients with a significant remnant stone were also successfully managed with additional procedures ( one patient underwent a second - look operation , and the remaining two patients were treated with shock wave lithotripsy ) . in the treatment of renal calculi , intermediate - supine pcnl may be a safe and effective choice that offers several advantages with excellent outcomes . thus , a prospective study with a larger population is needed to verify our outcomes .	percutaneous nephrolithotomy ( pcnl ) is an effective choice in the treatment of large kidney stones because of excellent outcomes and acceptably low morbidity , and it is usually performed with the patient in the prone position.1 this approach provides a larger surface area for the choice of puncture site and a wider space for instrument manipulation . however , the prone position has several disadvantages : respiratory and cardiovascular risks ; ventilatory difficulties , especially in obese patients and in elderly patients with compromised cardiopulmonary status;2,3 and the need for position changes during the procedure . for these reasons , many urologists have tried modified positions for pcnl ( flank , supine , and modified supine).4,5 supine pcnl was first reported by valdivia ura et al . in 19983 and is regarded as possessing several advantages . another advantage of the supine position is that there is no need for position changes to perform other endoscopic procedures such as cystoscopic or ureteroscopic operations.6 the supine position enables simultaneous retrograde ureteroscopic procedures during pcnl without any position change . a nephrostomy catheter has routinely been placed after pcnl , because it provides proper drainage of urine , prevents urinary extravasation , and enables tamponade of bleeding.7,8 the need for placement of a nephrostomy catheter has been questioned , however , owing to increases in postoperative discomfort and morbidity , and several studies in the past two decades have demonstrated the feasibility of tubeless pcnl . in the current study , we evaluated the feasibility and efficacy of intermediate - supine pcnl in our initial experiences with this approach . the study was approved by the institutional review board of our institution . between january 2012 and october 2012 , a total of 15 patients with renal stones underwent pcnl in the intermediate - supine position performed by a single surgeon ( k.s.c . ) . preoperative data , operative outcomes , and postoperative outcomes were retrospectively collected and evaluated . before surgery , the desired calyx was punctured under fluoroscopy guidance and a guidewire was inserted by an interventional radiologist . then a pcnl was performed with the patient under general anesthesia in the intermediate - supine position . for intermediate - supine pcnl , the patient was placed in the supine position with a 1-l saline bag below the ipsilateral flank . thus , the ipsilateral flank was elevated 20 , causing the posterior calyx to project more laterally ( fig . , a retrograde ureteral occlusion catheter was fixed through the ipsilateral ureteral orifice by use of cystoscopy . under the guidance of c - arm fluoroscopy , we accurately assessed the punctured calyx through a guidewire placed in the intervention port . after amplatz sheath insertion , the nephroscope was introduced , and the stones were fragmented by using a lithoclast lithotripter ( electro - medical systems , nyon , switzerland ) and were extracted by use of stone forceps or a suction catheter . after completion of stone removal , antegrade placement of a double - j ureteral stent was performed in most cases . if there was no significant bleeding and no significant injury to the renal pelvis or ureter , a tubeless pcnl was performed . in all cases , the stone size was measured with noncontrast computed tomography ( ct ) and was calculated by use of the following formula : widthlength0.25.9 complete stone - free status was defined as no stone fragments visible on the postoperative image study , and the operation was considered successful if the patient had either no or clinically insignificant residual stone fragments ( largest stone diameter less than 4 mm and asymptomatic , nonobstructive , and noninfectious).10,11 length of hospital stay was defined as the time interval between the day of surgery and the day of discharge from the hospital . perioperative complications were evaluated according to the dindo - modified clavien system validated in 2004.12 pain was evaluated every 8 hours by a trained nurse using the visual analogue scale ( vas ) for pain . patients with moderate to severe pain were given tramadol 50 mg intravenous ( iv ) or pethidine 25 mg intramuscular ( i m ) if postoperative pain was not controlled with nonsteroidal analgesics . all patients were monitored for levels of hemoglobin and serum creatinine preoperatively and by the seventh postoperative day . a plain x - ray or an abdomen - pelvis ct the patient was discharged when he or she was thought to be free of complications and when the patient 's pain was controlled with oral analgesics ( vas pain score lower than 3 ) . the preoperative and postoperative vas pain scores and serum creatinine variations among the subjects were analyzed by use of the wilcoxon signed rank test . comparisons with a p - value less than 0.050 were considered to be statistically significant . the study was approved by the institutional review board of our institution . between january 2012 and october 2012 , a total of 15 patients with renal stones underwent pcnl in the intermediate - supine position performed by a single surgeon ( k.s.c . ) . before surgery , the desired calyx was punctured under fluoroscopy guidance and a guidewire was inserted by an interventional radiologist . then a pcnl was performed with the patient under general anesthesia in the intermediate - supine position . for intermediate - supine pcnl , the patient was placed in the supine position with a 1-l saline bag below the ipsilateral flank . thus , the ipsilateral flank was elevated 20 , causing the posterior calyx to project more laterally ( fig . , a retrograde ureteral occlusion catheter was fixed through the ipsilateral ureteral orifice by use of cystoscopy . under the guidance of c - arm fluoroscopy , we accurately assessed the punctured calyx through a guidewire placed in the intervention port . after amplatz sheath insertion , the nephroscope was introduced , and the stones were fragmented by using a lithoclast lithotripter ( electro - medical systems , nyon , switzerland ) and were extracted by use of stone forceps or a suction catheter . after completion of stone removal , antegrade placement of a double - j ureteral stent was performed in most cases . if there was no significant bleeding and no significant injury to the renal pelvis or ureter , a tubeless pcnl was performed . in all cases , the stone size was measured with noncontrast computed tomography ( ct ) and was calculated by use of the following formula : widthlength0.25.9 complete stone - free status was defined as no stone fragments visible on the postoperative image study , and the operation was considered successful if the patient had either no or clinically insignificant residual stone fragments ( largest stone diameter less than 4 mm and asymptomatic , nonobstructive , and noninfectious).10,11 length of hospital stay was defined as the time interval between the day of surgery and the day of discharge from the hospital . perioperative complications were evaluated according to the dindo - modified clavien system validated in 2004.12 pain was evaluated every 8 hours by a trained nurse using the visual analogue scale ( vas ) for pain . patients with moderate to severe pain were given tramadol 50 mg intravenous ( iv ) or pethidine 25 mg intramuscular ( i m ) if postoperative pain was not controlled with nonsteroidal analgesics . all patients were monitored for levels of hemoglobin and serum creatinine preoperatively and by the seventh postoperative day . a plain x - ray or an abdomen - pelvis ct the patient was discharged when he or she was thought to be free of complications and when the patient 's pain was controlled with oral analgesics ( vas pain score lower than 3 ) . the preoperative and postoperative vas pain scores and serum creatinine variations among the subjects were analyzed by use of the wilcoxon signed rank test . comparisons with a p - value less than 0.050 were considered to be statistically significant . we performed a total of 15 pcnl procedures with patients in the intermediate - supine position ( table 1 ) . the mean patient age was 58.7717.33 years , and the patients ' mean body mass index was 26.174.22 kg / m . a nephrostomy catheter was inserted in two cases ( 13.3% ) ; significant venous bleeding was suspected in one case , and a second - look operation was considered in one case . in 13 cases ( 86.7% ) , an antegrade ureteral stent indwelling at the ipsilateral site was performed in 14 cases . in two patients , the retrograde procedures were simultaneously performed without a change in position ( one ureteroscopic ureterolithotomy and one transurethral placement of an occlusion catheter ) . the success rate was 80.0% , and the complete stone - free rate was 73.3% . one patient underwent a second - look operation owing to a large stone burden ( 26.00 cm ) and long operative time ( 191 min ) at the first operation . in the other two patients , it was anatomically difficult to obtain proper access to the remnant stones , which were relatively small , and the stones were successfully treated with shock wave lithotripsy . only two patients had mild complications according to the clavien - dindo classification ( grade i in one patient and grade ii in one patient ) . serum creatinine levels measured preoperatively , on the day of surgery , and on postoperative day one were 1.120.89 mg / dl , 1.100.91 mg / dl ( vs. preoperatively ; p=0.851 ) , and 1.090.87 mg / dl ( vs. preoperatively ; p=0.490 ) , respectively . generally , pain was tolerable within 2 to 3 days ; the vas scores for pain on the day of surgery , on postoperative day one , and on the day of discharge were 4.261.79 ( vs. operative day ; p=0.040 ) , 2.801.20 , and 1.730.79 ( vs. operative day ; p=0.003 ) , respectively . many urologists have attempted to overcome the disadvantages of conventional pcnl , and the technique of pcnl has largely evolved in terms of the placement of tubes and changes in position over the past two decades . many cases of both supine pcnl and tubeless pcnl have been accumulated , but data are lacking for supine pcnl in conjunction with the tubeless technique . our early experiences suggested that intermediate - supine and tubeless pcnl is a safe and effective choice that offers several advantages with excellent outcomes . these experiences are consistent with a previous report by rana et al.13 those authors maintained that pcnl with the patient in the supine position has several advantages , such as a lower rate of complications , a shorter operative time , and the simplicity and ease of performance of the procedure . therefore , the procedure can provide uniform comfort for the anesthesiologist , patient , and surgical team . when the pcnl procedure is performed with the patient in the prone position , a ureteral catheter is commonly fixed in the lithotomy position before the patient is turned ; however , pcnl in the supine position does not require turning . furthermore , pcnl in the intermediate - supine position facilitates the completion of ureteroscopic processes at the same time . therefore , the operative time for pcnl in the supine position is shorter than that for pcnl in the prone position.14 pcnl with the patient in the supine position saves 30 to 40 minutes of operative time and avoids the risk associated with a position change under general endotracheal anesthesia . de sio et al.14 reported that the operative time was 68 minutes ( range , 55 - 140 minutes ) in the prone position but 43 minutes ( range , 25 - 120 minutes ) in the supine position ( p<0.001 ) , and karami et al.15 reported that the operative time was reduced in the lateral position ( 74.426.9 minutes ) , prone position ( 68.737.4 minutes ) , and supine position ( 54.225.1 minutes ) ( p<0.040 ) . de sio et al.14 reported that in their randomized controlled study , the stone - free rate was excellent in both the supine ( 88.7% ) and the prone ( 91.6% ) position pcnl groups . shoma et al.16 reported similar success rates for the supine ( 89% ) and prone ( 84% ) positions in a prospective nonrandomized study , and karami et al.15 reported that the success rate was 92% in the prone position , 86% in the supine position , and 88% in the lateral position . the success rate of supine pcnl is also dependent on the stone size and location . hoznek et al.17 demonstrated that the success rate was 90% for single stones , 78% for multiple stones , and 43% for staghorn stones during supine pcnl . xu et al.18 also suggested similar stone - free rates of 85.7% overall , 92.2% for a single calculus , and 72.9% for staghorn calculi . in the supine and intermediate - supine positions , mobile fluoroscopy ( c - arm ) can be utilized without the interrupted artifacts ( spine , ribs , etc . ) that are present in the lateral position , which might be helpful in improving surgical outcomes such as the stone - free rate.16,19 on the contrary , there are some limitations in using fluoroscopic examination during pcnl in the lateral position because of the possibility of the aforementioned artifacts . for the pcnl procedure , several studies have reported that the rate of significant bleeding requiring transfusion was about 1.5% to 9%3,13,16,19 and was directly related to the stone size , procedure time , and creation of multiple tracts.20 basiri and mohammadi sichani21 suggested that the risk of bleeding might be less in the supine position than in the prone position because a backflow of blood to the renal vein is caused by obstruction of the inferior vena cava . also , there might be less risk of colonic injury during supine pcnl because of the more anterior displacement of the colon away from kidney in the supine position than in the prone position.3,22 valdivia ura et al.3 reported no damage to the peritoneum and colon in a ct study and confirmed that the colon was further away from the kidney in the supine position than in the prone position . in their report on 557 patients , the operation was successful in 93% of the cases , with a low complication rate and no colonic perforation.3 however , a different study reported similar colonic injury risk between the supine position and the prone position.18 meanwhile , severe anesthesia complications are rarely reported in the prone position , and it is generally accepted that the supine position is more satisfactory for the anesthetist , especially in obese or high - risk anesthesia patients.23 in the prone position , it is difficult for the anesthetist to observe the patient effectively , and the prone position may not be favorable for resuscitation when acute syndromes such as obstruction of respiratory passages or acute myocardial ischemia occur during the operation.24 nevertheless , pcnl in the supine position also has several disadvantages . because the angle between the surface of the operation table and the anterior calyxes is smaller than that in prone position , it is difficult to access stones in the anterior calyxes.14,16,21 approaching the upper calyx is more difficult in the supine position , especially if placed overly medially , and this problem is more obvious on the left side.13,21 another disadvantage in the supine position is the mobility of the kidneys , which is greater than in the prone position . therefore , the kidneys are easy to move anteromedially during tract formation in the supine position . finally , the pyelocaliceal system is constantly collapsed in this position ; thus , nephroscopic procedures might be more difficult.19 although there have been some debates on the efficacy and safety of tubeless pcnl , successful experiences by many urologists have rekindled clinical interest in tubeless or totally tubeless pcnl . according to recent studies , there is no significant difference in the rate of complications between standard pcnl and tubeless pcnl , and tubeless pcnl has several advantages.25,26 tubeless pcnl is associated with a shorter length of hospital stay ; thus , patients can return quickly to everyday life , experience less pain , and incur a lower cost . sofikerim et al.27 reported that tubeless pcnl is a safe and effective technique even after supracostal access and is associated with less postoperative pain and a shorter hospital stay . shen et al.28 suggested that the hospital stay of the tubeless group was less than that of the middle - tube and large - tube groups in a multiple center metaanalysis . they recommended that a nephrostomy catheter be placed in certain situations ( multiple access , major damage to the collecting system , possibility of a second look operation , severe intraoperative bleeding , complicated cases , and intrathoracic trauma).29 the disadvantages of the tubeless procedure , including the need to place a ureteral stent , should be considered ; patients may have bladder irritation symptoms such as flank pain , gross hematuria , urinary frequency , and urgency because of the placement of a ureteral catheter , and patients must undergo cystoscopy to remove the ureteral stent.30 there were several limitations to our study . in addition , we did not attempt multiple accesses or an upper pole approach , and we selected relatively simple cases , although there were also some difficult cases with large staghorn stones among our early experiences . nevertheless , intermediate - supine and tubeless pcnl might be a safe and effective option that offers several advantages with excellent outcomes in the management of renal calculi . thus , a prospective randomized study with a larger population is needed to confirm our observations . in conclusion , intermediate - supine pcnl is believed to be feasible and effective and is associated with several advantages . it may shorten the preparation period of the operation and the operative time , and it can result in a high stone - free rate with low complications . in our series , there was no increase in the bleeding risk with the intermediate - supine position ; therefore , the tubeless technique could be applied in most cases . the tubeless technique also seems to have contributed to the favorable surgical outcomes in our early experiences .
objective : to investigate the effect of autoimmune thyroiditis ( ait ) on risk of stroke and to assess whether any increased risk ( 1 ) varied by ait duration , and ( 2 ) was independent of classic cardiovascular risk factors.methods:this was a large historical cohort study using data from the health improvement network database . rates of first stroke during follow - up in thyroxine - treated patients with ait ( n = 34,907 ) were compared with those in matched individuals without ait ( n = 149,632 ) using random - effects poisson regression models.results:there was strong evidence for a slightly increased risk of stroke in patients with ait ( adjusted rate ratio = 1.10 , 95% confidence interval : 1.011.20 ) . the observed increase was partly independent of cardiovascular risk factors . higher effect sizes were identified in the first year after ait diagnosis ( rate ratio = 1.33 , 95% confidence interval : 1.141.56 ) but not in the long - term , consistent with a residual effect of hypothyroidism.conclusion:our results support the hypothesis of a slightly increased risk of stroke in patients with ait . the higher effect size found soon after ait diagnosis suggests an increased cardiovascular risk due to thyroid - hormone deficiency rather than a cumulative effect of autoimmune pathology . better screening and early treatment of patients with asymptomatic hypothyroid ait could help reduce excess risk of stroke in the first year after diagnosis .	this was a historical cohort study using data from the health improvement network ( thin ) database . thin is a population - based database of electronic health records of approximately 6 million patients from more than 300 general practices in the united kingdom . thin has been validated for a wide range of medical conditions , including stroke , and individuals contributing data to thin are representative of the uk population . data available include prescribed medications , medical diagnoses , lifestyle conditions , demographic / personal information , and feedback from specialist appointments and hospital admissions . the present study population was a subset of a larger thin study , in which patients with an autoimmune disease enrolled in thin between 1987 and 2007 had each been matched by age , sex , and general practice to up to 6 individuals without any autoimmune disease . ait was the exposure of interest ; unexposed individuals comprised patients without ait ( or any other autoimmune disease ) who had been matched on age , sex , and general practice to the patients with ait in the original thin dataset . the outcome of interest was first - ever stroke ( including ischemic and hemorrhagic ) or tia during follow - up . exposure and outcome were defined using prespecified read code lists ( appendix e-1 on the neurology web site at neurology.org ) . start of follow - up for individuals with ait and their unexposed counterparts was the date of the first thyroxine prescription in the patients with ait ( the index date ) . end of follow - up was defined as the day of the outcome ( stroke or tia ) or end of follow - up in thin ( death , transfer out , or the practice 's last data - collection date ) . patients with ait must have been first diagnosed with ait during active follow - up in thin and have been prescribed thyroxine during follow - up . individuals with other causes for hypothyroidism ( e.g. , previous thyroidectomy ) were excluded from analyses ( figure 1 ) . diagnoses for past or ongoing conditions ( including ait ) are sometimes recorded retrospectively in the first few months after a patient registers with a practice . to ensure that we enrolled incident cases , we excluded patients with ait who were diagnosed during their first year of follow - up in thin . all individuals ( exposed and unexposed ) who had a code for stroke or tia before start of follow - up , or who were younger than 18 years , were not considered for inclusion . also , because the unexposed ( non - ait ) individuals had no other autoimmune disorders , individuals with ait who had a preexisting or who developed a second autoimmune disorder were also excluded . unexposed individuals had to be actively enrolled in thin at the index date of their matched individual with ait ; to ensure that they were truly active , they had to have a consultation with the practice in the 6 months before or in the year after the index date of the patient with ait . aid = autoimmune disease ; ait = autoimmune thyroiditis . in the conceptual framework for this study ( figure 2 ) , cofactors were categorized as follows : ( 1 ) a priori confounders ( sex , current age , general practice ) ; ( 2 ) other potential confounders ( smoking , alcohol consumption , calendar year , time in study ) ; and ( 3 ) factors that could be either confounders or on the causal pathway from ait to stroke , depending on whether they occurred before or after ait diagnosis ( figure 2b ) . missing data for smoking , alcohol , and body mass index ( bmi ) were treated using the missing indicator method . an individually matched analysis is not needed in matched cohort studies , but inclusion of the matched variables in a multivariable analysis is advisable ; this also addressed any imbalances between exposed and unexposed patients regarding these variables after the application of exclusion criteria . ( b ) differentiation between confounding and mediating effects of factors hypothesized to be on the causal pathway . presence of factors at the index date was assumed to be attributable to confounding in the primary analysis . ait = autoimmune thyroiditis ; bmi = body mass index ; chd = coronary heart disease ; chf = congestive heart failure . statistical analyses were conducted using stata 11 ( statacorp , college station , tx ) . baseline characteristics were compared using cross - tabulation , means or medians as appropriate , and tests , 2-sided t tests , and wilcoxon rank - sum tests . main analyses were performed using random - effects multivariable poisson regression models allowing for clustering within general practice . , we assessed potential confounding by smoking , alcohol consumption , calendar year , and time in study , introducing these variables sequentially into the model and retaining them if they changed the effect estimate of ait on stroke incidence appreciably . the third model additionally assessed risk factors ( hypertension , af , hyperlipidemia , diabetes , chd , congestive heart failure [ chf ] , and bmi ) that were present at the index date and could therefore also confound the association between ait and stroke . in the final model , we examined time - updated values of these factors for individuals who developed these conditions during follow - up to assess potential causal pathways between diagnosed ait and stroke . we applied this analysis strategy to 2 main outcomes , first stroke , then stroke or tia . in all models , standard errors were examined for evidence of colinearity , and p values were obtained using likelihood ratio tests . we investigated effect modification by ait duration as a proxy for the effects of hypothyroidism ( likely to be evident in the early stages of disease , before adequate thyroxine replacement ) vs the possible cumulative effect of autoimmunity ( long - term increased risk , likely to occur later during follow - up ) . we also investigated effect modification by age ( < 60 , 6080 , > 80 years ) and performed several sensitivity analyses . first , analyses were repeated starting follow - up at the date of first ait code instead of first thyroxine script . second , cardiovascular conditions present among patients with ait at diagnosis could be early mediating factors for stroke rather than a confounding variable ; to assess their importance , we performed subgroup analyses for individuals without a history of any cardiovascular disease ethics approval was obtained from the south - east multicentre research ethics committee and from the london school of hygiene and tropical medicine ethics committee . this was a historical cohort study using data from the health improvement network ( thin ) database . thin is a population - based database of electronic health records of approximately 6 million patients from more than 300 general practices in the united kingdom . thin has been validated for a wide range of medical conditions , including stroke , and individuals contributing data to thin are representative of the uk population . data available include prescribed medications , medical diagnoses , lifestyle conditions , demographic / personal information , and feedback from specialist appointments and hospital admissions . the present study population was a subset of a larger thin study , in which patients with an autoimmune disease enrolled in thin between 1987 and 2007 had each been matched by age , sex , and general practice to up to 6 individuals without any autoimmune disease . ait was the exposure of interest ; unexposed individuals comprised patients without ait ( or any other autoimmune disease ) who had been matched on age , sex , and general practice to the patients with ait in the original thin dataset . the outcome of interest was first - ever stroke ( including ischemic and hemorrhagic ) or tia during follow - up . exposure and outcome were defined using prespecified read code lists ( appendix e-1 on the neurology web site at neurology.org ) . start of follow - up for individuals with ait and their unexposed counterparts was the date of the first thyroxine prescription in the patients with ait ( the index date ) . end of follow - up was defined as the day of the outcome ( stroke or tia ) or end of follow - up in thin ( death , transfer out , or the practice 's last data - collection date ) . patients with ait must have been first diagnosed with ait during active follow - up in thin and have been prescribed thyroxine during follow - up . individuals with other causes for hypothyroidism ( e.g. , previous thyroidectomy ) were excluded from analyses ( figure 1 ) . diagnoses for past or ongoing conditions ( including ait ) are sometimes recorded retrospectively in the first few months after a patient registers with a practice . to ensure that we enrolled incident cases , we excluded patients with ait who were diagnosed during their first year of follow - up in thin . all individuals ( exposed and unexposed ) who had a code for stroke or tia before start of follow - up , or who were younger than 18 years , were not considered for inclusion . also , because the unexposed ( non - ait ) individuals had no other autoimmune disorders , individuals with ait who had a preexisting or who developed a second autoimmune disorder were also excluded . unexposed individuals had to be actively enrolled in thin at the index date of their matched individual with ait ; to ensure that they were truly active , they had to have a consultation with the practice in the 6 months before or in the year after the index date of the patient with ait . in the conceptual framework for this study ( figure 2 ) , cofactors were categorized as follows : ( 1 ) a priori confounders ( sex , current age , general practice ) ; ( 2 ) other potential confounders ( smoking , alcohol consumption , calendar year , time in study ) ; and ( 3 ) factors that could be either confounders or on the causal pathway from ait to stroke , depending on whether they occurred before or after ait diagnosis ( figure 2b ) . missing data for smoking , alcohol , and body mass index ( bmi ) were treated using the missing indicator method . an individually matched analysis is not needed in matched cohort studies , but inclusion of the matched variables in a multivariable analysis is advisable ; this also addressed any imbalances between exposed and unexposed patients regarding these variables after the application of exclusion criteria . ( b ) differentiation between confounding and mediating effects of factors hypothesized to be on the causal pathway . presence of factors at the index date was assumed to be attributable to confounding in the primary analysis . ait = autoimmune thyroiditis ; bmi = body mass index ; chd = coronary heart disease ; chf = congestive heart failure . statistical analyses were conducted using stata 11 ( statacorp , college station , tx ) . baseline characteristics were compared using cross - tabulation , means or medians as appropriate , and tests , 2-sided t tests , and wilcoxon rank - sum tests . main analyses were performed using random - effects multivariable poisson regression models allowing for clustering within general practice . , we assessed potential confounding by smoking , alcohol consumption , calendar year , and time in study , introducing these variables sequentially into the model and retaining them if they changed the effect estimate of ait on stroke incidence appreciably . the third model additionally assessed risk factors ( hypertension , af , hyperlipidemia , diabetes , chd , congestive heart failure [ chf ] , and bmi ) that were present at the index date and could therefore also confound the association between ait and stroke . in the final model , we examined time - updated values of these factors for individuals who developed these conditions during follow - up to assess potential causal pathways between diagnosed ait and stroke . we applied this analysis strategy to 2 main outcomes , first stroke , then stroke or tia . in all models , standard errors were examined for evidence of colinearity , and p values were obtained using likelihood ratio tests . we investigated effect modification by ait duration as a proxy for the effects of hypothyroidism ( likely to be evident in the early stages of disease , before adequate thyroxine replacement ) vs the possible cumulative effect of autoimmunity ( long - term increased risk , likely to occur later during follow - up ) . we also investigated effect modification by age ( < 60 , 6080 , > 80 years ) and performed several sensitivity analyses . first , analyses were repeated starting follow - up at the date of first ait code instead of first thyroxine script . second , cardiovascular conditions present among patients with ait at diagnosis could be early mediating factors for stroke rather than a confounding variable ; to assess their importance , we performed subgroup analyses for individuals without a history of any cardiovascular disease ethics approval was obtained from the south - east multicentre research ethics committee and from the london school of hygiene and tropical medicine ethics committee . in total , 184,539 eligible individuals from 287 general practices were included in this study , of whom 34,907 had ait ( figure 2 ) and 149,632 had no evidence of ait . individuals with ait were slightly older than individuals without ait ( median 59.8 vs 57.0 years ) , were followed up for longer ( median 3.2 vs 3.0 years ) , and a slightly higher proportion was female ( 81.5% vs 80.0% ; table 1 ) . for cardiovascular risk factors , individuals with ait were more likely to have been diagnosed with hypertension , diabetes , hyperlipidemia , chd , or chf at baseline ( all p < 0.001 ) . this pattern remained after adjusting for the slight age and sex imbalances ( data not shown ) . baseline characteristics of individuals with and without ait individuals with ait had a 13% increased rate of stroke compared with individuals without ait after adjusting for current age and sex and allowing for clustering in practice ( model 1 , table 2 ) . the increased risk was very similar ( rate ratio [ rr ] = 1.14 , 95% confidence interval [ ci ] : 1.041.24 , p = 0.003 ) after adjusting for alcohol and smoking ( model 2 ) and was not affected by the duration of follow - up or calendar year . the rr decreased slightly to 1.10 ( 95% ci : 1.011.20 , model 3 ) after adjusting additionally for cardiovascular risk factors present at baseline ( hypertension , hyperlipidemia , af , and bmi ) and was not further changed by consideration of existing diabetes , chd , and chf . multivariable analysis of the effect of ait on stroke and tia adjusted for covariates ( and allowing for clustering in practice ) individuals with ait had an increased risk of developing chd , hyperlipidemia , chf , and diabetes during follow - up compared with unexposed individuals ( table e-1 ) , but not hypertension or af . after adjusting for these potential mediating factors between ait and stroke , the rr for stroke was further reduced to 1.06 ( 95% ci : 0.971.15 , model 4 ) . effect sizes were slightly higher for all models when the outcome definition was expanded to both stroke and tia ( table 2 ) . there was evidence that the effect of ait on stroke varied with duration of disease ( pinteraction = 0.078 , figure 3 ) . the effect of ait on stroke was largest in the first year after diagnosis ( rr = 1.33 , 95% ci : 1.141.56 ) and was increased both in the first 6 months ( rr = 1.44 , 95% ci : 1.171.78 ) and 7 to 12 months after start of treatment ( rr = 1.22 , 95% ci : 1.021.55 ) . similar results were obtained for analyses using tia or stroke and tia alone as the outcome . there was no evidence that the effect of ait varied with age ( pinteraction = 0.516 ) . * adjusted for current age , sex , alcohol , smoking , hypertension , atrial fibrillation , hyperlipidemia , and body mass index ( all at baseline ) and allowing for clustering in practice . * * using a likelihood ratio test to compare a model with against a model without an interaction term for time since diagnosis . all sensitivity analyses showed results compatible to those obtained from the main analyses ( data not shown ) . our study provides strong evidence for a slightly increased risk of stroke in patients with ait , particularly in the first year after ait diagnosis . our analyses indicated that people with ait were more likely to develop hyperlipidemia , chd , and chf , adding evidence to previous discussions about whether ait is associated with these factors , and that some , but not all , of the increased risk of stroke among patients with ait was mediated via these classic cardiovascular risk factors . our systematic review identified 7 studies , which showed effect sizes ranging from 0.8 to 1.6 ( table e-2 ) . all of these studies except for the scottish study from 2006 were small and had effect estimates with wide cis that overlapped with those from the present study . other methodologic limitations of previous studies included overadjustment for variables that could be mediators of increased stroke risk , and residual confounding ( for example , the scottish study lacked information on smoking , alcohol use , and medication history ) . this could explain their slightly higher effect estimates , because thyroid hormone status is often assessed at stroke units , resulting in better ait ascertainment in individuals with a stroke history ( who are at higher risk of a subsequent stroke ) . three of the studies were conceptually different from this study because they used asymptomatic patients identified as hypothyroid by laboratory tests who were mostly untreated with thyroxine . we found differences between individuals with and without ait in the prevalence of traditional cardiovascular risk factors at baseline . individuals with ait were more likely to have af and hypertension at baseline compared to those without ait ; however , they were not at increased risk of developing these conditions during follow - up . this finding is consistent with that from a recent population - based study from germany , which reported that hypothyroidism was associated with prevalent but not incident hypertension . hypertension and af may not be on the causal pathway between ait and stroke , although the differences we observed at baseline could represent early changes induced by undiagnosed ait . differences in af ( and possibly hypertension ) at baseline between individuals with ait and those without could also be explained by differences in ascertainment of ait , if individuals with af were more likely to have thyroid - stimulating hormone levels measured . our main model of interest , adjusted for these baseline variables , is based on the assumption that pathologic mechanisms on the causal pathway to stroke did not start until after ait diagnosis and initiation of thyroxine treatment . however , it is likely that the date of ait diagnosis / start of treatment did not accurately capture start of disease . given that patients with ait often present with nonspecific symptoms , diagnosis of ait can be delayed . thus , it is plausible that cardiovascular causal processes could have started before formal diagnosis of ait . if so , model 3 ( which adjusted for these variables ) may have provided a conservative estimate of relative stroke risk , and model 2 might provide an estimate closer to the true effect . however , the effect estimates produced by these 2 models were similar ; also , the sensitivity analysis excluding all individuals with cardiovascular disease related conditions at baseline showed results similar to the main analysis . our finding of a higher effect of ait on stroke risk in the first year after diagnosis is compatible with the hypothesis of increased cerebrovascular risk in patients with ait due to prediagnosis hypothyroidism and the time taken to reduce this risk after thyroid hormone replacement . there was no evidence for an increasing effect with increased length of follow - up ( a possible proxy for a long - term effect mediated by autoimmune processes ) . an alternative explanation is that the observed effect of ait on stroke was caused by thyroxine treatment because thyroxine can have a procoagulant effect . however , if the observed effect was due to thyroxine , we might expect to have seen the excess stroke risk restricted to the period soon after start of treatment , whereas the risk remained increased 6 to 12 months after diagnosis in our study . moreover , a thyroxine - mediated effect might also be expected to generate short - term differences in incidence of af between patients with ait and individuals without ait because thyroxine in high doses can induce af ; this was not the case . nevertheless , we can not exclude that some of the effect attributed to hypothyroidism could have been due to thyroxine , and we were unable to examine this formally because all patients with ait received thyroxine . we used a considerably larger study population than all previous studies , and thus had greater power to ascertain effect estimates . we had information on a variety of potential confounding factors that could be considered in the analysis . unlike previous studies , we excluded individuals with a history of stroke , avoiding introduction of bias . moreover , excluding people with other autoimmune diseases from our study population enabled us to observe the effect of ait unmodified by other autoimmune disorders . this might not have been the case in other studies , because patients with ait are at higher risk of acquiring other autoimmune disorders , some of which ( e.g. , rheumatoid arthritis , type 1 diabetes ) are associated with considerably increased risk of cardio- and cerebrovascular disease . almost all noniatrogenic hypothyroidism in the united kingdom is due to ait , and we excluded individuals who had other reasons for hypothyroidism . however , our read code list included nonspecific hypothyroidism codes , which increased sensitivity of ait diagnosis but could have included a few non - ait cases . because hypothyroidism can remain undiagnosed for an unspecified time this is likely to have happened largely independently of subsequent stroke risk , and thus may have driven the rr toward 1.0 . those with preexisting af could have been more likely to have their ait diagnosed , which could have resulted in some overestimation of the effect size of ait on the risk of stroke . however , our sensitivity analyses excluding individuals with af at baseline indicate that this is unlikely to have been a major problem . differential misclassification of the outcome was unlikely in this study because stroke is a serious condition and should have been recorded equally in both groups independent of ait status , or health - seeking behavior . however , we could not differentiate between cases of ischemic and hemorrhagic stroke , because 85% of stroke codes did not specify the stroke subtype , consistent with previous studies of stroke using electronic health data . we hypothesized that ait might be associated specifically with ischemic stroke because hypothyroidism is linked with classic risk factors for ischemia . moreover , carotid artery intima - media thickness and af , 2 large risk factors for ischemic stroke , have been linked to ait . ischemic stroke causes approximately 80% of stroke cases in adults , and effect sizes for tia ( an ischemic process ) were comparable to those shown for stroke in our study , further supporting that ait is associated with ischemic stroke . nevertheless , ait could also affect the risk of hemorrhagic stroke , because hypothyroidism has been shown to be associated with the development of von willebrand syndrome and bleeding events . it is therefore possible that they were more likely to be diagnosed and treated for conditions such as hyperlipidemia or hypertension . monitoring and prompt treatment of these conditions could have decreased stroke risk associated with these conditions and might partly explain the relatively small increase in stroke risk associated with ait . nevertheless , this study provides a realistic picture of the residual effect of ait after standard treatment and surveillance in the united kingdom . data concerning the potential confounders of ethnicity and socioeconomic status were not available . however , socioeconomic status was partly accounted for by allowing for clustering by practice . although thin has been shown to be representative for the uk population , the results of this study might not be generalizable to all patients with ait . if hypothyroidism is on the causal pathway between ait and stroke , the observed effect relates to treated rather than all patients with ait . excluding individuals with ait who had a second autoimmune disorder allowed us to pinpoint the specific effect of ait , but may have excluded some patients with ait who had stronger autoimmune processes and thus might not have captured all of the long - term risk of stroke mediated by autoimmune pathology . our study has demonstrated a 10% to 14% increased risk of stroke among patients with hypothyroid ait after extensive consideration of confounding and potential biases . given the relatively high prevalence of ait and the morbidity and mortality associated with stroke , even small increases in stroke risk might be of high clinical relevance . the results of our study highlight the potential for regular screening for cardiovascular risk factors and preventive treatment ( e.g. , with statins ) in patients with ait . both authors developed the study design . a.k . conducted the data management , analyzed the data , and wrote the manuscript , with contributions from s.t . both authors interpreted the findings , contributed to critical revision of the manuscript for important intellectual content , and approved the final version . supported by a project grant from the wellcome trust ( 079482/z/06/z ) and by a postgraduate fellowship of the daad ( german academic exchange service , for a.k . ) .
bimetallic , initially spherical ag / au nanoparticles in glass prepared by ion implantation have been irradiated with intense femtosecond laser pulses at intensities still below the damage threshold of the material surface . this high - intensity laser processing produces dichroism in the irradiated region , which can be assigned to the observed anisotropic nanoparticle shapes with preferential orientation of the longer particle axis along the direction of laser polarization . in addition , the particle sizes have considerably been increased upon processing .	nano - sized metal particles embedded in glass are of great interest because of their potential application as non - linear material for photonic devices . the non - linear properties of nanocomposite glasses equipped with such particles are induced by the surface plasmon resonance at the interface between particles and glass matrix . this means that the optical effects in the spectral region around the surface plasmon resonance result from an electric field enhancement or a quantum confinement . thus , applications are possible as in integrated photonic networks , in nanoelectronics , for surface enhanced raman scattering , for up - conversion processes and laser materials . recently , the preparation of specific bimetallic nanoparticles like core - shell structures has been intensively investigated because of the far - reaching possibilities to modify the macroscopic properties [ 3 - 6 ] . a special way to extend the range of manipulating the optical properties of such nanocomposite glasses there are known some first examples for hollow nanoparticles in glass that were prepared by sequential implantation of two different metal ions [ 6 - 8 ] . a further degree of freedom introducing anisotropic optical properties is the method of femtosecond laser pulse - induced shape transformation of the nanoparticles which has been studied intensively in recent years [ 9 - 11 ] . depending on the actual irradiation parameters , uniformly oriented prolate or oblate nanoparticles can be prepared , whose orientation is controlled by the laser polarization . so far these effects have been demonstrated for ag nanoparticles at low concentration . in this letter , we will demonstrate that a similar shape modification of initially spherical bimetallic and hollow ag / au nanoparticles in soda - lime glasses is possible by irradiation with femtosecond laser pulses at high intensity , but below damage threshold ; in particular , it will be shown that anisotropic particle shapes or nearly linear arrangements of nanoparticles with preferential orientation along the direction of laser polarization can be fabricated with the help of this irradiation technique . the samples used for this study were sheets of soda - lime glass containing ( in mol% ) 72.4% sio2 and 14.4% na2o as main components , which were exposed subsequently to au ( 150 kev ) and ag ( 100 kev ) ion implantation at room temperature . by this sequential high - dose ion implantation of ag and au , metal particles have been formed in a surface - near region of the soda - lime silicate glass . the dose of implanted ions was 4 10 ions / cm for each type of ions ( for further details see ) . to characterize the surface plasmon resonance due to the metal nanoparticles formed in the implanted areas the optical density of glass samples was recorded by means of a perkin - elmer spectrometer in the wavelength range of 250900 nm . these samples were irradiated by linearly polarized laser pulses of 150 fs temporal width at a wavelength = 550 nm . this wavelength is the sum frequency of a 1 khz repetition rate ti : sapphire laser at = 800 nm and the idler ( = 1,760 nm ) of a travelling - wave optical parametric amplifier of superfluorescence ( topas ) . the laser beam was focused on the sample to a spot size of ~100 m . moving the sample continuously on a motorized x y translation stage , several parallel lines of ~1.5 mm length and 150 m lateral distance have been inscribed in the glass at a velocity of 0.5 mm / s , corresponding to , on average , 200 laser pulses hitting each spot within the lines . the polarization direction of the laser was parallel to the lines ( writing direction ) . the effect of irradiating the sample in the described way with average single pulse energy of 20 j is shown in figs . the lines prepared by high intensity fs laser irradiation exhibit a certain subdivision into a few parallel traces of decoloration as well as , at a few positions along the line center , some damage at the very glass surface . in the central region there is still a color change visible , but the dichroism decreases continuously to that of the original glass region . optical micrographs of the au / ag nanoparticles containing glass after laser irradiation recorded usinganon - polarized , andb , cpolarized light , respectively optical density within the central region of irradiated glass measured before ( original glass , solid line ) and after laser irradiation with polarized light , parallel ( dotted ) or perpendicular ( dashed ) with respect to laser pulse polarization and line direction ( see fig . 1 ) . the spectra of non - irradiated samples are identical for parallel and perpendicular polarization to evaluate the nanoscopic background of the observed optical changes , the parameters mean size , size distribution , shape , and penetration depth of the metal particles have been examined by transmission electron microscopy ( tem ) using a jem 1010 operating at 100 kv and a jem 4010 operating at 400 kv . for this purpose , planar and cross - section preparation were applied including mechanical grinding , polishing and argon ion - beam etching followed by deposition of a thin carbon film on both sides . for the samples of this study , prepared by applying an ion dose of 4 10/cm for both of the metals , a particle - containing region has been obtained that extends from the very glass surface to a depth of about 135 nm . tem imaging of cross - section samples reveals that this particle layer exhibits a non - uniform spatial distribution of particles as already reported earlier . in the middle of the particle layer mainly larger particles are situated whose anomalous image contrasts point to the presence of voids in their interior as may be recognized from the tem image of a planar preparation sample shown in fig . while for the entire set of particles present in the layer a mean size of 5.34 3.89 nm has been determined , the mean outer diameter of the void - containing particles amounts to 16.2 nm . altogether the particle sizes range from about 1.524 nm . the frequency of the surface plasmon resonance of the non - irradiated glass ( see fig . 2 ) appearing between those of pure ag and au particles demonstrates the formation of ag au alloy nanoparticles . the relation of concentrations of both elements incorporated into the nanoparticles should be ~1:1 corresponding to calculations of theoretical spectra and to experiments by anomalous small angle x - ray scattering . 4 10/cmauion implanted sample the above - described ultrashort laser pulse processing of this sample resulted in a totally different appearance of the structural characteristics of this particles - in - glass composite material which , however , is restricted to those regions where the laser lines have been inscribed into the particle layer . by careful target preparation we achieved to place the position of the hole at the edge of one of these lines ; this allowed us to image within one specimen regions without laser irradiation as well as regions where the maximum laser pulse intensity had been applied . the tem examination reveals that ultrashort laser pulse processing causes fundamental changes in size , shape , arrangement and configuration of the metal nanoparticles in the irradiated regions ( see fig . 4 ) , whereas no changes can be observed in non - irradiated regions . in the center of the irradiated regions most of the larger particles are elongated nearly along a common direction parallel to the laser lines inscribed ( and thus along the laser polarization vector ) . the elongated particle shapes may be described as spheroidal , but with a certain degree of irregularity . altogether the particle dimensions range from about 4.4 to 52.6 nm for the minor axis ( mean value 16.68 8.69 ) and from 5.6 to 79.6 nm for the major axis ( mean value 20.98 13.47 ) . the aspect ratio ( i.e. , the ratio of major to minor axes lengths ) of the particles increases nearly linearly from a value of 1 for particles of about 4.7 nm diameter to a value of 1.5 for particles of about 60.4 nm diameter ( these diameters refer to spheres of the same volume as the spheroids have ) . the total increase in particle size compared to the initial situation in the sample before irradiation corresponds to a more than tenfold volume increase for the individual particle . so , the laser processing resulted in a totally different appearance , not only the particle dimensions have increased by a factor of 3 , but also the size distribution has become a little bit narrower and lost part of its asymmetry , and , what is the important issue in this result , shape and arrangement of the particles deviate from the previous isotropic appearance . on the other hand the optical absorption remains fairly constant or even decreases indicating that the total amount of silver and gold does not grow . so it can be concluded that the volume increase of the particles is compensated by a simultaneous decrease of their number . the optical spectra also show that the composition of alloy nanoparticles should be similar to that before the irradiation . there are only slight shifts due to variations in sizes of particles and concentrations of elements within the particles . tem micrograph of the above sample upon ultrashort laser pulse irradiation finally , we want to discuss briefly which physical mechanisms may lead to the observed shape changes of bimetallic nanoparticles upon intense fs laser irradiation , in particular explaining the preferential orientation of elongated particles more or less arranged parallel to the laser polarization . the shape changes observed here can be compared to similar previous results obtained on two quite different types of metal - dielectric nanocomposites . the first type of material is glass containing low concentration of ag nanoparticles . for such systems it has been found that only the individual particle and its immediate surroundings are affected by laser - induced modifications . the sequence of processes there starts with field - driven electron emission from the particle , followed by electron trapping in the glass matrix , ion emission , their local recombination with trapped electrons , and diffusion and precipitation of ag atoms at the poles of the particle in the transiently heated nearest shell of the matrix . the second type of material studied previously is plasma polymer embedding a quasi 2-dimensional metal island film . irradiating these systems with similar parameters as in this work , a grating - like superstructure oriented along the laser polarization with a typical period of 2/3 of the laser wavelength has been observed , where stripes of unchanged metal nanostructure ( percolation region ) are alternating with stripes of coagulated larger , spherical particles . the explanation for these self - organized structures comprises spatially modulated energy input in the metal layer by interference of the incoming laser light with the scattered surface wave , where statistical inhomogeneities of the sample provide a feedback , so that the structures are becoming more pronounced and regular shot by shot . comparing the situation with the plasma polymer samples , we here also observe a large increase of particle sizes , but no regular superstructure . comparing with isolated ag nanoparticles in glass , we also find individual non - spherical shapes with the long axes oriented preferentially along the laser polarization ; but , in addition , particles are growing and sometimes merging , preferentially in situations where together they form a coagulated particle with longer axis along the laser polarization . from these considerations we conclude that the laser - induced shape changes of bimetallic nanoparticles are initiated by the same mechanisms as in the case of low - concentrated ag nanoparticles , i.e. , directional electron emission and capture in the glass matrix , followed by the processes listed above . the main difference appears to come from the higher metal concentration leading to spatially and temporally more extended regions of high temperature around metal particles in the matrix , enabling much larger diffusion distances of electrons and metal ions or atoms . it can not be decided at present if , in addition to the basic mechanism of particle reshaping during laser irradiation ( migration of individual atoms or ions ) , also processes like migration and coalescence of very small particles contribute to the observed particle growth . the reason is that all processes are started by an ~100 fs laser pulse ; then the surrounding glass is heated within a few ps and cools down again by heat conduction within a few ns . particle formation or growth under such strongly non - equilibrium conditions has , to the best of our knowledge , so far not been modeled theoretically . still , however , the local trapping sites for electrons in glass are obviously a necessary prerequisite for shape anisotropy of the particles . this is confirmed by the lack of similar shape anisotropy of the metal particles after fs irradiation in plasma polymers . furthermore , the temperature increase during laser irradiation within the particle regions explains the transformation of hollow particles into solid ones because of their thermal instability . the vacancies leave the central void toward the outer surface and the hollow region disappears at elevated temperatures . the laser - induced changes of shape and configuration of nanoparticles described above can also explain the slight blue shift of the surface plasmon resonance observed for perpendicularly polarized light as well as the lacking red - shift for parallel polarization ( as shown in fig . , one can conclude a reduced concentration of precipitated particles compared with the nanocomposites before irradiation . that is , obviously the recombination of emitted ions with trapped electrons is not completed . while , however , the probability for emission during interaction with an ultrashort laser pulse is the same for au and ag ions , the mobility of au in the glass matrix is considerably less than that of ag species . this difference should also affect the amount of both elements being incorporated into the particles again ; so in the end the concentration ratio in the particles will be shifted toward ag atoms , and this will shift their plasmon resonances toward shorter wavelengths . in conclusion , we have shown that spherical , bimetallic au / ag nanoparticles in glass at high concentration can be transformed to anisotropic shapes ( accompanied by size increase ) preferentially oriented along the direction of the linear laser polarization . the mechanism appears to be similar to that observed for silver nanoparticles in glass at low concentration , but with additional effects like coalescence caused by the close proximity of the particles . overall , the demonstrated high - intensity laser processing is a promising and flexible technique to design the linear and non - linear optical properties of metal - glass nanocomposites . the authors would like to thank the institute of solid state physics of the friedrich schiller university of jena for implantation of glass samples .
purposeto compare the two transscleral fixation ( tsf ) techniques of intrascleral pocket and conventional scleral flap with conjunctival division techniques in terms of short - term clinical effects.methodsthis retrospective cohort study included all consecutive patients with aphakia in gyeongsang national university hospital in jinju , korea , who underwent tsf between january 2012 and december 2014 . the medical records of all patients were retrospectively reviewed , and the endothelial cell count ( ecc ) , refraction , best - corrected visual acuity ( bcva ) , intraocular pressure , slit lamp , and fundus examination results before and 1 day and 6 months after surgery were recorded . the postoperative complications and visual outcomes were also recorded.resultsthe intrascleral pocket and conventional - flap groups did not differ significantly in terms of demographics , presurgical bcva , or ecc . however , the intrascleral pocket group had a significantly lower bcva at 1 day and 6 months after surgery compared to the conventional - flap group . the two groups did not differ in terms of ecc 6 months after surgery . the intrascleral pocket group had no postoperative complications , but five patients in the conventional - flap group complained of irritation . in both groups , the intraocular lens was well positioned without tilting or subluxation , and astigmatism was significantly reduced at 1 day and 6 months after surgery.conclusionsthe intrascleral pocket technique of tsf does not involve conjunctival dissection and is a successful method of sulcus fixation . it stably corrects the intraocular lens and is easy to perform , which helps to reduce operation time . it also reliably yields rapid visual acuity recovery without complications .	the institutional review board of gyeongsang national university hospital approved the study protocol ( no . 2015 - 06 - 031 ) , and the protocol complied with the tenets of the declaration of helsinki . all consecutive patients in a tertiary referral hospital , gyeongsang national university hospital in jinju , korea , who underwent tsf for aphakia due to various causes , including iol dislocation , retinal detachment , trauma , zonulysis , endophthalmitis , phacomorphic , and acute angle closure glaucoma , between january 2012 and december 2014 were identified by a review of the medical records . before surgery and 1 day and 6 months after surgery all patients underwent examinations to measure refraction , best - corrected visual acuity ( bcva ) , intraocular pressure , and specular microscopy , as well as slit lamp and fundus examinations . the power of iol was calculated using the sanders - retzlaff - kraff ii formula . was conducted by one surgeon ( ksj ) , and the surgical procedures were as follows : at the limbus , two points 180 from each other were marked ( fig . to make a 2-mm - sized pocket , lamellar dissection without conjunctival dissection was performed with a crescent blade , then the blade was advanced about 1 to 1.5 mm ( fig . polypropylene was passed through the transconjunctival transcleral passage , which was located 1.5 mm posterior from the limbus . anterior to the limbus , a clear corneal incision ( cci ) was made using a keratome , and the prolene sutures were exteriorized through the cci pocket . an ophthalmic viscosurgical device was inserted into the anterior chamber . a three - piece foldable acrylic iol ( sensar soft acrylic iol ; abbott medical optics , santa ana , ca , usa ) was injected through the cci , and the prolene sutures for each haptic were tied ( fig . the iol was placed in the anterior chamber , and its orientation was adjusted by pulling the prolene sutures . the iol was centralized , and the ends of the prolene were sutured in the intrascleral pocket ( fig . the cci and other paracentesis wounds were then hydrated , and the conjunctival edges were joined without suture . the scleral flap with conjunctival division technique was as follows : after conjunctival dissection , a triangular - shaped scleral flap was made , a 2.8-mm cci was generated at the anterior limbus using a keratome , and a three - piece foldable acrylic iol was injected . the haptic ends were exteriorized through the cci , after which each haptic was tied . continuous data were presented as mean standard deviation , and categorical variables were presented as number ( % ) . the two surgical groups were compared in terms of bvca , endothelial cell count ( ecc ) , spherical equivalent , and astigmatism using the paired t - test . 19.0 ( ibm co. , armonk , ny , usa ) . a p - value less than 0.05 the institutional review board of gyeongsang national university hospital approved the study protocol ( no . 2015 - 06 - 031 ) , and the protocol complied with the tenets of the declaration of helsinki . all consecutive patients in a tertiary referral hospital , gyeongsang national university hospital in jinju , korea , who underwent tsf for aphakia due to various causes , including iol dislocation , retinal detachment , trauma , zonulysis , endophthalmitis , phacomorphic , and acute angle closure glaucoma , between january 2012 and december 2014 were identified by a review of the medical records . before surgery and 1 day and 6 months after surgery all patients underwent examinations to measure refraction , best - corrected visual acuity ( bcva ) , intraocular pressure , and specular microscopy , as well as slit lamp and fundus examinations . the power of iol was calculated using the sanders - retzlaff - kraff ii formula . the intrascleral pocket technique for tsf was conducted by one surgeon ( ksj ) , and the surgical procedures were as follows : at the limbus , two points 180 from each other were marked ( fig . to make a 2-mm - sized pocket , lamellar dissection without conjunctival dissection was performed with a crescent blade , then the blade was advanced about 1 to 1.5 mm ( fig . polypropylene was passed through the transconjunctival transcleral passage , which was located 1.5 mm posterior from the limbus . anterior to the limbus , a clear corneal incision ( cci ) was made using a keratome , and the prolene sutures were exteriorized through the cci pocket . an ophthalmic viscosurgical device was inserted into the anterior chamber . a three - piece foldable acrylic iol ( sensar soft acrylic iol ; abbott medical optics , santa ana , ca , usa ) was injected through the cci , and the prolene sutures for each haptic were tied ( fig . the iol was placed in the anterior chamber , and its orientation was adjusted by pulling the prolene sutures . the iol was centralized , and the ends of the prolene were sutured in the intrascleral pocket ( fig . knots were buried under the sclera flaps ( fig . 1 g and 1h ) . the cci and other paracentesis wounds were then hydrated , and the conjunctival edges were joined without suture . the scleral flap with conjunctival division technique was as follows : after conjunctival dissection , a triangular - shaped scleral flap was made , a 2.8-mm cci was generated at the anterior limbus using a keratome , and a three - piece foldable acrylic iol was injected . the haptic ends were exteriorized through the cci , after which each haptic was tied . continuous data were presented as mean standard deviation , and categorical variables were presented as number ( % ) . the two surgical groups were compared in terms of bvca , endothelial cell count ( ecc ) , spherical equivalent , and astigmatism using the paired t - test . 19.0 ( ibm co. , armonk , ny , usa ) . a p - value less than 0.05 in total , 40 consecutive patients with aphakia underwent tsf in our hospital between january 2012 and december 2014 . the mean age of the cohort was 59.8 4.1 years , and there were 28 males and 12 females . the mean age of the intrascleral pocket and conventional - flap groups was 59.7 9.2 and 59.8 9.7 years , respectively . in both groups , the main cause of aphakia was iol dislocation . other causes were phacoemulsification , lens dislocation , zonulysis , and vitrectomy with lensectomy , which occurred in the two groups with similar frequencies ( table 1 ) . the two groups did not differ significantly in terms of preoperative bcva and ecc or ecc at 6 months after surgery ( table 2 ) . however , the intrascleral pocket group had significantly better bcva at 1 day and 6 months after surgery compared to the conventional - flap group ( p = 0.03 and 0.04 , respectively ) . in contrast , five of the 20 patients in the conventional - flap group complained of irritation after surgery ( table 2 ) . one day and 6 months after surgery , the intrascleral pocket group exhibited significant decrease in spherical diopter ( p = 0.018 and 0.005 compared to preoperative values , respectively ) and astigmatism ( p = 0.027 and 0.028 , respectively ) . this was also observed for the conventional - flap group for spherical diopter ( p = 0.000 and 0.007 , respectively ) and astigmatism ( p = 0.009 and 0.007 , respectively ) ( fig . tsf of an iol is widely used for aphakic patients with poor support of the posterior chamber or ciliary body . many recent studies have reported modifications of the tsf technique that was first introduced by malbran et al . in 1986 . however , all of these methods can cause suture - related complications , including knot exposure or iol dislocation due to suture decomposition or breakage . of these complications , knot exposure is one of the most common and can have deleterious outcomes as it can act as a pathway for bacterial invasion into the eye , thereby causing endophthalmitis . when szurman et al . retrospectively analyzed the results of 45 eyes that underwent tsf of pciol using z - sutures and 22 eyes that underwent tsf of an iris prosthesis , they found one case of ciliary body hemorrhage during the 22.4 months of follow - up , but no complications such as knot exposure , suture looseness , conjunctival atrophy , or chronic inflammation . ma et al . also analyzed the clinical outcomes of a knotless external fixation technique of pciol tsf in five patients who had to undergo this technique because of adhesion between the conjunctiva and cornea or because the location of the conjunctival flap was near a corneal wound caused by trauma . therefore , this study introduced the sutureless intrascleral pocket technique and compared the effects and complications including astigmatism or postoperative visual prognosis . compared with conventional techniques , this technique resulted in less irritation and astigmatism since knots were buried in the intrascleral pocket , and vertical and regular wounds were formed without a gap of wound . in addition , this technique led to rapid visual recovery and return to daily life , since the visual acuity the day after the operation was comparable to the bcva before the surgery . during the follow - up period , there were no complications except for mild ciliary body hemorrhage in one case and iol tilting immediately after the operation due to excessive knot tightness in another case . the bcva in 16 of 20 cases ( 80% ) improved , while the remaining case did not exhibit a change in bcva . thus , the outcomes of the intrascleral pocket technique in the present study were generally as successful as those of the knotless external fixation technique of pciol in a previous study . the intrascleral pocket technique of tsf is especially useful for patients who previously underwent vitrectomy . patients after vitrectomy , especially those who underwent surgery with a 20-gauge needle , usually have a relatively thin conjunctiva or conjunctiva - tenon - scleral complex due to previous conjunctival dissection . this makes conjunctival dissection more difficult and increases the possibility of conjunctival defects and scleral injury . in addition , since this technique does not involve conjunctival dissection , the operation time and bleeding risk are reduced . moreover , subsequent glaucoma surgery can improve the success rate of the intrascleral pocket technique because the conjunctiva and tenon 's space maintain their native integrity . the intrascleral pocket technique also allows for rapid visual acuity recovery and return to daily life because the associated irritation is low . in addition , the vertical and regular wound without a gap helps to reduce astigmatism . in a previous study using the sutureless intrascleral pocket technique this prompted us to reassess patients at 6 months after the surgery in this study , and the wounds were found to be clean without dehiscence or knot exposure ( fig . unlike the reverse pocket technique , the intrascleral pocket technique leaves the corneal limbus intact , which reduces the chance of injury to the corneal nerve . moreover , it is possible to perform the intrascleral pocket technique with topical anesthesia because it is associated with less pain than other techniques . in addition , the intrascleral pocket technique is quite similar to glaucoma or flap - making procedures , which means that surgeons can rapidly become proficient in this technique . , the present study showed that the intrascleral pocket technique of tsf had better clinical results than the conventional scleral flap with conjunctival division technique , as determined by ophthalmological examinations before and 1 day and 6 months after the operation . the improvement in bcva was more marked in the intrascleral pocket group , and no postoperative complications were observed . these observations , together with the fact that this technique is relatively easy , suggest that this technique is a good method of tsf . . however , the simplicity and good outcomes of this technique suggest that it is a good method of tsf .
abstractaimto compare longterm effects of brushing with an oscillating rotating power toothbrush or an ada reference manual toothbrush on preexisting gingival recession.materials and methodsin this controlled , prospective , singleblind , parallelgroup study , healthy subjects with preexisting recession were randomized and brushed with a power toothbrush ( n = 55 ) or an ada reference manual toothbrush ( n = 54 ) for a 3year study period . subjects were required to brush their teeth twice daily for 2 min . using a standard fluoride toothpaste . during the study , subjects were assessed for clinical attachment loss and probing pocket depths to the nearest mm at six sites per tooth by the same calibrated examiner . gingival recession was calculated at preexisting sites as the difference between clinical attachment loss and probing pocket depths . hard and soft oral tissues were examined to assess safety.resultsafter 35 2 months , mean gingival recession did not differ significantly between groups , but was significantly reduced from baseline ( p < 0.001 ) , from 2.35 0.35 mm to 1.90 0.58 mm in the power and from 2.26 0.31 mm to 1.81 0.66 mm in the manual group.conclusionsgingival recession in subjects with preexisting recession was significantly reduced after 3 years of brushing with either a power or manual toothbrush .	as reported previously ( drfer et al . 2009 ) , the study had been approved by the imdec international medical & dental ethics commission freiburg ( approval number 01 17 04 03 ) . subjects were recruited from the general population between november 10th and 14th 2003 , to which the research was advertized by flyers at points highly frequented by the public around the campus . subjects who gave their written informed consent to participate were required to satisfy the study inclusion and exclusion criteria . to qualify for inclusion , the subjects were required to be 1870 years of age , in good general health , with a minimum of 18 scorable teeth ( excluding third molars , teeth with orthodontic appliances , bridges , crowns or implants ) and with two or more teeth showing recession on the facial surface of at least 2 mm . other main exclusion criteria were as follows : any condition that might preclude normal oral hygiene procedures or study participation ; medical condition requiring prophylactic antibiotic coverage prior to dental treatment ; therapy with any drugs for at least three consecutive days within the previous 28 days that might affect study outcome ; neglected dental health ; major hard or soft tissue lesions or trauma at the baseline visit ; known allergy to the test products ; or participation in any other oral hygiene clinical study within the previous 30 days . if applicants fulfilled all inclusion criteria and had no exclusion criteria , they were consecutively included in the study and randomized to either group . this was a singlecentre , randomized , examinerblind , parallelgroup study of gingival recession that compared the effects on oral tissues of power and manual brushing . subjects brushed twice daily and were assessed after 6 0 months ( drfer et al . 2009 ) , and after 12 0 and 18 1 and 35 2 months . an oscillating rotating and pulsating power brush ( d17u , oralb professionalcare , procter & gamble , cincinnati , oh , usa ) , and an american dental association reference flat trim manual brush were used . a standard sodium fluoride dentifrice ( blendamed ; however , they had to be informed about the aim of the study and the hypothesized interaction between toothbrushing and recession formation . at baseline , subjects were stratified based on initial preexisting gingival recession , gender and smoking status , and were randomized by the principal investigator to either the power brush group or the manual brush group . also , at this first visit , all subjects were instructed to brush their teeth for 2 min . twice daily throughout the study period using the assigned toothbrush and supplied a standard dentifrice . participants using the manual brush were told to continue brushing as they normally do . in the power group , participants were referred to the written instructions from the toothbrush manufacturer . subjects were instructed to return to the study centre every 3 months and clinical assessments were carried out at baseline , 6 0 , 12 0 , 18 , 1 and 35 2 months . at baseline , the medical history and details of concomitant medication and oral hard and soft tissue were assessed and any adverse events reported . plaque , gingival health , and periodontal measurements were also made at scheduled clinical assessments . every 3 months , standard fluoride toothpaste ( blendamed regular , procter & gamble , germany ) was dispensed along with a new manual brush or power brushhead , and questions about participation were answered . additional supplies of standard toothpaste were provided throughout the study at the study centre whenever requested . clinical measurements for all subjects at all time points were made by the same clinical examiner ( dw ) , who was blinded with respect to the assigned treatment , familiar with the clinical measurements used in the study , and had been calibrated ( drfer et al . 2009 ) . study measurements were made in the following order : oral safety assessments of soft and hard tissue , gingival index , plaque index , and periodontal measurements . periodontal measurements were carried out on each scorable tooth at six sites : mesiobuccal , midbuccal , distobuccal , mesiolingual , midlingual and distolingual . the oral cavity structures examined included the lips , tongue , gingivae , sublingual area , inner surfaces of the cheeks , mucobuccal folds , hard and soft palate , pharyngeal area , and cervical areas of all the teeth . any abnormal findings including observed or voluntarily reported adverse events were recorded . plaque and gingival health were also assessed , using the turesky modification of the quigley and hein plaque index ( turesky et al . 2007 ) and the le and silness gingival index ( le & silness 1963 ) respectively . in order to assess clinical recession , at each site periodontal pocket depths and clinical attachment levels were measured . a periodontal probe marked at each mm ( pcpunc15 , hufriedy , chicago il , usa ) was used to measure probing pocket depths ( ppd ) and clinical attachment level ( cal ) . after gently positioning the probe , measurements were averaged upward where the gingival margin or the cementoenamel junction was between mm markings . the gingival recession was calculated as difference between cal and ppd . if the cementoenamel junction was covered by a crown or cervical restoration the measurement was taken from the most apical margin of the restoration . the primary outcome variable was the mean recession change at preexisting recession sites at baseline . as secondary endpoints , recession changes were analysed at both the tooth and site level . cal and ppd were reported also as mean standard deviation as well as plaque and gingival index . no power calculation was made prior to the start of this study as no preliminary data were available , but the intention was for 50 subjects per group to have completed 6 months of the study ( drfer et al . clinical measurements were made at baseline , 6 0 , 12 0 , 18 1 , and 35 2 months for periodontal pocket depth and clinical attachment levels , and for the plaque and gingival indices for all sites . interim analyses were performed at 6 , 12 , and 18 months for quality assessment purposes . missing values at all time points were imputed by the baseline data of the respective participant . the statistical unit for the primary outcome was the participating subject ( firstlevel analysis ) . changes in recession between visits for each group were analysed using anova with post hoc bonferroni corrections for multiple testing . group differences in recession at 12 , 18 , and 35 months were tested for statistical differences by ttest . a twostep logistic regression analyses was performed to indicate the influence of relevant factors on the results in our data set . on the first level , relevant factors from the literature such as age , gender , and smoking status as well as the brush used were entered into the model . on the secondlevel , the local factors type of tooth , mandibular or maxillary , preexisting recession at baseline , local plaque , and local gingivitis at the final visit were added . risks due to the tooth type were reported relative to the second molar , due to the jaw relative to the mandibular and due to the brush used relative to the manual toothbrush respectively . the dependent variable ' recession ' was dichotomized into either having not changed or increased compared to being improved . statistical testing was twosided and a significance level of = 0.05 was used . as reported previously ( drfer et al . 2009 ) , the study had been approved by the imdec international medical & dental ethics commission freiburg ( approval number 01 17 04 03 ) . subjects were recruited from the general population between november 10th and 14th 2003 , to which the research was advertized by flyers at points highly frequented by the public around the campus . subjects who gave their written informed consent to participate were required to satisfy the study inclusion and exclusion criteria . to qualify for inclusion , the subjects were required to be 1870 years of age , in good general health , with a minimum of 18 scorable teeth ( excluding third molars , teeth with orthodontic appliances , bridges , crowns or implants ) and with two or more teeth showing recession on the facial surface of at least 2 mm . other main exclusion criteria were as follows : any condition that might preclude normal oral hygiene procedures or study participation ; medical condition requiring prophylactic antibiotic coverage prior to dental treatment ; therapy with any drugs for at least three consecutive days within the previous 28 days that might affect study outcome ; neglected dental health ; major hard or soft tissue lesions or trauma at the baseline visit ; known allergy to the test products ; or participation in any other oral hygiene clinical study within the previous 30 days . if applicants fulfilled all inclusion criteria and had no exclusion criteria , they were consecutively included in the study and randomized to either group . this was a singlecentre , randomized , examinerblind , parallelgroup study of gingival recession that compared the effects on oral tissues of power and manual brushing . subjects brushed twice daily and were assessed after 6 0 months ( drfer et al . 2009 ) , and after 12 0 and 18 1 and 35 2 months . an oscillating rotating and pulsating power brush ( d17u , oralb professionalcare , procter & gamble , cincinnati , oh , usa ) , and an american dental association reference flat trim manual brush were used . a standard sodium fluoride dentifrice ( blendamed ; however , they had to be informed about the aim of the study and the hypothesized interaction between toothbrushing and recession formation . at baseline , subjects were stratified based on initial preexisting gingival recession , gender and smoking status , and were randomized by the principal investigator to either the power brush group or the manual brush group . also , at this first visit , all subjects were instructed to brush their teeth for 2 min . twice daily throughout the study period using the assigned toothbrush and supplied a standard dentifrice . participants using the manual brush were told to continue brushing as they normally do . in the power group , participants were referred to the written instructions from the toothbrush manufacturer . subjects were instructed to return to the study centre every 3 months and clinical assessments were carried out at baseline , 6 0 , 12 0 , 18 , 1 and 35 2 months . at baseline , the medical history and details of concomitant medication and oral hard and soft tissue were assessed and any adverse events reported . plaque , gingival health , and periodontal measurements were also made at scheduled clinical assessments . every 3 months , standard fluoride toothpaste ( blendamed regular , procter & gamble , germany ) was dispensed along with a new manual brush or power brushhead , and questions about participation were answered . additional supplies of standard toothpaste were provided throughout the study at the study centre whenever requested . clinical measurements for all subjects at all time points were made by the same clinical examiner ( dw ) , who was blinded with respect to the assigned treatment , familiar with the clinical measurements used in the study , and had been calibrated ( drfer et al . study measurements were made in the following order : oral safety assessments of soft and hard tissue , gingival index , plaque index , and periodontal measurements . periodontal measurements were carried out on each scorable tooth at six sites : mesiobuccal , midbuccal , distobuccal , mesiolingual , midlingual and distolingual . the oral cavity structures examined included the lips , tongue , gingivae , sublingual area , inner surfaces of the cheeks , mucobuccal folds , hard and soft palate , pharyngeal area , and cervical areas of all the teeth . plaque and gingival health were also assessed , using the turesky modification of the quigley and hein plaque index ( turesky et al . 2007 ) and the le and silness gingival index ( le & silness 1963 ) respectively . in order to assess clinical recession , at each site periodontal pocket depths and clinical attachment levels were measured . a periodontal probe marked at each mm ( pcpunc15 , hufriedy , chicago il , usa ) was used to measure probing pocket depths ( ppd ) and clinical attachment level ( cal ) . after gently positioning the probe , measurements were averaged upward where the gingival margin or the cementoenamel junction was between mm markings . if the cementoenamel junction was covered by a crown or cervical restoration the measurement was taken from the most apical margin of the restoration . the primary outcome variable was the mean recession change at preexisting recession sites at baseline . cal and ppd were reported also as mean standard deviation as well as plaque and gingival index . no power calculation was made prior to the start of this study as no preliminary data were available , but the intention was for 50 subjects per group to have completed 6 months of the study ( drfer et al . clinical measurements were made at baseline , 6 0 , 12 0 , 18 1 , and 35 2 months for periodontal pocket depth and clinical attachment levels , and for the plaque and gingival indices for all sites . interim analyses were performed at 6 , 12 , and 18 months for quality assessment purposes . final analyses were performed based on the principle of intention to treat . missing values at all time points were imputed by the baseline data of the respective participant . the statistical unit for the primary outcome was the participating subject ( firstlevel analysis ) . changes in recession between visits for each group were analysed using anova with post hoc bonferroni corrections for multiple testing . group differences in recession at 12 , 18 , and 35 months were tested for statistical differences by ttest . a twostep logistic regression analyses was performed to indicate the influence of relevant factors on the results in our data set . on the first level , relevant factors from the literature such as age , gender , and smoking status as well as the brush used were entered into the model . on the secondlevel , the local factors type of tooth , mandibular or maxillary , preexisting recession at baseline , local plaque , and local gingivitis at the final visit were added . risks due to the tooth type were reported relative to the second molar , due to the jaw relative to the mandibular and due to the brush used relative to the manual toothbrush respectively . the dependent variable ' recession ' was dichotomized into either having not changed or increased compared to being improved . statistical testing was twosided and a significance level of = 0.05 was used . out of a total of 156 individuals that were screened , 109 subjects fulfilled the inclusion criteria and were enrolled at baseline . a number of subjects withdrew during the course of the study for a variety of reasons , which included pregnancy ( 2 ) , moving to another city ( 11 ) , and no further interest in participating in the study ( 21 ) . in total , 75 subjects ( 36 female , 39 male ) completed the study ; 38 in the manual group and 37 in the power group . table 1 shows the mean ages of both groups at baseline and the numbers of subjects assessed at each time point . age of subjects at baseline and numbers of subjects assessed at each study time point sd , standard deviation . varied between 33 and 37 months for individual subjects depending on visit schedule . the mean values at baseline and at 12 , 18 , and 35 months for periodontal pocket depth , clinical attachment level , plaque scores and gingivitis scores are summarized for all sites in table 2 . assessments at all time points for six sites per tooth : ppd ( mm ) , clinical attachment levels ( cal , mm ) , plaque ( tqhi ) , and gingivitis scores ( gi ) sd , standard deviation ; ppd , probing pocket depth ; cal , clinical attachment level ; tqhi , turesky modification of the quigley heinindex ; gi , le and silness gingival index . varied between 33 and 37 months for individual subjects depending on visit schedule . gingival recession measurements at preexisting recession sites for both groups are given in table 3 . for both groups , analysis of the differences in values between all time points showed a significant reduction in recession for both brushes . no statistically significant differences between groups were seen for any of the comparisons of changes in recession between study time points . gingival recession for sites with initial recession ( mm ) : mean values at all study time points and changes in recession during study sd , standard deviation ; n.s . , not significant . group difference non significant for all comparisons ( ttest ; p > 0.05 ) . varied between 33 and 37 months for individual subjects depending on visit schedule . the results of the logistic regression analysis showed on the tooth level statistically significant increases in risk for canines and first premolars ( odds ratios ( or ) 1.46 ( 95%ci 1.101.69 ) , and 1.25 ( 1.061.47 ) respectively ) and decreased risks at maxillary teeth compared to mandibular teeth ( or 0.66 ( 0.480.92 ) . on the tooth level , also the use of the power toothbrush reduced the risk of progression in preexisting recessions statistically significant ( or 0.81 ( 0.690.95 ) , p = 0.011 ) compared to the use of manual toothbrushes . multiple logistic regression analysis of increasing recession over 35 months in a multivariate model on the site level included variables : gender , age , smoking , recess base ( recession at baseline ) , ging 35 ( gingivitis score at 35 months ) , plaq 35 ( plaque score at 35 months ) , manual tb / power tb ( manual or power brush with manual as reference ) , manidbular / maxillary ( mandibular or maxillary with mandibular as reference ) , tooth types from central incisor to second molar ( with second molar as reference ) . examination of the oral cavity at each assessment visit revealed no adverse effects on hard or soft tissues in either group . this controlled , parallelgroup study compared the effect on preexisting gingival recession of brushing with an oscillating rotating power toothbrush ( oralb professionalcare ) or a manual brush over a period of approximately 3 years and showed no significant group differences in preexisting gingival recession . an unexpected finding from this study was a significant reduction in recession over time that occurred in both groups and led to significantly improved recession values over the complete observation time . the absence of a difference between groups in gingival recession corroborates the findings of the 6month report ( drfer et al . although , after 12 months a tendency towards a slight relapse was observed , but the recessions after nearly 3 years were still statistically improved compared to the baseline findings . the stability of the periodontal conditions during the complete course of the study can be seen by the overall probing depths , attachment levels and plaque and gingivitis scores . this is , therefore , the first controlled clinical longterm study showing existing localized gingival recessions may improve when patients ' attention is directed to the potential relationship between toothbrushing and gingival recession . although this is the first study to examine the course of preexisting gingival recessions under different conditions of brushing over such a long time in a randomizedcontrolled trial , the findings are consistent with previous studies on surrogate parameters , none of which recorded greater gingival abrasion or gingival trauma with powered than with manual brushes ( niemi et al . ( 2009 ) showed for sonic toothbrushes no difference in recession development compared to manual toothbrushes over a period of 12 months . the results from the present clinical study showing no group difference can be , therefore , considered a robust finding for reasons stated previously ( drfer et al . 2009 ) . mainly , the selection of subjects with preexisting recession , and hence the likelihood of further recession , would have improved measurement sensitivity and enhanced the opportunity for revealing any group differences . 1993 ) , which reported recession as a secondary outcome , appear to show that tooth brushing itself using either manual or power brushes does not cause gingival recession . the results of those studies together with the improvements found in this study at 6 months ( drfer et al . 2009 ) , and shown here to be maintained after 3 years of brushing , for subjects having preexisting recession , clearly contradict the results of studies that show tooth brushing per se may play a causal role in the development of gingival recession ( serino et al . those studies that have related tooth brushing to gingival recession have , however , been observational and generally not designed to report a causal relationship ; instead they have derived their conclusions from correlational findings ( rajapakse et al . for example , in a study with a population that had a high standard of oral hygiene , the subjects were examined at baseline and reexamined after 5 and 12 years ( serino et al . 1994 ) and although recession was observed to increase with age , the amount of tooth brushing over that study period may not have been the cause of increased recession . instead , an unsatisfactory brushing habit , such as a horizontal scrub technique ( tezel et al . 2001 ) , or an ageing process could have been responsible . in support of this suggestion it is worth noting the results of an observational study of 100 dental students , who also maintained a high level of oral hygiene ( carlos et al . , the authors concluded that the gingival recessions could have been attributed to the wrong brushing technique . other factors identified were too much strength exerted while brushing , over brushing , and the use of hard toothbrush bristles . in a study that considered a history of hard toothbrush use and gingival recession , subjects with a history of hard toothbrush use showed more pronounced gingival recession and had more surfaces with recession associated with increased brushing frequency , than subjects without a history of hard brush use ( khocht et al . 1993 ) . however , in a study again with dental students over 5 years of dental education ( daprile et al . 2007 ) it was shown that recession increased although the use of hard brushes was significantly reduced and an almost perfect brushing technique was achieved over 5 years of observation . the authors conclude that other than the suspected factors such as wrong brushing techniques or too hard brushes may explain their observation . they speculate that pressure , time , and amount of toothpaste may be responsible for progressive recession despite the elimination of the above mentioned factors . it is possible , therefore , that aggressive brushing , rather than the amount of brushing per se , could have caused the recession seen in observational studies . there are clear limitations when attempting to establish causality based on correlational evidence from observational studies alone . in contrast , wellcontrolled prospective experimental studies are able to provide convincing evidence of causation . this was the intention of the present controlled study that had a number of design features to allow findings to be interpreted with greater confidence , while also accounting for apparent discrepancies between the conclusions drawn from observational and experimental studies . in this study , subjects were selected in terms of predefined inclusion and exclusion criteria , they were randomly assigned to groups and assessments were made by a trained , calibrated examiner , who was blinded to treatment . the reliability of this methodology was emphasized in the earlier report on the 6month data ( drfer et al . scores for probing pocket depth , clinical attachment level , and plaque and gingivitis all reveal the periodontal health of participants , and any changes in periodontal condition over the course of the study can be expected to be reflected in these scores . low plaque and gingivitis scores were seen at baseline and this study did not seek , or expect , evidence for significant changes over time or for group differences . evidence that certain power toothbrushes are superior compared to manual brushes with respect to gingivitis reduction in longterm studies is already available ( yaacob et al . 2014 , van der weijden & slot 2015 ) . preexisting gingival recession , however , was of primary interest in this study , and its reversal seen both at 6 months ( drfer et al . 2009 ) and in this report requires an explanation , possibly in terms of some behavioural change on the part of the participating subjects . it is wellknown that the general public does not brush for the recommended 2 min . twice a day ( macgregor & rugggunn 1979 ) and brushing technique is commonly less than ideal ( saxer & yankell 1997 ) . in any group of subjects , therefore , there is likely to be plenty of opportunity for improvement in oral hygiene . if the subjects had adopted better brushing behaviour in this study , then this could account for reduced gingival recession . as was proposed to account for the reduction in recession seen in the 6month study , this behavioural change could have arisen simply because these subjects knew they were participating in an investigation . this is an example of the hawthorne effect , familiar to behavioural scientists , according to which , in a wide variety of experiments , the behaviour of human subjects is modified purely as a result of knowing that they are experimental subjects ( adair 1984 ) . its relevance here is supported by a study showing that by deliberately inducing the hawthorne effect it was possible to improve oral hygiene in a group of noncompliant adolescent orthodontic patients with poor oral hygiene ; improvements were seen at both 3month and 6month observation periods ( feil et al . , it should be noted that although brushing instructions had not been given at any visit during the study , the regular and repeated visits in circumstances likely to command their attention alone could be expected to change their brushing behaviour towards a gentler attitude . this seemed to work better than a systematic brushing exercise ( slot et al . the hypothesis that changes in brushing behaviour may lead to a decrease in recessions has been at least anecdotally reported ( everett 1968 ) . although it is unlikely due to the amount of effect , the stability over time and the validity of the measurements , the effect seen after 6 months may be influenced in parts by regression to the mean(egelberg 1989 ) . in summary , power toothbrushes with an oscillating rotating action have been shown to be more effective than manual brushes in plaque removal and control of gingivitis ( heanue et al . although the total amount of tooth brushing over time has been thought to play a causal role in the development of gingival recession ( hirschfeld 1931 ) , in the present 3year study no group differences emerged and neither the use of a power brush nor a manual brush was accompanied by increased gingival recession . on the contrary it seemed likely that the procedures followed in this study resulted in improved tooth brushing behaviour and that this , in turn , reduced gingival recession . it has been suggested that with an improved technique , along with some slight reduction in gingival inflammation , there can be an element of creeping buccal attachment more usually seen after mucogingival surgery ( rajapakse et al . this offers a possible explanation for the reversal of gingival recession found in this study . the findings of this study of the effects of toothbrushing on preexisting gingival recession are clear and compelling : over a period of approximately 3 years of regular twicedaily brushing , there was a significant and sustained reduction in gingival recession in both the group using a power toothbrush and the group using a manual brush.over the same period , there was no difference in the amount of gingival recession between the two groups.longterm reductions in gingival recession may have been caused by sustained improvements in brushing technique due to the manual tooth brushing carried out daily , appears to have no adverse effects on gingival recession ; it may even serve to improve the condition . over a period of approximately 3 years of regular twicedaily brushing , there was a significant and sustained reduction in gingival recession in both the group using a power toothbrush and the group using a manual brush . over the same period , there was no difference in the amount of gingival recession between the two groups . longterm reductions in gingival recession may have been caused by sustained improvements in brushing technique due to the power or manual tooth brushing carried out daily , appears to have no adverse effects on gingival recession ; it may even serve to improve the condition .
this paper takes a public health approach to briefly examine : ( i ) the concept of community health care need assessment ; ( ii ) the roles of academic institutions in health needs assessment ; ( iii ) jazan study to address the health care needs in jazan region , saudi arabia . the methods included an analysis of the literature , distillation of experience from the recently jazan health need assessment survey , and who reports . the most important perceived health problems in jazan region are shortage of health care providers , increased prevalence of communicable diseases and poor environmental health . the academic institutions , ministry of health and other health care institutions need to work together to look for innovative approaches , especially to increase the awareness of the society on public health issues , and give more support to increase national and regional funding for community based studies.the findings of the assessment of the health needs of jazan presented in this review could be utilized as a baseline and reference information for policy formulation , subsequent planning and cost effective intervention programs . it could also be utilized for the curriculum development or review for a community oriented medical schools .	health needs assessment is a process of determining the health and health care needs of any given population or sub - group in an area . it is a complex task requiring epidemiological expertise , the ability to work across organizational boundaries as well as an understanding of , and an ability to engage with all appropriate population groups . health needs assessment process needs to take account of the diversity within these populations . health improvement programs provide opportunities to engage in such assessments in the national and regional population . research has shown that preventive programs can improve community health when properly implemented.1 there are examples of successful prevention programs in local communities . however , many still have significant challenges , which demonstrate a gap between science and practice . though in the united states , there are such common strategies ( training programs ) , to address this issue , outcomes are still unsatisfactory . building the capacity of the community to implement high quality prevention can help communities achieve positive health outcomes , thereby narrowing the gap between theory and practice . while there is ample research on the efficacy of evidence - based programs , there is little on how to improve community capacity to improve the quality of prevention . what is being proposed is a new model of research : one based on community science that improves the latest theoretical understanding of community capacity and evaluates technologies designed to enhance it . in most developing countries , the evolution of health services has been dominated by western models of health care2 . these have rarely taken into account how local people explain illness , seek advice , or use traditional healing methods . the emphasis has been on hospitals and curative care rather than on trying to address local health needs equitably and effectively . since the alma ata declaration on primary health care , more attention has been given to increasing coverage of basic services and preventing common diseases . health care needs are changing and new challenges arising from chronic diseases and hiv infection must be faced . better coverage of preventive and essential healthcare services has led to greater emphasis on improving the quality of health care to ensure the efficient and judicious use of scarce resources . for example , infant mortality has fallen dramatically in the past two decades through such interventions as oral rehydration for diarrhea and immunization programs . with fewer children dying , there has been greater emphasis on the need to tackle the causes of infant and child morbidity . the size of families can be reduced with the improvement of the availability of family planning . if health services are to respond to the changing health needs of their local populations , planners and managers would need useful and timely information about the health status of these populations . some of this information can come from routine data sources or may be collected from large , one - off population studies . a confusing number of terms describe similar methods : rapid evaluation methods , rapid appraisal methods , rapid community surveys , rapid rural appraisal , relaxed rural appraisal , participatory rural appraisal3 . the development of rapid appraisal methods in the 1980s came in recognition of the time consuming and rigid nature of traditional epidemiological and questionnaire surveys . experience with these appraisal methods showed that when they were perfectly executed , they provided valuable , reliable , and timely information on health status , knowledge , attitudes , and behaviors . more recently , emphasis has been placed on encouraging people to participate in their own appraisal ( for example , participatory rural appraisal)4 . the hardest part of any needs assessment is translating the results into policies and practices to elicit and ( or ) initiate beneficial change . the involvement of health care workers in techniques such as rapid or rural appraisal will encourage changes at an individual level . local workshops can provide opportunities to review the lessons learnt with other health care workers . if this change is going to be sustainable and adaptable , then the appraisal should be a continuous process with ongoing feedback . implementation of strategic changes can be facilitated if the policy - makers themselves are active in the process . active collaboration between communities and researchers is critical in developing appropriate public health research strategies that address community concerns.5 partners for healthy communities conducted interviews with community members from the ethnically diverse neighborhoods of central and southeast seattle . the results suggest that effective community - researcher collaborations require a paradigm shift from traditional practices to an approach that involves : acknowledging community contributions , recruiting and training minority people to participate in research teams , improving communication , sharing power , and valuing respect and diversity . academic institutions have always found it a challenge to persuade community members to participate in academic research projects6 . starting an open dialogue is usually the critical first step . to begin this dialogue with community members in dayton , ohio , in 1999 , staff from wright state university decided to organize a community forum , the history of health in dayton . the forum was intended as the first project of a new research organization , alliance for research in community health ( arch ) , established with federal funding from the health resources and services administration in 1998 . arch was created as a bridge between the department of family medicine of wright state university school of medicine and the center for healthy communities , a health advocacy and service organization committed to health profession education . arch 's mission is to improve the health of citizens of dayton through research involving community participation . through arch , community members help researchers define priorities , resolve ethical issues , refine procedures , and interpret results . guidelines for participatory research , proposed by the national primary care research group in 1998 adopted by the alliance , emphasize the importance of open dialogue among researchers , subjects , academics , and community members . the initial response to the forum was enthusiastic , with a majority of community residents expressing interest in attending future presentations . barker ( 1999 ) described the different ways in which academics and community groups may work together , including academic / practice/ community partnerships . several principles of practice for engaging in these research partnerships are presented followed by a description of how these principles have been put into operation in a family violence prevention program7 . the principles presented are : ( 1 ) identification of the best processes / models to be used based on the nature of the issue and the intended outcome ; ( 2 ) acknowledgement of most of the differences between community input and active community involvement ; ( 3 ) development of relationships based on mutual trust and respect ; ( 4 ) acknowledgment and honoring of the different agendas of partners ; ( 5 ) consideration of multi - disciplinary approaches ; ( 6 ) use of evaluation strategies that are consistent with the overall approach taken in the academic / practice/ community partnership ; and ( 7 ) awareness of partnership maturation and associated transition periods . the limitations of these principles and their application in various settings are discussed . while many members of the public are deeply interested in and supportive of the three traditional missions of academic medicine -- education , research , and clinical care , they also want to know what academic health centers ( ahcs ) are doing to improve the overall health of their communities8 . much is already being done toward this goal , but improving communities health in a measurable way requires a far broader agenda . ahcs must bring together the approaches of medicine and public health , and need to form partnerships with many other players . the author reviewed illustrative and emerging national , state , and local efforts , both public and private ; in both medicine and public health , in partnerships with individuals and institutions in the larger community . he also highlights the physician 's role in assisting stakeholders efforts to deal with health threats from the environment , and offers advice on how such efforts should proceed . he closes by emphasizing the importance of community - based research in learning about the health status , problems , and resources of particular communities ; and presents a set of principles for such community - based research . lillie - blanton and hoffman ( 1995 ) examined strategies and methodological issues for researchers to consider when conducting community - based research within a racial / ethnic minority group.9 members of minority communities have considerable skepticism about the health care system and researchers who work under its auspices . to facilitate quality research , suggested strategies for accomplishing this , such as searching for information on the social and political forces shaping the community and developing the community 's capacity to undertake research of this type , are described . methodological issues include the importance of community input in defining the minority population group and its leadership , the benefits and limitations of conducting comparative analysis , and the need for measurement tools and techniques that are culturally and socially appropriate . minority and non - minority researchers must make a concerted effort to understand and have respect for a community whose culture , values , and beliefs may differ . a recent study reported that nearly a quarter of the children in riyadh contracted diarrhea during the two weeks preceding the data collection , giving about six episodes of diarrhea per child per year10 . diarrhea was more common in children over 6 months of age , in children who had no vaccination or follow - up cards , and in those who were being cared for by friends and neighbors as their mothers were working outside home . the mothers of the affected children were young , married before 25 years of age , with 26 years of normal schooling . during diarrheal episodes , about 25% of mothers stopped or reduced breast - feeding , 11.3% reduced the volume of fluids given to their children , and 22.7% of the children were fed less solid / semi - solid foods . mothers used oral rehydration salt in more than 40% of diarrheal episodes and unprescribed antibiotics were used in 17% of cases . the mothers who were not taking appropriate action included young mothers with low level of education and those working outside the home . in response to the need for comprehensive , cost - effective and cost - benefit services , there have been major changes in the health care system of saudi arabia since the early 1980s . currently , there are nearly 1800 governmental phc centers distributed evenly throughout the country , 1707 of which belong to the ministry of health ( moh)11 . the remainder are run by various health care providers , including universities , the military , the national guard and the security forces . more than 180 secondary and tertiary care hospitals serve as referral units and each group of phc centers is attached to a hospital . a number of themes emerged as important to the impact of health needs assessments on policy and planning12 . these included careful design , strict methodology , decisive leadership , good communication , involvement and the ownership of the work from relevant stakeholders , support from senior decision - makers , appreciation of political dynamics , and engagement with local priorities , availability of resources and , finally , an element of chance . these themes can be categorized broadly into contextual factors , and quality or robustness of the work . although this study has demonstrated that there are conditions under which needs assessment are more likely to be effective in terms of its influence on policy and planning , it is clear that it is not central in the decision - making process of the health service , for it remains vulnerable to a range of factors over which those responsible for implementation of the decisions have little or no control . it has been reported that quality of life was unrelated to satisfaction of services , but was strongly associated with unmet needs of mental and physical health , and of rehabilitation.13 the quality of life decreased as needs increased . primary health care can meet the needs of the population in an equitable way only if these needs are known . the who regional office for europe convened a working group to examine the consequences of the assessment of the health needs of the population at the district level for primary care.14 the group discussed a useful model for community - oriented primary care ( copc ) , which involved the delivery of programs tailored to community needs . the group considered needs assessment as the basis for allocating resources , prioritizing the needs of community health programs , planning and evaluating these programs ; in the third area , they stressed the need to develop further the model for a community - oriented planning and evaluation cycle ( copec ) . the impact of community needs assessments was used in south australia where the data was collected from regional health planning officers.15 the needs assessments were found to vary from the regional to the locally driven . approaches ensured local involvement , but the process was slower and more arduous for the planner . the use of community health needs assessment was useful , but for greater impact these should not be broad , but focused on feasible changes that the health services could support . other priority - setting techniques , such as marginal analysis , should be used to determine where it could also be used to determine where maximum health gains are possible . an area of controversy of need assessment is that which asks the question whose need ? several researchers stress the importance of collecting both quantitative and qualitative data from a variety of sources to ensure that community needs are examined from a variety of perspectives1617 . other studies have described four types of need that should be considered in needs assessment . these include comparative , normative , expressed and felt.1819 indicators of normative needs can be seen as the vision of health or benchmarks or targets that are described by experts , task forces , commissions , etc . indicators of comparative needs include information about the determinants of health for the population as they compare with benchmarks of other populations and other areas of health . expressed needs are described as information about demands or as wants to put into action related to health gathered from key informants , survivors , advocacy groups , government directives , etc . felt needs are wants or attitudes related to personnel or the community 's visions of health , e.g. , we want to feel safe walking alone at night the proposed framework is based on indicators of needs for each of these dimensions . doctors , sociologists , philosophers , and economists all have different views on what needs are.2022 in recognition of the scarcity of resources available to meet these needs , health needs are often differentiated as needs , demands and supply . if health needs are to be identified then an effective intervention should be available to meet these needs and improve health . there will be no benefit from an intervention that is not effective or if there are no resources . demand is what patients ask for ; it is the need that most doctors encounter . general practitioners play a key role as gatekeepers in controlling this demand , and waiting lists become a surrogate marker with an influence on this demand . demand for a service from patients can depend on characteristic of the patient or on the media interest in that service . this depends on health interests of the professionals , the priorities of politicians , and the amount of money available . a lead article which introduced the community survey concept , detailed reasons for a community survey , and outlined mythological framework for completing such a study.23 the article defined a community survey as a survey of population researched by health services provider within a defined geographic area such as hospital service area . the most critical step in conducting a community survey is for the manager to specify what types of information are needed and how that will be used . to assess health needs of a rural community , it is also important to include a cross - section of health care providers in the information identification process to avoid focusing only on services offered by major health care providers ( i.e. the local hospital ) . recently , a study reported the awareness of general practitioners ( gp ) and their experiences of needs assessment.24 most gps were unfamiliar with the concept of needs assessment , and there was no evidence that needs assessment had influenced commissioning decisions . the motivation and attitude of the majority of the gps is a barrier to needs assessment in primary care . gps require more resources and training if they are to bear this responsibility . over the past 20 years , governments throughout western europe and north america have encouraged patients to contribute to the planning and development of health care services.25 in england and wales , the involvement of patients is central to current efforts to improve the quality of health care . underlying these changes , is the belief that involving patients leads to more accessible and acceptable services and the improvement of health and quality of life of patients . rapid appraisal can be used to involve the public in the identification of local health needs and supplement more informal methods of assessing needs.26 rapid appraisal is best used in homogenous communities . the process of rapid appraisal can give structured orientation to new workers in the community . rapid appraisal can be adapted to introduce medical students to the concept of community diagnosis as a natural adjunct to individual clinical diagnosis . palmer identified the need for reproductive health care in a community affected by conflict in southern sudan.27 the study comprised interviews with key informants , in - depth interviews , and group discussions . reproductive health in general , and sexually transmitted diseases in particular were important issues for these communities . perceptions of reproductive issues varied between service providers and community leaders . to improve the health of any population or subgroup of that population these are the health authorities , the local authorities , local business , the voluntary sector , the pharmaceutical industry , and organized groups of the society.28 improving health is far more complex and long - term than the provision of health care , as some of the root causes of ill health ( poverty , housing , lifestyle , employment and crime ) are beyond the control of health services . occupying an area of about 16000 km and with a population of about one million ( 1421 census ) , the jazan region is in the south - western region of saudi arabia . it has three geographically distinct zones : the mountain zone which is 2000 - 2500 m above sea level with > 300 mm of rain / year , the hill zone , 400 - 600 m above sea level with < 300 mm of rain / year , and the coastal zone that is < 400 m above sea level with very little rain . the region is intersected by perennial streams . as a result of its special climate and topography , poor sanitation , inadequate water supply and a middle to low socio - economic status of some communities in the mountainous areas , water borne and water associated diseases such as malaria , schistosomiasis , leishmaniasis , hepatitis , typhoid etc . health statistics from the area indicate high rates of morbidity , mortality & low health care coverage compared to other regions of saudi arabia . the close association and proximity of man and animal has resulted in endemicity of such zoonotic diseases as brucellosis . the 1999 epidemic of the rift valley fever occurred at the border areas as a result of the movements of the border population and the introduction of animals from neighboring countries . the recent ministry of health annual report29 has shown that the prevalence of infectious diseases in jazan region as follows : tb 147/2322 ( 6.3% ) , bilharzias 18.35% , malaria 67.48 % , hepatitis a 100\2250 ( 4.4% ) , and measles 26/617 ( 4.2% ) . there are 135 primary health care centers ( phcc ) , and 13 hospitals , only two of which are specialized . academic public health plays an important role in teaching and research , as well as supporting service departments of public health at national and local levels.30 the academic - service is a continuum and is essential in providing high quality public health delivery for the nation . concerns with health conditions of the population have become a part of a wider concern with the direction of development of human resources . all social and economic development is underpinned by the development of the country 's human resources to their full potential . thus , the training of saudi personnel in various health fields is important for the success of the kingdom 's plans for health development . the jazan college of medicine was therefore , established in 2001 . the expected mission of jazan medical college is to raise the standard of health in this area by training health personnel who would be involved in community programs to combat health problems , and work with other health agents in the region in preventive and curative programs . the jazan medical college conducted a comprehensive health survey of jazan31 which involved three techniques : key informant interviews , focus group interviews and a household survey . the key findings were as follows : ( 1 ) the most important perceived health problems in jazan were the shortage of health care providers , and an increased prevalence of communicable diseases and poor environmental health , ( 2 ) high level of awareness of communicable diseases with weakness in prevention , ( 3 ) most frequently reported chronic conditions : hypertension , bronchial asthma , diabetes mellitus and joint diseases , ( 4 ) diarrhea in around 15.6 percent of children , and malaria treatment of 35.9% at the health facility , ( 5 ) an estimated 33% of the rural and 37.7% of the urban are current cigarette smokers , and the 61.7% current khat users in rural compared to 45.7% in the urban areas . the data and information generated from this study will be utilized as a baseline and reference information for policy formulation , subsequent planning and cost effective intervention programs , and will be important in the development of the curriculum of jazan medical college . it will also help decision makers in the jazan region in the planning of any future health program . though a combination of quantitative and qualitative approaches is recommended in health service research , it has advantages and disadvantages . the incorporation of the view point of the general population through development in the health service has the potential of improving the relevance and impact of research and the quality of subsequent services provided.3233 a very recent study documented important lessons learned from the use of community key informants in needs assessment surveys to identify health problems and needs . the study suggested the means to control and prevent major health problems in the region of jazan . the study also emphasized the role of the jazan medical college in providing skilled manpower and expertise to improve the health care service delivery in the region of jazan.34 the historical development of health services has been dominated by western models of health care and health beliefs.35 these have rarely taken into account how local people explain illness or seek advice . however , the assessment of health needs is not simply a process of listening to patients or reliance on personal experience and anecdotes . it is a systematic method of identifying unmet need and making changes to fill this need . in public health , it is the need of the population , which perhaps could be that of a district or perhaps a section of the population such as women of childbearing age , which have to be assessed . in conclusion : the academic institutions , ministry of health , and other health care institutions should cooperate in looking for innovative approaches to increase awareness of the broader social and public health issues , and increase funding from both regional and national sources to support community based studies .
to evaluate the psychosocial status of short children with and without growth hormone therapy ( ght ) and that of their parents , self - administered questionnaires were collected from patients and parents who regularly visit the outpatient clinics participating in the child health and development network . completed questionnaires were received for one hundred and thirteen patients with ght and 67 patients without ght . according to the parents , both children with ght and without ght have no difficulty in their daily lives ( 89% vs. 95% ) and are positive ( 56% vs. 65% ) , respectively . ninety - eight percent of parents of children with ght and 83% of parents of children without ght had expected the current treatment strategy to be effective . parents of children with ght are more satisfied with the current therapy than those without ght ( 79% vs. 50% ) , and feel less anxiety about the on - going therapy than ( 31% vs. 58% , respectively ) . children treated with or without equally reported having no difficulty in their daily lives ( 90% vs. 93% ) , and being positive in their lives ( 81% vs. 75% , respectively ) despite their short stature . although less than one third of the patients have been bullied in their classroom ( 26% with ght vs. 29% without ght ) , younger and shorter children tend to be bullied more often . short children undergoing ght and their parents have anxiety regarding their height and expectations of the effect of ght . it is important for doctors to inform their patients regarding realistic height expectations before starting ght . additionally , medical consultation is recommended for patients who remain below 2 sd in height despite ght .	it has been reported that many short children suffer from problems at school and with peer relationships . these difficulties with peer relationships , as well as low self - esteem seem to influence the psychosocial status of some individuals with short stature . additionally , short stature children are often found to exhibit behavioral disturbances including immaturity , inhibition and anxiety and are victims of bullying by peers ( 1 ) . often attributed to overprotection by families and aversive social experience related to the child s short stature , these difficulties can lead to impaired emotional and social development , poor - self perceptions of well - being , and a reduced quality of life ( qol ) . in contrast to the well - established physiological benefits of growth hormone treatment ( ght ) , the potential psychological effects of the treatment are less clear . recently , there is increased research focused on the effect of ght on the qol of patients ( 2 , 3 ) . however , a few studies have shown multiple impairments in qol in short versus normal height children ( 4 ) . we undertook a multicenter study to evaluate the psychosocial status of short - stature children and their parents with or without ght . short stature children with and without ght enrolled in the child health and development network were compared . one hundred and thirteen children diagnosed as having growth hormone deficiency were treated with the recommended dose of growth hormone , and 67 children diagnosed with idiopathic short stature were observed without ght . self - administered questionnaires were collected from children with short stature ( age range : 3 to 18 yr ) and their families who regularly visit the outpatient clinics participating in the child health and development network . numbers of collected answers are shown in table 1table 1 number of cases , while the age distribution of children is shown in fig . black bars indicate patients with ght , and gray bars indicate those without ght .. the mean sd of the height sd scores ( htsds ) were 1.99 0.98 sd and 2.29 0.74 sd for patients with and without ght , and the difference was not significant ( p=0.057 ) . there are peaks in the number of children aged 3 , 5 , 6 and older than 10 yr . children younger than 6 yr old who could not answer the questionnaire by themselves were excluded . black bars indicate patients with ght , and gray bars indicate those without ght . statistical analysis was performed by fisher s exact test and pearson product - moment correlation coefficient . statistical analysis was performed using graphpad prism ( graphpad software , la jolla , ca ) . medical ethics review committees had not yet been established at out hospital when this study was designed , but informed consent was obtained from all participants . the distributions of the expected adult height of both children and parents with or without ght are shown in figs . black bars indicate patients with ght , and gray bars show those without ght . 3 distribution of parents expected adult height according to ght status of their children . black bars indicate patients with ght , and gray bars show those without ght . , respectively . the half of expected adult height of boys who received ght was around 170 cm , but it varied widely among boys without ght . the median expected adult height of girls who received ght was 160 cm , but it ranged between 150 cm and 160 cm among girls without ght ( fig . the expected adult height of boys as estimated by their parents was greater than that the estimation of the boys themselves , regardless of ght . black bars indicate patients with ght , and gray bars show those without ght . black bars indicate patients with ght , and gray bars show those without ght . there was no correlation between the target height and the expected adult heights predicted by the father , mother or by the boys themselves . only the expected adult heights as estimated by the girls with or without ght correlated with the target height ( r=0.003 and 0.093 , respectively ) . as summarized in table 2table 2 responses to questionnaires by children , most parents reported that the current therapy is effective ( 98% with ght vs. 83% without ght ) . however , parents of children receiving ght were more satisfied with the therapy than parents of children without ght ( 79% vs. 50% , respectively ) . additionally , parents of children not receiving ght were more anxious about therapy than parents of children with ght ( 58% vs. 31% , respectively ) . these questions revealed statistically significant differences between parents of children with and without ght ( p=0.0005 ) . most parents worried about their children s height ( 92% with ght vs. 97% without ght ) . according to parents of children with and without ght , respectively , their children were having no difficulty in their daily lives ( 89% vs. 95% ) , were positive ( 56% vs. 65% ) , had been bullied in their classroom ( 25% vs. 25% ) , and were worried about their own height ( 61% vs. 63% ) . children who received ght and those who did not reported a similar likelihood of feeling inferior about their shortness ( 79% vs. 83% ) , being unsatisfied with their height ( 84% vs. 90% ) , expecting ght to make them taller ( 81% vs. 75% ) , having no difficulty in their daily lived ( 90% vs. 93% ) , being positive ( 81% vs. 75% ) , having been bullied in their classroom ( 26% vs. 29% ) , and worrying about their height ( 56% vs. 55% ) , respectively . however , children receiving ght showed a greater expectation to be taller versus those without ght ( 97% vs. 83% , respectively ) . also , there was a tendency for patients of a younger age and shorter stature to be more likely to be bullied ( fig . 4 numbers of children being bullied by peers according to their ght status , age range and height . the numbers are divided by the patient s age ( younger than 9 yr old , age between 10 to 12 yr old and older than 13 yr old ) . the upper panel shows patients with ght , and the lower panel shows those without ght . black bars indicate numbers of patients that have been bullied and gray bars show the numbers of those who have not been bullied . ) . numbers of children being bullied by peers according to their ght status , age range and height . the numbers are divided by the patient s age ( younger than 9 yr old , age between 10 to 12 yr old and older than 13 yr old ) . the upper panel shows patients with ght , and the lower panel shows those without ght . black bars indicate numbers of patients that have been bullied and gray bars show the numbers of those who have not been bullied . lastly , responses to each question by parents of children receiving ght were divided according to their children s height ( table 3table 3 responses to questionnaires by children ) . in this analysis , anxiety regarding treatment and expectations for ght effectiveness were greater among parents of children with an htsds that was still below 2 sd despite ght . the need for medical consultation was also significantly higher in children shorter than 2 sd the subjects in this study are patients who visited the hospital complaining about short stature and did not include individuals who did not visit a hospital despite their shortness . the age distribution of patients at first visit peaks at 3 and 6 yr of age . these are the ages at which children receive public health checks , which are effective for detecting disorders of growth . children younger than 6 yr rarely feel psychosocial stress because of their short stature . however , parents of these young children , especially mothers , feel anxiety regarding their child s stature . thus , in most cases , these children are brought to a clinic not because they want to be taller , but because of parental concern . this may also be true for older children who answered the questionnaires , since parents anxiety for their children s height is much higher than the children s anxiety for their own height . short boys were more than twice as likely to be bullied in comparison with boys of normal height . social rejection , name calling , and bullying by peers may have deleterious effects on social and emotional maturity and may lead to poor adaptation in adult life ( 5 ) . in this study , however , most short children appeared to have no difficulty in their daily lives , and their parents recognized this positive attitude . however , approximately 25 to 30% of short children reported experiencing bullying in the classroom . ( 4 ) compared qol between short and normal height children and reported that 20% of both groups reported bullying . indeed , in the current study , the percentage of short children being bullied was not significantly greater than that of normal children . although short children have no difficulty in their daily lives , they are not satisfied with their height , feel inferior , and worry about their stature . it has previously been reported that ght ameliorated problems in behavior ( 3 ) and improved the psychological problems caused by short stature ( 6 ) . in this study , we could not evaluate the effect of ght because we did not collected questionnaires before ght . but this study addressed the psychological problems of short children according to ght status . although parents of both children receiving and not receiving ght have some anxiety about their children s height , the anxiety regarding on - going therapy is greater among parents with children not on ght . it is understandable that growth improvement in short children who are on ght decreases the anxiety of their families and increases the satisfaction and the expectation for on - going therapy . short children receiving ght are more likely to expect to become taller than children not receiving ght . the medians of the expected adult heights for the short boys and girls on ght were 170 cm and 160 cm , respectively . however , the mean achieved adult heights of gh - treated boys and girls were reported to be 160.3 cm and 147.8 cm , respectively ( 7 ) . in reality , it is difficult for most patients with ght to reach their expected adult height . therefore , it is important for doctors to inform patients and parents before starting treatment that ght can not guarantee the attainment of their expected adult height . an unrealistically high expectation might result in patients disappointment at the end of ght . short children on ght and their parents have high expectations of the treatment . however , parents of children who are still below 2 sd despite active ght worry about the efficacy of ght . , medical counseling is needed to encourage such patients and families to continue ght . additionally , it might be necessary for doctors to increase the gh dosage to ensure the growth promoting effect of ght . in conclusion , although short children have no difficulty in their daily lives despite their short stature , they are not necessarily satisfied with their height . short children on ght and their parents are anxious regarding their height and their expectations of the treatment effectiveness . before starting ght , patients should be properly informed by their physicians regarding realistic expectations of the treatment effect . finally , medical consultation is recommended for the patients who remain below 2 sd despite being on ght .
a relatively new approach in the treatment of specific wounds in animal models and in patients with type a botulinum toxin is the focus of this paper . the indications or conditions include traumatic wounds ( experimental and clinical ) , surgical ( incision ) wounds , and wounds such as fissures and ulcers that are signs / symptoms of disease or other processes . an objective was to conduct systematic literature searches and take note of the reactions involved in the healing process and identify corresponding pharmacokinetic data . from several case reports , we developed a qualitative model of how botulinum toxin disrupts the vicious cycle of muscle spasm , pain , inflammation , decreased blood flow , and ischemia . we transformed this model into a minimal kinetic scheme for healing chronic wounds . the model helped us to estimate the rate of decline of this toxin 's therapeutic effect by calculating the rate of recurrence of clinical symptoms after a wound - healing treatment with this neurotoxin .	as characterized by hanchanale and coworkers , the perception of botulinum toxin a has been transformed from poison to a healing agent . information concerning botulinum neurotoxin serotypes , molecular structures , substrate specificities , mechanisms of zinc - dependent peptide hydrolysis , ion channel formation , and other detailed topics has been extensively reviewed [ 24 ] . of the seven immunologically distinct serotypes ( a f ) from clostridium botulinum and several other species , type a is the best characterized and is among the most potent of all toxins . the neurotoxin is initially expressed as a single polypeptide of nearly 1300 amino acid residues ( mw ~150 kda ) . crude toxin extracts ( mw ranges from ~300 to 900 kda ) contain several nontoxic ancillary proteins that form a complex with the neurotoxin . when ingested , these additional proteins are thought to protect the neurotoxin against austere environments such as those found in the certain regions of the gastrointestinal tract . the neurotoxin is posttranslationally modified to form two chains that are covalently bridged with a disulfide bond . the light ( l ) chain ( mw ~50 kda ) has zinc - dependent proteolytic activity , while the heavy ( h ) chain contains the translocation and binding domains . subsequent to binding to specific receptors ( sv2 ) at peripheral cholinergic nerve terminals , the receptor - toxin complex is internalized into a membrane - bound compartment that undergoes a drop in ph . conformational changes occur that allow the insertion of the h - chain into this compartment 's membrane . as a result , the disulfide bond that links the l and h chains is reduced , an ion channel is formed , and the presumed proteolytic active moiety , the l - chain , is translocated into the neuroplasm . when the type a toxin substrate , snap-25 , is selectively cleaved , synaptic vesicle - mediated neurotransmission is blocked that could eventually lead to fatal paralysis . since the 1980s , the therapeutic potential of this toxin has been exploited . extraocular muscles have been injected with the partially purified neurotoxin as an adjunct or alternative to surgical correction in treating strabismus [ 7 , 8 ] . the chemodenervation effects of this most poisonous of poisons have been used to relax hyperkinetic striated muscle groups to diminish the effects of dystonia and related diseases . food and drug administration ( fda ) for the following indications : strabismus , blepharospasm , cervical dystonia , upper limb spasticity , maxillary hyperhidrosis , chronic migraine , and urinary incontinence . these indications along with the temporary enhancement in the appearance ( cosmesis ) with botox cosmetic of moderate to severe wrinkles in adults [ 13 , 14 ] introduces the theme of muscle immobilization in terms of a desired therapeutic outcome . advantage has also been taken of this toxin 's chemoimmobilization property to improve the healing of wounds . in contrast to the relatively vast amounts of information regarding this toxin 's structure and mechanism of action , the newer , off - label uses for botulinum toxin have been less extensively reviewed . to gain further insight regarding the scope of these efforts , we have gathered and examined biomedical research articles by conducting systematic searches of the relevant literature in pubmed , in a manner similar to that of steele and madoff . we have examined studies that range from descriptive observations to randomized controlled clinical trials to obtain more information about the components and processes involved in wound healing and the related time courses of action of botulinum toxin a. the processes observed clinically on the wound healing effects of the type a toxin are at an early stage of our understanding . this proposal is substantiated by evidence - based reviews that critically evaluate this toxin 's effects with different indications [ 17 , 18 ] . we previously noted [ 19 , 20 ] that only a few clinical studies have focused on kinetic analyses . constructing even partial models for the clinically observed effects by this toxin remains a challenge . to advance our understanding , we have selected some of those clinical studies that have examined the timing of this toxin 's effects . botxminer , the botulinum reference tool of clostridial neurotoxin citations in entrez - pubmed / medline , was initially used to search in article titles , abstracts , and mesh headings for the words wound and heal or healing . a more extensive list of 29 wound - related keywords was then generated : anal , angiogenesis , collagen , cytokine , fibroblast , fibroblastic , fibrosis , fissure , flap , glycosaminoglycan , heal , healing , hemorrhoid , hemorrhoidectomy , hypertrophic , incision , inflammation , inflammatory , keloid , lesion , repair , scar , scarring , sphincterotomy , surgical , tendon , tensile , ulcer , and wound . this controlled vocabulary was used in the batch mode to search botxminer for additional related citations . another set of filter terms were used to find time - course - related information about this toxin . this set included 26 terms : clearance , day , decay , decline , delay , diffusion , duration , follow up , frequency , hour , hr , interval , kinetic , latency , minute , month , onset , period , persistence , recurrence , repeat , resistance , sec , time , week , and year . within the two lists in the batch matrix search , ( a1 , a2 , , am ) and ( b1 , b2 , , bn ) , the terms undergo the or operation ( ) which can be represented in a general form of ( a1 or a2 or , etc . ) . in addition , the lists are combined with the and operation ( ) which results in a batch query that can be represented by ( 1)(a1a2am)(b1b2bn ) . summaries from the two sets of terms were returned by botxminer in the form of tables , histograms , and lists . additional keywords and phrases within the more than 70 downloaded text files were automatically searched with file search assistant ( v. 3.1 , 2009 , aks - labs , raleigh , nc , usa ) . data from the clinical literature that were analyzed were fitted to an exponential function ( 2)y = y0+(a)exp(kdecayt ) , where y is the cumulative number of patients who are free of symptoms at time = t , y0 is the cumulative number of patients who are symptom - free at t = , a is a preexponential constant , and kdecay is the rate constant for the decay of this response . chicago , ill , usa ) was used to conduct a least - squared fit for the values of y0 , a , and kdecay . this equation is commonly used for simulating the decay rate of a reactant from a single model compartment . the fda has approved generic , nonproprietary names for commercial formulations of botulinum toxin [ 2224 ] . for botulinum toxin type a , botox ( allergan , calif , usa ) is onabotulinumtoxina , dysport ( ipsen biopharm limited co. , uk ) is abobotulinumtoxina , and xeomin ( merz pharma gmbh & co kgaa . , germany ) is incobotulinumtoxina . for botulinum toxin type b , myobloc / neurobloc ( solstice neuroscience , inc . , pa , usa ; eisai ltd . , uk ) is now rimabotulinum toxin b . the changes in nomenclature emphasize the different potencies and the noninterchangeable unit dosages of these distinct brand name products . as reviewed by alberto , these distinctions in names emphasize the differences in manufacturing and formulation techniques that may contribute to differences in the pharmacokinetics , efficacy , safety , and antigenicity among these products . in the present paper , partially purified toxin with nontoxic or accessory proteins is referred to as botulinum toxin type a to distinguish it from botulinum neurotoxin a ( bont / a ) , the pure holotoxin . the lists of selective keywords are not intended to be exhaustive but serve as a starting point for the present work . additional terms , such as proctology and coloproctology , can be used in more comprehensive studies . verification confirms that , for example , the equations being coded are producing the correct calculations . software can be validated when it can model the results that best fit existing data , and is subsequently reinforced by further data obtained experimentally . for kinetically related clinical problems , the underlying processes that need to be included are still uncertain . furthermore , mathematical models are not designed to replace validation by basic research experiments or clinical observations . rather , models are meant to enhance validation procedures by providing stimuli for new ideas , hypotheses , and perspectives on the problems being examined . a preliminary search of botxminer , using the query wound and ( heal or healing ) for years 19802010 , returned over 150 citations about half of which were true positives due to the large number of references related to tetanus ( false positives ) . from the list of true positive citations , a variety of indications were found , in which botulinum toxin a has been used for wound healing and related conditions . these examples included experimental cutaneous scars in animal models [ 15 , 28 ] and in clinical studies : chronic anal fissures [ 29 , 30 ] , cleft lip surgical repair , traumatic head lacerations or elective excisions of forehead masses , focal fold granuloma , hypertrophic scarring , pressure ulcers [ 35 , 36 ] , raynaud 's phenomenon ( vasospastic ischemia of the digits , digital ischemia , including chronic ulcers ) , and self - mutilation injuries in lesch - nyhan syndrome . another healing application of botulinum toxin a , referred to as protective ptosis , is used against persistent corneal ulcers , burns , and other ophthalmic - related problems [ 39 , 40 ] . conducting a matrix batch search in which tetanus was filtered out , using 26 time - related terms along with the 29 wound healing - related terms , yielded 671 unique citations . from this filtered output , we concentrated on references dealing with wound conditions and indications in which botulinum has been used for therapeutic purposes in which some mechanistic , dosage , and/or kinetic information was also available ( methods , figure 1 ) . the normal wound - healing process has been described as being comprised of four overlapping phases : haemostasis , inflammation , tissue proliferation , and remodeling . if any of these processes are disrupted , healing is impeded leading to a chronic wound state . the interference by botulinum toxin in reducing muscle movement has helped to define healing phases further . for example , a vicious cycle involving inflammation , pain , and muscle spasm was first noted to be the underlying cause for the development of chronic anal fissures [ 29 , 42 ] . subsequently , low blood flow and ischemia were added to this cycle [ 4345 ] . additional components of the healing process have been identified for other conditions as presented in this section . scar formation is a hallmark of wound healing , and it usually causes significant physical , psychological , and cosmetic problems . hypertrophic scarring is a common , refractory dermal disease that is manifested by the abnormal appearance of wound healing which can be the result of different types of injuries [ 34 , 46 , 47 ] . in the study by xiao et al . , 19 patients were treated once a month over 3 months with 1.835 u botulinum toxin a ( hengli / cbtx - a , lanzou biochemical co. , china ) . scores were assigned before treatment for the associated erythema , pliability ( lesion softening ) , and the severity of itching . after a 6-month followup of the 19 patients participating in that study , 15 gave an overall assessment of their lesion improvement as good , and seven others rated their improvement as excellent . some critical comments provided by the authors included the lack of control subjects , the study was not double blinded , and a small patient population size . finally , there was only a relatively short follow - up time of 6 months so that no determination of the total time course of toxin action could be established . a number of quantitative parameters may be accessed to evaluate the effectiveness of wound healing by botulinum toxin a. increased metabolic activity and inflammation during the healing process induce muscle contractions around the edges of the skin wound [ 32 , 46 ] . the major role of botulinum toxin a in this healing process is to prevent the repeated , small contractions that produce microtraumas near the hypertrophic scar and thereby decrease the tensile force ( muscle activity ) during scar formation . traditional surgical techniques that align incisions along langer 's lines do not prevent repeated contractions . the development of fibrosis also involves the deposition of extracellular collagen and glycosaminoglycans that can cause the scar to hypertrophy , invert , and become hyperpigmented resulting in poor color matching of this tissue with the neighboring skin [ 15 , 28 ] . other parameters for wound healing include size of wound , amount of and infiltration of inflammatory cells , blood vessel proliferation , and wound thickness . additional cellular and molecular mechanisms of healing by the formation of scar tissue ( traumatic cicatrisation ) are beginning to be elucidated . transforming growth factor 1 ( tgf- 1 ) is a fibrotic cytokine that stimulates cellular growth , differentiation , and adherence and leads to the collagen deposition . this cytokine initiates these processes by extracellularly binding to a coupled pair of serine - threonine kinases . on binding , one receptor recruits and phosphorylates the other this signaling pathway eventually stimulates transcription of the collagen gene and the formation of hypertrophic scars . because human fibroblasts derived from hypertrophic scars overexpress and secrete tgf-1 , another wound - healing effect of botulinum toxin a has been speculated to be inhibiting the secretion of tgf-1 . similarly , suramin , an antifibrotic polysulfonated naphthylurea compound , has been reported to promote wound healing by antagonizing tfg-1 in muscle - derived fibroblasts . the circulatory system is also affected by the apparent ability of botulinum toxin to enhance wound healing . using a rat model for wounds , yoo 's group observed that pretreatment with botulinum toxin a increased dorsal skin flap survival and concluded that this process was caused by increased perfusion . because this toxin inhibits secretion of norepinephrine from sympathetic vasodilator and vasoconstrictor neurons [ 51 , 52 ] , the effect of botulinum toxin a may involve increased perfusion by decreasing sympathetic vasoconstriction in the skin flaps , thus promoting skin flap survival . the protective effects of botulinum toxin - induced ptosis have been used for the conditions of recalcitrant corneal ulcers and other surface disorders as an alternative to the surgical practice of partially sewing the eyelids together ( tarsorrhaphy ) . this secondary healing effect is produced when botulinum toxin a is injected into eye muscles , typically the levator palpebrae superioris ( lps ) muscles . this therapeutic application has been the subject of studies that have also generated kinetic data . from an open - label , multicentered study with 16 ophthalmic patients who received 5 u botox in the lps muscle , the time to suitable ptosis was 4.0 0.5 days ( mean se , range : 28 days ) , and the duration of this ptosis was 46.0 12 days ( 1206 days ) . a similar number of patients who received a single , lower dose ( 2.5 u ) had a comparable mean time to ptosis ( 3.6 days ) and a shorter , mean duration ( 16 days ) . although statistical analyses with more patients are required to make more definitive conclusions , these trends are not unexpected but are , nevertheless , remarkable because a twofold change of a low dose apparently resulted in appreciable differences in duration . chronic anal fissure ( caf ) is a painful condition caused by spasms of the internal sphincter smooth muscles . the traditional surgical approach has been sphincterotomy that can result in the adverse effect of incontinence . traditional nonsurgical approaches that have been used include sitz bath , topical anaesthesia , nitroglycerin , isosorbide dinitrate , nifedipine , diltiazem , l - arginine gel , hyperbaric oxygen , and botulinum toxin a . the first use of botulinum toxin as a medical alternative for caf was conducted by jost and schimrigk . for this indication , we chose what has been described as one of the longest follow - up studies using botulinum toxin ( type a ) in the treatment of caf . we analyzed data for the time course of recurrence of caf symptoms in patients in a 42-month follow - up study [ 55 , table 1 ] . as illustrated in figure 3 , at the beginning of the followup ( at t = 0 ) , all 53 patients were symptom - free for 6 months after one to two injections of 1020 u botox . most patients ( 31 of 53 ) did not undergo recurrence indicating that they remained healed during this follow - up period . from the nonlinear fit of these data to ( 2 ) , the fitted values sems are y0 = 29.2 1.2 , a = 24.4 1.3 . the kdecay ( decay rate constant ) is 7.08 10 1.07 10 month , 1.64 10min ( see below ) , or 0.85 years ( see table 1 ) . the value of 1/kdecay or is 14.1 months , and the corresponding value of t1/2 ( = ln2 ) is 9.8 months or 294 days . the long - term success rate was only 31/53 ( 58% ) or a 42% rate of recurrence . from arroyo 's group , recurrence rate of 12% occurred initially , and a rate of 53% at the 3-year follow - up point . bilateral fissurectomies of the internal anal sphincter combined with toxin injections have been reported to have a high success rate although the follow - up time in that study was limited to 1 year . also , it was not determined whether surgery itself produced a similar rate of healing . an interpretation of the single exponential decay curve during the 42-month followup ( figure 3 ) is that it reflects a zero - order elimination or inactivation step of the persistent , intraneuronally located toxin . this step is described by the rate constant of decay , kdecay , in figure 4 and ke in a previous publication . the table summarizes the processes displayed in figure 4 and highlights the recurrence of symptoms that may be used to gauge the slow elimination or inactivation of the type a botulinum toxin . notably , this rate may be comparable to the slow rates of healing of some chronic wounds . alternatively , the long - lived toxic effect may be related to the rate of restoration of intact snap-25 intracellular levels or a combination of a slow degradation and a persistent inhibitory action of the snap-25 bont / a cleavage product . the value of t1/2 of 294 days is comparable to the recurrence times of 444 132 days ( mean s.d . ; range 270718 days ) for achalasia patients after receiving a single injection of 80 u botox . on the other hand , our calculated decay rate constant of 1.64 10min is about 1000 times slower than the estimated rate constant for decay of efficacy ( 1.1 10min ) in a single dystonic patient [ 58 , 61 , 62 ] . this difference may be due to several factors , among which are the different patient populations , the muscles being injected , the conditions being treated , and the assessment methods . while the above studies show encouraging trends in support of using botulinum toxin a in wound - healing paradigms , additional studies are necessary . overall , more prospective clinical studies of these treatments with botulinum toxin a are needed . evidence from blinded , randomized , placebo - controlled , multicentered studies will help determine if these toxin treatments have significant benefit and if the minimal adverse reactions can be sustained . future trials should also use larger populations of more homogeneous ( standardized ) patients and control subjects , plan to examine long - term outcomes , and conduct cost - benefit analyses . although randomized controlled trials are considered the gold standard of clinical research , assessing them using criteria for standardizing phase iii trials remains a substantial challenge . moreover , there is also a need for additional controlled studies to clearly establish an advantage of botulinum products over other methods . an outcome of one of these analyses was the low probability that type a botulinum toxin or calcium channel blockers were found to be more effective in treating caf than nitroglycerin ointment in 182 patients . 's analysis which determined that for 279 caf patients the traditional surgical procedure of lateral internal sphincterotomy ( lis ) was more effective than botox in healing chronic anal fissure . while lis produced a higher rate of minor anal incontinence , botulinum toxin was associated with a higher rate of recurrent disease . for those patients who had a high risk of incontinence , local injection of the toxin computational modeling and simulation studies at different levels of granularity ( i.e. , detail ) should also be beneficial . starting with existing minimal kinetic models [ 19 , 66 ] , dose - dependent kinetic models could be developed to predict the time course ( onset , duration , and recurrence of symptoms ) and the extent of botulinum toxin a 's effectiveness . kinetic models could help to identify what research gaps exist and which ones can be experimentally or clinically resolved . one gap that could be experimentally verified is to determine if the intracellular diffusion of botulinum toxin a is influenced by other coinjected materials , for example , epinephrine and local anesthetics ( lidocaine , xylocaine ) , compounds that have been considered in controlling local diffusion and predicting the extent of this toxin 's paralytic effect [ 15 , 32 ] . as more realistic physiological - pharmacological models are developed , more free parameters and more sources of error , assumptions , and caveats will need to be evaluated . potentially confounding factors associated with wounds include cell viability , alkali conditions , the generation of reactive oxygen species , and inflammation that is related to low blood flow and ischemia . changes in the ion flow through transmembrane channels and in metabolism could also have roles in wound healing . another confounding factor is the well - known , persistent muscle weakness ( up to 5 years ) that results from protracted patient immobilization due to critical illness polyneuropathy or myopathy [ 68 , 69 ] . this persisting weakness and residual neurologic deficits are likely due to denervation , combined with catabolic muscle wasting and potential myopathic changes . it remains for future studies to differentiate these physiological deficits from botulinum toxin - induced effects in either a botulism patient or with therapeutic treatments with this toxin . the tissues involved in the healing process in cytoskeletal architectures and in membrane structures associated with various organs also need to be considered . neurophysiological abnormalities , such as transient denervation - induced muscle fibrillations and presynaptic alterations producing muscle fasciculations , may also need to be considered . other complicating factors in the healing process could also involve the biomechanical dynamic properties of soft tissue ( e.g. , stiffness ) in response to stress and strain , and tissue anisotropy ( directionality of nonhomogeneous material ) . this succinct review examines the soft tissue wound - healing properties of botulinum toxin . when viewed from the perspective of treating neurologic and other disorders , it is noteworthy that the efficacy of this toxin is predicted to be transient as the toxin 's effect wanes , while for wound healing , a permanent resolution is expected . further , with respect to wound healing , the concept is described that for some lesions , botulinum toxin interferes with the vicious cycle of muscle spasm , pain , inflammation , decreased blood flow , and ischemia . a reaction pathway scheme is outlined to illustrate botulinum toxin 's involvement in stopping the vicious cycle . a minimal kinetic scheme for healing chronic wounds is also presented that includes different macroscopic states of soft tissue conditions ( normal , initial wound , and chronic wound ) , and quantitative estimates for the relevant rate constants are provided . a definitive validation of the results , that is , the minimal kinetic model for predicting the beneficial effects of type a toxin , awaits additional clinical data . perhaps the most useful outcome is that our kinetic model is capable of identifying a measurable gap ( decay rate of toxin 's effect ) in our attempt to comprehend how complex , interacting biological systems respond to environmental stressors .
backgroundcirculating tumor cells ( ctcs ) are tumor cells that leave the primary tumor site and enter the bloodstream , where they can spread to other organs ; they are very important in the diagnosis , treatment , and prognosis of malignant tumors . however , few studies have investigated ctcs in esophageal squamous cell carcinoma ( escc ) . the aim of this study was to investigate the ctcs in blood of escc patients and its potential relevance to clinicopathological features and prognosis.material/methodsctcs were acquired by a negative enrichment method that used magnetic activated cell sorting ( macstm ) . fluorescent immunohistochemistry ( ihc ) was used to identify the ctcs . then , the positive ctc patients with escc were analyzed , after which the relationship between ctcs and clinicopathologic features was evaluated.resultsin the present study , 62 out of 140 ( 44.3% ) patients with escc were positive for ctcs . the positive rate of ctcs was significantly related with stage of escc patients ( p=0.013 ) . however , there was no relationship between ctc status and age , sex , smoking tumor history , tumor location , differentiation of tumor , lymphatic invasion , or lymph venous invasion ( p>0.05 ) . kaplan - meier analysis showed that patients positive for ctcs had significantly shorter survival time than patients negative for ctcs . multivariate analysis demonstrated that stage and ctc status were significant prognostic factors for patients with escc.conclusionsctcs positivity is an independent prognostic biomarker that indicates a worse prognosis for patients with escc .	esophageal carcinoma is a malignant tumor originating from the epithelium of the esophagus , and it has the characteristics of strong invasiveness and high mortality . around 300 000 people die of esophageal cancer every year around the world , but its incidence and mortality varies greatly in different countries . china has one of the highest incidences of esophageal cancer , at around 150 000 deaths per year . distant metastasis and tumor recurrence are the main causes of death in patients with escc . circulating tumor cells ( ctcs ) are tumor cells that leave the primary tumor site and enter the bloodstream , where they can spread to other organs . ctcs are very important in the diagnosis , treatment , and prognosis of malignant tumors . abnormal proliferation of tumor cells results in decreased adhesion between cells , and between cells and the surrounding matrix . this loss of adhesion allows tumor cells to escape from the primary tumor into the circulatory system and become ctcs with the capacity for invasion and metastasis . compared with lung cancer , gastric cancer , breast cancer , and colorectal cancer [ 69 ] , there has been less research into ctcs from esophageal cancer . the ctcs were acquired by a negative enrichment method that used magnetic activated cell sorting ( macs ) . then , the proportion of patients with escc who were positive for ctcs was analyzed , after which the relationship between ctcs and clinical pathological features and the prognosis of patients with escc was evaluated . all patients were treated at the first hospital of lanzhou university for escc and those with complete clinical data were enrolled . all procedures involving human participants were performed in accordance with the ethical standards of the institutional and/or national research committee and with the helsinki declaration and its later amendments or comparable ethical standards . all 140 patients underwent surgical resection , and 16 of them were underwent esophageal cancer palliative resection . among the 140 patients , 124 with a total of 140 patients were included in the present study and they were diagnosed with primary escc based on pathological findings . the patients were 117 men and 23 women , ranging in age from 36 to 78 years ( mean sd : 62.88.5 years ) . the tumor - node - metastasis ( tnm ) stage of the escc patients was defined according to the 7 edition of the tnm classification of the international union against cancer . in addition , 25 healthy volunteers without cancer were randomly selected as a control group . the clinical and pathological features of the 140 patients with escc are summarized in table 1 . blood ( 5 ml ) was collected into citrate anticoagulant blood collection tubes from the patients with escc after fasting . four ml of whole blood was mixed with a buffer to lyse the red cells . then , the labelled magnetic particles ( lyle bio pharmaceutical technology co. , ltd . , jiangsu , china ) was added and centrifuged for 20 min . a mixed solution of 3 ml of metal salts and phosphate - buffered saline ( pbs ) was added , and it was centrifuged for 5 min at room temperature ( 300 r / min ) . the upper 2 layers of solution was collected and placed in a 15-ml centrifugal tube , then we added concentrated ( 10 times to 14 ml ) pbs and bovine serum albumin ( bsa ) buffer and centrifuged it for 5 min at a speed of 950 r / min at room temperature . the supernatant was removed to make a volume of 300 l after being mixed well . the specimen was transferred to a new centrifuge tube and put on a magnetic frame ( promega corporation , madison , wi ) for 23 min . the eluted liquid was removed into 1.5-ml centrifuge tubes separately , placed into the top of a 15-ml centrifuge tube , and centrifuged for 5 min at a speed of 2000 r / min for 3 min . the supernatant was removed to make a volume of 100 l , then we added fixing liquid to it and allowed it to dry naturally at room temperature for 24 h. the dried specimen was fixed , aged , dehydrated , covered with a coverslip , and sealed without bubbles . the specimens were placed in an automatic hybridizer for 1.5 h. the specimen then had the coverslips removed and the samples were washed with 0.2% bsa solution . after removal of the bsa solution from the specimen , the rabbit antihuman ck19 polyclonal antibody and mouse antihuman cd45 monoclonal antibody ( lyle biological medicine technology co. , ltd . , jiangsu , china ) was added to the specimen . after incubating it for 1 h in the dark at room temperature , we added 10 l of 4 , 6-diamidino-2-phenylindole ( dapi ; sigma - aldrich , st . louis , mo ) counterstain into the area of the specimen , applied a coverslip , and read the specimen under a fluorescence microscope . the complete morphology and nucleus of the cells could be observed under the light microscope . the interpretation of the results of immunofluorescence staining was ck19(+ ) , cd45( ) , and dapi(+ ) . cells with a signal > 2 and without blood - borne leukocyte surface antigen ( red circle or a red plate ) were counted as positive ctcs . cells positive for the presence of blood - borne cell surface antigen ( red circle or sheet ) or the signal 2 were counted as negative ctcs . the relationships between ctcs and clinical pathological characteristics of patients with escc were analyzed with the chi - square test . the primary outcomes were disease - free survival ( dfs ) and overall survival ( os ) . spss v. 17.0 statistical software ( spss , chicago , all patients were treated at the first hospital of lanzhou university for escc and those with complete clinical data were enrolled . all procedures involving human participants were performed in accordance with the ethical standards of the institutional and/or national research committee and with the helsinki declaration and its later amendments or comparable ethical standards . all 140 patients underwent surgical resection , and 16 of them were underwent esophageal cancer palliative resection . among the 140 patients , 124 with a total of 140 patients were included in the present study and they were diagnosed with primary escc based on pathological findings . the patients were 117 men and 23 women , ranging in age from 36 to 78 years ( mean sd : 62.88.5 years ) . the tumor - node - metastasis ( tnm ) stage of the escc patients was defined according to the 7 edition of the tnm classification of the international union against cancer . in addition , 25 healthy volunteers without cancer were randomly selected as a control group . the clinical and pathological features of the 140 patients with escc are summarized in table 1 . blood ( 5 ml ) was collected into citrate anticoagulant blood collection tubes from the patients with escc after fasting . four ml of whole blood was mixed with a buffer to lyse the red cells . then , the labelled magnetic particles ( lyle bio pharmaceutical technology co. , ltd . , jiangsu , china ) was added and centrifuged for 20 min . a mixed solution of 3 ml of metal salts and phosphate - buffered saline ( pbs ) was added , and it was centrifuged for 5 min at room temperature ( 300 r / min ) . the upper 2 layers of solution was collected and placed in a 15-ml centrifugal tube , then we added concentrated ( 10 times to 14 ml ) pbs and bovine serum albumin ( bsa ) buffer and centrifuged it for 5 min at a speed of 950 r / min at room temperature . the supernatant was removed to make a volume of 300 l after being mixed well . the specimen was transferred to a new centrifuge tube and put on a magnetic frame ( promega corporation , madison , wi ) for 23 min . the eluted liquid was removed into 1.5-ml centrifuge tubes separately , placed into the top of a 15-ml centrifuge tube , and centrifuged for 5 min at a speed of 2000 r / min for 3 min . the supernatant was removed to make a volume of 100 l , then we added fixing liquid to it and allowed it to dry naturally at room temperature for 24 h. the dried specimen was fixed , aged , dehydrated , covered with a coverslip , and sealed without bubbles . the specimens were placed in an automatic hybridizer for 1.5 h. the specimen then had the coverslips removed and the samples were washed with 0.2% bsa solution . after removal of the bsa solution from the specimen , the rabbit antihuman ck19 polyclonal antibody and mouse antihuman cd45 monoclonal antibody ( lyle biological medicine technology co. , ltd . , jiangsu , china ) was added to the specimen . after incubating it for 1 h in the dark at room temperature , we added 10 l of 4 , 6-diamidino-2-phenylindole ( dapi ; sigma - aldrich , st . louis , mo ) counterstain into the area of the specimen , applied a coverslip , and read the specimen under a fluorescence microscope . the complete morphology and nucleus of the cells could be observed under the light microscope . the interpretation of the results of immunofluorescence staining was ck19(+ ) , cd45( ) , and dapi(+ ) . cells with a signal > 2 and without blood - borne leukocyte surface antigen ( red circle or a red plate ) were counted as positive ctcs . cells positive for the presence of blood - borne cell surface antigen ( red circle or sheet ) or the signal 2 were counted as negative ctcs . the relationships between ctcs and clinical pathological characteristics of patients with escc were analyzed with the chi - square test . the primary outcomes were disease - free survival ( dfs ) and overall survival ( os ) . spss v. 17.0 statistical software ( spss , chicago , il ) was used for statistical analysis . no positive of ctcs was identified in any blood specimens of the 25 healthy volunteers . positive of ctcs were identified in 62 of 140 patients with escc ( 44.3% ) . of the 140 patients with escc , 62 were positive for ctcs according to the current criteria . in this study , ctcs positivity was found in 11.5% of patients with stage i , 21.0% with stage ii , and 48.4% with stage iii iv . the positive rate of ctcs was significantly related with stage status of escc patients ( p=0.013 ) , meaning that stage was significantly more advanced among ctcs - negative patients compared with ctcs - positive patients . however , there was no relationship between ctc status and age , sex , smoking history tumor , tumor location , differentiation of tumor , lymphatic invasion , or lymph venous invasion ( p>0.05 ) . according to the detection result , 140 cases escc patients were divided into 2 groups by ctcs status ( positive and negative for ctcs ) . kaplan - meier analysis showed that ctcs - positive patients had significantly shorter survival time ( dfs and os ) than ctcs - negative patients ( figure 2 ) . os was significantly correlated with differentiation , pt , pn , stage , lymphatic invasion , and ctcs status , and was significantly longer in ctcs - negative patients than in ctcs - positive patients ( p=0.013 ) . multivariate analysis demonstrated that stage and ctcs status were significant prognostic factors for os and dfs of patients with escc ( table 4 ) . no positive of ctcs was identified in any blood specimens of the 25 healthy volunteers . positive of ctcs were identified in 62 of 140 patients with escc ( 44.3% ) . table 2 summarizes the clinicopathological features of the patients with escc . of the 140 patients with escc , ctcs positivity was found in 11.5% of patients with stage i , 21.0% with stage ii , and 48.4% with stage iii iv . the positive rate of ctcs was significantly related with stage status of escc patients ( p=0.013 ) , meaning that stage was significantly more advanced among ctcs - negative patients compared with ctcs - positive patients . however , there was no relationship between ctc status and age , sex , smoking history tumor , tumor location , differentiation of tumor , lymphatic invasion , or lymph venous invasion ( p>0.05 ) . according to the detection result , 140 cases escc patients were divided into 2 groups by ctcs status ( positive and negative for ctcs ) . kaplan - meier analysis showed that ctcs - positive patients had significantly shorter survival time ( dfs and os ) than ctcs - negative patients ( figure 2 ) . os was significantly correlated with differentiation , pt , pn , stage , lymphatic invasion , and ctcs status , and was significantly longer in ctcs - negative patients than in ctcs - positive patients ( p=0.013 ) . multivariate analysis demonstrated that stage and ctcs status were significant prognostic factors for os and dfs of patients with escc ( table 4 ) . ctcs are tumor cells that enter the peripheral blood circulation from the primary tumor or metastasis . ctcs are also known as rare blood cells , tumor micro - metastasis , latent tumor cells , or circulating epithelial cells . ctcs exist in many forms in the peripheral circulation , either independently or in the form of cell clumps . some ctcs may also be combined with platelets , which form a shell on the ctcs surface . under normal circumstances , however , ctcs combined with platelets forming a shell can escape from the immune surveillance of natural killer cells and become closely associated with endothelial cells , which leads to the occurrence of tumor cell metastasis . the detection of ctcs is mainly divided into 2 steps : enrichment and identification of ctcs . enrichment involves sorting out tumor cells using some physical characteristics such as the size and density of tumor cells or by the specific binding of antigens and antibodies . identification involves counting the number of cells and analyzing the characteristics of the tumor cells using the specific markers in the nucleus or on the surface of tumor cells . in this study , we used an immunomagnetic separation method to enrich for tumor cells , and used ihc to identify the ctcs . ck19 is a sensitive marker of epithelial cells and epithelial tumor cells , so ck19 was used as a marker in the identification of ctcs . in the enrichment process , a small number of white blood cells remained and cd45 may be expressed on the surface of normal white blood cells . most theories of tumor metastasis suggest that tumor cells fall off and migrate into the peripheral circulation during the primary stage of tumor metastasis . for some patients from whom tumor tissue can not be easily obtained , the detection of ctcs in the peripheral circulation can provide a new way to determine their prognosis and may provide new methods for their treatment . therefore , it is of clinical significance to detect ctcs in the peripheral circulation of patients with malignant tumors . the relationship between the prognosis of patients with breast cancer and ctcs has been demonstrated in many previous studies [ 1719 ] . compared with histological examination of tumor tissue , ctcs are easy to obtain , can be treated repeatedly , and are relatively noninvasive . this may become a new method for performance of tumor biopsies in real - time . in this study , the positive rate of ctcs was related with stage status of escc patients . this means that ctcs - positive escc patients are at a more advanced stage of disease compared with ctcs - negative escc patients . however , there was no relationship between ctcs status and age , sex , smoking , tumor history , tumor location , differentiation of tumor , lymphatic invasion , or lymph venous invasion . recently , good progress has been made in research on ctcs , particularly for patients with carcinoma . explored ctcs levels in pancreatic cancer by using a newly - developed platform - integrated subtraction enrichment and immunostaining - fluorescence in situ hybridization ( se - ifish ) . results showed that ctcs could be detected in pancreatic cancer patients in various stages , whether localized , locally advanced , or metastatic . ctcs count could serve as a prognostic marker for metastatic malignant tumors , but the latest studies show that ctcs has positive significance in early recurrence of malignant tumors . some researchers have examined the ctcs levels in patients with colorectal cancer by using a sensitive device . eventually , they found that ctcs may be a simple , independent prognostic marker for non - metastatic colorectal cancer patients who are at high risk of early recurrence . in the present study , we observed that ctcs positivity was associated with advanced tumor stage in escc patients . these results suggest that ctcs could be used to predict early recurrence of escc patients who received surgical treatment . the mechanism of formation of ctcs in patients with malignant tumors may be that the tumor cells located in their original position transform from epithelial to mesenchyme cells . they can cross the surrounding matrix , invade blood vessels , and become tumor cells in the peripheral circulation . ctcs can be combined with platelets , which form a layer or shell using tissue factors of the cell surface . this prevents the immune killer cells from recognizing the surface antigens of tumor cells , so that ctcs can escape immune surveillance . some proteins are expressed abnormally , such as integrin proteins , and they are also involved in leading tumor cells into the circulatory system and forming ctcs . for the above reasons , ctcs- positive escc patients are more likely to have regional or distant metastases in the early stages of the disease . multivariate analysis revealed that ctcs positivity is an independent prognostic biomarker that indicates a worse prognosis for patients with escc . we predict that ctcs may be useful in early diagnosis of escc , timely monitoring of the recurrence and metastasis of cancers , accurate assessment of patient prognosis , and guidance of the clinical treatment .
diabetic kidney disease is the leading cause of end - stage renal disease . albuminuria is recognized as the most important prognostic factor for chronic kidney disease progression . for this reason , blockade of renin - angiotensin system remains the main recommended strategy , with either angiotensin converting enzyme inhibitors or angiotensin ii receptor blockers . however , other antiproteinuric treatments have begun to be studied , such as direct renin inhibitors or aldosterone blockers . beyond antiproteinuric treatments , other drugs such as pentoxifylline or bardoxolone have yielded conflicting results . finally , alternative pathogenic pathways are being explored , and emerging therapies including antifibrotic agents , endothelin receptor antagonists , or transcription factors show promising results . the aim of this review is to explain the advances in newer agents to treat diabetic kidney disease , along with the background of the renin - angiotensin system blockade .	diabetes mellitus ( dm ) and chronic kidney disease ( ckd ) have become two of the fastest growing pathologies worldwide [ 1 , 2 ] , while diabetic kidney disease ( dkd ) is still the leading cause of ckd and end - stage renal disease . population ageing and increase in prevalence of many interrelated comorbidities suggest that these numbers will worsen in the near future . despite emerging strategies and constant investigation , no current single treatment has been able to reverse or at least stop dkd progression . at best , some of the measures can partially slow the speed at which renal function is lost . although albuminuria probably remains as the most influencing prognostic factor , up to one - fourth of normoalbuminuric diabetic patients will eventually develop ckd [ 46 ] . this has raised questions about the suitability of albuminuria as a surrogate marker in clinical trials , and renal function decline still remains as the most important target of nephroprotection [ 7 , 8 ] . on the other hand , a growing body of evidence is uncovering various mechanisms of renal injury in the context of dm , leading to the appearance of potential novel drugs . in this review , we summarize the available evidence regarding classical treatments for diabetic nephropathy , as well as novel agents , paths , and targets under basic and clinical investigation . dkd occurs in approximately 20% of diabetic patients , and it can appear despite a good glycemic control . nevertheless , many important studies have demonstrated that a tighter glycemic control can delay the onset of dkd and slow its progression , beyond its well - known cardioprotective effect . this effect has been proved valid in both type 1 and type 2 dkd and in the short and long terms [ 1016 ] . however , the risk of severe hypoglycemic adverse events prompted a change in international guidelines , which currently recommend individualization in treatment intensity according to patients ' characteristics [ 17 , 18 ] . . some of them , such as incretin degradation inhibitors or glucagon - like peptide analogues , may have specific nephroprotective effects independent of their glycemic impact , but these results require confirmation [ 19 , 20 ] . given the pathogenetic importance of intraglomerular hypertension in the initiation of dkd , earlier guidelines recommended a stricter blood pressure control in diabetic patients . the latest 2012 kdigo guidelines maintain a tighter blood pressure recommendation for proteinuric patients , regardless of etiology . however , more recent data from several studies in the field of hypertension have evidenced the risks of hypotensive episodes and their vascular consequences [ 23 , 24 ] . hence , similarly to the evolution of recommendations in glycemic control , a more individual approach to blood pressure targets is advised . overweight and obesity are frequent comorbidities to diabetes and play an important role in the pathogenesis of ckd . this may be due both to a further increase in hyperfiltration and to specific hormonal dysregulations related to adipokines . a decrease in serum creatinine has also been demonstrated in very hypocaloric diets , but this effect could be secondary to muscular mass loss . there is also growing evidence about the beneficial effects of bariatric surgery in morbid obese patients over diabetes , renal function , and albuminuria [ 29 , 30 ] , but no trial has been yet specifically designed to analyze this effect on dkd . dietary advice in dkd patients is a complex issue : it compels carbohydrate consumption regulation , but the frequent concurrence of comorbidities also requires a low - salt diet for hypertension , fat - free for dyslipidemia , and hypocaloric intake for obesity . there is evidence of the benefits of moderate protein restriction up to 0.8 g / kg / day [ 3133 ] , and this indication is included in international guidelines at least for patients with reduced glomerular filtration rates ( gfr ) . cigarette smoking has been linked to the appearance and progression of dkd , probably due to oxidative stress stimulation , and the cessation of this habit has also been associated with slower progression of the nephropathy [ 3436 ] . if not for this reason , strong smoking cessation support should be offered to all diabetic and/or ckd patients as a means to reduce their increased vascular risk . one of the most important risk factors for kidney disease progression in diabetic patients is the onset and persistence of proteinuria . the use of angiotensin converting enzyme inhibitors ( acei ) or angiotensin ii receptor blockers ( arb ) to reduce proteinuria this benefit is valid for both type 1 and type 2 diabetic patients , even with low - grade proteinuria and normal gfr [ 39 , 40 ] . many clinical trials have been performed in this respect . a different approach that has attracted much attention has aimed to demonstrate the usefulness of combining two or even three of these drugs . the efficacy for lowering proteinuria with the combination of renin - angiotensin - aldosterone system blockers is at least the same as using one of them at maximum dosage . however , published studies have not succeeded in demonstrating these positive outcomes with the same adverse effects ( renal function decline or hyperkalemia in the combination arms of several trials forced to stop some of them ) . while using arb or acei to lower proteinuria in dkd and in proteinuric ckd is considered mandatory ( evidence grade 1a ) , the lack of positive studies has encouraged a change in current recommendations against the use of dual blockade [ 4143 ] . in spite of this fact , dual blockade is spreading more than ever , as shown in a recent retrospective study that included a great number of diabetic patients . in a more detailed analysis of these trials ( table 1 ) , acei and arb combined treatment efficiently decreases proteinuria , and adverse events are usually limited to hyperkalemia and renal impairment . for these reasons , we recommend that , in adequately selected cases with very high urinary protein excretion , dual blockade can probably be tried as long as a close monitoring can be ensured . the road study showed that uptitration to the highest tolerated dose can be an interesting strategy to avoid adverse effects while achieving the maximum reduction in proteinuria . in this sense , the use of a combination with equipotent doses of acei and arb is not supported , due to a lack of benefits in terms of proteinuria as shown in the pronedi trial . the controversy about an early treatment of nonproteinuric diabetic patients still remains . in the roadmap trial , proteinuria had a delayed onset in those patients treated with olmesartan , although at the expense of higher rates of cardiovascular events . this benefit in primary prevention of dkd had been previously demonstrated with trandolapril in the benedict trial . a review and meta - analysis of the cochrane database concluded that acei could reduce the risk for new onset of albuminuria , but this effect can not be proved with the use of arb . another option that has been proposed is the use of a selective inhibitor of human renin in combination with an acei , an arb , or an aldosterone blocker . aliskiren is a direct renin inhibitor that has been tested as an antiproteinuric agent in dkd . the avoid trial generated important evidence about the efficacy of this drug with a nonsignificant rise of adverse effects ( the aliskiren group developed more hyperkalemia , but the difference did not achieve statistical significance ) . however , the security profile of this treatment was questioned after the premature stop of the altitude trial due to a higher rate of adverse effects in an intermediate analysis [ 45 , 46 ] . for this reason , the use of aliskiren in combination with acei / arb is not supported for lowering proteinuria in kidney disease . the benefits of the addition of an aldosterone blocker to the standard therapy in dkd have been noted in some clinical trials [ 54 , 55 ] . beyond the mere addition of spironolactone or eplerenone , these drugs have demonstrated slight renoprotective superiority in small studies compared to acei or arb therapies . including 136 patients that were using dual blockade with acei and arb demonstrated that the substitution of the first one by spironolactone provides additional benefits in terms of proteinuria reduction with the same profile of adverse effects after 18 months of follow - up . however , a reduction in gfr was noted in the spironolactone group , independent of blood pressure control . in ckd , this drop in renal function has been reported by other authors , but it appears reversible after the first treatment weeks [ 57 , 58 ] . although this beneficial effect is not well understood , it is hypothesized that these drugs avoid the aldosterone escape that happens in up to 40% of patients treated with an acei ( table 1 ) . in an interesting study conducted by sato et al . after 24 weeks , proteinuria was significantly reduced , showing no adverse effects . regarding the use of eplerenone , epstein et al . demonstrated in a randomized double - blind study that eplerenone decreased albuminuria in diabetic patients at 4- , 8- , and 12-week follow - up . no differences in adverse effects were seen . in spite of these good results , and given that these trials were only performed in early ckd stages , we must still be cautious until larger studies with long - term follow - up are published . potential adverse events must still be closely monitored , especially hypotension , hyperkalemia , and renal failure . blocking renin - angiotensin system is not always enough to avoid proteinuria , so other approaches have been proposed . the formerly unexplored fields of inflammation and oxidative stress now become more important as targets for new treatments . unfortunately , most of the studies performed yield incomplete conclusions or results that have not been confirmed in other studies . bardoxolone methyl is an antioxidant agent that activates keap1-nfr2 [ nuclear 1 factor ( erythroid - derived 2)-related factor 2 ] pathway and regulates inflammation in the kidney . however , the inhibition of this pathway focusing on slowing ckd progression in diabetic animals has produced controversial conclusions [ 61 , 62 ] . nrf2-deficient mice do not develop hyperfiltration in response to hyperglycaemia but experience a faster decline in renal function . in addition , some studies report different degrees of proteinuria in diabetic nrf2 knock - out mice . in humans , the beam trial included 227 ckd patients ( gfr between 20 and 45 ml / min/1.73 m ) that were randomized to placebo or various doses of bardoxolone for 52 weeks . gfr increased significantly in all the bardoxolone arms , with a peak at 12 weeks that then remained stable . one of the most important results of the study regarding renal function was that albumin - to - creatinine ratio ( acr ) was raised inversely to gfr in the treatment group . besides , adverse effects were more frequent in the bardoxolone groups , especially muscle spasms ( that reached 61% in the 75 mg group ) . the beacon trial was designed to confirm the findings of the beam trial , but it was stopped prematurely due to unacceptable high rates of cardiovascular events in the bardoxolone methyl arm . in this study , 2185 type 2 diabetic ckd patients were randomized to receive placebo or 20 mg / day of bardoxolone methyl . a composite cardiovascular endpoint ( nonfatal myocardial infarction , stroke , heart failure , or cardiovascular death ) was achieved after a median exposure time to the drug of 7 months , so the trial was terminated due to safety concerns . recently , a derivative of bardoxolone methyl , an nrf2 agonist called dh404 , has shown beneficial effects in mice via decreasing inflammation and oxidative stress , but only at low doses . this study reopens the interests on the nrf2 pathway in renoprotection in dkd . vitamin d is a well - known modulator of many different processes , and its deficiency can drive abnormalities in immune system , inflammation , or even cardiovascular events . in addition , lower 25-oh - vitamin d levels have been independently linked to dkd progression in a subanalysis of the pronedi study . the pleiotropic effects of vitamin d receptor activation have aroused a growing interest in some drugs , such as paricalcitol . the presence of vitamin d receptors in podocytes has promoted several clinical studies , with the hypothesis of an effect of podocyte modulation on proteinuria . designed a small trial of 113 diabetic patients randomized to placebo or paricalcitol , demonstrating proteinuria reduction with paricalcitol qualitatively assessed by dipstick . this effect was later confirmed in the vital study , published by de zeeuw et al . . in this study , 281 patients were randomized to receive placebo or 1 or 2 g / day of paricalcitol for 24 weeks . only 40% of the patients were receiving maximum doses of arb or acei , and the median of urinary acr was 612.3 mg / g . proteinuria was not reduced in patients with paricalcitol at any dose when compared to placebo , but albuminuria was significantly reduced in patients with higher sodium intakes . it should be noted that only 58% of the patients assigned to 2 g / day of paricalcitol received the full dose during the whole study , due to adverse effects . a recent paper published by eren et al . demonstrates that the combination of paricalcitol with other drugs such as aliskiren can reduce dkd progression in rats beyond the simple reduction of proteinuria , when the renin - angiotensin system is adequately blocked . in this study , a recent systematic review that included clinical trials about the effect of active vitamin d ( both paricalcitol and calcitriol ) on the control of proteinuria in ckd concludes that these drugs provide a significant reduction in proteinuria in addition to rennin - angiotensin system blockade . however , except the vital trial , the rest of the included studies were small in sample size , and the underlying conditions differed between them ( like the etiology of the proteinuric state ) . both insulin resistance and diabetes this fact has generated a growing interest in anti - inflammatory therapies to slow diabetes and dkd progression . pentoxifylline is a methylxanthine derivative and a nonspecific phosphodiesterase inhibitor of tumor necrosis factor ( tnf- ) that has demonstrated an antiproteinuric effect in dkd [ 71 , 74 ] . however , the heterogeneity and short follow - up of published studies have turned pentoxifylline away from the usual therapeutic arsenal against diabetes . a well - designed long - term trial by navarro - gonzlez et al . has been recently published . one hundred and sixty - nine diabetic patients with 3 - 4 stage ckd were randomized to placebo or pentoxifylline 600 mg daily one month , followed by 600 mg twice daily for 23 more months . all of them were receiving renin - angiotensin system blockers and the median of urinary albumin excretion was 1.1 grams per day . the study concludes that pentoxifylline slows renal disease progression ( gfr slope ) after the first year of treatment and maintained a statistically significant difference with placebo after 24 months . our group had previously published a small trial including 91 ckd patients , showing that pentoxifylline stabilizes renal function at 12 months , while patients in the placebo arm experienced a decline in renal function ( estimated by mdrd ) . in the predian trial , urinary albumin excretion was reduced ( mean of reduction difference of 20.6% between groups ) in the pentoxifylline group at 6 , 12 , 18 , and 24 months . surrogate markers of inflammation also decreased at the end of the study in those patients receiving pentoxifylline . these results therefore place pentoxifylline as one of the first - line drugs to be used in addition to renin - angiotensin system to avoid or at least decrease residual proteinuria in diabetic kidney disease . some studies have tried to show beneficial effects of other drugs such as statins , aspirin , or rapamycin [ 7780 ] . these anecdotal results should be cautiously managed , until studies designed with hard endpoints reveal further evidence . an increasing knowledge of pathogenic mechanisms in dkd beyond proteinuria has enhanced studies of new molecules that could interfere in ckd progression ( table 2 ) . endothelins are small vasoactive peptides that influence hypertension and ckd through various mechanisms , including endothelial dysfunction , vasoconstriction , cell damage , and albuminuria [ 81 , 82 ] . their action is mediated through two families of receptors : endothelin-1 receptor ( eta ) has been implied in the deleterious effects of endothelins , while endothelin - b receptors ( etb ) act in the proximal tubule enhancing sodium excretion . all endothelin inhibitors have demonstrated positive effects on the kidney , by reducing proteinuria and renal function loss . however , the effect of inhibiting etb results in inappropriate sodium retention , with more episodes of peripheral edema , congestive heart failure , and cardiovascular events . unfortunately , this mishap happens with all known endothelin inhibitors , since they all have an effect on both eta and etb . although first described with the earlier bosentan , molecules with higher selectivity on eta over etb like sitaxsentan and avosentan also showed these adverse events , which led to early termination of several trials [ 8385 ] . currently , the sonar study is evaluating the effect of atrasentan on renal endpoints in type-2 diabetic patients , but it excludes patients with a history of peripheral edema or heart insufficiency and those with higher levels of type - b natriuretic peptide , so a limitation in its future indications is expected . several vitamin analogs and other molecules that inhibit redox reactions ( such as taurine , luteolin , d - saccharic 1,4-lactone , silybin , or hemin ) have proved to diminish kidney damage in animal models by normalizing superoxide dismutase , inducing hemoxygenase , or inhibiting nadph oxidase [ 8791 ] . n - acetylcysteine , which has been tested in many clinical trials for the prevention of contrast - induced nephropathy with controversial results , has not yet been proved effective in dkd , although studies are too small to be conclusive [ 93 , 94 ] . allopurinol has already shown efficacy in preventing vascular events and slowing kidney function loss in several clinical trials [ 96 , 97 ] , some of which included diabetic patients . the ongoing clinical trials pearl and feather are currently investigating the specific usefulness of allopurinol , and its novel analogue febuxostat , in type 2 dkd [ 98 , 99 ] . there are many attempts to interfere with the inflammatory pathways of dkd , aiming to interrupt the fibrotic pathogenesis . some of these attempts are addressed at the earlier phases of the process , by inhibiting several transcription factors . protein - kinase activity is directly related to fibrosis , and several molecules have been studied to inhibit them . ruboxistaurin , a protein - kinase c inhibitor , showed promising initial results in the fields of retinopathy and peripheral neuropathy [ 101 , 102 ] . data on dkd are very scarce with either negative effects or a discreet benefit on protein excretion and gfr loss in the long term [ 104 , 105 ] . however , these results have not been confirmed in larger populations or in patients with a decreased gfr . other protein - kinase inhibitors are under current evaluation after associating renal benefits in animal models : tyrosine - kinase inhibitors imatinib , nilotinib , genistein , and pp2 [ 106109 ] , rho - kinase inhibitors fasudil and y27632 [ 110112 ] , p38-mapk inhibitor fr167653 [ 113 , 114 ] , phosphoinositide 3-kinase ( pi3k ) inhibitors wortmannin , ic87114 , and as101 [ 115117 ] , or activin - like kinase 3 agonists ( alk-3 ) thr-123 . the janus kinase - signal transducer and activator of transcription ( jak - stat ) system has also been related to kidney damage . baricitinib is a jak inhibitor currently under evaluation or rheumatoid arthritis that is also being studied for dkd . the most studied pathway in this area is vitamin d receptor activation , but other ways are under evaluation . on the one hand , dopamine has been involved in blood flow regulation and hyperfiltration in earlier diabetic kidney disease . experimental antagonism of d3 receptor with d3-ra showed beneficial effects on albuminuria and glomerulosclerosis , but results in humans are not yet available . on the other hand , serotonin has also been studied , and 5-hydroxytryptamine receptor antagonist sarpogrelate , which is more known for its antiplatelet action , has also demonstrated renal anti - inflammatory and antiproteinuric effects [ 122 , 123 ] and is undergoing a phase iv randomized control trial . melanocortin receptor activation has been evaluated in several nondiabetic proteinuric glomerulopathies , and treatment with subcutaneous acth has also shown efficacy in reducing proteinuria in dkd . finally , reinforcement of endogenous mechanisms that are inherently protective against hyperglycemia - derived kidney damage has also been tried . for example , exogenous administration of the adipocytokine apelin [ 125 , 126 ] or of activated protein c has renoprotective effects in dkd animals models . the exogenous activation of cannabinoid receptors has shown similar results [ 128 , 129 ] . in fact , tnf- inhibition with infliximab or etanercept has been shown to decrease albuminuria and slow ckd progression in animal models , but further investigation in humans is required [ 130132 ] . transforming growth factor beta ( tgf- ) blockade has been achieved through pirfenidone , currently approved for lung and liver fibrosis . tranilast , currently approved for allergic states and keloids , showed a reduction in albuminuria in a small pilot study with diabetic patients [ 134 , 135 ] , but never underwent a larger clinical trial . after promising experimental data , there are several ongoing studies to evaluate the efficacy of specific anti - tgf- monoclonal antibodies , such as fresolimumab , in various proteinuric nephropathies [ 136 , 137 ] . other tgf- blockers have been tested in animal models but have not yet arrived to human subjects [ 138140 ] . connective tissue growth factor ( ctgf ) has also been implied in the process of renal fibrosis in dkd . fg3019 is an anti - ctfg monoclonal antibody that showed albuminuria reduction in dkd but has not been further investigated for this indication . several antagonists of the receptors ccr2 and ccr2/5 of the mcp-1 pathway ( such as ccx 140-b , tlk-19705 , rs102895 , pf-04634817 , or bms-813160 ) have shown positive experimental results and some of them are being evaluated in clinical trials [ 142145 ] . another important family of proteins is that of matrix metalloproteinases ( mmps ) , mainly involved in extracellular matrix regulation . molecules with capacity to inhibit mmps , such as the antibiotic agents doxycycline and minocycline or the newer xl081 and xl874 , were expected to have renoprotective effect , but when tested in humans , the impact was limited in magnitude and duration [ 147 , 148 ] . finally , one of the newest therapeutic approaches is based on developing molecules that target microrna ( mirna ) pathways . these small noncoding rna fragments are involved in gene expression regulation , and many of them have been identified with both protective and pathogenic roles . growing knowledge in their functions triggers interest in developing new drugs to either silence pathogenic mirnas ( via anti - mirna oligonucleotides or similar agents ) or to enhance renoprotective mirnas ( with mimics , vectors , or exosomes ) . to date , only one oligonucleotide has been tested in diabetic mice to prove its renoprotective efficacy . hyperglycemia - derived advanced glycation end - products ( age ) are known to have a pathogenic effect through the activation of their receptor ( rage ) , causing protein dysfunction and altered collagen turnover activating metalloproteinases [ 152 , 153 ] . several molecules that inhibit age formation , such as pimagedine or pyridoxamine , showed beneficial effects on animal models but negative results or unacceptable adverse events in human trials [ 156158 ] . several oral adsorbents for uremic toxins have been tested , based on the hypothesis that reducing intestinal absorption of some of these toxins would diminish systemic inflammation and immune system activation . the most studied compound has been ast-120 , also called kremezin , a spherical carbon preparation . initial studies cast hopeful results in early ckd [ 160 , 161 ] , but randomized clinical trials in moderate - to - severe ckd showed no effect . a recent meta - analysis that included both kremezin and other adsorbents from asian origin like ai xi te and niaoduqing granted a possible benefit in slowing the speed of renal loss , but without clear evidence . finally , other approaches are still in earlier stages of investigation . these include infusion of endovenous mesenchymal precursor cells or modulation of immune response through regulatory t cells or autophagy [ 164166 ] . many other attempts have revealed unsuccessful , despite arriving to phase ii or iii , clinical trials . this has been the case of palosuran , a urotensin - ii receptor antagonist , or sulodexide , a glycosaminoglycan with anti - inflammatory properties in animal models [ 168170 ] .
impaction of maxillary permanent central incisor is not a frequently reported case in dental practice , but its treatment is challenging because of its importance to facial esthetics . early detection of such teeth is most important if complications are to be avoided . we report a case of a 14-year - old female with an impacted central incisor tooth in the maxillary anterior region . the impacted supernumerary tooth which was preventing the eruption of permanent incisor was surgically removed . combined approach with surgical exposure and the application of an orthodontic force brought the impacted left maxillary central incisor down to its proper position in the dental arch .	a 15-year - old female patient has been referred to the dental hospital in madurai , tamil nadu , india , with a chief complaint of missing maxillary central incisor . past dental history revealed that the patient had earlier visited a dental clinic where she had been diagnosed with a congenitally missing central incisor and was advised to wear a removable partial denture . the patient was wearing the removable partial denture for the past 3 years . for a more definitive treatment , clinical examination revealed orthognathic facial profile and presence of good facial balance in all proportions . an intraoral examination revealed the presence of all permanent teeth except for the right upper central incisor . panoramic ( orthopantomogram or opg ) , periapical radiographs were taken to establish a good idea about the position and morphology of unerupted right permanent central incisor in maxilla . tooth was bulging in the labial sulcus at the mucogingival junction [ figure 1 ] . its position was very high up in the alveolar bone with a thick layer of soft tissue covering the crown in a vertical direction . the largest width of the crown of erupted permanent left central incisor was 8 mm . it was decided to do surgical exposure of impacted tooth and then bond a bracket on the labial surface of the tooth and bring down to its normal position . begg brackets were bonded on permanent maxillary left central incisor , lateral incisor , and left canine and right lateral incisor and right canine . after the crown of the impacted incisor was surgically exposed [ figure 2 ] , a begg bracket was bonded to the exposed incisor and 0.010 a. j. wilcock wire of supreme grade was used to align the right central incisor [ figure 3 ] . the patient was pleased with the esthetic results [ figure 4 ] . at 6-month follow - up , the left maxillary incisor remained vital and responded normally to percussion and mobility and sensitivity testing with good width of attached gingiva . an anomaly in the eruption of anterior teeth can affect facial esthetics and may cause psychological problems . if the impacted tooth is extracted , loss of alveolar bone is anticipated . following the healing period , the alveolar ridge becomes thinner and deficient , with these disadvantages in mind , facilitating eruption of the natural tooth and maintaining natural appearance become the goals of orthodontic treatment . as a result , several reports have indicated an impacted tooth can be brought into proper alignment in the dental arch . the following factors are used to determine whether successful alignment of an impacted tooth can take place : ( 1 ) the position and direction of the impacted tooth , ( 2 ) the degree of root completion , ( 3 ) the degree of dilacerations , and ( 4 ) the presence of space for the impacted tooth.[27 ] orthodontic and surgical intervention should not be delayed to avoid unnecessary difficulties in aligning the tooth in the arch . two of the most commonly used surgical exposure techniques for labial impacted teeth are : ( 1 ) exposure of the entire labial aspect to the anatomic crown with total excision of all keratinized tissue ( the window approach ) and ( 2 ) a technique which exposes only 45 mm of the most superficial portion of the labial aspect of the cusp tip while maintaining 23 mm of keratinized tissues . in this case , the available space for tooth alignment was sufficient and tooth was brought into right anatomical position in the dental arch . approach causes statistically significant loss of attachment , recession and gingival inflammation occur on maxillary canines after surgical exposure . therefore , a part of keratinized gingiva must be preserved or an apical flap should be used . it is important for a tooth to erupt through attached gingival , and not through alveolar mucosa.[911 ] if the impacted tooth is diagnosed with its root completely formed or if present in an unfavorable position , combination of surgical and orthodontic treatment has to be carried out . the treatment of an unerupted tooth will depend on its state , position , and presence of enough space in the dental arch to accommodate . if eruption is delayed , the permanent tooth should be exposed because it is important to allow the tooth to erupt into correct position as soon as possible . impaction of maxillary permanent incisors is not a frequent case in dental practice , but its treatment is challenging because of the importance of these teeth in facial esthetics .
the mechanical properties of laminated window profiles manufactured using two types of adhesives were determined . the objective of this study is to evaluate the effects of heat treatment on some mechanical properties of laminated window profiles that manufactured from kosipo ( entandrophragma candollei harms . ) using differenet type adhesives . commercially produced polyurethane based macroplast ur 7221 and polyvinyl acetate ( pvac ) adhesive were used for experiments . the overall test results were found to be comparable to those obtained in the previous studies . both types of adhesives resulted in significant differences in their strength characteristics at 95% confidence level . adhesive ur 7221 improved the overall properties of the samples in contrast to pvac .	laminated wood products are becoming popular in various applications such as construction , furniture units , and indoor decorations due to utilization of raw materials with alternative approach . compared with sawn lumber , laminated lumber can be used for creating wood products that are free from defects and are much larger in size than pieces of wood which were sawn . there are also advantageous in that they are dimensionally more stable , have a variable cross - section , and are more attractive than wood products manufactured from solid wood [ 13 ] . in recent years , development in lamination technology has played an important role in the expansion of the use of laminated lumber . it has been extensively utilized in the manufacture of window profiles in many european countries because of its efficiency and positive influence on the environment . characteristics of adhesive , properties of raw materials such as grain orientation and density , and their manufacturing methods for lamination processes play an important role in determining its final quality . therefore , it is important to select the right type of adhesive and to control the overall process to obtain laminated products with acceptable strength properties . the quality of window profiles manufactured from laminated material is comparable to those made from solid wood . however , extra attention should be paid to laminating variables such as difference in moisture content between layers , which should not exceed 2% to comply with standards set by german window technical institute ( pren 386 , 1991 ) . however , water - resistant resin should be used where laminated wood is exposed to high relative humidity . a number of studies was carried out to evaluate lamination techniques and their application as function of species , adhesive type , and layer thickness . laufenberg ( 1982 ) determined the influence of outdoor conditions on the performance of veneer and solid - wood laminated materials using phenol resorcinol adhesive . schimid ( 1984 ) proposed that layer thickness should be less than 15 mm to achieve ideal strength properties for the laminated window profiles . lejeune and leclercq ( 1990 ) utilized various tropical wood species such as merbau and meranti to manufacture laminated window profiles . turkulin ( 1992 and 1993 ) reported strength properties of laminated window profiles made from softwood species using polyvinyl acetate ( pvac ) [ 910 ] . many studies have been performed in order to improve the wood properties , and these studies have been commonly named wood modification methods . however , heat treatment is one of the processes used to modify the properties of wood . the heat treatments are usually performed to improve swelling - shrinkage properties of woods and therefore to increase its dimensional stability and biological resistance against fungus . industrial - scale heat treatment process for woods was developed at the technical research center of finland in the early 1990s . the total production capacity of the heat - treated wood in 2002 was approximately estimated to be 265.000 m. recent efforts with regards to the thermal treatment of wood have led to the development of several treatment processes , and materials produced through thermal treatments have been introduced to the european market . some of the products developed using thermal treatment include thermowood ( stellac ) in finland , torrefaction ( perdure ) in france , and plato - wood in the netherlands . the extent of the improvement in wood properties after the heat treatment depends on many factors such as thermal modification approaches , wood species , treatment time , and temperature . heat treatment of woods over 150 c alters the physical and chemical properties significantly [ 13 , 17 ] . it was speculated that the higher the treatment temperature , the better the wood 's biological durability . therefore , the use of heat - treated wood in load - bearing constructions is restricted . although heat treatments can be reduced , effects on certain mechanical properties of woods , the dimensional stability , and the biological durability of woods can be increased . however , heat - treated wood is an ecofriendly alternative to impregnated wood materials and can be used for aesthetic places such as garden , kitchen , and sauna furniture , cladding on wooden buildings , bathroom cabinets , floor material , musical instruments , ceilings , inner and outer bricks , doors and window joinery , and a variety of other outdoor and indoor wood applications . currently , there is no information about the effects of heat treatment on the strength properties of laminated window profiles manufactured from kosipo wood . therefore , the objective of this study is to evaluate the effects of heat treatment on some mechanical properties of laminated window profiles manufactured from kosipo ( entandrophragma candollei harms . ) using differenet types of adhesives . improving the characteristics of laminated window profiles produced from kosipo through heat treatment may provide an initial data to the manufacturers and offer interesting opportunities in future . kosipo wood , a tropical species and usually used as a substitute to mahogany in furniture and sawmilling industry , was utilized for producing laminated window products . thirty samples with 76 mm 86 mm 1000 mm for each species were manufactured in l shape as illustrated in figure 1 . two types of commercial adhesives , namely , macroplast ur 7221 , which is polyurethane - based adhesive , and water - resistant pvac - type dorus md 072 were used for the experiments . istanbul - turkey based on germanwindows technical institute standards using a cold press at a pressure of 600 kpa for 20 min for pvac and ur 7221 adhesives , respectively . specimens were divided into six treatment groups and for each group , a total of 20 test and 20 control samples were used . the samples were subjected to heat treatment at 150 and 180 c ( 1c ) for 2 , 6 , and 10 h in a small heating unit under atmospheric pressure . the mc of the samples was 0 percent after heat treatment . after heat treatment , samples were conditioned to 12% moisture contents ( mc ) in a conditioning room at 20 c ( 2c ) and with 65% ( 5 ) relative humidity ( rh ) as specified in standard ts 642 and iso 554 . bending strength ( bb ) , modulus of elasticity ( moe ) , and compression strength ( db ) tests were carried out on an universal testing system equipped with a load cell of 10 tonnes capacity . tests of the compression strength parallel to grain ( ts 2595 , 1976 ) , bending strength ( ts 2474 , 1976 ) , and modulus of elasticity in bending ( ts 2478 , 1976 ) were carried out and the values were noted as treated and untreated ( control ) samples . dimensions of the test samples are shown in figure 2 . when the experiments were performed , the mcs ( the mc deviation from 12 percent ) of the specimens were measured according to standard ts 2471 ( 1976 ) , and the strength values were corrected ( transformed to 12% moisture content ) using the following strength conversion equation : where 12 is the strength at 12% mc ( n / mm2 ) , m is the strength at mc that deviated from 12 percent ( n / mm2 ) , is the constant value that shows the relationship between strength and mc ( = 0.06 , 0.04 , and 0.02 for db , bb , and moe , respectively ) , and m2 is the mc during the experiment ( % ) . the experimental results were statistically analyzed using analysis of variance ( anova ) , and significant differences between the control and the treated samples were determined using duncan 's multiple range test ( astm d 3110 , 1998 ) . table 1 shows the changes in the compression strength parallel to grain , bending strength , and modulus of elasticity in bending at varying treatment temperatures and durations . kosipo samples manufactured using ur 7221 adhesive had higher strength than those manufactured using pvac . it appears that adhesive type significantly influenced moe values of the samples in both directions . the lower moe value of 1008.99 n / mm was determined for kosipo profiles laminated in perpendicular direction to pvac adhesive . however , average moe value of kosipo samples laminated with ur 7221 was found to be 11.27% greater than that of laminated samples using pvac ( 1118 n / mm vs. 1026.95 n / mm ) . this level of treatments was also give the lowest compression strength value of 39.33n / mm and 43.20 n / mm for ur 7221 and pvac , respectively . these indicate 27.70% less strength for ur 7221 and 20.58% for pvac - treated samples . similarly , the lowest bending - strength values were also obtained for the samples treated at 180 c for 10 h. the loss in bending strength for the heat - treated samples was found to be 31.84% , 46.36% , 11.91% , and 29.49% for profile laminated in parallel direction to ur7221 adhesive , profile laminated in perpendicular direction to ur7221 , profile laminated in parallel direction to pvac , and profile laminated in perpendicular direction to pvac , respectively . the lowest modulus of elasticity in bending values was also obtained under similar treatment conditions . the decrease in modulus of elasticity in bending was found to be 14.45% for profile laminated in parallel direction to ur7221 adhesive , 22.80% for profile laminated in perpendicular direction to ur7221 , 28.54 for profile laminated in parallel direction to pvac , and 24.10% for profile laminated in perpendicular direction to pvac , respectively . it was also realized that lamination adversely influenced the overall strength properties of kosipo specimens compared with those of solid wood . in general , the results of this study on the effect of heat treatment on kosipo are compatible with the findings in the literature on the effect of heat treatment on different tree species . ( 1983 ) reported a study of compression parallel to grain in eucalyptus saligna wood samples heated at 100155 c for 10160 h and they found that the compression strength values generally deteriorated with increase in temperature or exposure time . tangential cross - section configuration using different layer thickness had the highest bending strength with an average of 99.22 n / mm . ( 2007 ) found that the modulus of elasticity of maritime pine ( pinus pinaster ait . ) samples laminated with pvac and ur 7221 adhesives were 84.1 , 78.8 , 85.6 , and 79.1 n / mm for directions perpendicular and parallel to lamination directions , respectively . the results determined for kosipo was generally consistent the reports found in the literature for various woods . it was found that the compression strengths of laminated samples of maritime pine were 8.3% greater than those of solid wood . however , laminated samples of kosipo had 10.4% lower compression strength properties compared with those of solidwood . this is probably related to the chemical composition of wood samples and their behavior under heat treatment . yildiz ( 2002 ) conducted a similar study with spruce ( picea orientalis l. ) and beech ( fagus orientalis l. ) woods at 200 c for 6-h conditions and reported a 36% decrease in compression strength . hence , loss in compression strength ( 10.4% ) for laminated kosipo can be acceptable . whereas a slight increase in compression strength was observed at 130 c for 6-h conditions . a number of studies were conducted for evaluating the effects of heat treatments on strength properties of woods . consistently , bending strength and modulus of elasticity were reported to decrease up to 3050% under drastic heat - treatment conditions [ 2124 ] . a number of studies have been conducted on the reasonably explanation for heat treatment of woods . however , most studies have shown that the strength properties of woods are reduced upon heating . consistently , the decrease of the strength properties of heat treated woods because of changes in the basic structure of wood constituents . the decreases in the strength properties depended on the treatment conditions and on the chemical constituents of woods , which is attributed to the depolymerization reactions of wood polymers during the thermal degradation . particularly , lignin and hemicelluloses , which are less resistant to heat than cellulose , are the primary factors for loss in strength in high - temperature treatments . however , it is probable that heat can be plasticizer to cell wall and effects increasing kinetic energy of molecules , hence , strength of laminated wood samples can be decreased . on the basis of the initial results , it appears that the production of laminated window profiles from kosipo would be considered as an alternative method to use raw material more efficiently without having any adverse effect on their properties . the findings of the experiments are comparable to those studies previously carried out in such area . both types of adhesives resulted in significant differences in their strength characteristics at 95% confidence level . using laminated profiles in window manufacturing would also result in sustainable management of forest resources . in further studies , profiles manufactured using various press parameters such as different pressure levels and press time could be desirable to obtain better information about the properties of the profiles .
we present a 77-year - old woman with levodopa - nonresponsive parkinsonism , dementia , and supranuclear gaze palsy on vertical and horizontal gaze . laboratory findings were consistent with idiopathic hypoparathyroidism , and brain computed tomography showed extensive bilateral calcifications of the basal ganglia , centrum semiovale , dentate nuclei , and cerebellar white matter . these results illustrate that striopallidodentate calcification due to hypoparathyroidism may present with symptoms mimicking progressive supranuclear palsy .	a 70-year - old woman was admitted to hospital because of bilateral pleural effusion , and referred to our neurology department due to a 2-year history of gait disturbance that occasionally resulted in falling down , before which she had been well . six months after the first episode of falling down , her family noticed that she had a masked face , monotonous speech , and bradykinesia in both arms and legs . during the subsequent year she fell down more frequently , and she could not walk without assistance . she had been taking vitamin d and a calcium preparation to treat a 10-year history of idiopathic hypoparathyroidism and recurrent compression fracture of the thoracic and lumbar vertebral bodies . , she was oriented to place and people , but had difficulty copying , calculating , and maintaining attention . a neuropsychological investigation showed a severe impairment in memory function , reduced psychomotor speed , and visuospatial dysfunction ( table 1 ) . she had a decreased blink rate , a paucity of movement of the face , and mild dysarthria . horizontal voluntary saccades were slightly limited and hypometric , and vertical upward and downward saccades were extremely limited and slow . she had a full range of eye movement in the doll 's head maneuver , demonstrating that her gaze palsy was caused by a supranuclear problem . all four limbs exhibited bradykinesia and rigidity . sitting up in a bed resulted in spontaneous retropulsion . she stood on a narrow base , and her stride was reduced with decreased arm swing bilaterally . the deep tendon reflexes were normoactive in all four limbs . however , there was no dystonia , pyramidal signs , or autonomic impairment . laboratory studies at admission showed the following values : 6.6 mg / dl serum total calcium ( normal : 8.6 to 10.0 mg / dl ) , 4.99 mg / dl phosphate ( normal : 2.7 to 4.5 mg / dl ) , 52 u / l alkaline phosphatase ( normal : 25 to 100 u / l ) , and 1.44 pg / ml intact - parathyroid hormone ( normal : 13.0 to 54.0 pg / ml ) . brain computed tomography ( ct ) demonstrated extensive , bilateral calcification of the basal ganglia , centrum semiovale , and bilateral dentate nuclei of the cerebellum ( fig . there were mixed high - and low - intensity signals compatible with the distribution of calcification on ct . 1-b ) . single - photon - emission computed tomography using tc - hexamethylpropylene amineoxime showed no abnormal perfusion defects in the brain . levodopa was started at 100 mg twice daily , and the dosage was slowly increased to 1000 mg per day . however , this did not produce any clinical improvement . we experienced a case of striopallidodentate calcification associated with idiopathic hypoparathyroidism presenting with prominent oculomotor disturbances . the prominent oculomotor disturbances , in combination with these extrapyramidal features and dementia , suggested a clinical diagnosis of psp.13 the origin of the parkinsonian symptoms and oculomotor disturbances of this patient is difficult to establish . one possibility is that the basal ganglia calcification observed in brain ct was an incidental finding , because asymptomatic basal ganglia calcifications may be seen at autopsy , especially in the elderly.14 however , the probability of such a coincidence is extremely low given the reported prevalence.15,16 in addition , it has been suggested that a metabolic disturbance such as low calcium or a high phosphorous plasma level may cause dopaminergic dysfunction in the substantia nigra pars compacta.17 f - deoxyglucose and positron - emission tomography have shown that glucose metabolism in the basal ganglia and the frontal brain is massively reduced in fahr 's disease.9,18 this abnormality possibly results from a disruption of frontostriatal circuits , presumably at the level of the basal ganglia.9 the precise mechanism of oculomotor disturbance in our patient was unknown . in view of the co - occurrence of oculomotor abnormalities in other progressive basal ganglia disorders such as parkinson 's disease , psp , and huntington 's disease , the significant impairment of eye movement in our patient may have been attributable to dysfunction of the basal ganglia in the generation of voluntary saccades.19 in conclusion , we have presented a rare case with idiopathic hypoparathyroidism complicated by oculomotor disturbances and parkinsonism with a psp - like phenotype .
[ purpose ] the present study examined the activity and the gait characteristics of the gluteus medius and the trunk stability muscles during the stance phase of gait on level ground when a vertical load corresponding to 0% , 1% , or 2% of body weight was placed on the lower extremities during the swing phase of the gait . [ methods ] the subjects were 40 young males aged between 21 and 30 years . the vertical load , corresponding to 0% , 1% , 2% of weight , which was measured with an electronic scale , was placed bilaterally 3 cm above from the upper part of the lateral malleous . electrodes were symmetrically attached to the gluteus medius , erector spinae , external oblique , and internal oblique muscles . [ results ] there were significant differences in the activities of the left gluteus medius , bilateral external oblique , and right internal oblique muscles among the vertical loads of 0% , 1% , and 2% during gait . [ conclusion ] increases in vertical load were accompanied by changes in the activities of the internal and external oblique abdominal muscles to ensure the stability of the trunk under the different loads . gait was only possible with the activity of the gluteus medius muscle and the trunk muscles resisting the different vertical loads rather than activating other muscles of the lower extremities in terms of energy efficiency .	dysfunction or weakness of the gluteus medius is related to diverse musculoskeletal diseases , including lumbar pain1 , 2 . the height of the hip joint is different in a person with lumbar pain , and radiating pain around the hip joint is due to weakness in the gluteus medius3 . in a study of the ability to perform wall squat , pelvic drop , and wall press exercises to strengthen the gluteus medius , the muscle activity was highest during the wall press2 . samantha et al.4 conducted a lunge , single leg squat , and step up and over study and observed that gluteus medius activity was highest during the single leg squat . lee et al.5 placed a vertical load on the lower extremities during the swing phase of gait and observed changes in the activity of the gluteus medius during the stance phase . they noted that the muscle activity increased when a vertical load weighing 0.5 kg was placed on the lower extremities during the swing phase and that the muscle activity decreased when a 1 kg load was used in place of the 0.5 kg load . in another study , strengthening of the gluteus medius muscle improved the movement of the lower extremities , preventing injury , and possibly decreased pain6 . the present study examined the activity and the gait characteristics of the gluteus medius and the trunk stability muscles during the stance phase of gait on level ground when a vertical load corresponding to 0% , 1% , or 2% of body weight was placed on the lower extremities during the swing phase of gait . the exercise methods used in the study can easily be performed in everyday life to selectively strengthen the gluteus medius . the subjects were 40 young male university students aged between 21 and 30 years . a vertical load corresponding to 0% , 1% , or 2% of body weight , which was measured with an electronic scale , was placed bilaterally 3 cm above the upper part of the lateral malleous . the order of the load was decided randomly , and the load was affixed to each subject prior to the measurement . five steps were taken , and after discarding the data of the first and last cycles , the average of the remaining three cycles were calculated . sufficient exercise for natural gait a wireless surface emg telemyo 2400 t ( noraxon co. , usa ) was used to obtain the emg values . the electrodes were symmetrically attached to the gluteus medius , erector spinae , external oblique , and internal oblique muscles7 . the activities of the bilateral gluteus medius , erector spinae , external oblique , and internal oblique were compared among and within the different breathing patterns . one - way analysis of variance was used to examine the muscle activity of the lower extremities during the stance phase . as a post hoc test there were significant differences in the activities of the left gluteus medius , bilateral external oblique , and right internal oblique during gait with vertical loads of 0% , 1% , and 2% of body weight ( table 1table 1 . comparison of muscle activations of various vertical loads ( unit : % rvc)0%1%2%gmleft483.74 48.70706.97 67.75597.88 48.70right340.58 41.21329.40 36.58365.91 43.51esleft345.39 33.88357.18 37.63361.48 37.56right344.04 27.35358.18 31.27373.27 33.17eoleft211.53 13.14221.89 15.67265.65 19.40right216.79 14.21222.87 18.36276.51 21.94ioleft192.74 16.20181.95 9.46213.81 16.91right176.76 13.82190.38 10.17221.34 14.29p<0.05 , mean se , gm : gluteus medius , es : erector spinae , eo : external oblique , io : internal oblique , vl : vertical load . significant difference between 0% and 1% ( p<0.05),significant difference between 1% and 2% ( p<0.05 ) , significant difference between 0% and 2% ( p<0.05 ) . ) . * p<0.05 , mean se , gm : gluteus medius , es : erector spinae , eo : external oblique , io : internal oblique , vl : vertical load . significant difference between 0% and 1% ( p<0.05),significant difference between 1% and 2% ( p<0.05 ) , significant difference between 0% and 2% ( p<0.05 ) . lee et al.5 investigated changes in the activity of the gluteus medius muscle during the stance phase by placing a vertical load on the lower extremities during the swing phase . they noted that when a vertical load of 0.5 kg was placed on the lower extremities during the swing phase , the muscle activity increased compared to gait with no load , and that muscle activity with a 1 kg load decreased compared to that during gait with a 0.5 kg load . this study also compared the activity of the gluteus medius muscle during the stance phase when a 1 kg load corresponding to 1% body weight was placed on the lower extremities during the swing phase . we found that the gluteus medius muscle was significantly higher during 1% vertical load gait compared to 0% vertical load gait . the muscle activity during gait with the 2% vertical load was higher than during gait with the 0% vertical load , but lower than during the gait with the 1% vertical load . however , there was no significant difference in the gluteus medius activity between the 1% vertical load and the 2% vertical load . this result suggests that the activity of the gluteus medius muscle does not increase in proportion to the increase in the vertical load . in contrast , the activities of the internal and external oblique abdominal muscles significantly increased during gait with the 2% vertical load compared to gait with the 0% and 1% vertical loads . the external oblique abdominal muscle plays a role in the bending of the trunk , when it acts bilaterally8 . in the present study , the action of the external oblique abdominal muscle was significantly different between gait with the 0% and 2% vertical loads . during gait with the 0% and 1% vertical load , the activity of the gluteus medius muscle may have been more significant than that of the external oblique abdominal muscle , enabling gait with a vertical load on the lower extremities . however , under the 2% vertical load , providing a vertical load that was possibly greater than the muscle strength needed to move the gluteus medius muscle , may have forced the alignment of the trunk to the right in order to maintain gait , making the bilateral external oblique abdominal muscle act as trunk flexors , moving the trunk anteriorly . when the external oblique abdominal muscle and the internal oblique abdominal muscle contract as individual muscles , the external oblique abdominal muscle acts as a rotator muscle on the opposite side of the trunk . simultaneously , the internal oblique abdominal muscle acts in the same direction as the trunk . however , when the two muscles act together , the distance between one shoulder and the iliac crest on the opposite side decreases8 . in the present study , the right internal oblique abdominal muscle showed significant activity differences between gait with the 0% and 2% loads . when the left lower extremity was in the stance phase and the right extremity was in the maximal stance phase , the right internal oblique abdominal muscle and the right external oblique abdominal muscle acted together . thus , the gluteus medius muscle regulated the pelvis of subjects at loads corresponding to 1% of their weight . however , when the load on the lower extremity during the swing phase was increased to 2% of the subject s weight , in addition to the gluteus medius muscle regulating the pelvis , the external oblique abdominal muscle and the internal oblique abdominal muscle acted together to stabilize the trunk and to raise the lower extremities . the gluteus medius muscle , which contracts simultaneously with the external oblique abdominal muscle and the internal oblique abdominal muscle , is a postural muscle that is necessary for stability when raising the heels during gait ; it also regulates the pelvis in relation to the movement of the lower limbs9 . the activation of the gluteus medius was accompanied by changes in the activity of the internal oblique abdominal muscle and the external oblique abdominal muscle to ensure the stability of the trunk under the different conditions of gait at the various vertical loads . gait was only possible with the activity of the gluteus medius muscle and the trunk muscles resisting the different vertical loads rather than activating other muscles of the lower extremities in terms of energy efficiency .
the objective of the study is to evaluate a range of potential risk factors in the etiology of varicose veins with superficial venous reflux . forty - nine patients attending a cardiovascular surgery clinic for the management of varicose disease between 2009 and 2010 were enrolled for the study . the age range of the patient group was 44,04 15,05 years and female / male ( f / m ) ratio was 30/19 . twenty - six normal , healthy volunteers with the age of 40,94 13,60 years and with the female / male ratio of 15/11 acted as control subjects . we investigated several parameters including body mass index , age , birth number > 1 , standing for a long time ( standing for 8 or more hours without taking a break ) , systemic diseases , family history , venous doppler fndings , the levels of homocysteine , ferritin , vitamin b12 , and hemoglobin , sedimentation rate , mean corpuscular volume , low density lipoprotein , and rheumatoid factor of the patient group and the control group . we also determined the contribution of the methylene tetrahydrofolate reductase 677 c > t and 1298 a > c gene polymorphisms and fv leiden in both groups . in this small study , there appears to be no association between varicose veins and body mass index , smoking , type 2 dm , hypertension , family history , and birth number . a history of increased standing duration period ( > 8 hours ) and rheumatoid factor positivity have association with varicose veins with superficial venous reflux .	varicose veins occur commonly in the general population but despite much research the etiology of venous disease is still poorly understood . obesity , age , parity , standing for long times , and family history are risk factors . incidence of varicose veins in adult population has been shown to vary among populations ( between 10% and 60% ) and to increase by age in various studies [ 16 ] . the main factors in the etiology of varicose vein are venous dilation and valvular insufficiency that are started by unknown factors [ 13 , 7 , 8 ] . a number of epidemiological studies have shown that , in addition to environmental factors , genetic mechanisms may play a role in determining susceptibility to vascular disease . in particular , abnormalities in the enzymes that control homocysteine metabolism have been shown to cause atherosclerotic vascular disease . since genetic mechanisms may play a role in determining susceptibility to vascular disease , we studied several mutations in patients with varicose veins to look for any association between varicose veins and homocysteine level , protein c , protein s , fv , fviii , d - dimer , vitamin b12 level , folic acid level , mthfr , fvr2 , b fibrinogen , fv leiden , and prothrombin mutations besides other possible factors . the present study is planned to determine various risk factors and to analyze the methylenetetrahydrofolate reductase polymorphisms ( mthfr-677 and mthfr-1298 ) and fv leiden mutations in patients with primary varicose veins . the patients considered for the study were those attending ankara education and research hospital for the management of venous disease . the following data were collected : age , height , weight , smoker or nonsmoker , personal and family medical history with a special focus on varicose veins and birth number > 1 ( women only ) , standing for a long time before , and additional diseases . informed written consent this study protocol was approved by the ethics committee of ankara education and research hospital . the diagnosis of primary varicose vein was performed by the combination of clinical and duplex scanning examination . the exclusion criteria for the study were deep vein thrombosis , postthrombophlebitic syndrome , and recent infection . there were 45 patients with superficial venous reflux , 2 patients with superficial and perforator venous reflux , and 2 patients with superficial venous reflux and healed venous ulcer . the effects of the grade and type of the reflux on the results were not studied in the paper . we could not evaluate the location of valvular reflux at the various levels , sfj , upper thigh , midthigh , lower thigh , or below the knee , because of outpatient density in the radiology department . since there were not enough patients in the study group , the differences in the gene polymorphism or the procoagulant parameters between c2 and c3 patients versus the control group were not analysed . the control subjects did not have any disease established by careful history , examination , and routine laboratory test including the levels of homocysteine , ferritin , vitamin b12 , hemoglobin , sedimentation rate , mean corpuscular volume , low density lipoprotein , and rheumatoid factor . the patients in the observation group and the control group were similar regarding gender , body mass index , smoking habits , medical history , family medical history , and age . we asked patients one by one about how long they stand up all through the day and recorded the duration of time in hours for each patient separately . in the physical examination , subjects stood on a raised platform with their feet in three standard positions during inspection of the legs : facing towards the examiner with heels together and forefeet spread wide apart , facing away from the examiner in a similar position , and facing away from the examiner with feet parallel . they were asked to remain in a standing position for a minimum of two minutes before examination of their veins , to allow the blood to pool in the legs . real - time images of the common femoral , deep femoral , femoral , and popliteal veins were obtained before and after compression . all patients were evaluated with color duplex scanning in a warm , comfortable examination room by a radiology physician . a 512 mhz linear transducer was used to measure the diameters of the great saphenous vein ( gsv ) and the small saphenous vein ( ssv ) and to rule out acute or chronic deep venous thrombosis ( dvt ) in supine position . using color flow imaging in the longitudinal view , the valvular function of the gsv was evaluated at the sfj , upper thigh , midthigh , lower thigh , and below the knee . the valvular function of the ssv was evaluated at the level of the popliteal fossa . the reflux was quantified based on valve closure time , with the doppler spectral tracings obtained in a longitudinal plane . reflux was defined as being present if the valve closure time was greater than 0.5 seconds . examination for reflux was made with the patients standing , with upper body elevation of more than 45 , or in reverse trendelenburg position . blood samples were collected into citrate tubes ( for protein c , protein s , fibrinogen , and homocysteine ) and centrifuged at 4000 rpm ( revolutions per minute ) for 4 min to obtain plasma and serum fractions . quantitative determination of functional protein c depended on the prolongation of activated partial thromboplastin time . protein s was determined in accordance with the inhibition of activated factor v. the normal ranges of protein c and s antigens in plasma were 70130% and 65140% , respectively . the assay kit is designed to quantitate the true functional fxiii and fxiiia activity in plasma by measuring transglutaminase activity . in this assay , the thrombin - activated fxiiia from the patient plasma binds to a substrate coated plate . next , a horse radish peroxidase- ( hrp- ) conjugated fxiii is cross - linked to the captured fxiiia . the cross - linked hrp is then detected with a hrp chromogenic substrate at 450 nm . the data was analyzed to evaluate the correlation between fxiii activity and other dic markers which included platelet count , fibrinogen activities , prothrombin time ( pt ) , activated partial thromboplastin time ( aptt ) , and fibrin degradation product ( d - dimer ) . pt , aptt , fibrinogen , and d - dimer results were generated with an automated clotting analyzer ( sta - r ) . for the genetic testing , a sample of 10 ml 0.05 m edta - anticoagulated blood was taken . dna was extracted according to a standard salting - out procedure . for dna analysis , commercially available kits were used and the procedure was performed in accordance with the manufacturer 's instructions . mutation status ( normal , heterozygous , or homozygous ) the analysis of factor v leiden and prothrombin g20210a mutations was done . for determination of the mutation status of the mthfr c677 t and a1298c mutations , we used pcr amplification and restriction fragment length polymorphism analysis that was performed according to kim et al . , frosst et al . [ 9 , 10 ] , frosst et al . , and van der put et al . since the control group has less than 30 patients , all parameters in the study were assumed nonparametric and the statistical analyses were chosen for nonparametric tests . nominal parameters were described using frequency analysis , whereas scale parameters were described using mean and standard deviations . comparison between groups was tested by mann - whitney u at 95% confidence interval ( ci ; = 0,05 level ) . gender distribution , age , bmi , smoking , diabetes mellitus , rheumatoid factor positivity , hypertension , family history , long standing duration , and birth of more than one child for patient and control groups are gathered as baseline characteristics of the respondents . the ages of the forty - nine subjects with varicose veins included in the study group ranged between 15 and 85 years ( mean 44.04 15,05 years ) and the ages of the sixteen subjects in the control group ranged between 22 and 68 years ( mean 40,94 13,60 years ) . the women / men ratio was 30/19 in the study group and 15/11 in the control group . diabetes mellitus , hypertension , family history of varicosis , and birth of more than one child of the study group were higher than of the control group . differences between the two groups were found statistically insignificant ( p > 0,05 ) except for standing for long durations ( p < 0,001 ) and rheumatoid factor positivity that were found to be statistically significant ( p < 0,05 ) . it is possible that increasing severity of c class might result in greater differences in hematological or biochemical parameters . since the classification of the patient group in the study was most notably between class 2 ( c2 ) and class 3 ( c3 ) , the differences were not noted in this study with the increasing c class . according to table 2 , ceap class of the study group was changing between class 2 ( c2 ) and class 5 ( c5 ) . comparison of hematological and biochemical parameters in the study and the control groups was given in table 3 . no statistically significant difference was observed between both groups with respect to haematological and biochemical parameters , including whole blood count , erythrocyte sedimentation rate , prothrombin time , partial thromboplastin time , c - reactive protein level , glucose level , cholesterol profile , electrolyte levels , liver and kidney function tests , homocysteine , vitamin b12 , folic acid , factor v , factor viii , and protein c and protein s levels . table 4 shows the comparison of point mutations in the study group and the control group . when we look at the comparison of point mutations between the study group and the control group , heterozygote mutations were more dominant in the study group , but the difference was not statistically significant . homozygote mutations for all parameters were seen to be rare and scattered in both study and control groups . we could say that heterozygote mutations were dominant in the study group compared to the control group , but the difference was not statistically significant . incidence of varicose veins in adult population has been shown to vary among populations ( between 10% and 60% ) and to increase by age in various studies [ 16 ] . the main factors in the etiology of varicose vein are venous dilation and valvular insufficiency that are started by unknown factors [ 13 , 7 , 8 ] . on the basis of experimental and clinical evidence collected for the past decade , it is possible to suggest that the cause of dilatation of the varicose vein is in the vascular wall [ 12 , 13 ] . valvular theory , suggesting valvular incompetence , has been criticized in a number of biochemical and morphological studies . despite the evidence of a primary defect in the vein wall javien 's study showed that varicose veins were more common in women , but female sex was not found to be a strong risk factor . among the risk factors most closely associated with chronic venous insufficiency ( cvi ) were age , family history of varicose veins , and constipation . this is in keeping with findings from recent epidemiologic studies [ 16 , 17 ] . obesity and lack of physical activity were strongly associated with cvi in women , more so than in men . a modest association was found with female sex , previous injury in legs ( dvt ) , and remaining in the standing position for a long time , although these parameters are usually among those mostly agreed on as being risk factors . a number of epidemiological studies have shown that , in addition to environmental factors , genetic mechanisms may play a role in determining susceptibility to vascular disease . in particular , abnormalities in the enzymes that control homocysteine metabolism have been shown to cause vascular disease . methylenetetrahydrofolate reductase ( mthfr ) plays an important role in the folate cycle and contributes to the metabolism of the aminoacid homocysteine . it catalyses the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate , thus generating the active form of folate required for remethylation of homocysteine to methionine . if there is a genetic mutation in the mthfr gene , homocysteine levels may not be regulated properly . genetic mutations in mthfr are the most commonly known inherited risk factor for elevated homocysteine levels . the most common mthfr mutation is called the mthfr c677 t mutation , or the thermolabile mthfr mutation . even when 2 mthfr mutations are present ( e.g. , 2 c677 t mutations , or one c677 t mutation and one a1298c mutation ) , not all people will develop high homocysteine levels . although these mutations do impair the regulation of homocysteine , adequate folate levels essentially cancel out this defect . the mthfr polymorphism , which is associated with a predisposition for elevated plasma concentrations of homocysteine , has been reported to represent a genetic risk factor for occlusive vascular diseases , carotid atherosclerosis , silent brain infarction , and small artery occlusion with ischemic stroke , although these associations remain controversial . in our study we studied whether these mutations are associated with another type of vascular disease in the form of varicose veins because the observation of vascular complications in patients with homocystinuria led to the hypothesis that mild to moderate elevation of plasma homocysteine may be related to changes in the vascular wall . whether mild hyperhomocystinuria is causally related to the development of varicose veins was not known . since genetic mechanisms may play a role in determining susceptibility to vascular disease , we studied several mutations in patients with varicose vein to look for any association between varicose veins and homocysteine level , protein c , protein s , fv , fviii , d - dimer , vitamin b12 level , folic acid level , mthfr , fvr2 , b fibrinojen , and fv leiden and prothrombin mutations . in our study , we did not observe high levels of homocysteine in our varicose disease patients compared to control group . the results of our study showed that there was no statistically significant difference between patients and control groups based on their baseline characteristics except for standing durations of the patients and rheumatoid factor positivity . age , bmi , gender distribution , ht , family history , and birth of more than one child were not different between groups . although some researches reported that family history of the patients has an effect on varicose veins , we found it noneffective in the study . in addition , it was seen that heterozygote mutation was dominant in both male and female patients in the patient group , but the difference was not significant . a study done on a population of 1684 individuals around turkey demonstrated that the frequency of the c677 t in turkey was 42.9% ; of c677c , 47.4% ; and of t677 t , 9.6% . the frequency in turkey of a1298c was 43.7% ; of a1298a , 46.3% ; and of c1298c , 10.0% . the allelic frequencies of the t allele of mthfr 677 and the c allele of mthfr 1298 were 33.34 and 33.16% , respectively . the frequency of c677t / a1298c compound heterozygosity is highest in turkey ( 21.6% ) , as compared to canada ( 15% ) , the united states ( 17% ) , and the netherlands ( 20% ) . genetic abnormalities specific to factor v , prothrombin , and homocysteine metabolism were shown to increase the risk for myocardial infarction and ischemic stroke , particularly among younger patients and women in a study . unlike our results sverdlova am demonstrated an association between the mthfr genotype and the risk of developing varicose veins in the lower limbs . they found a significantly higher prevalence of subjects with at least one c677 t mthfr allele among those with varicose veins than among a control group ( or = 1.74 ; p < 0,005 ) . studies including higher numbers of cases and controls could show more significant relations between them . since the number of cases in the patient and control group is small , we did not take the effect of the type ( superficial and deep reflux ) and the degree of reflux into consideration in the study . economical factors and the type of rheumatic diseases were not evaluated in the study also . we only studied rheumatic factor positivity which was found to be statistically significant compared to the control group . while there was a trend towards an increase in certain biochemical parameters in our study , ultimately there was no statistically significant difference in hematological or biochemical markers between the subject group and the control group . in this small study , there appear to be no significant genetic differences related to folate metabolism or procoagulant predisposition or medical risk factors including bmi value , smoking , diabetes mellitus , hypertension , family history of varicosis , and birth of more than one child . a history of increased standing and rheumatoid factor positivity were found to be associated with varicose veins . further studies , perhaps with a larger cohort , could show significance of parameters that were found to be insignificant in our study . further studies should also be conducted to find any association between rheumatic diseases and varicose vein development .
the advent of computer - assisted technology has revolutionized planning for complex craniofacial operations , including orbital reconstruction . orbital reconstruction is ideally suited for virtual planning , as it allows the surgeon to assess the bony anatomy and critical neurovascular structures within the orbit , and plan osteotomies , fracture reductions , and orbital implant placement with efficiency and predictability . in this article , we review the use of virtual surgical planning for orbital decompression , posttraumatic midface reconstruction , reconstruction of a two - wall orbital defect , and reconstruction of a large orbital floor defect with a custom implant . the surgeon managing orbital pathology and posttraumatic orbital deformities can benefit immensely from utilizing virtual planning for various types of orbital pathology .	over the past 20 years , advances in radiographic imaging and computer technology have allowed for application of image - guided surgery in orbital reconstruction . conversely , volumetric analyses of anatomical structures have been utilized for the design of standardized anatomic implants for orbital reconstruction and custom patient - specific implants for complex orbital and midfacial defects . these advances have allowed for improved efficiency , accuracy , and safety in the surgical management of orbital pathology . the purpose of this review is to discuss the applications of computer - guidance and advanced computed tomography ( ct)-imaging in the management of various conditions affecting the bony orbit . four illustrative case examples are presented : orbital decompression for thyroid - eye disease , midface reconstruction following a zygomaticomaxillary complex ( zmc ) injury , reconstruction of a posttraumatic two - wall orbital defect , and reconstruction of a large orbital floor defect with a custom alloplastic implant . thyroid eye disease ( ted ) is an autoimmune inflammatory disorder of the orbit that results in proptosis , diplopia , eyelid retraction , exposure keratopathy and in severe cases , optic nerve compression.1 the pathogenesis of ted has not been entirely elucidated however it is most likely due to the activation of thyroid stimulating receptors by circulating thyrotropin receptor antibodies . activation of these receptors in the orbit results in deposition of hyaluronan and resulting extraocular muscle enlargement as well as hypertrophy of orbital adipose tissue.123 as the orbital contents enlarge , the patient is at risk for sight - threatening compressive optic neuropathy . various treatment methodologies including steroids , orbital radiation , immunomodulators , and selenium have been used . however , refractory or sight - threatening cases of ted usually require surgical decompression.3 orbital decompression has been a mainstay of ted treatment since the 1950s . the variability in outcomes particularly in terms of proptosis and ocular alignment has resulted in the development of numerous surgical techniques for decompression with no clear consensus on which is most effective.4 the recent development of computer - assisted surgical planning and execution has the potential to result in more efficacious , consistent and predictable decompressions . multiplanar ct scans with three - dimensional ( 3d ) reconstruction allow the surgeon to carefully evaluate the individual anatomy and identify specific bone segments for resection . intraoperatively , the ct images and the patient 's anatomy are virtually overlapped allowing for stereotactic navigation throughout the case.5 this allows the surgeon to check his / her distance from important anatomical landmarks and to measure the extent of bony resection in order to carefully match preoperative planning.6 postoperative imaging and assessment may be used to objectively quantify the magnitude of decompression and correlate this with clinical outcomes . the objective data provided by computer - assisted orbital decompression has the potential to result in safer , more efficacious and consistent decompressions . in the clinical example shown [ figures 1 and 2 ] , real - time image guidance was utilized to allow for extensive , asymmetric decompression of the bilateral orbits ( left : lateral orbital wall , right : medial and lateral walls ) . preoperative ( top panels ) and postoperative ( bottom panels ) frontal and submentovertex views of a male patient with thyroid orbitopathy who underwent computer - assisted orbital decompression preoperative ( left panels ) and postoperative ( right panels ) demonstrating the three - dimensional computer planning for orbital decompression and superimposition of the expected result over the postoperative images ( right middle and right bottom panels ) injuries to the bony orbit are common among patients sustaining craniomaxillofacial trauma.789101112 the external orbital framework is disrupted in several different types of facial fractures ( e.g. zmc , frontal bone , le fort iii ) . the integrity of the internal orbit can likewise be disrupted , either in isolation or as a component of complex midfacial or upper facial injury . the orbital skeleton provides support for the globe and ocular adnexa and houses neurovascular structures critical to normal visual sensory function . while disruption of the orbital framework can compromise these structures , such injuries are , fortunately , rare . conversely , surgical management of orbital skeletal injuries is fraught with potential peril , as dissection , bony mobilization , or placement of intra - orbital hardware may damage the globe or critical neurovascular structures . in this regard , advances in computer imaging have enhanced the surgeon 's ability to safely dissect the internal orbit , have allowed for the design and manufacture of standard implants for orbital reconstruction , planning for correction of secondary deformities , quantitative assessment of fracture reduction and volume restoration , and have improved the ability to visualize , in real - time , the orbital anatomy during dissection.13141516171819202122232425262728293031 zmc fractures sometimes termed zygoma fractures or orbitozygomatic fractures , are among the most common type of facial fracture.78910 high - or low - energy mechanisms can disrupt the articulations of the zygomatic bone with the frontal bone ( frontozygomatic suture ) , temporal bone ( zygomatic arch ) , sphenoid bone ( sphenozygomatic suture ) , and maxilla ( zygomaticomaxillary buttress ) . the 3d relationship between these articulations was not initially understood in the management of these injuries , which were commonly referred to as tripod fractures . advances in ct have improved the understanding of these injuries and the complex 3d anatomy that needs to be restored for successful management.78910111213 restoration of the sphenozygomatic articulation in the lateral orbit remains the most reliable predictor of a successful reduction but is difficult to assess without surgical access to the internal orbit.712272829 furthermore , management of the orbital floor remains controversial among patients with zmc fractures.2829 the use of real - time imaging ( mini c - arm , ct ) has allowed for assessment of anatomic alignment and orbital floor integrity following reduction , improving operating times and decreasing patient morbidity from unnecessary orbital exploration.13202123 in the case presented , an orbitozygomatic fracture was repaired utilizing virtual planning to establish the appropriate position of the right zygoma . to accomplish this , the left zygoma complex was digitally rendered from a 3d ct reconstruction and subsequently mirrored and placed onto the right midface [ figure 3 ] . the reconstituted virtual position of the zygoma served as a template and was then used to guide intra - operative positioning of the displaced fracture . virtual planning also allowed for visualization of the sizable orbital floor defect , which required orbital exploration and reconstruction using an anatomical plate . three - dimensional and axial computed tomography , as used for operative planning for a displaced orbitozygomatic fracture . in the preoperative images ( top panels ) , the displaced zygoma ( top left ) was repositioned by mirroring the left zygoma and virtually positioning the mirrored bone ( top middle and top right panels ) . the postoperative images ( bottom panels ) demonstrate anatomic alignment of the zygomatic articulations and reconstruction of the orbital floor injuries to the orbital floor are commonly encountered among surgeons affiliated with trauma centers that treat facial injuries . orbital floor injuries may occur in isolation or in conjunction with higher level le fort injuries , zmc fractures , naso - orbital - ethmoid fractures , panfacial trauma , and often occur with fractures of the medial orbital wall . the primary goal for orbital reconstruction is to restore the premorbid orbital volume . in this regard , image - guidance technology has been useful for the design of anatomic orbital implants , particularly for two - walled defects involving the floor and medial wall.14151617181924 custom implants can also be utilized for reconstruction of irregular defects or when there is a significant bone loss . real - time intra - operative image guidance is a useful to aid in dissection of the orbital floor and medial wall , allowing the surgeon to assess accurately position relative to the superior orbital fissure , optic canal , and the anterior and posterior ethmoidal foramina , thereby decreasing the risk of damage to critical arterial , venous , and nervous structures in these areas . in the first clinical example , virtual planning was used to assess the size and position of the orbital implant required for reconstruction of a posttraumatic defect involving the right medial orbital wall and floor [ figures 4 and 5 ] . the unaffected orbit was used for comparison to allow for adequate reconstitution of orbital volume . the preoperative enophthalmos was predictably corrected ; the postoperative image fusion demonstrates appropriate positioning of the implant along the orbital floor and medial wall , with a stable landing on the posterior ledge . in the second clinical example , a patient with a large , posttraumatic orbital floor defect with significant volume change and marked enophthalmos underwent presurgical planning and design of a custom titanium / polyethylene implant for orbital reconstruction [ figures 6 and 7 ] . the postoperative result demonstrates marked improvement in globe position , with excellent positioning of the implant along the fracture margins . preoperative ( top panels ) frontal and submentovertex views of a male patient with an orbital floor and medial wall fracture who underwent computer - assisted planning for orbital reconstruction . in the postoperative ( bottom panels ) photos , the enophthalmos has been corrected and the globe position is symmetric preoperative ( left panels ) and postoperative ( right panels ) demonstrating the three - dimensional computer planning for orbital reconstruction with an anatomic implant . the implant is rendered within the software and virtually placed within the defect . this allows the surgeon to position the implant and compute the orbital volume relative to the unaffected side . intra - operatively , placement of the implant in the appropriate position can be ensured with the use of real - time navigation preoperative ( top panels ) frontal and submentovertex views of a female patient with a large right orbital floor fracture with significant enophthalmos . computer - assisted planning was utilized to design a custom implant for reconstitution of orbital volume for enophthalmos correction . postoperatively ( bottom panels ) , the patient has has no orbital dystopia or enophthalmos preoperative ( left panels ) and postoperative ( right panels ) images , demonstrating the three - dimensional computer planning and subsequent reconstruction of a large orbital floor defect with an anatomic titanium - alloplastic hybrid implant . the preoperative images are utilized to compute the volume of the defect relative to the unaffected side . the custom implant is then designed to restore the orbital volume and correct the globe position advances in computer technology and imaging have improved the accessibility and efficacy of orbital reconstruction for defects involving the bony orbit . due to the complex shape of the internal orbit , proximity of critical soft tissue structures , and small margins of error , the orbital skeleton is an ideal anatomic region for virtual planning and real - time intra - operative navigation . utilization of this technology has the potential to improve the surgical treatment of common problems , make challenging clinical cases more accessible and predictable , and has enormous utility as an adjunct for trainee instruction .
purposephotothermal therapy is an emerging cancer treatment paradigm which involves highly localized heating and killing of tumor cells , due to the presence of nanomaterials that can strongly absorb near - infrared ( nir ) light . in addition to having deep penetration depths in tissue , nir light is innocuous to normal cells . little is known currently about the fate of nanomaterials post photothermal ablation and the implications thereof . the purpose of this investigation was to define the intratumoral fate of nanoparticles ( nps ) after photothermal therapy in vivo and characterize the use of novel multidye theranostic nps ( mdt - nps ) for fractionated photothermal antitumor therapy.methodsthe photothermal and fluorescent properties of mdt - nps were first characterized . to investigate the fate of nanomaterials following photothermal ablation in vivo , novel mdt - nps and a murine mammary tumor model were used . intratumoral injection of mdt - nps and real - time fluorescence imaging before and after fractionated photothermal therapy was performed to study the intratumoral fate of mdt - nps . gross tumor and histological changes were made comparing mdt - np treated and control tumor - bearing mice.resultsthe dual dye - loaded mesoporous nps ( ie , mdt - nps ; circa 100 nm ) retained both their nir absorbing and nir fluorescent capabilities after photoactivation . in vivo mdt - nps remained localized in the intratumoral position after photothermal ablation . with fractionated photothermal therapy , there was significant treatment effect observed macroscopically ( p = 0.026 ) in experimental tumor - bearing mice compared to control treated tumor - bearing mice.conclusionfractionated photothermal therapy for cancer represents a new therapeutic paradigm enabled by the application of novel functional nanomaterials . mdt - nps may advance clinical treatment of cancer by enabling fractionated real - time image guided photothermal therapy .	photothermal ablation is an exciting noninvasive cancer paradigm,15 which is just beginning to enter the clinical arena for human cancer therapy.6 numerous nanomaterials including nanoshells,1,2 polyhydroxy fullerenes,5 gold speckled silica nanoparticles ( nps),7 gold nps,8 gold nanorods,3 nanotubes,9,10 nanocages,11 and quantum dots12 have been investigated as anticancer photothermal agents . numerous studies have demonstrated the efficacy of these nanomaterials for tumor ablation in vitro.2,7,8,13 several studies have focused on the effect of photothermal therapy using these nanomaterials using in vivo models.1,10,13 these in vivo studies have demonstrated that a single photothermal treatment results in partial but incomplete tumor destruction based on long term follow - up of tumor growth.10,13 approaches and strategies to improve the efficacy of the photothermal antitumor strategy are needed . understanding the intratumoral fate of nanomaterials after photothermal therapy is essential for furthering the clinical application of nanoparticle - mediated photothermal therapy . to investigate this issue in vivo requires the development and application of nanomaterials that are not only robust photothermal reagents , but also stable bioimaging contrast agents . magnetic resonance imaging,1 optical coherence tomography,13 and photoacoustic imaging14 have been used to guide photothermal treatments in vivo . near - infrared ( nir ) fluorescence imaging has not been utilized commonly to track photothermal nanomaterials in vivo due to difficulties in combining plasmonic materials with a fluorescent component into single , biocompatible nanoconstructs.15 in this report , novel multidye theranostic nps ( mdt - nps ) were employed to address the fate of nanomaterials following photothermal ablation . these recently developed nps are biocompatible , have strong and stable nir fluorescence , and robust nir photothermal properties.16 the unique multi - functionality and stability of these novel nanomaterials favor their application to answer these questions regarding the fate of nanomaterials of photothermal therapies . in this report , the ability of mdt - nps to mediate enhanced photothermal tumor destruction when utilized for multiple fractionated ablations is demonstrated . the findings suggest fractionated photothermal therapy as a new paradigm for noninvasive image - guided cancer therapy . nir fluorescent ( nirf ) mesoporous silica nps16 were synthesized by incorporating a modified , silane - conjugated heptamethine cyanine dye ( ir780 ; sigma - aldrich corporation , st louis , mo ) during the synthesis of mesoporous silica nps , as described elsewhere.16 briefly , 0.5 g of cetyltrimethylammonium bromide ( c16tab ; sigma - aldrich ) and 1.75 ml of 2 m sodium hydroxide ( fisher scientific co , pittsburgh , pa ) were added to 240 ml of nanopure water ( fisher scientific ) and heated to 80c . once thermal equilibrium was reached , 2.5 ml of tetraethyl orthosilicate ( fisher scientific ) was added dropwise to this solution . after 23 minutes , the transparent solution turned milky due to the nucleation of silica nps . at this time , 800 l of silane conjugated dye was added . the particle suspension was allowed to stir for 3 hours at constant temperature of 80c . for the removal of surfactant , particles were dispersed in 3 wt% methanolic solution of calcium chloride ( fisher scientific ) and the suspension was refluxed for 4 hours at 60c . for 1 g particles , 100 ml of calcium chloride - methanol solution was used . these particles were centrifuged ( 7000 g , 20 min ; beckman model j2 - 21 , ape - bridgepath scientific , frederick , md ) and resuspended in the calcium chloride - methanol solution with the aid of sonication ( crest tru - sweep model 575d ; crest ultrasonics , trenton , nj ) . the refluxing/ centrifugation / resuspension steps were repeated three times in order to completely remove the surfactant from the pores . finally , these particles were suspended in nanopure water by centrifugation and resuspension using sonication . to confirm the complete removal of surfactant , the nps were incubated with cells and a lactate dehydrogenase assay ( roche diagnostics , indianapolis , in ) was performed ; the assay demonstrated no deleterious effects on cells . for the synthesis of mdt - nps , nirf mesoporous particles were dispersed in chloroform ( fisher scientific ) to encapsulate the photothermal dye inside the pores . in a typical synthesis , nirf mesoporous silica nps ( 10 mg ) were dispersed in 8 ml chloroform . then , 2 mg of silicon 2,3-naphthalocyanine dihydroxide ( sigma - aldrich ) in chloroform ( concentration 1 mg / ml ) was added to the nanoparticle dispersion . the dispersion was stirred overnight to allow for the maximum loading of the dyes into the np pores . measuring the absorbance of the supernatant allowed an estimate of the amount of dye incorporated inside the pores of the nps ( 11.1% ) . nanoparticles were characterized for absorbance , emission , and heating using techniques detailed elsewhere.16 the 4t1 murine mammary tumor cell line ( tufts university , medford , ma ) was orthotopically implanted ( 1 10 cells ) into female 6-week - old balb / c mice ( charles river laboratories , wilmington , ma ) . on postimplantation day eight , 27 mice were equally and randomly distributed to nine groups ( three mice per group ) : mdt - np / no ablation , mdt - np / one ablation , mdt - np / two ablations , mdt - np/ three ablations , mdt - np / four ablations , control / one ablation , control / two ablations , control / three ablations , control / four ablations . only one ablation was performed per day per mouse . at the start of the experiment , mice were initially imaged via fluorescence ( 710 nm excitation/820 nm emission ) with the ivis spectrum ( caliper life sciences , hopkinton , ma ) . for in vivo imaging experiments , 820 nm wavelength was chosen as it provides the greatest signal to noise ratio . this was followed by administration of mdt - nps via intratumoral injection ( 15 mg / ml , 20 l ) or 0.9% sterile saline solution ( 20 l ; fisher scientific ) for control mice . mice then underwent photothermal ablation ( continuous wave laser , 785 nm , 625 mw / cm , 5 minute duration , 1.5 cm source - tumor distance ) . fluorescence imaging was also obtained after each photothermal ablation . for imaging and ablation experiments , mice were anesthetized using 2%3% isoflurane ( baxter healthcare corporation , deerfield , il ) . on day nine , whole animal digital photographs ( d90 ; nikon , melville , ny ) were obtained before mice were euthanized . severity of treatment effect was based on gross tumor inspection ( experiment day nine ) and scored in a blinded fashion using a scale of zero ( no treatment ) to four ( maximal treatment effect ) . tumors were preserved in 10% neutral buffered formalin ( fisher scientific ) for histological analysis . histological tumor analysis was performed in a blinded fashion by a board certified pathologist ( jak ) . region of interest quantification of fluorescent signal was performed at all imaging points with living image 3.2 or 4.0 software ( caliper life sciences ) . all experiments were performed under protocols approved by the university of florida institutional animal care and use committee . mdt - nps were synthesized using a two - step approach.16 first , nirf mesoporous silica nps were synthesized by incorporating a modified , silane - conjugated heptamethine cyanine dye ( ir780 ) during the surfactant - templated synthesis of mesoporous silica nps . second , for the synthesis of mdt - nps , these nirf mesoporous nps were dispersed in chloroform to encapsulate the nir photothermal silicon 2,3-naphthalocyanine dihydroxide dye inside the pores of the silica matrix . the final nanoconstructs were washed and dispersed in water for the subsequent sequential photothermal ablation experiments and visualization . the photophysical properties ( absorbance and fluorescence ) of nirf nps are presented in figure 1a . the broad excitation spectra and large stoke s shift of the particles enabled use of a broad range of excitation wavelengths and decreased the extent of self - quenching , respectively . having a porous interior structure , these nps were circa 100 nm , as measured by transmission electron microscope ( jeol 2010f , tokyo , japan ) ( figure 1d ) . upon loading of a silicon 2,3-naphthalocyanine dihydroxide dye into the pores of nirf nps , their absorption cross - section in the nir region increased ( figure 1b ) ; the broad extinction spectra of these mdt - nps displayed their ability to absorb light over the entire nir region . the ability of mdt - nps to absorb nir light and generate heat was tested by measuring the temperature increase of an aqueous dispersion of mdt - nps upon illumination with a low power laser source ( 785 nm , 625 mw/ cm ) . the temperature of a solution of mdt - nps ( 1 mg / ml ) increased by approximately 15c after 5 minutes of continuous irradiation . the repeated heating over a period of 3 days resulted in only partial loss ( ~4% decrease in temperature on day three relative to day one ) of their heat generating capacity ( figure 1c ) . next , a murine orthotopic model of breast cancer was used to study the fate of mdt - nps following photothermal ablation in vivo . after intratumoral injection of mdt - nps , mammary tumors in mice were exposed to nir laser light once daily for 1 , 2 , 3 , or 4 days . after each nir irradiation event , the fluorescence signal was plotted by normalizing each signal to the intensity of the original injected mdt - nps ( day one before ablation , figure 2 ) . following a single injection of mdt - nps , all experimental groups ( both with and without laser irradiation ) showed a consistent intratumoral fluorescence pattern for 9 days . importantly , mdt - nps could easily been seen in the intratumoral position for the entire 9-day observation period in all groups . figure 3 is a fluorescence imaging sequence over a time course of 9 days of a representative mouse from the mdt - np / four ablations group . after 9 days , mdt - nps retained high fluorescence imaging capacity and remained within the tumor . in order to determine if mdt - nps were accumulating in reticuloendothelial organs ( liver and spleen ) following photothermal ablation therapy , whole animal organ in situ fluorescence imaging was performed following primary tumor removal on day nine . no mdt - nps were detected beyond the intratumoral injection site in these mice ( figure 3 ) . the persistence of mdt - nps within the tumor for 9 days following administration suggests the possibility for fractionated image - guided photothermal therapy after a single administration of mdt - nps to a tumor . increasing the number of ablation fractions corresponded to a significant ( p = 0.026 ) increase in treatment effect as assessed by gross tumor inspection on experiment day nine ( figures 4 and 5 ) . control groups demonstrated no effect from irradiation , and tumors in the mdt - np treated / no ablation group were unaltered by the presence of mdt - nps ( figure 5 ) . histologically , a congruent pattern was observed : no evidence of thermal injury or other tissue damage was noted in control mice ( figure 6a ) , whereas mice treated with mdt - nps and exposed to four fractionated photothermal ablations demonstrated significant thermal injury with coagulative tumor necrosis , hemorrhage , and edema ( figure 6b ) . for fractionated photothermal therapy to be possible , photothermal nps must maintain their intratumoral location following photothermal activation . to the authors knowledge , the intratumoral fate of photothermal nps following one or more photothermal treatments has not been previously well characterized . mdt - nps are a novel tool which has allowed the intratumoral fate of photothermal nps following photoablation to be studied . multimodal mdt - nps can easily be both tracked using fluorescence imaging and repeatedly excited for photoablation with a nir laser . in the reported experiments , mdt - nps were clearly able the presence of mdt - nps within the tumor following photothermal treatments ( as demonstrated by nir imaging ) and their ability to be repeatedly heated in vitro ( figure 1c ) suggests that they may be used for additional photothermal treatments ( fractionated photothermal therapy ) . the ability to image mdt - nps within the tumor over time it was observed that mdt - nps remain in the intratumoral position for days following photothermal treatment in the 4t1 murine mammary carcinoma model . it is possible that the kinetics of nanomaterial extravasation are different for other tumor types with different lymphatic and vascular patterns . intratumoral persistence and function of mdt - nps following photoablation may allow the use of lower laser intensities to treat tumors compared to single ablation strategies . it is noteworthy that in the present study , very low laser intensity ( 625 mw / cm ) was required to achieve fractionated photothermal ablation compared to previous reports of photothermal ablation ( 4 w / cm).1,10 the minimal number of ablations needed to achieve complete tumor destruction is not investigated in the present study and very likely depends on many factors , such as tumor type , tumor size , tumor location , nanoparticle construct ( eg , nir - absorbing component used for ablation ) , intratumoral distribution of nps , and concentration of mdt - nps within tumors.17 such investigations would represent the next step in advancing the concept of fractionated tumor photothermal ablation toward clinical application . foremost , if complete tumor destruction is not obtained on the initial photothermal treatment , then subsequent successive treatments can be performed using the fractionated approach . absolute dosing of nanomaterials for photothermal therapy may be minimized with the fractionated treatment paradigm . mdt - nps are unique nanomaterials that maintain their fluorescent and photothermal properties after photoactivation . in the 4t1 murine breast cancer model , it has been demonstrated mdt - nps can therefore be utilized as mediators of fractionated photothermal therapy . fractionated treatments can be guided by the ability to image mdt - nps within the tumor using fluorescence imaging techniques . the concept of image - guided , fractionated photothermal ablation is one which has the potential to expand the efficacy of photothermal therapy for clinical application while minimizing the morbidity of cancer care . this new paradigm should allow photothermal treatments to be delivered and modulated depending on tumor response to achieve the desired clinical effect .
in the aftermath of spinal cord injury , glial restricted precursors ( grps ) and immature astrocytes offer the potential to modulate the inflammatory environment of the injured spinal cord and promote host axon regeneration . nevertheless clinical application of cellular therapy for the repair of spinal cord injury requires strict quality - assured protocols for large - scale production and preservation that necessitates long - term in vitro expansion . importantly , such processes have the potential to alter the phenotypic and functional properties and thus therapeutic potential of these cells . furthermore , clinical use of cellular therapies may be limited by the inflammatory microenvironment of the injured spinal cord , altering the phenotypic and functional properties of grafted cells . this report simulates the process of large - scale grp production and demonstrates the permissive properties of grp following long - term in vitro culture . furthermore , we defined the phenotypic and functional properties of grp in the presence of inflammatory factors , and call attention to the importance of the microenvironment of grafted cells , underscoring the importance of modulating the environment of the injured spinal cord .	astrocytes are the most abundant cell type in the central nervous system ( cns ) , providing crucial support for the development and maintenance of cns function . nevertheless , astrocytes are also involved in the injury process and in a variety of neurological disorders , in which they become activated or dysfunctional ( sloan and barres , 2014 ; ben haim et al . , 2015 ; khakh and sofroniew , 2015 ) . given this dichotomy , it is important not only to define their phenotypic and functional properties , but also to identify the molecular mechanisms and develop protocols for the preparation of supportive astrocyte subtypes , particularly if these cells are to be used as beneficial transplants in the cns . these supportive astrocytes have numerous beneficial effects in the aftermath of cns injury promoting host axonal regeneration , limiting the inflammatory response , and reducing the inhibitory microenvironment of the injury site to create a permissive environment for axon growth and synaptic connectivity ( hill et al . , 2004 ; bonner et al . , 2011 ; jin et al importantly , restoration of functional connectivity after spinal cord injury ( sci ) has focused on promoting host axon regeneration and using neuronal relays with graft - derived neurons ( bonner and steward , 2015 ) , strategies that are facilitated by the use of supportive astrocytes ( figure 1 ) . the objective of this paper is to summarize recent findings pertaining to the properties of glial restricted precursors ( grp ) and grp - derived astrocytes and discuss how these findings can be translated into a therapeutic strategy that promotes connectivity and recovery following sci . glial restricted precursor ( grp ) transplants provide a supportive environment following spinal cord injury ( sci ) and can be used in combinatorial strategies to restore connectivity . the lesion border contains reactive astrocytes and a scar of chondroitin sulfated proteoglycans , while the lesion core is composed of activated microglia and infiltrating macrophages . damaged host axons retract from the site of injury and the physical , chemical , and inflammatory inhibitors of axon growth . ( b ) transplantation of grp creates a permissive environment that provides for : 1 ) reduction of glial scar formation , 2 ) reduced activation of microglia / macrophages , and 3 ) support for axon growth and regeneration into lesion . ( c ) transplantation of grp together with nrp allows for the formation of synaptic connections between regenerating sensory host axons and graft - derived neurons . in this case , grp transplants also support : 4 ) survival and differentiation of nrp into neurons and 5 ) synaptic connectivity for the formation of a neuronal relay . during cns development , astrocytes originate from a variety of progenitor cells including grp , which give rise to both astrocytes and oligodendrocytes ( rao and mayer - proschel , 1997 ) . grp , as well as neuronal restricted precursors ( nrp ) , can be isolated from the rat spinal cord during embryonic development at day ( e ) 13.514 or derived in culture from multipotent neuroepithelial stem cells ( cai et al . , 2002 when grp are grafted into normal adult spinal cord they show robust survival , proliferation , and migration as well as differentiation into both astrocytes and oligodendrocytes , confirming their progenitor properties ( han et al . , 2004 ; lepore and fischer , 2005 ) . our previous studies have focused on the ability of grp and grp - derived astrocytes to support regeneration and connectivity following sci ( figure 1 ) . we and others found that grp and grp - derived astrocytes have remarkable supportive properties that include : 1 ) reduction of glial scar formation as indicated by preventing the upregulation of host glial fibrillary acid protein ( gfap ) and chondroitin sulfated proteoglycan ( cspg ) expression , 2 ) supporting axon growth and regeneration , 3 ) promoting the survival and neuronal differentiation of nrp , and 4 ) supporting structural and functional synaptic connectivity and the formation of a neuronal relay ( lepore and fischer , 2005 ; bonner et al . , 2011 ; jin et al . , 2011 ; haas and fischer , 2014 ) . in summary , transplantation of grp generates supportive astrocytes , which can be used as a therapeutic platform in strategies designed for sci repair in animal models and eventually translate into human clinical trials . grp are characterized by a refractive , short stellate morphology , the expression of the surface marker a2b5 , and the neural precursor marker nestin ( rao and mayer - proschel , 1997 ) . traditionally , astrocytes have been defined by their multi - process stellate morphology and expression of gfap , which is commonly used as a marker of mature astrocytes , although other markers such as s100 , aldh1l1 , and glast have also been used . however , there is growing evidence for a heterogeneous population of astrocytes capable of performing a diverse array of temporally , regionally , and contextually specific functions ( zhang and barres , 2010 ; khakh and sofroniew , 2015 ) . our previous studies demonstrated that grp cultured in serum - free , defined media supplemented with basic fibroblastic growth factor ( bfgf ) maintained their phenotypically undifferentiated state ( haas et al . , 2012 ) . furthermore , under such conditions , these cells were capable of extensive proliferation , allowing for the expansion and cryopreservation of large , near - homogenous cell stocks . we found that in vitro differentiation of grp into astrocytes with bmp4 or cntf induced distinct morphological and phenotypic changes , with bmp4 generating a more mature phenotype and cntf an intermediate state that retained progenitor markers . the use of grp for preparation of astrocytes provides considerable advantages over isolation and culturing primary astrocytes from the cns . these advantages are underscored by comparable protocols developed for human grp and the availability of embryonic stem ( es ) and induced pluripotent stem ( ips ) cells for preparation of neural progenitors ( haas and fischer , 2013 ; roybon et al . , 2013 ; palm et al . , 2015 ) . in vitro co - culture assays are useful tools to evaluate the interactions between cell populations and elucidate the mechanisms of these interactions . such interactions can be studied by a direct co - culture model ( analyzing cell - cell interactions ) or an indirect model with multi - compartment slides or the use of conditioned medium ( analyzing secreted factors ) . studies using co - cultures of astrocytes and dorsal root ganglion ( drg ) neurons have demonstrated the permissive properties of astrocytes with respect to their ability to support axon growth ( smith et al . , 1990 ) . importantly , in these studies , only immature astrocytes , freshly isolated from early post - natal cerebral cortices , but not mature astrocytes , expanded in culture in the presence of fetal bovine serum or those isolated from the mature cortex , were able to support axon growth ( smith et al . , 1990 ) . similar to the effects of immature astrocytes on axon growth , grp and astrocytes pre - differentiated with either bmp4 or cntf also supported axon growth of embryonic and adult rat drg neurons ( haas et al . , in addition , conditioned media harvested from high - density grp cultures facilitated the growth of drg axons on an inhibitory cspg - enriched substrate and allowed them to cross onto a cspg - enriched border ( ketschek et al . , 2012 ) . these data indicate that grp and immature astrocytes , isolated from fetal tissue or derived from grp , secrete trophic factors that support and promote axon growth even in the presence of an inhibitory environment , underscoring their potential use in sci . to test the data obtained from in vitro experiments in animal models , we examined the properties of grp using a c4 dorsal column hemisection of sci and found that ascending sensory axons , labeled with cholera toxin b , regenerated into the injury / transplant site as shown in figure 1 ( hill et al . importantly , both grp and grp - derived astrocytes prepared with bmp4 or cntf showed comparable effects , creating a permissive environment for host axon growth into but not out of the injury site . the finding that grp transplants were capable of inducing only a modest regenerative response , which did not extent beyond the injury site , may be related to the limited regenerative response of axons in the cns and the permissive nature of the grp for axon growth in absence of directional guidance molecules outside the transplant . it is important to note that the regenerative capacity of the motor system , particularly that of the corticospinal tract ( cst ) , is poor even in the presence of grp or astrocytes ( hill et al . , 2004 , and unpublished data ) . in this setting , grp were only able to prevent the significant cst retraction that occurs in the aftermath of sci . taken together , grp and grp - derived astrocytes create an improved supportive environment for axon regeneration in both in vitro and in vivo systems . the clinical use of cellular products for transplantation requires large - scale preparation , cryopreservation , and a strict process of quality assurance to ensure the standardized properties of cellular therapeutics with minimal lot - to - lot variability . to guarantee a supply of supportive astrocytes it will be necessary to expand grp cultures while ensuring that the therapeutic properties of these cells have not changed . there have been several reports examining the phenotypic and functional properties of neural cells cultured for extensive periods of time with respect to their supportive properties . for example , multipotent neural stem cells isolated from embryonic human spinal cord and grown as neurospheres can be expanded for up to 25 passages ( over 350 days ) in vitro and maintain their multipotent capacity in vivo when grafted into an sci model ( akesson et al . , 2007 ) . similarly , oligodendrocyte precursor cells ( opc ) can be maintained as oligospheres for several months and retain their ability to differentiate into functional oligodendrocytes capable of myelinating axons of primary cortical neurons in a co - culture assay ( zhu et al . , 2014 ) . in contrast , olfactory ensheathing cells ( oec ) , specialized glial cells with schwann cell and astrocyte - like properties , can lose their ability to remyelinate the injured cord after 6 weeks in culture ( radtke et al . , 2010 ) . these examples emphasize the importance of testing and identifying the appropriate temporal parameters and culture conditions for expansion of cells to be used in transplantation studies . in a recent study , we examined the long - term properties of grp following expansion of adherent cultures in serum - free , defined media supplemented with the bfgf mitogen ( hayakawa et al . , 2015 ) . specifically , we compared the morphological , phenotypic , and functional properties of early ( 1020 days in culture ) and late ( 120140 days in culture ) passage grp . our results demonstrated that late passage grp were maintained in an undifferentiated state , and more importantly , showed comparable axon growth - promoting effects on adult drg axon growth in both direct co - culture and indirect conditioned medium experiments ( figure 2a ) ( hayakawa et al . , 2015 ) . taken together , we established an in vitro culture protocol that enables grp to maintain their progenitor characteristics and supportive properties relative to axon growth even after long - term culture . it is now important to confirm that late passage grp maintain their permissiveness in vivo and support axon growth when transplanted into animal models of sci . differences in the phenotypic and the functional properties of grp using in vitro co - culture experiments with dorsal root ganglion ( drg ) neurons . both early and late passage grp were characterized by high expression of a2b5 and low expression of glial fibrillary acid protein ( gfap ) typical of an immature state . early and late passage grp promoted comparable axon growth relative to control cultures consisting of drg neurons alone . ( b ) grp treated with inflammatory factors and their ability to support axon growth . grp were exposed to various concentrations of lipopolysaccharide ( lps)/interferon gamma ( ifn ) for 24 hours and tested in co - culture experiments . grp treated with low concentrations of lps / ifn retained their axon growth supportive properties . in contrast , grp treated with high concentrations of lps / ifn showed a transient gfap expression that was followed by a loss of a2b5 expression , and displayed an attenuation of their axon growth supporting properties . traumatic injury to the spinal cord initiates an inflammatory cascade mediated by a multitude of signaling molecules in a stage - specific manner ( alexander and popovich , 2009 ; bowes and yip , 2014 ) . astrocytes respond to injury by undergoing a spectrum of morphological , biochemical , and functional alterations in a process known as reactive astrocytosis ( fitch and silver , 2008 ; sofroniew , 2009 ) . during this process , astrocytes become hypertrophic , upregulate gfap expression , and form a glial scar . the consequences of reactive astrocytosis depend on the location , severity , and temporal relationship to the injury , ranging from reversible alterations with preservation of cellular domains and tissue structure to , in severe cases , irreversible changes resulting in scar formation with permanent rearrangement of tissue structure . accordingly , there is growing evidence for the presence of different types of reactive astrocytes with diverse morphological , molecular , and functional properties ( bowes and yip , 2014 ; khakh and sofroniew , 2015 ) . given the dramatic effects of inflammatory factors on the properties of mature astrocytes , we examined how the inflammatory environment may directly affect the morphological , phenotypic , and functional properties of grp using the drg co - culture system ( figure 2 ) . we used a combination of lipopolysaccharide ( lps ) and interferon gamma ( ifn ) , maximizing the pro - inflammatory effects by signaling through two divergent pathways ( schroder et al . , 2006 ) . we found that grp exposed to either low or high concentrations of lps / ifn for 24 hours resulted in transient gfap expression , which returned to normal levels by 72 hours after treatment cessation ( hayakawa et al . , 2015 ) . although expression of gfap was transient and normalized following withdrawal of the inflammatory factors , the expression of a2b5 , a marker of grp , was significantly reduced in grp treated with high concentrations of lps / ifn. these results suggest that the exposure of grp to a severe pro - inflammatory microenvironment induces progressive changes in the phenotypic characteristics of these cells in a dose dependent manner . importantly , these phenotypic alterations were mirrored by the reduced ability of grp to support axon growth after exposure to high concentrations of lps / ifn as evaluated by direct co - culture assays ( figure 2b ) . these observations are consistent with reports demonstrating alterations in transcriptional profiles of astrocytes following lps exposure , consistent with reactive astrogliosis ( zamanian et al . , 2012 ) . they also highlight the importance of the microenvironment on the properties of transplanted cells and suggest that modulation of the injury environment may be important in creating more favorable conditions for cell transplantation . as we relate these results to our previous studies , which demonstrated that grp create a permissive environment when grafted in an acute sci model ( bonner et al . , 2011 2012 ) , we need to consider : 1 ) the dynamic nature of the injury environment characterized by a complex cascade of pro - inflammatory and anti - inflammatory signaling molecules , 2 ) the diversity of cells present and/or recruited in the injury site ( e.g. , resident microglia and recruited macrophages ) and their plasticity in response to inflammation that provides both beneficial and detrimental effects , and 3 ) the complex bi - directional interactions between grafted cells and the injured environment . while this complex environment is difficult to recreate in culture conditions , in vitro experiments serve as a model to define not only the direct interactions between cells , but also with signaling molecules , contributing to an understanding of the mechanism of the inflammatory process . the complexity of sci is underscored by changes that occur in distinct spatial and temporal patterns , variations in injury models and experimental design , and the need for combination therapy targeting multiple aspects of the injury . it is therefore important to continue research using not only in vitro systems to identify potential therapeutic candidates and evaluate their mechanism of action , but also in vivo models of sci to validate promising strategies with respect to functional recovery . our work with grp has emphasized the value of grp as a therapeutic platform in the repair of sci . the in vitro co - culture studies identified the axon growth - supporting properties of grp , the means to expand grp cultures to prepare sufficient stocks for transplantation experiments , and the direct impact of pro - inflammatory factors on the phenotypic and functional properties of these cells . subsequently , grp have been evaluated as a therapeutic strategy in animal models of sci , which confirmed the therapeutic potential of these cells and their derived astrocytes with respect to modulation of the injured environment , host axon growth , and synaptic connectivity . it is likely that grp transplants will be used as a therapeutic platform in combination with additional strategies to reconnect the injured nervous system , such as : 1 ) nrp , for relay formation , 2 ) trophic molecules , to enhance grp migration or guide axon regeneration , 3 ) neuronal modification , to enhance the intrinsic regenerative capacity of injured neurons , 4 ) anti - inflammatory agents , to limit the inflammatory microenvironment , 5 ) anti - scar agents , to reduce glial scarring in order to achieve more efficient cell survival and axonal regeneration , and 6 ) biomaterials , to create a niche capable of providing a more permissive environment for transplantation . finally , it is also important to note that the lessons and experience gained from sci studies could also be applied to the treatment of various other neurological diseases , such as amyotrophic lateral sclerosis ( als ) , where transplantation of grp and immature astrocytes could be used to provide neuroprotection and slow progression of the neurodegenerative process ( lepore et al . , 2008 ) .
backgroundthe close relationship between human health , performance , well - being and the thermal environment is obvious . nevertheless , most studies of climate and climate change impacts show amazing shortcomings in the assessment of the environment . populations living in different climates have different susceptibilities , due to socio - economic reasons , and different customary behavioural adaptations . the global distribution of risks of hazardous thermal exposure has not been analysed before.objectiveto produce maps of the baseline and future bioclimate that allows a direct comparison of the differences in the vulnerability of populations to thermal stress across the world.designthe required climatological data fields are obtained from climate simulations with the global general circulation model echam4 in t106-resolution . for the thermo - physiologically relevant assessment of these climate data a complete heat budget model of the human being , the perceived temperature procedure has been applied which already comprises adaptation by clothing to a certain degree . short - term physiological acclimatisation is considered via health related assessment of the thermal environment.resultsthe global maps 19711980 ( control run , assumed as baseline climate ) show a pattern of thermal stress intensities as frequencies of heat . the heat load for people living in warm humid climates is the highest . climate change will lead to clear differences in health - related thermal stress between baseline climate and the future bioclimate 20412050 based on the business - as - usual greenhouse gas scenario is92a . the majority of the world 's population will be faced with more frequent and more intense heat strain in spite of an assumed level of acclimatisation . further adaptation measures are crucial in order to reduce the vulnerability of the populations.conclusionsthis bioclimatology analysis provides a tool for various questions in climate and climate change impact research . considerations of regional or local scale require climate simulations with higher resolution . as adaptation is the key term in understanding the role of climate / climate change for human health , performance and well - being , further research in this field is crucial .	the heat exchange between the human body and the thermal environment can be described in the form of the energy balance equation , an application of the first theorem of thermodynamics applied to the body 's heat sources ( metabolism and environmental ) and the various avenues of heat loss to environment ( see method box 1 ) ( 24 ) . among the advanced heat budget models , fanger 's pmv - equation ( 6 , 26 ) can be considered as appropriate if gagge et al . 's ( 27 ) improvement in the description of latent heat fluxes by the introduction of pmv * is applied . together with a radiation model this approach combines the operational thermal assessment procedure klima michel-model ( kmm ) ( 2830 ) of the german national weather service deutscher wetterdienst ( dwd ) with the output parameter ( descriptive term ) perceived temperature , pt ( 31 ) . pt follows the pet approach ( 22 , 32 ) , standard effective temperature ( set ) ( 27 ) , or outdoor standard effective temperature ( out_set ) ( 33 , 34 ) in order to achieve a descriptive term in c . pt is defined as the air temperature of a standard environment ( wind calm , air temperature = mean radiant temperature , relative humidity ( rh ) = 50% , metabolic rate 2.3 met = 135 wm , which means walking at 4 kmh ) that would produce the same thermal stress as the actual environment ( table 1 ) . additionally , optimal behavioural adaptation by clothing ( along the scale from the thermal resistance of summer clothing ( 0.5 clo ) to winter clothing ( 1.75 clo ) ( 1 clo = 0.155 kmw ) is considered based on iso 9920 ( 35 ) . the pt scale ( table 1 ) refers to the ashrae - scale ( iso 7730 ) ( 26 ) extended to a nine - step scale . metabolic rate ( activity ) storage ( change in heat content of the body ) turbulent flux of sensible heat turbulent flux of latent heat ( diffusion of water vapour ) turbulent flux of latent heat ( sweat evaporation ) respiratory heat flux ( sensible and latent ) mean radiant temperature wind velocity relative to the body water vapour pressure the meteorological input variables include air temperature t a , water vapour pressure e , wind velocity v and mean radiant temperature t mrt including short and long - wave radiation fluxes , in addition to metabolic rate and clothing insulation . in eq . ( 1 ) the appropriate meteorological variables are attached to the relevant energy fluxes in w / m . the physiological ( internal ) variables , such as the temperature of the core or the skin , the sweat rate , and the skin wetness , which all interact with the environmental heat conditions , are not mentioned here . an overview on the basics in thermo - physiology and heat exchange modelling with respect to adaptation is given e.g. in ( 25 ) . perceived temperature , predicted mean vote ( according to ( 6 ) ) , and corresponding thermal stress ( initial conditions without acclimatisation ; negative values are heat load ) the pt procedure incorporates the acclimatisation approach health related assessment of the thermal environment ( herate ) ( 36 ) . herate is a conceptual model of short - term acclimatisation based on findings in adaptation studies . the procedure modifies the absolute pt thresholds of table 1 by superimposing the ( relative ) historic experience of the population in terms of pt of the previous weeks . 2 shows how the actual thresholds for the different thermal stress categories change from the initial values of table 1 . this procedure has the advantage that the index can be used without modification in different climate regions and during different times of the year without the need to artificially define seasons and calibrate it to a particular locale . example of daily values of perceived temperature at 12 utc ( black line ) for the weather station lisbon ( portugal ) from march to september 2003 ( 36 ) . compared to table 1 the coloured lines indicate the variable thresholds for the different thermal stress categories specifying acclimatisation . for global bioclimate maps however , the necessary meteorological input data for the global scale can be taken either from global gcms or from reanalyses of numerical weather prediction ( nwp ) models ( e.g. european centre for medium - range weather forecasts ( ecmwf ) , reading / uk , or national centers for environmental prediction ( ncep ) , camp springs / usa ) . in this investigation we use two runs of the global gcm echam4 ( 38 ) at the relatively high resolution t106 ( 39 ) , about 100 km grid - point distance in middle latitudes ( see method box 2 ) . due to restricted computer power , only two time slices with a length of 10 years each are available in the high - resolution version of the gcm . in the first experiment ( control run ) the concentration of greenhouse gases is set constant at the observed level 19711980 ( recent climate conditions ) . in the second run the concentration of greenhouse gases is taken from the ipcc - scenario is92a business - as - usual ( 40 ) for the period 20412050 . this scenario is between the two middle range scenarios a1b and a2 used in the report of the intergovernmental panel on climate change ( ipcc ) ( 41 ) . using the model output data of echam4/t106 , the values of pt were calculated for the different time periods for each land - grid point and four universal times ( utc ) at 00.00 , 06.00 , 12.00 and 18.00 . in order to obtain comparable pt values , the predominant effect of the daily thermal variation was considered by recalculating corrected pt values for 12 mean local time ( mlt ) for all grid points by a nonlinear interpolation procedure centred around the four neighbouring pt values at the given utc times . for the given six - hour intervals the maximum mean error of this procedure is in the order of 1 k ( = 1c ) , which was tested with observational data of a couple of european weather stations with a time resolution of one hour . the global gcm echam4 applies the relatively high resolution t106 , about 100 km grid - point distance in middle latitudes ( 39 ) . in both experiments ( time slices ) , the surface of the earth is represented by 320160 grid points which means a distance of about 1.1 in zonal and meridional direction . the gcm - data are available at fixed times 00.00 , 06.00 , 12.00 and 18.00 universal time ( utc ) . the meteorological variables required for the assessment are air temperature t a , dew point t d ( to derive water vapour pressure ) and wind velocity v , which are directly used to calculate the sensible and latent heat flux . in addition to these , for the parameterisation of the long - wave and short - wave radiant fluxes , cloudiness information such as total cloud cover n and cloud cover in the different tropospheric levels ( low , middle and high ) are used to calculate mean radiant temperature tmrt . the maps of pt values are related to people staying outdoors at noon ( 12 mlt ) . because ptmax usually occurs one or two hours later , the pt value at 12 mlt can be considered as a mean value over a certain time period starting some time before 12 mlt until some time after the same pt value as at 12 mlt occurs once more in the afternoon . from these 12 mlt pt - time - series at every land - grid point the mean annual frequency ( number of days p.a . ) of comfort conditions ( no thermal stress ) or different intensities of thermal stress , respectively , has been derived for the two echam4 time slices 19711980 and 20412050 , taking acclimatisation into account according to herate . the actual pt value for a certain thermal stress level at a given point and a given date always depends on the previous thermal conditions . the difference between the time periods ( 20412050 ) and ( 19711980 ) can be interpreted as the change in the bioclimate . the heat exchange between the human body and the thermal environment can be described in the form of the energy balance equation , an application of the first theorem of thermodynamics applied to the body 's heat sources ( metabolism and environmental ) and the various avenues of heat loss to environment ( see method box 1 ) ( 24 ) . among the advanced heat budget models , fanger 's pmv - equation ( 6 , 26 ) can be considered as appropriate if gagge et al . 's ( 27 ) improvement in the description of latent heat fluxes by the introduction of pmv * is applied . together with a radiation model this approach combines the operational thermal assessment procedure klima michel-model ( kmm ) ( 2830 ) of the german national weather service deutscher wetterdienst ( dwd ) with the output parameter ( descriptive term ) perceived temperature , pt ( 31 ) . pt follows the pet approach ( 22 , 32 ) , standard effective temperature ( set ) ( 27 ) , or outdoor standard effective temperature ( out_set ) ( 33 , 34 ) in order to achieve a descriptive term in c . pt is defined as the air temperature of a standard environment ( wind calm , air temperature = mean radiant temperature , relative humidity ( rh ) = 50% , metabolic rate 2.3 met = 135 wm , which means walking at 4 kmh ) that would produce the same thermal stress as the actual environment ( table 1 ) . additionally , optimal behavioural adaptation by clothing ( along the scale from the thermal resistance of summer clothing ( 0.5 clo ) to winter clothing ( 1.75 clo ) ( 1 clo = 0.155 kmw ) is considered based on iso 9920 ( 35 ) . the pt scale ( table 1 ) refers to the ashrae - scale ( iso 7730 ) ( 26 ) extended to a nine - step scale . metabolic rate ( activity ) storage ( change in heat content of the body ) turbulent flux of sensible heat turbulent flux of latent heat ( diffusion of water vapour ) turbulent flux of latent heat ( sweat evaporation ) respiratory heat flux ( sensible and latent ) mean radiant temperature wind velocity relative to the body water vapour pressure the meteorological input variables include air temperature t a , water vapour pressure e , wind velocity v and mean radiant temperature t mrt including short and long - wave radiation fluxes , in addition to metabolic rate and clothing insulation . in eq . ( 1 ) the appropriate meteorological variables are attached to the relevant energy fluxes in w / m . the physiological ( internal ) variables , such as the temperature of the core or the skin , the sweat rate , and the skin wetness , which all interact with the environmental heat conditions , are not mentioned here . an overview on the basics in thermo - physiology and heat exchange modelling with respect to adaptation is given e.g. in ( 25 ) . perceived temperature , predicted mean vote ( according to ( 6 ) ) , and corresponding thermal stress ( initial conditions without acclimatisation ; negative values are heat load ) the pt procedure incorporates the acclimatisation approach health related assessment of the thermal environment ( herate ) ( 36 ) . herate is a conceptual model of short - term acclimatisation based on findings in adaptation studies . the procedure modifies the absolute pt thresholds of table 1 by superimposing the ( relative ) historic experience of the population in terms of pt of the previous weeks . 2 shows how the actual thresholds for the different thermal stress categories change from the initial values of table 1 . this procedure has the advantage that the index can be used without modification in different climate regions and during different times of the year without the need to artificially define seasons and calibrate it to a particular locale . example of daily values of perceived temperature at 12 utc ( black line ) for the weather station lisbon ( portugal ) from march to september 2003 ( 36 ) . compared to table 1 the coloured lines indicate the variable thresholds for the different thermal stress categories specifying acclimatisation . however , the necessary meteorological input data for the global scale can be taken either from global gcms or from reanalyses of numerical weather prediction ( nwp ) models ( e.g. european centre for medium - range weather forecasts ( ecmwf ) , reading / uk , or national centers for environmental prediction ( ncep ) , camp springs / usa ) . in this investigation we use two runs of the global gcm echam4 ( 38 ) at the relatively high resolution t106 ( 39 ) , about 100 km grid - point distance in middle latitudes ( see method box 2 ) . due to restricted computer power , only two time slices with a length of 10 years each are available in the high - resolution version of the gcm . in the first experiment ( control run ) the concentration of greenhouse gases is set constant at the observed level 19711980 ( recent climate conditions ) . in the second run the concentration of greenhouse gases is taken from the ipcc - scenario is92a business - as - usual ( 40 ) for the period 20412050 . this scenario is between the two middle range scenarios a1b and a2 used in the report of the intergovernmental panel on climate change ( ipcc ) ( 41 ) . using the model output data of echam4/t106 , the values of pt were calculated for the different time periods for each land - grid point and four universal times ( utc ) at 00.00 , 06.00 , 12.00 and 18.00 . in order to obtain comparable pt values , the predominant effect of the daily thermal variation was considered by recalculating corrected pt values for 12 mean local time ( mlt ) for all grid points by a nonlinear interpolation procedure centred around the four neighbouring pt values at the given utc times . for the given six - hour intervals the maximum mean error of this procedure is in the order of 1 k ( = 1c ) , which was tested with observational data of a couple of european weather stations with a time resolution of one hour . the global gcm echam4 applies the relatively high resolution t106 , about 100 km grid - point distance in middle latitudes ( 39 ) . in both experiments ( time slices ) , the surface of the earth is represented by 320160 grid points which means a distance of about 1.1 in zonal and meridional direction . the gcm - data are available at fixed times 00.00 , 06.00 , 12.00 and 18.00 universal time ( utc ) . the meteorological variables required for the assessment are air temperature t a , dew point t d ( to derive water vapour pressure ) and wind velocity v , which are directly used to calculate the sensible and latent heat flux . in addition to these , for the parameterisation of the long - wave and short - wave radiant fluxes , cloudiness information such as total cloud cover n and cloud cover in the different tropospheric levels ( low , middle and high ) are used to calculate mean radiant temperature tmrt . the maps of pt values are related to people staying outdoors at noon ( 12 mlt ) . because ptmax usually occurs one or two hours later , the pt value at 12 mlt can be considered as a mean value over a certain time period starting some time before 12 mlt until some time after the same pt value as at 12 mlt occurs once more in the afternoon . from these 12 mlt pt - time - series at every land - grid point the mean annual frequency ( number of days p.a . ) of comfort conditions ( no thermal stress ) or different intensities of thermal stress , respectively , has been derived for the two echam4 time slices 19711980 and 20412050 , taking acclimatisation into account according to herate . the actual pt value for a certain thermal stress level at a given point and a given date always depends on the previous thermal conditions . the difference between the time periods ( 20412050 ) and ( 19711980 ) can be interpreted as the change in the bioclimate . the map of the frequency distribution of thermal comfort ( i.e. no thermal stress ) 19711980 shows that the mid - latitude regions are most comfortable , in particular in the maritime affected areas ( fig . this is true for western europe , new zealand , the southern parts of australia , chile and argentina as well as a narrow coast strip in the west of north - america where 300 days with thermal comfort at 12 mlt can be taken as typical . similar conditions can be found in higher elevations of the andes and in the area of the foothills of the himalayas . with increasing continental influence in mid - latitudes or increasing subtropical influence , the number of days with comfort conditions decreases considerably . in the lowlands of the humid tropics thermal comfort this is also true for the ice shield regions of antarctica and greenland due to the limit to maximum clothing insulation of 1.75 clo which is used here . mean annual frequency of comfortable conditions ( no thermal stress ) at 12 mlt ( = noon ) taking acclimatisation into account based on echam4/t106-data 19711980 . the general relationship of heat load conditions to latitude ( solar climate ) is evident ( fig . heat load is the predominant thermal state in the tropics throughout the year . with increasing latitude the heat load probability declines , but even in moderate climates a considerable number of days with heat load ( a d ) mean annual frequency of exceeding the threshold for ( a ) heat load , ( b ) moderate heat load , ( c ) strong heat load and ( d ) extreme heat load at 12 mlt taking acclimatisation into account , based on echam4/t106-data 19711980 . from a health point of view , higher heat load intensities become more relevant . 4b ) is of course smaller and now almost covers those regions of the world known for tropical or subtropical climate . in the equator region it is interesting , for example , that saudi arabia shows the most days with extreme heat load ( > 170 days ) while the threshold for at least strong heat load is more frequently exceeded in the amazon basin and in indonesia ( > 300 days ) . the zonal variation in the thermal conditions is also found in the distribution of cold stress ( in spite of winter clothing with 1.75 clo! ; fig . while almost every day cold stress occurs in antarctica , followed by less than one - third of the time span of a year over the southern tip of south - america ( tierra del fuego ) , the northern hemisphere shows a more differentiated pattern due to superimposed topography and position relative to the sea . the frequency of cold stress in the tropical and subtropical regions is zero or close to that . when the thresholds for cold stress are tightened in the given definitions ( fig . extreme cold stress can only be found some distance away from the coast over the ice shields of antarctica almost every day ( fig . 5d ) . in central greenland , the most extreme region in the northern hemisphere , with respect to health consequences it is interesting to note that the regions showing at least moderate cold stress ( fig . ( a d ) mean annual frequency of exceeding the threshold for ( a ) cold stress , ( b ) moderate cold stress , ( c ) strong cold stress and ( d ) extreme cold stress at 12 mlt taking acclimatisation into account , based on echam4/t106-data 19711980 . the spatial distribution of the thermal conditions across europe looks very similar to maps based on data from 918 european weather stations 1971/2000 ( 20 , 21 ) . the echam4/t106 simulation of the climate in the period 20412050 ( future ) uses the ipcc is92a business - as - usual scenario . difference of the mean annual frequency of comfortable conditions ( no thermal stress ) at 12 mlt between future and recent climate taking acclimatisation into account , based on echam4/t106-data ( 20412050 and 19711980 ) . ( a d ) difference of the mean annual frequency of exceeding the threshold for ( a ) heat load , ( b ) moderate heat load , ( c ) strong heat load and ( d ) extreme heat load at 12 mlt taking acclimatisation into account , based on echam4/t106-data ( 20412050 and 19711980 ) . ( a d ) difference of the mean annual frequency of exceeding the threshold for ( a ) cold stress , ( b ) moderate cold stress , ( c ) strong cold stress and ( d ) extreme cold stress at 12 mlt taking acclimatisation into account , based on echam4/t106-data ( 20412050 and 19711980 ) . both the humid tropics and antarctica are characterised by already lacking comfort conditions ( permanent heat load or cold stress conditions , respectively ) ( fig . 6 ) , and these will remain . the most pronounced decrease in comfort will happen in the subtropics up to the middle latitudes ( moderate climate ) . an increase in comfortable conditions is mainly limited to cold areas with low population density , maybe with the exception of southern scandinavia , parts of russia , china , the northwest of the usa , and british columbia in canada . the distribution of the changes of the annual frequencies of heat load ( fig . 7a 7b ) as these are already exceeded . there is a considerable increase in heat load in the subtropics and higher latitudes . while the affected areas are becoming smaller with tightening thresholds although the characteristics are different , almost all densely populated areas are affected by the increase of adverse thermal conditions due to climate change . as is nowadays the case , no cold stress will occur in the tropics and subtropics also in a future climate ( fig . d ) . the most densely populated areas will never be affected by extreme or even strong cold stress . in moderate climates a considerable reduction in the frequency of cold stress the at least moderate cold stress situation in the area of the great lake district in north - america , scandinavia and russia will be improved ( fig . 8b ) while a significant decrease in stronger cold stress will occur in practically non - populated regions ( fig . 8c and d ) . the map of the frequency distribution of thermal comfort ( i.e. no thermal stress ) 19711980 shows that the mid - latitude regions are most comfortable , in particular in the maritime affected areas ( fig . this is true for western europe , new zealand , the southern parts of australia , chile and argentina as well as a narrow coast strip in the west of north - america where 300 days with thermal comfort at 12 mlt can be taken as typical . similar conditions can be found in higher elevations of the andes and in the area of the foothills of the himalayas . with increasing continental influence in mid - latitudes or increasing subtropical influence , the number of days with comfort conditions decreases considerably . in the lowlands of the humid tropics thermal comfort this is also true for the ice shield regions of antarctica and greenland due to the limit to maximum clothing insulation of 1.75 clo which is used here . mean annual frequency of comfortable conditions ( no thermal stress ) at 12 mlt ( = noon ) taking acclimatisation into account based on echam4/t106-data 19711980 . the general relationship of heat load conditions to latitude ( solar climate ) is evident ( fig . heat load is the predominant thermal state in the tropics throughout the year . with increasing latitude the heat load probability declines , but even in moderate climates a considerable number of days with heat load ( a d ) mean annual frequency of exceeding the threshold for ( a ) heat load , ( b ) moderate heat load , ( c ) strong heat load and ( d ) extreme heat load at 12 mlt taking acclimatisation into account , based on echam4/t106-data 19711980 . from a health point of view , higher heat load intensities become more relevant . 4b ) is of course smaller and now almost covers those regions of the world known for tropical or subtropical climate . in the equator region it is interesting , for example , that saudi arabia shows the most days with extreme heat load ( > 170 days ) while the threshold for at least strong heat load is more frequently exceeded in the amazon basin and in indonesia ( > 300 days ) . the zonal variation in the thermal conditions is also found in the distribution of cold stress ( in spite of winter clothing with 1.75 clo! ; fig . while almost every day cold stress occurs in antarctica , followed by less than one - third of the time span of a year over the southern tip of south - america ( tierra del fuego ) , the northern hemisphere shows a more differentiated pattern due to superimposed topography and position relative to the sea . the frequency of cold stress in the tropical and subtropical regions is zero or close to that . when the thresholds for cold stress are tightened in the given definitions ( fig . extreme cold stress can only be found some distance away from the coast over the ice shields of antarctica almost every day ( fig . 5d ) . in central greenland , the most extreme region in the northern hemisphere , with respect to health consequences it is interesting to note that the regions showing at least moderate cold stress ( fig . ( a d ) mean annual frequency of exceeding the threshold for ( a ) cold stress , ( b ) moderate cold stress , ( c ) strong cold stress and ( d ) extreme cold stress at 12 mlt taking acclimatisation into account , based on echam4/t106-data 19711980 . the spatial distribution of the thermal conditions across europe looks very similar to maps based on data from 918 european weather stations 1971/2000 ( 20 , 21 ) . the echam4/t106 simulation of the climate in the period 20412050 ( future ) uses the ipcc is92a business - as - usual scenario . figs . difference of the mean annual frequency of comfortable conditions ( no thermal stress ) at 12 mlt between future and recent climate taking acclimatisation into account , based on echam4/t106-data ( 20412050 and 19711980 ) . ( a d ) difference of the mean annual frequency of exceeding the threshold for ( a ) heat load , ( b ) moderate heat load , ( c ) strong heat load and ( d ) extreme heat load at 12 mlt taking acclimatisation into account , based on echam4/t106-data ( 20412050 and 19711980 ) . ( a d ) difference of the mean annual frequency of exceeding the threshold for ( a ) cold stress , ( b ) moderate cold stress , ( c ) strong cold stress and ( d ) extreme cold stress at 12 mlt taking acclimatisation into account , based on echam4/t106-data ( 20412050 and 19711980 ) . both the humid tropics and antarctica are characterised by already lacking comfort conditions ( permanent heat load or cold stress conditions , respectively ) ( fig . 6 ) , and these will remain . the most pronounced decrease in comfort will happen in the subtropics up to the middle latitudes ( moderate climate ) . an increase in comfortable conditions is mainly limited to cold areas with low population density , maybe with the exception of southern scandinavia , parts of russia , china , the northwest of the usa , and british columbia in canada . the distribution of the changes of the annual frequencies of heat load ( fig . 7a there is a considerable increase in heat load in the subtropics and higher latitudes . while the affected areas are becoming smaller with tightening thresholds although the characteristics are different , almost all densely populated areas are affected by the increase of adverse thermal conditions due to climate change . as is nowadays the case , no cold stress will occur in the tropics and subtropics also in a future climate ( fig . d ) . the most densely populated areas will never be affected by extreme or even strong cold stress . in moderate climates a considerable reduction in the frequency of cold stress the at least moderate cold stress situation in the area of the great lake district in north - america , scandinavia and russia will be improved ( fig . 8b ) while a significant decrease in stronger cold stress will occur in practically non - populated regions ( fig . 8c and d ) . climate modelling provides global - wide time series of grid data for every day at several ( here four ) hourly time points , and here we used echam4 time - slice modelling assuming the business - as - usual green house gas scenario . in order to estimate impacts of climate or climate change on human health and well - being , a specific physiologically relevant assessment of the climate data this assessment must be based on heat exchange theory taking all mechanism of heat exchange into account , which results in heat budget models such as the pt procedure . such models have the ability to correctly assess the climatological variables : air temperature , mean radiant temperature , wind velocity and water vapour pressure . it is likely that our assessment procedure can be applied across the world because thermo - physiology functions are fundamentally the same in all populations . at the population level ( neglecting age , gender and inter - individual differences ) the susceptibility to thermal stress is mainly controlled by physiological and behavioural adaptation . the psychological aspects of how people assess and prefer climate sensation ( see e.g. 42 , 43 ) are not subject of this study . thermal comfort in our study stands for no stress which means no physiological strain . societies have always adapted their cultures to meet the climate requirements ( 11 , 44 ) . clothing ( see e.g. adaptive model by 45 , 46 ) , building design , food and drinking habits , working hours ( siesta ) , avoiding outdoors activities during the hottest time of the day , lowering metabolic rate , etc . if acclimatisation were perfect no adverse health effects of climate would be expected , but numerous publications report health impacts of climate indicating that societies are not able to adapt completely . acclimatisation ( = adaptation to climate ) is here considered firstly by assuming reasonable behaviour of the target group general population with respect to clothing . the range of clo - values between 0.5 and 1.75 refers to moderate climate experience . although there is some evidence that in cold winter climates the general population is not used to wearing more protective clothing thus limiting obviously their time for staying outdoors , this is certainly not true for people ( not considered here ) with particular activities such as soldiers , rangers , hunters , skiers who are able to avoid any cold stress by appropriate special clothing . typical inuit clothing ( 4.0 clo ) would actually produce approximate comfort under the predominant conditions in extreme cold winter climates . shorts instead of jeans as often used by people in the tropics reduce the clo - value to 0.4 which would slightly reduce heat load . secondly , the acclimatisation approach herate adjusts the initial thermal thresholds automatically to the prevailing local climate . in the warm humid tropics and in the hot subtropics this often results in an increase of the thresholds for the different heat load intensities of 68c . however , possible long - term ( over years and decades ) acclimatisation has not been considered due to the lack of a quantitative approach . additionally the assessment procedure focuses on the population level which means people walking outdoors with a given metabolic rate of 2.3 met ( 135 wm ) . reducing the met activity could be assumed under heat load conditions as an adaptation measure of the population if no specific work intensities are required . on the other hand there are many outdoor working conditions requiring much higher metabolic rates . in tropical and sub - tropical climates the obstruction of heat release from the body can very quickly produce extreme heat load conditions in a working individual which significantly differ from the assessments in our analysis for an assumed general public . with the help of pt and herate , the current and predicted thermal environment is described in health - related frequencies of exceeding locally adapted thresholds based on daily values at a fixed time ( noon = 12 mlt ) which is representative for a few hours around noon . this makes a big difference to the usual consideration of climate and climate change based on monthly or annual mean values of air temperature . in terms of both comfort and the absence of intense thermal stress , privileged while comfortable or only slight heat load conditions only rarely occur in the humid tropics . the distribution of heat load follows the solar climate to a great extent , while it is modified by maritime or continental influences and altitude . in the tropics and subtropics a huge number of human beings are affected by heat stress , particularly in asia and africa . in tropical and sub - tropical areas , where extreme heat load is already common extreme with significant impacts on health and well - being of populations living in these areas . of course these societies apply some behavioural adaptation measures but the dramatically increased and from a greenhouse gas point of view contra productive use of air - conditioning systems in these countries by those who have the financial capabilities indicates that they are not satisfied with the existing climate . the problem increases because in rapidly growing cities traditional buildings which relied on local experience in climate - related building design , is no longer realised . in addition , the urban heat island effect in large cities is already greater than the predicted climate change until the end of this century , and the public health risks are significant . when considering the fact that ( a ) almost the entire population of the world lives in areas indicated by yellow or red colours in the presentation of the changes ( see fig . 7c ) and ( b ) already exceeding the threshold for moderate heat load would increase mortality ( at least based on studies in moderate climates ) , the public health issue of this aspect of climate change becomes evident . this would also be true if , due to an underestimation of the effect of acclimatisation in the applied procedure , the thresholds for the different heat load intensities were actually somewhat higher . the predominant part of the world 's population will belong to the climate losers, being faced with more frequent and more intense adverse thermal conditions , while few countries or areas will belong to the climate winners, a view on the problem that first was introduced by auliciems in 1994 ( 47 ) . the described assessment procedure can also be applied to regional climate models ( see e.g. 48 ) are nested into global models , thus providing bioclimatological conditions with higher resolution ( down - scaling ) . further improvements of these assessments can be expected by applying the almost finalised universal thermal climate index ( utci ; ( cost 730 ) ( 49 ) ) , which is based on human response - related thermo - physiological modelling with the help of a multi - segmental , multi - layered representation of the human body ( 50 , 51 ) , to the new generation of climate simulation models including down - scaling . the application of the thermo - physiological assessment procedure pt to climate data broadens the usual consideration of climate maps presenting only single climate data such as air temperature . the approach results in a spatial assessment of thermal stress categories which relate climate to the health and well - being of humans . this provides specific problem - oriented information to the climate and climate change , and human health relationships in general and for the estimation of the vulnerability of different populations . optimal conditions in terms of no or at most slight thermal stress differ significantly between the individual continents . privileged while particularly africa and australia suffer from heat load with highest frequency at the extremes . asia and both the americas lie approximately in between . in the predicted future climate , based on the echam4 simulations , the most pronounced changes will occur at the extreme heat load category which will then become the most frequent condition for america as it is already now for africa and australia . favourable conditions will decrease basically all over the world . a better understanding of acclimatisation as the adaptation to climate and climate change in different time scales remains an important research issue . the authors have not received any funding or benefits from industry to conduct this study .
background : the palmaris longus is a degenerating weak flexor muscle in the anterior of the forearm . many techniques for clinically determining the presence of the palmaris longus have been described . ethnic variations in the prevalence of the absence of the palmaris longus are well known.objectives:this study considered the prevalence of absence of the palmaris longus muscle tendon in the north of iran.patients and methods : the presence of the palmaris longus was clinically determined in 562 men and women from the guilan population , using the standard technique ( schaeffer s test ) . in subjects with an absent palmaris longus , three other tests ( thompson , pushpakumar and mishra tests ) were performed to confirm the absence.results:the overall prevalence of right , left , bilateral and total absence of the palmaris longus were 4.1% , 5.2% , 3.9% and 13.2% , respectively . there was no significant difference in its absence with regard to the body side or gender ( p > 0.05).conclusions : this study demonstrated that the presence of the palmaris longus muscle tendon in the guilan population was considerably higher than the absence of the palmaris longus tendon . the overall prevalence of right , left , bilateral and total absence of the palmaris longus was not significantly different between men and women . the prevalence of the left - absent palmaris longus was more common in the present study .	the palmaris longus muscle is one of the flexor muscles of the forearm that originates from the medial epicondyle of the humerus , terminates on the flexor retinaculum and contributes to the palmar fascia . although in the upper limbs its function is considered insignificant , in the event of tendon grafting , it is considerably important . in the anatomical and surgical texts , occurrence of absence of the said muscle is reported to be about 15% ( 1 , 2 ) . but this prevalence level may not be an indicator for all the world populations . in most races , variation of the occurrence of absence of the palmaris longus muscle has been reported ( 3 ) . reported the prevalence of absence of the palmaris longus muscle ( 30.7% ) in the south of iran ( 4 ) . in another study , the prevalence of palmaris longus muscle agenesis in tehran ( the capital of iran ) was reported as 22.8% in medical students ( 5 ) , and in an additional study this prevalence was reported as 21.0% in patients referred to one health center ( 6 ) . however , no report has been presented in relation to the occurrence of absence of palmaris longus muscle in north of iran . therefore , considering the importance of providing anatomic data that could serve to enhance clinical applications , the aim of this study is to determine the prevalence of agenesis of the palmaris longus muscle and its association with gender in the guilan population in the north of iran . this study was performed on 1,124 upper limbs in 562 consecutive outpatients of the orthopedic ward of poursina hospital in rasht . the patients , who had a record of surgery , abnormality and inborn deformation of the upper limbs , and also children under the age of 10 , were excluded . for examining the palmaris longus muscle tendon , standard clinical methods were used . after explaining the study aim to the patients , they were asked to lay their thumb against their little finger and at the same time flex their wrist ( schaeffer s test ) . in this state , the palmaris longus muscle tendon in the front of the wrist will be raised under the skin ( figure 1a ) . if in this test the tendon is not observable or touchable , other tests such as pushpakumar s test ( figure 1b ) , thomson s test ( figure 1c ) and mishra s test ii ( figure 1d ) ( 7 - 10 ) were used for confirmation of the muscle absence . with these methods , existence or absence of the palmaris longus muscle tendon in the two upper limbs were registered . in this study , occurrence of absence of the palmaris longus muscle tendon in men and women , and symmetry of the right and left side were considered . the categorical variables were compared using the chi - squared test . a p value of < 0.05 was considered statistically significant . informed consent was obtained from each patient included in the study , and the study protocol conformed to the ethical guidelines of the 1975 declaration of helsinki . the age of the patients ranged from 15 to 70 year old ; the average age being 35 years . the overall prevalence of palmaris longus agenesis , according to gender and limb results , is summarized in table 1 . ( % ) . out of a total of 562 patients examined , at least one palmaris longus tendon was present in 540 patients ( 96.1% ) , and both the tendons were present in 488 ( 86.8% ) . out of those with unilateral agenesis , 29 ( 5.2% ) had left - sided agenesis and 23 ( 4.1% ) had right - sided agenesis . hence , agenesis was seen more commonly on the left side than on the right , and the difference was statistically significant ( p < 0.001 ) . on the whole , female subjects had a prevalence of agenesis of the palmaris longus tendon ( unilateral and bilateral combined ) of 36 out of 281 ( 12.8% ) , while in male subjects this prevalence was 38 out of 281 ( 13.5% ) . although the proportions of patients with unilateral and bilateral agenesis were comparable ( 22 patients or 3.9% had bilateral agenesis and 52 patients or 9.3% had unilateral agenesis , p=0.759 ) , the pattern of agenesis of the palmaris longus was different in the two genders . in male subjects , 26 ( 68.4% ) were unilateral , compared to 12 ( 31.6% ) who were bilateral ( p = 0.076 ) . female subjects had unilateral absence of the palmaris longus more commonly ; 26 ( 72.2% ) were unilateral , compared to 10 ( 27.8% ) who were bilateral ( p = 0.040 ) . this implies that there is a 31.6% probability of a male subject with one - sided agenesis to have agenesis on the opposite side as well . in contrast , if the palmaris longus tendon in a female subject is absent on one side , there is a 27.8% chance that it will also be absent on the other side . the palmaris longus is considered one of the superficial flexor muscles of the forearm and one of the most variable muscles of the body . most researchers consider this muscle equivalent to the plantaris muscle in the lower limbs . the palmaris longus muscle is well developed in the animal species , which tolerates more weight on the upper limbs than other species . but in the human species , in which the role of the upper limbs in toleration of body weight has been decreased , the palmaris longus muscle is less developed and uncompleted ( 11 ) . despite the fact that this muscle exists in humans , its role in hand function is decreased , and in most cases of limb surgery it is used as a tendon graft ( 12 ) . various studies have shown that absence of the palmaris longus muscle will create no disorder in hand function ; therefore , in necessary cases , the tendon of this muscle is used in tendon grafting without fear that patients will have decreased ability to clench their fist or perform other hand functions . the ability to estimate palmaris longus muscle absence in different population groups is an added value to most surgeons around the globe , wherever they are increasingly dealing with patients of different ethnic backgrounds . compared with the other muscles , because of its unique specifications including length and raised nature , the palmaris longus muscle tendon is well observable and available and has been studied by researchers more frequently than others ( 13 ) . absence of the palmaris longus muscle has been the subject of studies performed by many researchers in living and deceased people , and the occurrence percentage was variable in different populations and races . in the current research , absence of the palmaris longus muscle , both unilaterally and bilaterally , in the guilan population in the north of iran has been studied . in addition , the prevalence of absence of the muscle was compared between the two genders . in this study , the overall prevalence of absence of the palmaris longus muscle was 13.2% , which was lower than three studies performed by karimi et al . ( 4 ) ( 13.2% vs. 30.7% , p < 0.001 ) , ashouri et al . ( 5 ) ( 13.2% vs. 22.8% , p < 0.001 ) and kamrani et al . ( 6 ) ( 13.2% vs. 21% , p = 0.002 ) in iran ( 3 - 5 ) . compared to other studies , the prevalence of palmaris longus absence in serbia ( 22.4% and 37.5% in two studies ( 14,15 ) ) , america ( 25.0% , ( 16 ) ) , turkey ( 26.6% , ( 17 ) ) , india ( 17.2% to 28.0% in three studies in indian population ( 7 , 18 , 19 ) ) and bahrain ( 36.4% , ( 20 ) ) was higher than our result . other studies showed a lower value such as 1.5% in zimbabwe ( 21 ) , 4.1% in korea ( 22 ) , 5.5% in japanese subjects ( 23 ) , 6% in chinese subjects ( 3 ) , 7.1% in native american subjects ( 1 ) , 11.3% in malaysia ( 13 ) and 11.5% in a south african population ( 24 ) . therefore , it can be said that the study of one population can not be considered for another population , and absence of the palmaris longus muscle is dependent on race ( 3 , 25 ) . most researchers reported occurrence of palmaris longus absence in women more than in men and in the left side more than in the right side ( 13 ) . but our findings showed absence of the palmaris longus muscle in men was not significantly higher than in women ( 51.4% vs. 48.6% , p > 0.05 ) , and in the left side was not - significantly higher than in the right side ( 5.2% vs. 4.1% , p > 0.05 ) . also , there was not a significant difference between the two genders in most previous studies , which is concordant with our findings ( table 1 ) . one limitation of our study was that the presence of a palmaris longus muscle ( left , right and bilateral existence ) was determined by clinical exam , which can be examiner dependent , rather than cadaveric dissection . to mitigate this issue
il-33 is a multifunctional cytokine that is released in response to a variety of intrinsic and extrinsic stimuli . the role of il-33 in candida albicans infections is just beginning to be revealed . this cytokine has beneficial effects on host defense against systemic c. albicans infections , and it promotes resistance mechanisms by which the immune system eliminates the invading fungal pathogens ; and it also elevates host tolerance by reducing the inflammatory response and thereby , potentially , tissue damage . thus , il-33 is classified as a cytokine that has evolved functionally to protect the host from damage by pathogens and immunopathology .	candida albicans is a major commensal fungus residing in the mucosal surfaces of our bodies . it is mainly found in the gastrointestinal tract , skin and genital tract . when the mucosal barrier is disturbed , c. albicans penetrates into the epithelium and causes systemic infection . conditions associated with immunocomprimise , particularly neutropenia , are a major risk factor for invasive candidiasis ( 1 ) . this deep - seated infection occurs , as hyphae spread to internal organs , among which the kidney is the major target ( 2 ) . the mortality rate of patients with systemic candidiasis exceeds 30~40% even when potent antifungal agents are administered ( 3 ) . like many other pathogens , the host defense against invasive c. albicans infections is a function of immune detection and elimination of the pathogen ( 1 ) . this defense strategy is referred to as resistance ( 4 ) . however , mortality of the host following c. albicans infection may result largely from immunopathology , in which the immune response against c. albicans inflicts tissue damage . mice are known to tolerate high fungal burdens , even though uncontrolled fungal proliferation is lethal . thus , it is important to understand defense mechanisms that reduce host susceptibility to tissue damage caused by fungal infection or related immunopathologies , but that do not affect the fungal burden . , we discuss the role of il-33 in host resistance and tolerance to systemic c. albicans infections . il-33 is a member of the il-1 family of cytokines that delivers its signaling through st2 receptors ( 5 ) . endothelial cells and epithelial cells rapidly produce il-33 in response to a variety of intrinsic and environmental insults ( 6 ) . however , there are many sources of il-33 , and the kinetics of il-33 production vary across types of cells and stimuli ( 7 ) . interestingly , the il-33 receptor , st2 , is expressed on virtually all types of immune cells and numerous types of nonhematopoietic cells , suggesting that it exhibits a functional diversity ( 8) . il-33 function is context - dependent and can have opposing effects ( e.g. , inflammatory or anti - inflammatory ) on an immune response . for example , il-33 initiates type 2 immune responses through secretion of il-4 , il-5 and/or il-13 by th2 cells and type 2 innate lymphoid cells ( ilc2s ) ( 9 ) . il-33 is also associated with autoimmune and inflammatory diseases mediated by th1 and th17 cells ( 6 ) . in addition , anticancer and antiviral effects of il-33 require cd8 t cells , nk cells and ilc2s ( 10111213 ) . recent studies have identified anti - inflammation functions of il-33 : it plays a critical role in regulatory t cell expansion in acute colitis ( 14 ) , asthma ( 15 ) , and obesity ( 16 ) . one effector molecule produced by regulatory t cells is amphiregulin , which plays a role in tissue repair without influencing antiviral immune responses ( 17 ) . thus , the seemingly anti - inflammatory effect of il-33 is in fact a result of enhanced tolerance mechanisms . il-33 also may exert its anti - inflammatory effects by suppressing th1 or th17-mediated immune responses . this outcome can be particularly important in inflammatory or infectious diseases in which th1 or th17 cells mediate immunopathology . recent studies have begun to reveal the molecular mechanisms behind these observations . specifically , ifn and il-27 have been shown to counteract the activity of ilc2s mediated by il-33 ( 181920 ) . the participation of il-33 in host resistance to c. albicans infection was initially demonstrated in a fungal peritonitis model ( 21 ) . il-33 priming before infection with c. albicans resulted in effective fungal clearance and subsequently decreased mortality caused by fungal peritonitis ( 21 ) . this priming by il-33 acts on multiple steps of an anti - candida neutrophil response . first , il-33 priming enhances recruitment of neutrophils to the site of infection through two mechanisms : 1 ) it increases production of neutrophil - chemotactic cxcr2 ligands ( cxcl1 and cxcl2 ) by peritoneal macrophages ; and 2 ) it suppresses the internalization of surface cxcr2 in neutrophils . il-33 does so by inhibiting tlr2-induced grk2 expression that is required for cxcr2 internalization ( 22 ) . the increased phagocytosis of neutrophils occurs because of specific upregulation of the complement receptor cr3 through the synergistic activation of the tlr2 and dectin-1 signaling pathways by il-33 . increase in production of reactive oxygen species ( ros ) is correlated with yeast killing of il-33-primed neutrophils . it is not known why il-33 specifically promotes clearance of opsonized c. albicans , but this occurred under physiological conditions ( our unpublished data ) . mononuclear phagocytes , including resident macrophages and inflammatory monocytes , also play a critical role in candida clearance ( 2324 ) . administration of il-33 before and during c. albicans infection markedly induces m2 macrophage polarization ( 25 ) . il-33-induced m2 polarization is mediated by cd4 t cell - derived il-13 ( 25 ) . surprisingly , il-33 stimulates bone marrow - derived m2 macrophages to efficiently phagocytize and kill c. albicans . the mechanism of action is not known , but one possibility is that il-33 increases killing by directly upregulating dectin-1 and mannose receptor or by indirectly inducting il-13 production ( 2627 ) . massive emergency hematopoiesis occurs after infections to satisfy the need for myeloid cells in the periphery . under these conditions , in addition , hematopoietic stem and progenitor cells ( hspcs ) emigrate from the bone marrow to the periphery and active extramedullary hematopoeisis occurs . injection of il-33 induces massive expansion of myeloid cells , particularly eosinophis , in the periphery ( 5 ) . recently , we provided an explanation for how il-33 mediates this phenomenon ( 28 ) . vascular endothelial cells produce high levels of ccl7 in response to il-33 , and ccl7 , in turn , induces emigration of hspcs out of the bone marrow with the help of ccr2 . on the other hand , il-33 directs the differentiation of hspcs toward myeolopoiesis in both the bone marrow and periphery by an unknown mechanism . systemic c. albicans infections induce tubular epithelial cells of the kidney to produce il-33 ( our unpublished data ) and st2 is highly expressed on hspcs ( 8) . thus , it is tempting to propose that il-33 may be involved in hspc differentiation during c. albicans infection . this hypothesis is substantiated by the observation that c. albicans stimulates differentiation of hspcs toward monocytes ( 29 ) . altogether , these results strongly suggest that il-33 may control emergency granulopoiesis to supply sufficient numbers of granulocytes to the site of infection , thus , indirectly increasing host resistance to c. albicans infections . although administration of il-33 increases host survival by promoting fungal clearance , the anti - inflammatory activity of il-33 can also contribute to host survival during systemic c. albicans infections ( 25 ) . there were significantly lower levels of proinflammatory mediators ( il-6 , tnf- , il-1 , cxcl1 , and cxcl2 ) , relatively little neutrophil infiltrate , and m2 macrophage polarization in the kidneys of il-33-primed mice throughout the course of infection . as mentioned previously , il-13 mediates il-33-induced m2 polarization . interestingly , m2 polarization is required not only to keep fungal burden in check in the kidneys but also to maintain persistent immunosuppression associated with reduced candida - induced immunopathology that leads to renal damage . because hyperimmune responses are associated with more fatalities than high fungal burden in systemic c. albicans infections ( 29 ) consistent with these results , il-33 mice are more susceptible to systemic candida infection compared to wild - type mice , showing impaired fungal clearance and severe renal immunopathology ( our unpublished data ) . severe renal inflammation is associated with a dysregulated differentiation of tnf-- and inos - producing dendritic cells ( tip - dcs ) in the kidney of candida - infected il-33 mice . in contrast , these mice have a lower number of il-12- and il-23-producing mature dendritic cells ( dcs ) in their infected kidneys . as reducing tip - dcs decreases renal immunopathology without influencing fungal proliferation in il-33 mice , il-33 is a unique example of a factor that helps to reduce host vulnerability to damage by promoting pathogen clearance and reducing immunopathology ( our unpublished data ) . one important outstanding issue to explore is whether il-33 is involved in types of tolerance other than immunopathology - related tolerance . il-33 for example , il-33 can have direct protective effects on parenchymal cells of organs during a stress response ( 30 ) , or il-33 may exert tolerance through activation of cells that have a protective role against tissue injury . il-33 administration expands ilc2s and regulatory t cells that can produce amphiregulin , a molecule critical for tissue repair and host tolerance ( 1731 ) . two other types of cells that can be targeted by il-33 for tissue protection are m2 macrophages and th17 cells . however , dysregulation of il-33 can result in chronic inflammation or fibrosis ( 3435 ) . whether the tolerance mechanisms mentioned above operate in host defenses against systemic candida infection needs to be tested . we propose that il-33-induced tolerance is associated with an increased capacity for tissue repair and decreased immunopathology . il-33 production reaches a peak in the kidney at day 3 of systemic c. albicans infection , when maximum inflammation and active fungal proliferation are occurring . at this time , il-33 seems to prevent overactivation of an inflammatory response on one hand and to promote fungal clearance on the other hand ( fig . 1 ) . the former process may result mainly from blocking inflammatory dcs , while the latter may occur at multiple stages of innate and adaptive immune responses to c. albicans infection . il-33 also promotes il-23 production by dcs which results in activation of the nk cell - gm - csf - neutrophil axis . finally , dc - derived il-12 and il-23 may be critical in il-33-mediated fungal clearance through the induction of th1 and th17 differentiation , respectively . these beneficial activities of il-33 have therapeutic implications for patients with invasive candidiasis . the ability of il-33 to promote th1 and th17 responses against c. albicans also indicates that il-33 has merits as an anti - candida vaccine adjuvant .
backgroundin the subgroup of children with chronic cough , distinguishing children with allergic asthma from those with non - specific respiratory symptoms is difficult . we have focused on determination of diagnostic efficiency of serum total ige , sige , and skin prick test in differentiation of asthmatic children from children with nonspecific respiratory symptoms.material/methodsa total of 131 children with median age of 7.5 years were enrolled in study and divided into 2 groups ; children with allergic asthma ( n=71 ) and children with chronic cough ( n=60 ) . participants underwent the standard allergological examination , including skin prick test and measurement of total ige , and following 3 allergen - specific ige antibodies against aeroallergens : dermatophagoides pteronyssinus , ambrosia artemisiifolia , and phleum pratense.resultsthe percentage of patients with elevated level of total and sige was higher in children with allergic asthma than in children with chronic cough syndrome ( p=0.0001 ) . in children with asthma , sige had a better diagnostic value than total ige . the best diagnostic efficiency of cut - off values for sige was shown for der p sige . skin prick test to all allergens had 78.82% sensitivity and 91.3% specificity in differentiating the 2 tested groups . the highest sensitivity and specificity in skin prick test was proved for dermatophagoides pteronyssinus.conclusionsthe sensitization profile consisting of total ige , sige levels , and spt clearly distinguishes children with allergic asthma from children with chronic nonspecific cough , but still with overlap . therefore , diagnosis should always be confirmed by a thorough allergy investigation .	among the many different symptoms encountered in every day practice , cough is the most common symptom in children visiting the pediatrician . chronic nonspecific cough is defined as a nonproductive cough in the absence of identifiable respiratory disease or known cause persisting for more than 3 to 8 weeks . routine diagnostic tests , such as total serum ige determination , allergen - specific serum ige determination , and skin prick test , can help significantly in diagnosing allergic asthma and distinguishing it from other respiratory diseases as well as from chronic nonspecific cough syndrome . although sensitization is a major risk factor for asthma development , the role of sensitization remains poorly understood and thus is continuously debated in scientific literature [ 57 ] . the importance of total serum ige levels in diagnosis of asthma has been verified throughout a number of studies . it is also reported that asthma below certain levels of total ige does not exist . in addition , increased levels of total serum ige at birth and in early life have been associated with an increased risk for the development of persistent asthma later in life . while total serum ige determination is one of the key methods to reveal atopy , the determination of allergen - specific ige ( sige ) to environmental allergens has diagnostic and therapeutic importance ; reveals the patient s sensitization to a certain allergen and enables monitoring of the success of specific immunotherapy ( sit ) . among specific allergens , high sensitization rate have been reported for the house dust mites der p ( dermatophagoides pteronyssinus ) and der f ( dermatophagoides farinae ) [ 1416 ] . early sensitization and levels of sige to perennial respiratory allergens have likewise been associated with increased risk of childhood asthma . similarly reported that mite sensitization early in infancy ( measured by sige levels ) accounts for an up to 19.7-fold increased risk for allergic asthma . also found house dust mite sensitization to be the most important risk for allergic asthma . have shown that asthmatic children with higher asthma severity have a higher serum concentration of both total ige and specific ige to der p. the atopy can also be defined by positive result to skin prick test ( spt ) to a standard panel of allergens [ 2325 ] . investigating the association of skin test reactivity , total serum ige levels , and peripheral blood eosinophilia with asthma , khadadah et al . proved spt has the best positive predictive value and best efficiency in diagnosing respiratory atopic diseases . comparing spt to total serum ige levels , fajraoui et al . found that the 2 tests agree in 80% of cases . because chronic cough is one of the most common presentations of allergic asthma in children and being sensitized does not automatically include diagnosis of asthma , differentiation between allergic asthma and chronic ( nonspecific ) cough syndrome remains a relevant clinical problem . even though allergic diseases and diagnostic methods have been investigated during recent years , conclusive guidelines on how to distinguish allergic asthma from chronic ( nonspecific ) cough in the population of children have not been available . the aim of this study was to determine the diagnostic value of sensitization profile ( including serum total , sige determination , and spt ) in children with persistent respiratory symptoms to differentiate between children with allergic asthma and children with chronic ( nonspecific ) cough . this study is an analysis of data collected from patients at children s hospital srebrnjak , department of allergology and pulmonology , zagreb , croatia , during a 6-month period . a total of 131 children , aged 115 years , were included in the study . all of the patients included in the study experienced respiratory symptoms and were sent to our department for further diagnosis . participants underwent the standard allergological examination , including spt to the standard set of inhalatory allergens common for the region , lung function tests , and in vitro diagnostic tests . participants were tested for total ige and 3 allergen - specific ige antibodies against the most prevalent aeroallergens in children in continental region in croatia : hose dust mites ( dermatophagoides pteronyssinus ) , common ragweed ( ambrosia artemisifoliae ) , and timothy grass ( phleum pratense ) pollen . study participants were divided into 2 groups : children with clearly diagnosed allergic asthma , ie , having at least 3 episodes of wheezing and/or a positive bronchodilatation test ( nih gina 2009 ) ; n=71 , age 215 years , =8 years , 49 ( 69.01% ) males . children with chronic cough , ie , having less than 3 episodes of wheezing , with persistent cough lasting for more than 6 weeks ; n=60 , age 114 years , =7 years , 40 ( 66.67% ) males . total serum ige concentration was determined by fluoroimmunochemical method ( abbot , usa ) using an automatic analyzer imx ( abbot , usa ) . for data comparison , 95% central range was used and serum ige concentration was compared to in - house reference values . der p ( dermatophagoides pteronyssinus ) , phl p ( timothy grass pollen ) , and amb a ( short ragweed ) were determined by immunocap ( pharmacia , uppsala , sweden ) . analysis was performed using a unicap 100 analyzer ( pharmacia , uppsala , sweden ) . the spt was performed with standardized allergens produced by allergopharma , comprising the standard set of inhalatory allergens common for croatia . total and specific ige concentration showed asymmetric distribution and were presented by range and median , and the statistical significance of the difference between the 2 groups was tested using the wilcoxon test . the diagnostic efficiency of total ige and sige determination sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) were estimated for the groups of patients with allergic asthma and chronic cough . receiver operating characteristic ( roc ) curve analysis was performed using statistica for windows , version 6.0 ( statsoft , inc . , this study is an analysis of data collected from patients at children s hospital srebrnjak , department of allergology and pulmonology , zagreb , croatia , during a 6-month period . a total of 131 children , aged 115 years , were included in the study . all of the patients included in the study experienced respiratory symptoms and were sent to our department for further diagnosis . participants underwent the standard allergological examination , including spt to the standard set of inhalatory allergens common for the region , lung function tests , and in vitro diagnostic tests . participants were tested for total ige and 3 allergen - specific ige antibodies against the most prevalent aeroallergens in children in continental region in croatia : hose dust mites ( dermatophagoides pteronyssinus ) , common ragweed ( ambrosia artemisifoliae ) , and timothy grass ( phleum pratense ) pollen . study participants were divided into 2 groups : children with clearly diagnosed allergic asthma , ie , having at least 3 episodes of wheezing and/or a positive bronchodilatation test ( nih gina 2009 ) ; n=71 , age 215 years , =8 years , 49 ( 69.01% ) males . children with chronic cough , ie , having less than 3 episodes of wheezing , with persistent cough lasting for more than 6 weeks ; n=60 , age 114 years , =7 years , 40 ( 66.67% ) males . total serum ige concentration was determined by fluoroimmunochemical method ( abbot , usa ) using an automatic analyzer imx ( abbot , usa ) . for data comparison , 95% central range was used and serum ige concentration was compared to in - house reference values . der p ( dermatophagoides pteronyssinus ) , phl p ( timothy grass pollen ) , and amb a ( short ragweed ) were determined by immunocap ( pharmacia , uppsala , sweden ) . analysis was performed using a unicap 100 analyzer ( pharmacia , uppsala , sweden ) . the spt was performed with standardized allergens produced by allergopharma , comprising the standard set of inhalatory allergens common for croatia . total and specific ige concentration showed asymmetric distribution and were presented by range and median , and the statistical significance of the difference between the 2 groups was tested using the wilcoxon test . the diagnostic efficiency of total ige and sige determination sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) were estimated for the groups of patients with allergic asthma and chronic cough . receiver operating characteristic ( roc ) curve analysis was performed using statistica for windows , version 6.0 ( statsoft , inc . median total ige in the group of children with allergic asthma was significantly higher than in the group of children with chronic cough ( 495.0 kiu / l in the allergic asthma group ; 59.0 kiu / l in the chronic cough group , p<0.05 ; table 1 ) the percentage of patients with elevated concentration of total and specific ige was higher in children with allergic asthma than in children with chronic cough ( p<0.0001 ; table 2 ) . comparing the number of patients with elevated total and sige level between the 2 groups , we found an elevated total ige level in 97.2% of children with allergic asthma , whereas elevated ige level was found in 31.7% children with chronic cough . level of sige was elevated in 100% of children with allergic asthma and in 21.7% children with nonspecific cough ( =116.258 ; df=2 ; p=0.0000 , * * =129.741 ; df=2 ; p=0.0000 ) comparing levels of sige in sensitized children , we discovered that children sensitized to der p had a higher concentration of sige than children sensitized to phl p and amb a ( p<0.05 ) ( table 3 ) . we established that at a cut - off value of 116.6 kiu / l of total ige had 96.8% sensitivity and 77.8% specificity in differentiating allergic asthma and chronic cough . when comparing sige to der p in the group of children with allergic asthma and those in group with chronic cough , we found 89% sensitivity and 97% specificity ( table 4 ) . the best diagnostic value of cut - off values for sige was found for der p between children with allergic asthma and children with chronic cough ( table 4 ) . the sensitization profile can be defined by total and specific ige determination in serum and skin prick test to common inhalatory allergens . data on exact diagnostic efficiency ( a combined measure of sensitivity and specificity ) of serum total and sige determination and clinical relevance of ige level determination in differentiating children with asthma and non - specific respiratory symptoms differ from study to study . this study showed that total ige , and particularly sige , clearly distinguishes children with allergic asthma from children with nonspecific respiratory symptoms . this confirms the reports of other groups , who found that ige testing improved the predictive accuracy and patient differentiation [ 3033 ] . the differences in total ige concentration between the 2 groups ( children diagnosed with asthma and children with chronic cough ) were confirmed by the values of ige concentration ( p<0.05 ) and roc curve analysis . cut - off values of total ige concentration ( 116.6 kiu / l asthma vs. chronic cough ) were determined with excellent diagnostic efficiency . regarding literature variations of referent values for total ige concentration , the variation is due to different populations included in the studies and the applications of different immunochemical methods for ige determination [ 11,28,29,3439 ] . another important diagnostic procedure in estimating sensitization of children is the measure of ige to specific allergens in blood . results of our study show that children sensitized to the perennial allergen der p showed a higher concentration of sige than in children sensitized to amb a and phl p , which are present only during the blooming season . it is believed that because house dust mites are the most widespread perennial inhalatory allergens , a greater exposure to der p in comparison with other allergens results in higher sige concentration . variations between normal and elevated ige in the same individual may occur , depending on the age , the exposure to the specific allergen , and disease development . diagnostic efficiency of sige to pollen allergens could therefore be influenced by the period during which blood was sampled ( during or before vs. after the blooming season ) . it was also confirmed that exposure and sensitization to certain allergens strongly depend on the region and climate . sensitization to aeroallergens in correlation to asthma development is currently a world - wide scientific focus . sensitization is most commonly defined by a positive skin prick test result , along with sige in serum . being sensitized does not imply the diagnosis of allergic asthma or any other atopic disease . in 2001 , crimi et al . showed that in a population of children proven to be sensitized , a third of them were without any allergic symptoms . it is also important to emphasize the correlation between sensitization , especially to the most common aeroallergens , and asthma in children . in 2010 craig used spt to define sensitization and concluded that about 95% of patients with mild asthma were sensitized , as well as the 90% of those with severe asthma . one of the most frequent sensitizations in children with asthma is to the house dust mite . as in croatia , in many other countries around the world this sensitization is the most common among asthmatic children [ 4749 ] . in florida , 89.6% of asthmatic children had positive sige to house dust mites , along with 81.9% of chinese asthmatic children and children in zimbabwe [ 5052 ] . many studies have observed that deteriorating asthma can be related to increased exposure to allergens , particularly allergens from house dust mites , cockroaches , cats , rodents , mold , or pollen . isaac , the international study of childhood asthma and allergies , showed that remission of asthma is 10% yearly and was inversely correlated to sensitization . carroll et al . confirmed that increasing atopic sensitization ( estimated by spt and total ige level ) is associated with increased disease severity in children with asthma . furthermore , elevated level total serum ige has also been linked to risk of hospital admission . in contrast , jedrychowski et al . related reversibility of asthma with lung function growth there are a number of factors that can influence sensitization , such as location of residence , sex , and ethnicity . with lower prevalence of sensitization , female sex and residence in a rural environment a great number of studies have investigated risk factors for allergen sensitization and asthma development . sporic et al . described a significant relationship between early life exposure to dust mite allergen and asthma at the age of 11 years . studies that are still ongoing , such as the prevention and incidence of asthma and mite allergy study ( piama ) , the manchester asthma and allergy study ( maas ) , and the childhood asthma prevention study , are expected to reveal new information about asthma prevention through prevention of sensitization to specific aeroallergens . in conclusion , it is important to emphasize that for a confirmed diagnosis of allergic asthma , determination of serum ige should always be supplemented with a thorough allergy investigation , including a detailed medical history ( especially concerning allergen exposure ) , the presence of other possible immediate hypersensitivity diseases , skin tests , challenges , examinations for eosinophilia in blood and mucous membrane secretions , and in some cases with excluding the ger as a possible risk for asthma development . the results of our study show that determination of total ige and sige is a good method for differentiating asthmatic children from those with nonspecific chronic cough . sige determination in children with clinical asthma symptoms had a better diagnostic value than total ige determination . the best diagnostic value of cut - off values for sige was shown for der p. in our study , spt was shown to have lower efficacy ( 78.82% sensitivity , 91.3% specificity ) in distinguishing asthmatic children from children with non - specific chronic cough in comparison with levels of sige . although the differentiation between children with non - specific respiratory problems and children with asthma can be difficult , sensitization profile ( total and specific ige levels and spt ) facilitates achieving diagnosis . after analyzing 131 patient aged 115 years , we conclude that total ige , and particularly sige , clearly distinguish children with allergic asthma from children with nonspecific chronic cough . the final diagnosis should , however , always be confirmed by a thorough allergy investigation . considering the results of our study , as well as the results of other studies of sensitization profile and its correlation to asthma , we conclude that standardized procedures such as spt , total ige level , and sige should be performed in patients presenting with chronic cough , while it significantly correlates with asthma and can lead the physician to the final diagnosis of asthma vs. nonspecific cough .
introduction and objective : nuclear renal scan is currently the gold standard imaging study to determine differential renal function . we propose helical ct as single modality for both the anatomical and functional evaluation of kidney with impaired function . in the present study renal parenchymal volume is measured and percent total renal volume is used as a surrogate marker for differential renal function . the objective of this study is to correlate between differential renal function estimation using ct - based renal parenchymal volume measurement with differential renal function estimation using 99mtc - dtpa renal scan.materials and methods : twenty - one patients with unilateral obstructive uropathy were enrolled in this prospective comparative study . they were subjected to 99mtc - dtpa renal scan and 64 slice helical ct scan which estimates the renal volume depending on the reconstruction of arterial phase images followed by volume rendering and percent renal volume was calculated . percent renal volume was correlated with percent renal function , as determined by nuclear renal scan using pearson coefficient.results and observation : a strong correlation is observed between percent renal volume and percent renal function in obstructed units ( r = 0.828 , p < 0.001 ) as well as in nonobstructed units ( r = 0.827 , p < 0.001).conclusion : there is a strong correlation between percent renal volume determined by ct scan and percent renal function determined by 99mtc - dtpa renal scan both in obstructed and in normal units . ct - based percent renal volume can be used as a single radiological tests for both functional and anatomical assessment of impaired renal units .	the plan for management of obstructive uropathy is based on precise delineation of both renoureteral units along with accurate estimation of the renal function . usual approach is to combine diagnostic tools which delineates the anatomy like ivu and radioisotope renal scan for assessment of the split renal function which is unfortunately time consuming , expensive and not freely available . nuclear renal scan is currently the gold standard imaging study to determine differential renal function . there are different methods to evaluate the renal function like 24 hours creatinine clearance and ct - estimated gfr . so , the residual parenchymal volume can very readily and accurately measured by 64 slice helical ct scan along with other anatomical informations . we propose helical computerized tomography as a single modality for both the anatomical and functional evaluation of kidney with impaired function . in the present study renal parenchymal volume is measured and percent total renal volume is used as a surrogate marker for differential renal function . the objective of this clinical study is to correlate between differential renal function estimation using ct based renal parenchymal volume measurement with differential renal function estimation using tc - dtpa renal scan . between february 2011 and august 2011 , 21 patients with unilateral obstructive uropathy were enrolled in this prospective comparative study . renal units with unilateral obstruction was diagnosed by history , clinical examination , routine laboratory investigations including s. creatinine , plain x - ray of the abdomen ( kub)/ultrasonography kub region , ivu and tc - dtpa renal scan . the cases with s.creatinine > 2 mg / dl and acute obstruction are excluded from the study . furthermore , they were subjected to 64 slice helical ct scan ( ge medical system , light speed vct ct99 ) . the volume estimation software comes preinstalled with the ge 64 slice helical ct scanner ( ge medical system , light speed vct ct99 ) workstation ( adw 4.3 ) . the three dimensional tools that was available in this software package have add object function that was used to build the three - dimensional virtual representation of the kidneys depending on the arterial phase images . first the area of interest of renal parenchyma was delineated manually then by selecting a piece of enhancing renal parenchyma from the area of interest allows the software to identify the remaining surrounding renal parenchyma that have similar voxel values which is then added into the volume voxel to create the three dimensional renal volume . the final 3d model of each kidney was compared with the cross sectional images to ensure that only the renal parenchyma was included and sometimes it was necessary to use the cutting tool to free the margins of kidney from adjacent structures . renal parenchymal volume estimation was done by one radiologist in all the cases who was blinded regarding the nuclear scan report . the right and left percent renal volume was calculated by dividing right and left volumes respectively by the combined right and left renal volume . this percent renal volume was compared with percent renal function determined by tc - dtpa renal scan [ figure 1 ] . correlations between percent renal volume and percent renal function is evaluated using pearson coefficient in all cases . in our study male : female ratio was 1:1.1 with and the age ranged from 20 to 55 years . we evaluated a total 21 ( 13 left and 8 right ) obstructed renal units and 21 nonobstructed renal units i.e 42 renal units . of 21 obstructed renal units 2 had relief of obstruction before dtpa and ct scan . the etiology of chronic obstruction was pelvi - ureteric junction obstruction in four cases , ureteric calculus in nine cases , renal obstructing calculi in seven cases and lower ureteric stricture in one case . the range of s. creatinine was 0.5 - 1.2 mg / dl ( 0.78 ) . the range of gfr estimated by tc - dtpa in obstructed renal units was between 0 and 27 mg / dl ( 7.35 ) and in unobstructed renal units ranged between 23.3 and 61.7 mg / dl ( 46.43 ) . the parenchymal volume as estimated by 64 slice helical ct scan in obstructed renal units are in the range of 0 - 147.5 cc ( 31.45 ) and in unobstructed renal units was 94.2 - 267cc ( 142.41 ) . the percent renal fuction as estimated by tc - dtpa in obstructed renal units ranges from 0%-39.33% ( 13.03 ) and in nonobstructed units ranges from 60.67 to 100% ( 86.89 ) . the percent renal volume estimated by 64 slice ct scan in obstructed unit ranges from 0 to 35.58% ( 16.01 ) and in nonobstructed units ranges from 64.44 to 100% ( 83.94 ) . we found a strong correlation between percent renal function estimated by tc - dtpa renal and percent renal volume estimated by 64 slice ct scan in obstructed renal units ( r = 0.828 , p < 0.001 , 95% ci : 0.618 - 0.928 ) [ figure 2 ] [ table 1 ] . positive correlation between % renal volume and % renal function in obstructed renal units raw data for scatter graph ( figure 2 ) we found a strong correlation between percent renal function estimated by tc - dtpa renal and percent renal volume estimated by 64 slice ct scan in nonobstructed renal units ( r = 0.827 , p < 0.001 , 95% ci : 0.615 - 0.927 ) [ figure 3 and table 2 ] . strong correlation is found between percent renal function estimated by tc - dtpa renal and percent renal volume estimated by 64 slice ct scan on combining both obstructed and non obstructed renal units ( r = 0.985 , p < 0.001 , 95% ci : 0.974 - 0.992 ) [ figure 4 ] . positive correlation between % renal volume and % renal function in nonobstructed renal units data for scatter graph ( figure 3 ) positive correlation between % renal volume and % renal function in both obstructed and nonobstructed renal units obstructive uropathy is a common entity that urologists encounters in daily practice and accurate assessment of differential renal function is vital in the management of these chronically obstructed renal units . twenty - four hours creatinine clearance is one of the methods to evaluate the total renal function but we need to have differential renal function for management of these obstructed units . at present nuclear renal scan is considered as the imaging modality of choice for differential renal function . but this also have some drawbacks like it is expensive and not freely available particularly in a country like india , also it does not give any information regarding the anatomy of the renal units which is of utmost importance for the management of these obstructed renal units . found a strong linear relationship between differential renal function by dynamic ct using modified patlak graphic analysis , nuclear renal scan and 24-hour creatinine clearance . but it has certain disadvantages like it needs assumption of functional renal homogeneity as it involves only a particular section of kidney , complex calculations and nonavailability of the software everywhere . ng et al . in his study compared differential renal parenchymal volume with 24-hr creatinine clearance by percutaneous nephrostomy in obstructed units and found correlation between differential renal function with differential creatinine clearance . herts et al . in their study proposed that estimated gfr by ct - based parenchymal volume can replace gfr measurement by i - iothalamate clearance imaging after studying 244 renal donors . observed contrast enhanced spiral ct is more sensitive than ivp for identifying the cause of chronic obstructive uropathy and it is as accurate as radioisotope renal scan for calculating the total and separate kidney function . so they recommend spiral ct with contrast medium as a single radiological diagnostic modality for the assessment of patients with chronic renal obstruction and normal serum creatinine . feder mt in their study predicting differential renal function using computerized tomography measurements of renal parenchymal area found differential renal parenchymal area measured by ct strongly correlates with differential function on renal scintigraphy and it may obviate the need for nuclear renal scan in some circumstances . morrisroe s n et al . found strong correlations between percent renal function and percent renal volume in all cases ( r = 0.90 , p < 0.001 ) . they concluded that differential renal volume measured from computerized tomography strongly correlates with differential renal function on nuclear renal scan for normal and chronically obstructed kidneys and ct may serve as a single radiological diagnostic study for anatomical and functional assessment in patients in whom a poorly functioning renal unit is suspected . tc - dtpa renal scan and ct angiography are the investigations that are mandatory to evaluate the donor for the assessment of split renal function and vascular anatomy of the donor kidney . in his study found split renal function based on 3d ct models can provide a one - stop evaluation of both the anatomical and the functional characteristics of the kidneys of living potential kidney donors . differential renal function estimation by ct - based parenchymal volume can not replace nuclear scan in cases of acute cortical dysfunction and for documentation of improved renal function after surgery . the disadvantages of ct estimated differential functions are its inability to be performed in renal insufficiency and false results in acute obstruction . according to the health physics society fact sheet 2010 : the effective dose of ivu is 3 msv , the effective dose of ct abdomen / pelvis is 10 msvand the effective dose in dtpa scan is 1.8 msv . if we combine ivu and dtpa the radiation dosage is half that of ct scan which is a disadvantage but other factors which are advantages in case of ct have to be considered as well . measurement of differential renal function by ct - based parenchymal volume correlates strongly with differential renal function estimated by tc - dtpa renal scan in both obstructed and nonobstructed renal units . it has the added advantage of precise delineation of the anatomical details of the renoureteral unit . so , helical ct scan can be used as a single modality to asses the functional and anatomical status of the obstructed kidneys . it will help urologists in better management of obstructed renal units and lowering the expenses incurred by the patients . although our study is small it throws light into the efficacy of helical ct in assessment of split renal function . larger studies are needed to ascertain the usefulness of cect as a functional study in patients with severely impaired renal function .
point - of - care ( poc ) ultrasonography is considered fundamental in emergency medicine training and recently has become a milestone in critical care fellowship programs as well . currently , there is no such standard requirement for internal medicine residency programs in the united states . we present a new case and briefly review another case at our institution a community hospital in which internal medicine house staff trained in ultrasonography were able to uncover unexpected and critical diagnoses that significantly changed patient care and outcomes . we also review the growing evidence of the application of ultrasound in the diagnosis of a myriad of conditions encountered in general internal medicine as well as the mounting data on the ability of internal medicine residents to apply this technology accurately at the bedside . we advocate that the literature has sufficiently established the role of poc ultrasonography in general internal medicine that there should no longer be any delay in giving this an official place in the development of internal medicine trainees . this may be particularly useful in the community hospital setting where 24-h echocardiography or other sonography may not be readily available .	a previously healthy 20-year - old man presented to our community emergency department with nausea , vomiting , and generalized weakness for 1 month . he had undergone an extensive outpatient evaluation with normal imaging and laboratory studies and an esophagogastroduodenoscopy revealing only reflux gastritis . vital signs included a blood pressure of 100/73 mmhg , heart rate 105 bpm , and 95% oxygen saturation on room air . poc echocardiography was performed by the medical resident , revealing a severely reduced left ventricular ejection fraction with multiple echogenic structures suspicious for thrombi ( fig . several hours later , standard echocardiogram confirmed an ejection fraction of 1015% with three apical thrombi . the patient was then transferred to a specialized cardiomyopathy service where inotropic and diuretic therapy led to resolution of his gastrointestinal symptoms which were presumed to be from intestinal venous congestion . we recently reported another case demonstrating the impact of poc ultrasonography that of an 84-year - old man with a history of esophageal cancer ( in remission ) and deep venous thrombosis on warfarin who presented with shortness of breath ( 12 ) . echocardiogram on the day of admission revealed a low normal ejection fraction with signs of elevated right - sided pressures and no pericardial effusion . the clinical diagnosis was congestive heart failure and his symptoms improved after several days of diuretics . on the fifth day of hospitalization , laboratory studies revealed a creatinine steadily rising to 3.5 mg / dl , an inr of 3.5 , and fractional excretion of sodium of 0.3% . the assessment was that of over - diuresis with prerenal acute kidney injury , but he did not respond to small boluses of intravenous fluids . due to progressive decompensation overnight , poc ultrasonography was performed by the internal medicine resident . unexpectedly , this revealed a moderate pericardial effusion with right atrial and right ventricular collapse consistent with tamponade physiology ( fig . the patient was urgently transferred to a tertiary care center where pericardiocentesis yielded 750 cc of frank blood . positron emission tomography / computed tomography ( pet / ct ) was negative for any sign of recurrence of his esophageal cancer . warfarin was discontinued and a repeat echocardiogram , 4 weeks later , showed no pericardial effusion . a pericardial effusion is noted with associated right atrial and right ventricular inversion consistent with tamponade physiology . in the last decade , there has been a steadily growing body of research on the utility of ultrasonography for a myriad of conditions commonly seen in internal medicine . in fact , when compared to standard imaging , ultrasound has often yielded similar if not better results . this is particularly well demonstrated in the realm of pulmonary disease . a recent meta - analysis including over 1,000 patients demonstrated a high sensitivity ( 94.1% ) and specificity ( 92.4% ) for ultrasonography in the diagnosis of pulmonary edema ( 13 ) . ultrasound has also proven to be better than chest x - ray ( cxr ) and comparable to ct for pneumonia and lung consolidation ( 14 , 15 ) . two recent meta - analyses , both including over 1,000 patients , confirmed these findings with a sensitivity of 9497% and specificity of 9496% for the diagnosis of pneumonia . the area under the receiver operating characteristic ( roc ) curve was consistently high ( 0.980.99 ) with useful positive and negative likelihood ratios ( 16 , 17 ) . lung ultrasound can also be used to follow patients for resolution of their pneumonia similar to cxr and ct ( 18 ) . with regard to pneumothorax , ultrasound can outperform radiography by several measures and is quite comparable to ct while being much faster ( 1921 ) . 22 ) have published a well - known algorithm that uses a variety of sonographic lung findings to help narrow the differential diagnosis in patients presenting with respiratory insufficiency . similarly , a recent , large multicenter study demonstrated that ultrasound can distinguish cardiogenic from non - cardiogenic dyspnea better than clinical assessment , cxr , or brain natriuretic peptide levels ( 23 ) . this can be particularly difficult to assess outside the intensive care unit ( icu ) where pulmonary artery catheters and stroke volume monitors are absent . however , multiple studies have demonstrated the usefulness of sonographic inferior vena cava ( ivc ) and cardiac evaluation in non - icu patients ( 24 , 25 ) . notably , guiotto et al . ( 26 ) showed that ultrasound assessment of the ivc can guide volume removal by ultrafiltration . this can be particularly valuable with hemodialysis patients on the general medical floor in whom the determination of how much volume to remove is not usually done via very objective or precise means . with regard to the abdomen , sonography has exhibited excellent sensitivity and specificity in diagnosis of small bowel obstruction ( 27 ) . in addition , sonographic techniques have long been described for diverticulitis and appendicitis and have very good specificity when compared to ct ( 28 , 29 ) . interestingly , inflammatory bowel disease can be distinguished from non - inflammatory disease by ultrasound as well . in a study by novak et al . ( 30 ) , sonography demonstrated high specificity and negative predictive value in comparison with gold standard endoscopy in patients presenting with abdominal pain and diarrhea . likewise , with regard to patients presenting with possible nephrolithiasis , poc ultrasound was similar in accuracy compared to both conventional radiology department ultrasound and ct scan without differences in outcomes ( 31 ) . clearly , many of the conditions that are encountered in the internal medicine setting can be evaluated quite well with ultrasonography . the question that naturally follows is whether providers on the general medical floor particularly internal medicine house staff are able to apply this technology at the poc . there is indeed growing literature demonstrating that poc ultrasound performed by medical residents can change the course of patient care . in nearly 200 patients undergoing cardiac and abdominal sonography , andersen et al . ( 32 ) showed that approximately one - third had an additional diagnosis uncovered or the primary diagnosis changed completely . in the pulmonary realm , filopei et al . ( 33 ) demonstrated how ultrasound increased residents diagnostic accuracy compared to clinical assessment alone particularly for chronic obstructive pulmonary disease ( copd ) , pneumonia , pleural effusion , and pulmonary edema . residents are also able to perform and interpret abdominal sonography of the kidneys comparable to radiologists ; they can evaluate the abdominal aorta comparable to vascular surgeons and sonographers as well ( 34 , 35 ) . similar findings have been shown with estimating volume status and diagnosing various cardiac pathologies compared to history and physical examination ( 36 , 37 ) . interestingly , in one study , the use of portable cardiac ultrasound by residents changed management in 40% of cases and solidified prior management decisions in 76% ( 37 ) . more specifically , residents are able to assess left ventricular function , pleural and pericardial effusion , and even valvular disease accurately compared to cardiologists ( 38 ) . what is particularly remarkable is that extensive training is not needed for residents to acquire and interpret images effectively . after training on 20 patients , house staff were able to accurately identify left ventricular dysfunction with sensitivity , specificity , and positive and negative predictive values close to or well over 90% ( 39 ) . after a 5-h module , medicine residents could diagnose certain kidney conditions with high sensitivity and specificity compared to attending radiologists ( 34 ) . similarly , with a half - day workshop , 84% of residents in one study were able to independently evaluate the abdominal aorta compared to only 16% beforehand ( 40 ) . in fact , many studies demonstrating accurate resident - performed ultrasound show that only a cumulative total training time of several hours to at most a few days is needed ( 35 , 36 , 40 , 41 ) . understandably , these skills need to be reinforced with regular practice lest they wane over time ( 41 ) . given the above , it is not surprising that poc ultrasonography can impact quality measures and costs . when lung ultrasound was implemented , peris et al . ( 42 ) demonstrated a clear reduction in the number of cxrs and ct scans performed without any decrement in outcomes in one population . when used to guide thoracentesis , ultrasonography decreases complication rates , costs of hospitalization , and lengths of stay ( 42 , 43 ) . furthermore , in an age where hospital readmission rates are closely monitored , ivc ultrasound by residents can be predictive and preventative , reducing rates from 30 to 4% in one recent study ( 44 ) . ( 39 ) demonstrated how residents are able to perform poc echocardiography not only accurately but also nearly a full 24 h before formal echocardiogram results are available to confirm their findings . delays in imaging are known to cause increased hospital lengths of stay ( 47 ) . synthesizing the aforementioned data , there is likely no more appropriate environment for poc ultrasound training of internal medicine house staff than the community hospital . in these settings , community hospitals are not usually designed to provide highly specialized services such as at a tertiary care center , but it is crucial , nonetheless , to identify patients in need of such therapies . our cases resulted in the appropriate and necessary transfer of the patient to a university center . given such experiences at our institution , we are currently working to formalize poc ultrasonography in our house staff curriculum and highly encourage other community hospital programs to do so as well . in the last decade , there has been a steadily growing body of research on the utility of ultrasonography for a myriad of conditions commonly seen in internal medicine . in fact , when compared to standard imaging , ultrasound has often yielded similar if not better results . this is particularly well demonstrated in the realm of pulmonary disease . a recent meta - analysis including over 1,000 patients demonstrated a high sensitivity ( 94.1% ) and specificity ( 92.4% ) for ultrasonography in the diagnosis of pulmonary edema ( 13 ) . ultrasound has also proven to be better than chest x - ray ( cxr ) and comparable to ct for pneumonia and lung consolidation ( 14 , 15 ) . two recent meta - analyses , both including over 1,000 patients , confirmed these findings with a sensitivity of 9497% and specificity of 9496% for the diagnosis of pneumonia . the area under the receiver operating characteristic ( roc ) curve was consistently high ( 0.980.99 ) with useful positive and negative likelihood ratios ( 16 , 17 ) . lung ultrasound can also be used to follow patients for resolution of their pneumonia similar to cxr and ct ( 18 ) . with regard to pneumothorax , ultrasound can outperform radiography by several measures and is quite comparable to ct while being much faster ( 1921 ) . 22 ) have published a well - known algorithm that uses a variety of sonographic lung findings to help narrow the differential diagnosis in patients presenting with respiratory insufficiency . similarly , a recent , large multicenter study demonstrated that ultrasound can distinguish cardiogenic from non - cardiogenic dyspnea better than clinical assessment , cxr , or brain natriuretic peptide levels ( 23 ) . this can be particularly difficult to assess outside the intensive care unit ( icu ) where pulmonary artery catheters and stroke volume monitors are absent . however , multiple studies have demonstrated the usefulness of sonographic inferior vena cava ( ivc ) and cardiac evaluation in non - icu patients ( 24 , 25 ) . notably , guiotto et al . ( 26 ) showed that ultrasound assessment of the ivc can guide volume removal by ultrafiltration . this can be particularly valuable with hemodialysis patients on the general medical floor in whom the determination of how much volume to remove is not usually done via very objective or precise means . with regard to the abdomen , sonography has exhibited excellent sensitivity and specificity in diagnosis of small bowel obstruction ( 27 ) . in addition , sonographic techniques have long been described for diverticulitis and appendicitis and have very good specificity when compared to ct ( 28 , 29 ) . interestingly , inflammatory bowel disease can be distinguished from non - inflammatory disease by ultrasound as well . in a study by novak et al . ( 30 ) , sonography demonstrated high specificity and negative predictive value in comparison with gold standard endoscopy in patients presenting with abdominal pain and diarrhea . likewise , with regard to patients presenting with possible nephrolithiasis , poc ultrasound was similar in accuracy compared to both conventional radiology department ultrasound and ct scan without differences in outcomes ( 31 ) . clearly , many of the conditions that are encountered in the internal medicine setting can be evaluated quite well with ultrasonography . the question that naturally follows is whether providers on the general medical floor particularly internal medicine house staff are able to apply this technology at the poc . there is indeed growing literature demonstrating that poc ultrasound performed by medical residents can change the course of patient care . in nearly 200 patients undergoing cardiac and abdominal sonography , andersen et al . ( 32 ) showed that approximately one - third had an additional diagnosis uncovered or the primary diagnosis changed completely . in the pulmonary realm , filopei et al . ( 33 ) demonstrated how ultrasound increased residents diagnostic accuracy compared to clinical assessment alone particularly for chronic obstructive pulmonary disease ( copd ) , pneumonia , pleural effusion , and pulmonary edema . residents are also able to perform and interpret abdominal sonography of the kidneys comparable to radiologists ; they can evaluate the abdominal aorta comparable to vascular surgeons and sonographers as well ( 34 , 35 ) . similar findings have been shown with estimating volume status and diagnosing various cardiac pathologies compared to history and physical examination ( 36 , 37 ) . interestingly , in one study , the use of portable cardiac ultrasound by residents changed management in 40% of cases and solidified prior management decisions in 76% ( 37 ) . more specifically , residents are able to assess left ventricular function , pleural and pericardial effusion , and even valvular disease accurately compared to cardiologists ( 38 ) . what is particularly remarkable is that extensive training is not needed for residents to acquire and interpret images effectively . after training on 20 patients , house staff were able to accurately identify left ventricular dysfunction with sensitivity , specificity , and positive and negative predictive values close to or well over 90% ( 39 ) . after a 5-h module , medicine residents could diagnose certain kidney conditions with high sensitivity and specificity compared to attending radiologists ( 34 ) . similarly , with a half - day workshop , 84% of residents in one study were able to independently evaluate the abdominal aorta compared to only 16% beforehand ( 40 ) . in fact , many studies demonstrating accurate resident - performed ultrasound show that only a cumulative total training time of several hours to at most a few days is needed ( 35 , 36 , 40 , 41 ) . understandably , these skills need to be reinforced with regular practice lest they wane over time ( 41 ) . given the above , it is not surprising that poc ultrasonography can impact quality measures and costs . when lung ultrasound was implemented , peris et al . ( 42 ) demonstrated a clear reduction in the number of cxrs and ct scans performed without any decrement in outcomes in one population . when used to guide thoracentesis , ultrasonography decreases complication rates , costs of hospitalization , and lengths of stay ( 42 , 43 ) . furthermore , in an age where hospital readmission rates are closely monitored , ivc ultrasound by residents can be predictive and preventative , reducing rates from 30 to 4% in one recent study ( 44 ) . ( 39 ) demonstrated how residents are able to perform poc echocardiography not only accurately but also nearly a full 24 h before formal echocardiogram results are available to confirm their findings . delays in imaging are known to cause increased hospital lengths of stay ( 47 ) . synthesizing the aforementioned data , there is likely no more appropriate environment for poc ultrasound training of internal medicine house staff than the community hospital . in these settings , community hospitals are not usually designed to provide highly specialized services such as at a tertiary care center , but it is crucial , nonetheless , to identify patients in need of such therapies . our cases resulted in the appropriate and necessary transfer of the patient to a university center . given such experiences at our institution , we are currently working to formalize poc ultrasonography in our house staff curriculum and highly encourage other community hospital programs to do so as well . our cases demonstrate the diagnostic impact of poc ultrasonography in the hands of trained medical residents especially in the community hospital setting . providers on the general medical floor particularly medical residents are able to use this highly adaptive technology quite effectively , including in settings where urgent formal studies may not be easily accessible . a thorough medical history and detailed physical examination will remain cornerstones of diagnosis but , nevertheless , technology that can improve accuracy should be embraced . rene laennec met some degree of resistance when he introduced the first stethoscope in 1,821 but soon enough this new technology became an icon of the medical profession ( 48 ) . similarly , in the training of its newest physicians , the internal medicine specialty needs to accept its newest the authors have not received any funding or benefits from industry or elsewhere to conduct this study .
this paper summarizes our research for herbal extracts with potent antioxidant activity obtained from a large scale screening based on superoxide radical ( o2 ) scavenging activity followed by characterization of antioxidant properties . firstly , scavenging activity against o2 was extensively screened from ethanol extracts of approximately 1000 kinds of herbs by applying an electron spin resonance ( esr)-spin trapping method , and we chose four edible herbal extracts with prominently potent ability to scavenge o2. they are the extracts from punica granatum ( peel ) , syzygium aromaticum ( bud ) , mangifera indica ( kernel ) , and phyllanthus emblica ( fruit ) . these extracts were further examined to determine if they also scavenge hydroxyl radical ( oh ) , by applying the esr spin - trapping method , and if they have heat resistance as a desirable characteristic feature . experiments with the fenton reaction and photolysis of h2o2 induced by uv irradiation demonstrated that all four extracts have potent ability to directly scavenge oh . furthermore , the scavenging activities against o2 and oh of the extracts of p. granatum ( peel ) , m. indica ( kernel ) and p. emblica ( fruit ) proved to be heat-resistant.the results of the review might give useful information when choosing a potent antioxidant as a foodstuff . for instance , the four herbal extracts chosen from extensive screening possess desirable antioxidant properties . in particular , the extracts of the aforementioned three herbs are expected to be suitable for food processing in which thermal devices are used , because of their heat resistance .	superoxide radical ( o2 ) is known to be harmful to cellular components and to function as a precursor of other reactive oxygen species ( ros ) , such as singlet oxygen ( o2 ) and hydroxyl radical ( oh ) . a dismutation reaction can result in the formation of hydrogen peroxides ( h2o2 ) and o2 from the reaction of o2 with water . a reactive non radical although h2o2 itself is not very reactive , it can convert into more reactive species such as oh , the most reactive and harmful ros , by ultraviolet irradiation , fenton like reactions and the metal ion catalyzed haber - weiss reaction . electron spin resonance ( esr ) using the spin trapping agent 5,5-dimethyl-1-pyrroline - n - oxide ( dmpo ) is a technique for the direct detection of unpaired electrons such as o2 and oh , both of which are identified by hyperfine coupling constants assigned to the spin adducts such as dmpo - ooh ( an adduct from dmpo and o2 ) and dmpo - oh ( an adduct from dmpo and oh ) . that is , the primary function is nutritional feature ( life support ) , and the secondary function is gustational feature ( taste , flavor , and texture ) . recently , antioxidant potency has been received much attention as one of the tertiary function of foodstuffs . for instance , human studies with de - alcoholized red but white wine showed short - term cardiovascular benefits , and the specific components of the de - alcoholized wine that are active on cardiovascular endpoints are the polyphenols found in red wine , especially resveratrol . in addition to its potent antioxidant properties , it has recently been reported that resveratrol mimics the anti - ageing effects of calorie restriction in lower organisms , and ameliorates insulin resistance , increases mitochondrial content , and prolongs survival in mice fed a high - fat diet . based on these backgrounds , we conducted a large scale screening to search for edible herbal extracts with potent antioxidant activity . in this review , we summarize our research for herbal extracts with potent antioxidant activity obtained from a large scale screening based on o2 scavenging activity followed by characterization of antioxidant properties . more than a thousand of herbal ethanol extracts were prepared as shown in table 1 . the assay used in this study was essentially identical to that described in our previous papers . in brief , 50 l of 2 mm hypoxanthine ( hpx ) , 30 l of dimethyl sulfoxide ( dmso ) , 50 l of sample dissolved in dmso , 20 l of 4.5 m dmpo , and 50 l of 0.4 u / ml xanthine oxidase ( xod ) were placed in a test tube and mixed . in the primary screening , all of the herbal extracts were served at a fixed concentration of 25 g / ml in the reaction mixture . the mixture was transferred to an esr spectrometry cell , and the dmpo - ooh spin adduct was quantified 100 s after the addition of xod . when a spin trapping agent , dmpo , was added to a solution of the hpx - xod reaction system , an esr signal with the hyperfine coupling constants of an = 1.37 mt , ah = 1.10 mt , ah = 0.12 this signal was assigned to the spin adduct , dmpo - ooh , by the hyperfine coupling constants . the reduction of the signal intensity of dmpo - ooh is likely reflected by the ability to scavenge o2 . polyphenol contents of some herbal extracts were determined by folin - denis method , and results were expressed as garlic acid equivalency . table 1 shows a list of all herbs tested and herbs that showed 80% or more reduction of signal intensity of dmpo - ooh measured by esr - spin trapping method at a fixed concentration of 25 g / ml . 1 shows the representative esr spectra of solvent control and ethanol extract with either poor or potent o2 scavenging activity . based on the data , we picked up four edible herbal extracts for further analyses . they are no.a-058 ( peel of punica granatum ) , no.c-037 ( bud of syzygium aromaticum ) , no.i-077 ( kernel of mangifera indica ) , and no.i-079 ( fruit of phyllanthus emblica ) . as shown in fig . 2 , these four extracts contained abundantly polyphenols , whilst herbs with poor o2 scavenging activity ( 5% or less reduction of signal intensity of dmpo - ooh at a fixed concentration of 25 g / ml ) contained a little amount of polyphenols . the results indicate that the o2 scavenging activity was apparently reflected by the polyphenols content . esr analyses of oh from fenton reaction and from photolysis of h2o2 by uv - irradiation were conducted . the assays used in this study were essentially identical to those described in a previous paper . in brief for the former assay , 50 l of 2 mm h2o2 dissolved in 0.1 m phosphate buffer ( ph 7.4 ) , 50 l of 8.9 mm dmpo dissolved in pure water , 50 l of sample dissolved in pure water and 50 l of 0.2 mm feso4 dissolved in pure water were placed in a test tube and mixed . each mixture was transferred to an esr spectrometry cell and the dmpo - oh spin adduct was quantified 113 s after the addition of feso4 . for the latter assay , a reaction mixture consisting of 440 l of 100 mm h2o2 prepared in 25 mm phosphate buffer ( ph 7.4 ) , 10 l of 111.25 mm dmpo dissolved in pure water and 50 l of sample dissolved in pure water was exposed to 254 nm uv irradiation at 4 w for 1 min at a distance of 12 cm . then the esr spectrum of dmpo - oh was measured . when dmpo was added to a solution of the fenton reaction system , the spin adduct dmpo - oh was formed . a reduction in the signal intensity of dmpo - oh by the addition of selected four herbal extracts likely reflects an ability to scavenge oh . this was also confirmed by the assay of photolysis of h2o2 by uv - irradiation . in the assay , the esr signal of dmpo - oh was reduced by adding any of the four herbal extracts , depending on the concentration . this indicates that any of the extracts has the ability to directly scavenge oh . as for the heat resistance of the antioxidant potency of the four herbal extracts , the effect of heat ( 100c ) exposure on the o2- and oh - scavenging activity of the extracts was examined . while the o2-scavenging activity of 6.25 g / ml l - ascorbic acid as a reference agent , at which concentration l - ascorbic acid exerted scavenging activity comparable to 25 g / ml herbal extract , was completely inactivated after heat exposure for 30 min , the activities of 25 g / ml extracts from p. granatum ( peel ) , m. indica ( kernel ) and p. emblica ( fruit ) were reduced by only about 20% even after heat exposure for 120 min . the extract of s. aromaticum ( bud ) was relatively heat - labile compared with the other three herbal extracts , as its activity was reduced by almost 40% after heat exposure for 60120 min . similar tendency was also observed in the oh - scavenging activity of the four herbal extracts exposed to heat ( 100c ) . these results indicate that the four herbal extracts chosen from extensive screening possess desirable antioxidant properties . in particular , the extracts of p. granatum ( peel ) , m. indica ( kernel ) and p. emblica ( fruit ) are expected to be suitable for food processing in which thermal devices are used , because of their heat resistance .
the purpose of this study was to investigate the protein expression pattern and the in vivo trichogenicity of dermal papilla - like tissues ( dplts ) made from canine adipose - derived mesenchymal stem cells ( ascs ) in athymic nude mice . canine ascs were isolated and cultured from adipose tissue , and differentiation was induced by culturing ascs in dermal papilla forming media . dplts were embedded in collagen gel , and their structural characteristics and protein expression were evaluated by hematoxylin and eosin stain and immunohistochemistry . athymic nude mice were divided into two groups ( control and dplts groups ) , and dplts were injected in skin wounds of mice in the dplts group . the trichogenicity of dplts was assessed by gross and histological evaluations for 30 days . the fate and the growth factor - secretion effect of dplts were evaluated by immunohistochemistry and western blotting . dplts have a compact aggregated structure , form extracellular matrix and highly express the protein specific for dermal papillae , including alp and versican . new hair follicle formation was remarkable in nude mice of the dplts group in gross findings and h&e stain . vascularization was increased in the dplts group , which was the effect of vascular endothelial growth factor secreted by dplts in vitro and in vivo . these data suggest that engineered canine dplts have characteristics of dermal papillae and have a positive effect on hair regeneration by secreting growth factors .	isolation and cultivation of adipose derived mesenchymal stem cells : the canine ascs used in this study were obtained in the same manner described in previous paper , and these were previously characterized . briefly , adipose tissues were aseptically harvested from gluteal region of 2-year - old beagle dogs , which were sacrificed for other objectives . the procedure was conducted with the approval of the institutional animal care and use committee of seoul national university ( snu-120306 - 5 ) . adipose tissues were washed extensively with phosphate - buffered saline ( pbs ) , minced with cut and digested with collagenase type i ( 1 mg / ml ; sigma - aldrich , st . the tissue samples were washed with pbs solution and centrifuged at 300 g for 10 min . the pellets were resuspended , filtered with a 100 m nylon mesh and incubated overnight in low - glucose dulbecco s modified eagle medium ( low glucose - dmem , gibco , life technologies , grand island , ny , u.s.a . ) supplemented with inactivated 10% fetal bovine serum ( fbs , gibco , life technologies ) and 100 units / ml penicillin-100 mg / ml streptomycin at 37.0c , in a 5% co2 incubator . after 24 hr , unattached cells and residual non - adherent red blood cells were removed by washing with pbs solution . the medium was changed every 2 days until the cells reached 8090% confluence . at passage 3 , the cells were used for the following experiments . reconstruction of dermal papilla - like tissues : after canine ascs were cultured until 80% confluence , the culture medium was changed from low glucose - dmem to dermal papilla forming media ( dpfm ) that contained 10 ng / ml hydrocortisone ( hydrocortisone , sigma - aldrich ) , 5 ml insulin - transferrin - selenium liquid media ( its , gibco , life technologies ) , 100 units / ml penicillin , 100 mg / ml streptomycin and 20 ng / ml recombinant human hepatocyte growth factor ( hgf , gibco , life technologies ) . the last changed medium contained green fluorescent nanoparticles ( cellstalker ii - csf , biterials , seoul , korea ) at concentration of 0.2 mg / ml to label the cells . after that , the cultured cells were treated with the cell dissociation reagent ( accutase , gibco , life technologies ) to detach the cells from the culture dish ( detach - attach step ) . cells were re - applied at concentrations of 8 10 cells / cm . cells were undergone redistribution , and aggregation appeared 24 hr after the detach - attach step . cell aggregates became suspended 2448 hr and then isolated from the culture medium by centrifugation at 500 rpm for 3 min . all procedures used for reconstruction of dplts were based on and modified from previous reports . immunohistochemistry of dplts : to observe the structure and protein expression state of dplts , they were harvested and embedded in collagen gel . according to the manufacturer s instructions , seven parts of 0.5% collagen solution ( matrixen - psc , bioland ltd . , gyeonggi , korea ) , two parts of 5x dmem ( gibco , life technologies ) and one part of 0.05 n naoh solution containing 2.2% sodium bicarbonate and 200 mm hepes were mixed and blended with reconstructed dplts on ice . this mixture was then placed in a humidified incubator ( 95% air/5% co2 at 37c ) for 1 hr until gelation . after the gel containing dplts was fixed with 4% paraformaldehyde solution overnight , that was embedded in paraffin . sections were cut in 4 m and stained with hematoxylin and eosin ( h&e ) . immunohistochemistry for laminin ( rabbit polyclonal antibody , ab11575 , 1:100 , abcam , cambridge , ma , u.s.a . ) , vimentin ( mouse monoclonal antibody , ab8069 , 1:100 , abcam ) , versican ( rabbit polyclonal antibody , ab19345 , 1:50 , abcam ) and vascular endothelial growth factor ( rabbit polyclonal antibody , ab46154 , 1:100 , abcam ) were performed . the sections were exposed to horseradish peroxidase ( hrp)-conjugated goat anti - rabbit igg ( g21234 , 1:500 , molecular probes , eugene , or , u.s.a . ) or goat anti - mouse igg ( g21040 , 1:500 , molecular probes ) and were visualized by a reaction with 3,3-diaminobenzidine ( dab ) tetrahydrochloride ( immpact dab peroxidase substrate , sk-4105 , vector laboratories , burlingame , ca , u.s.a . ) . canine skin tissue was used as the positive control , and 5% normal goat serum was applied instead of primary antibodies as the negative control . alkaline phosphatase activity : alkaline phosphatase ( alp ) activity of asc and dplts was measured with a colorimetric method using a tracp & alp assay kit ( # mk301 , takara bio , tokyo , japan ) . undifferentiated ascs were washed with 0.9% sodium chloride , and floating dplts were collected with culture medium and washed . equal parts of the cell lysate and substrate solution containing p - nitro - phenyl phosphate ( pnpp ) were mixed and reacted in a 96-well plate for 30 min . absorbance at 415 nm of each well was measured with a microplate reader ( model 680 , bio - rad laboratories , burlingame , ca , u.s.a . ) . protein concentration in the cell lysate was measured at the same time with a bradford assay for normalizing the alp activity . preparation of the experimental animal model : the hair inducing activity of dplts and their fate were evaluated in vivo . thirty athymic nude mice ( balb / cslc - nu / nu , six weeks old , female , 1720 g , japan slc inc . , shizuoka , japan ) were used in this study , which were divided equally in each group ( control or dplts group ; 10 , 20 and 30 days ) . mice were anesthetized by intraperitoneal injection of a tiletamine / zolazepam combination ( 30 mg / kg , zoletil50 , virbac korea , seoul , korea ) and xylazine ( 10 mg / kg , rompun , bayer korea , seoul , korea ) . the scalp of mice was prepared aseptically , and full thickness skin wounds were created with a 6 mm biopsy punch . in the dplts group , 400 dplts mixed with 50 l of pbs were injected into the inner - dermis at the border between the wound and the normal skin . on the other hand , only 50 l of pbs were injected in the same manner to mice in the control group . paul , mn , u.s.a . ) was used for wound protection until wound healing was completed . the animals were housed in a specific pathogen - free ( spf ) room at seoul national university , and all of the procedures were approved by the institutional animal care and use committee of seoul national university ( snu-140917 - 2 - 3 ) . evaluation of hair growth in gross and histological examinations : the wounds were photographed using a digital camera at 0 , 10 , 20 and 30 days after transplantation of dplts . the mice were euthanized by cervical dislocation at 10 , 20 and 30 days from the time of dplts injection . the skin around the wounds was harvested and divided into two parts with wound as a center : one part for histologic evaluation and the other part for western blotting . the sampled skin was fixed in 10% neutral formalin solution and embedded in paraffin . the number of hair follicles and sebaceous glands in the regenerated wounds , and vascularization were evaluated . immunohistochemistry for endothelial cells with anti - cd31 antibody ( rabbit polyclonal antibody , ab28364 , 1:50 , abcam ) was used to visualize the vessels . the positive control ( murine skin tissues ) and negative control were also applied in the same manner . vascularization was evaluated by the number of blood vessels in the high - power field ( 400 ) using five randomly selected fields from each skin section . for immunofluorescence , the sections were incubated with rabbit anti - vascular endothelial growth factor ( vegf ) antibody ( ab46154 , 1:200 , abcam ) . then , cytm3-conjugated affinipure donkey anti - rabbit igg ( h+l ) ( 711 - 165 - 152 , 1:500 , jackson immunoresearch , west grove , pa , u.s.a . ) was applied at room temperature for 1 hr . western blotting : the tissue extracts were prepared from the skin with the pro - prep protein extraction solution ( intron biotechnology , gyonggi - do , korea ) . the protein concentrations were determined with the bradford assay ( bio - rad laboratories ) . the extracted proteins ( 20 g ) were mixed with laemmli s sodium dodecyl sulfate ( sds)-sample buffer ( gendepot , baker , tx , u.s.a . ) and boiled for 5 min before loading . samples were run on a 12% sds - polyacrylamide gel and transferred onto a nitrocellulose membrane . the membranes were blocked with 5% skim milk for 1 hr at room temperature and incubated overnight at 4c with rabbit polyclonal anti - vegf antibody ( ab46154 , 1:1,000 , abcam ) . anti - rabbit igg - hrp was used as a secondary antibody for 1 hr at room temperature . anti--actin antibody ( sc-47778 , 1:1,000 , santa cruz biotechnology , santa gruz , ca , u.s.a . ) was used for a loading control . blots were visualized with an enhanced chemiluminescence kit ( ecl kit , invitrogen , life technologies ) and visualized under chemidoc . the optical densities were quantified using image analyzer software ( image j , u.a . statistical analysis : statistical analyses were performed using spss , version 22.0 ( spss inc . , tokyo , japan ) . a mann - whitney u test was used to assess the changes or differences between the control group and the dplts group . data are reported as the mean values , with the variability expressed as standard deviation . reconstruction of dplts : ascs underwent morphological changes by dpfm from a spindle shape to a polygonal shape , which started approximately 24 hr after changing media . after the detach - attachment step , cell distribution was changed , and groups of cells began to pull apart and form aggregates ; cell aggregates started to float after 2448 hr . the morphology and the size of dplts were consistent with the previous study [ 1 , 32 ] . structure and protein expression of dplts : the compact structure of the dplts , which had closely connected cells , was observed by h&e staining ( fig . 1.analysis of the structure and the protein expression of collagen embedded dplts by histological ( a ) , immunohistochemical ( ihc ) staining ( b d ) and alkaline phosphatase activity ( e ) . dplts were immunostained by versican ( b ) , laminin ( c ) and vimentin ( d ) . ( e ) intracellular alp concentration was significantly ( * * p<0.01 ) higher in dplts compared to ascs . reconstructed self - aggregated dplts had significantly higher levels of alp compared to the ascs ( fig . analysis of the structure and the protein expression of collagen embedded dplts by histological ( a ) , immunohistochemical ( ihc ) staining ( b d ) and alkaline phosphatase activity ( e ) . dplts were immunostained by versican ( b ) , laminin ( c ) and vimentin ( d ) . ( e ) intracellular alp concentration was significantly ( * * p<0.01 ) higher in dplts compared to ascs . hair inductivity : approximately 10 days after wound induction , wound healing was completed in all groups . new hair fibers on the regenerated skin were observed at 1520 days after injection , especially in the dplts group ( fig . photographs of the wounds at days 10 , 20 and 30 from the control and the dplts groups . hair growth around and at the wound bed was remarkable in the dplts group compared to the control group at day 20 . ) . regenerated hairs at the wound bed in the control group were short and thin as well as mainly observed around the periphery of the wound , whereas the central wound region had regenerated hairs in the dplts group . intriguingly , the number of hairs at the wound bed slightly decreased at day 30 . photographs of the wounds at days 10 , 20 and 30 from the control and the dplts groups . hair growth around and at the wound bed was remarkable in the dplts group compared to the control group at day 20 . the difference between the groups was more apparent in the histological evaluation . at day 10 and day 20 , the skin tissues from the dplts group ( 4.6 2.19 and 10.7 2.89/wound bed ) contained significantly more regenerated hair follicles in the wound bed than those from the control group ( 0.5 1.00 and 4.0 1.00/wound bed ) ( fig . microphotographs of wounds and outside the wound bed at days 10 , 20 and 30 ( a ) . trichogenicity was assessed by the number of regenerated hair follicles ( b ) and regenerated sebaceous glands ( c ) per wound bed at a section . the number of regenerated hair follicle and sebaceous gland was significantly ( * p<0.05 ) higher in the dplts group versus the control group at days 10 and 20 . at days 30 , the number of regenerated hair follicles was slightly decreased . and , between the groups , the differences in the number of hair follicles and sebaceous glands were unremarkable . ) . newly formed hair follicles increased until day 20 , and then , the trends changed by 30 days . the number of regenerated hair follicles was slightly decreased in both groups , and the difference between two groups was not significant at day 30 . microphotographs of wounds and outside the wound bed at days 10 , 20 and 30 ( a ) . trichogenicity was assessed by the number of regenerated hair follicles ( b ) and regenerated sebaceous glands ( c ) per wound bed at a section . the number of regenerated hair follicle and sebaceous gland was significantly ( * p<0.05 ) higher in the dplts group versus the control group at days 10 and 20 . at days 30 between the groups , the differences in the number of hair follicles and sebaceous glands were unremarkable . the number of sebaceous glands in the wound bed observed at days 10 and 20 was significantly higher in the dplts group ( 2.2 0.84 and 2.3 0.50/wound bed ) compared to the control group ( 0.0 0.00 and 0.3 0.50/wound bed ) ( fig . however , similar to the hair follicles , there was no significant difference between two groups in the number of sebaceous glands at day 30 . in addition , the majority of the pre - existing hair follicles outside the wound bed were in the anagen phase at day 20 , which consisted of multilayered cells and located deeply in the subcutaneous region . while , most pre - existing hair follicles at day 30 were in the telogen phase ( fig . angiogenesis was evaluated by counting the number of vessels in the slides , which were immunostained against cd31 at day 10 ( fig . microphotographs of the wounds at 10 day in the control group ( a ) and the dplts group ( b ) . the number of vessels at the wound per five fields at days 10 , 20 and 30 ( c ) . significantly ( * * p<0.01 ) more vessels were observed in wound bed from the dplts group than from the control group . ) . the number of cd31-positive vessels at the wound per five fields was significantly higher in the dplts group ( 48.0 15.05/hpf ) relative to the control group ( 16.2 5.54/hpf ) at day 10 . microphotographs of the wounds at 10 day in the control group ( a ) and the dplts group ( b ) . the number of vessels at the wound per five fields at days 10 , 20 and 30 ( c ) . significantly ( * * p<0.01 ) more vessels were observed in wound bed from the dplts group than from the control group . the role of dplts in hair inductivity : under fluorescence microscopy , it is confirmed that injected fluorescence - labeled dplts did not constitute regenerated dps . instead of structural function , we performed immunohistochemistry on collagen - embedded dplts and mice skin tissues at day 10 in order to identify the paracrine effect of dplts on hair regeneration . several vegf - positive dplts were detected in both samples , which indicates that dplts could secrete vegf in vitro and in vivo ( fig . 5a and 5bfig . 5.in vitro ( a ) and in vivo ( b ) vegf secretion of dplts and quantitative evaluation of vegf from the wound at day 10 ( c ) . ( a ) notice strong positive cytoplasmic immunostaining for vegf of collagen embedded dplts . ( b ) ihc of injected dplts ( green - labeled ) for vegf ( red ) at 10 day . ( c ) quantitative evaluation of vegf protein from the wound at day 10 by western blotting . western blotting was performed to quantify the expression level of vegf at day 10 . the larger amount of vegf was detected in the dplts group compared with the control group , though the difference in the thickness of the bands was not significant ( fig . ( a ) and in vivo ( b ) vegf secretion of dplts and quantitative evaluation of vegf from the wound at day 10 ( c ) . ( a ) notice strong positive cytoplasmic immunostaining for vegf of collagen embedded dplts . ( b ) ihc of injected dplts ( green - labeled ) for vegf ( red ) at 10 day . ( c ) quantitative evaluation of vegf protein from the wound at day 10 by western blotting . our previous study demonstrated that dplts could be successfully reconstructed from canine ascs . in succession , reconstructed dplts were evaluated as an alternative to actual dps by rna and protein expression through the previous and the present studies . our immunohistochemistry data revealed versican in the dplts , and alp activity is significantly elevated in dplts than in ascs . moreover , the compact spheroid - forming structure of the cells was observed in h&e staining , and the extracellular matrix formed during aggregation of dplts was immunostained by laminin and vimentin . versican and alp are key characteristic molecular markers of dps , which are specifically expressed in dps during the anagen phase and absent in the telogen phase [ 19 , 30 ] . consistent with our findings , the levels of versican and alp are closely associated with in vivo hair inducing activity of the dpcs [ 6 , 11 , 12 ] , and a distinct tendency to form cell aggregates of dermal papilla cells indirectly indicates the in vivo trichogenic property of cells [ 2 , 8 , 20 ] . in vivo trichogenesis was evaluated by injecting dplts into a full - thickness skin wound on the head of nude mice . athymic nude mice , which were used in this study , are characterized by disturbed development of hair follicles and dysgenesis of the thymus [ 17 , 18 ] , which is a suitable animal model to evaluate hair regeneration with a xenograft of canine cells . yoo et al . transplanted dplts to inner - dermis in nude mice with outer root sheath cells ( orscs ) as a source of epithelial cells . by contrast , we injected dplts as a mesenchymal component at the full - thickness skin wound , anticipating the wound epithelium would interact with dermal components as epithelial components as previously described . gross and microscopic findings at days 10 and 20 indicate that dplts play a crucial role in contributing to hair regeneration . regeneration of sebaceous gland was evaluated with that of hair follicle in this study . the results of the present study indicated that not only hair follicles , but also the complete pilosebaceous units were regenerated by dplts . the number of hair follicles was slightly decreased at day 30 compared to that of at day 20 , and the difference between the groups also decreased . there are two possible explanations for the decrease in regenerated hair follicles at day 30 . first , hair inducing role of dplts could be influenced by the hair cycle of mice . nude mice are not actually nude ; they also have hair follicles and a hair cycle , which is synchronized and repeated with a 3-week interval . this is consistent with our observation , and anagen phase proceeded during days 1020 , whereas at days 30 , most hair follicles outside the wound bed were in the telogen phase , which was characterized by shrunk hair bulb . in addition , our findings demonstrated small amounts of injected dplts could survive for 30 days , and the effect of dplts was minimal at day 30 . moreover , in the histological examination , the number of vessels in the wound bed was higher in the dplts group . especially , statistically significant difference between groups was observed at day 10 , which suggests that implanting dplts on the injection site may enhance the neovascularization . the decrease in the number of vessels in the dplts group could be explained by the decrease of granulation tissue as time passed . in addition , as the survival rate of injected dplts declined over time , the amount of vegf secreted from dplts also diminished . the transition of the hair cycle ( physiological hair loss ) and alopecia ( pathological hair loss ) were not only controlled but also characterized by dps and peri - papillary vascularization [ 23 , 31 ] . in addition , the close connection between hair regeneration and neovascularization around hair follicles has been identified under various treatments for alopecia including when medical treatments and laser therapy were applied [ 13 , 23 , 29 ] . while mesenchymal stem cells have been observed to have a stimulatory effect on hair regeneration due to the paracrine secretion of growth factors in previous studies [ 10 , 22 ] , we expected dplts to be the direct structural components of the hair follicle as a substitute for dps under control of emi . therefore , emi with reconstructed canine dplts is not fully evaluated in the present study , and a further study is needed . instead of a direct structural role , growth factor secretion of dplts although polyclonal antibody used in this study detected both murine and canine vegf in an in vivo study , merged signals of dplts and vegf could prove the presence of growth factors secreted from dplts . these results are consistent with the fact that the mesenchymal component , including dpcs , strongly expresses vegf compared to other components of the hair follicle . in other words , the vegf autocrine effect of dps is also identified on our reconstructed dplts . vegf stimulates the proliferation of dpcs in vitro as well as stimulates hair regrowth and increases the size of the hair follicles and hair shafts in vivo [ 15 , 31 ] . as a result of western blotting , the amount of vegf tended to be higher in the dplts group than in the control group , although no significant difference was observed between groups . these results could be explained by the survival rate of dplts on wound bed . a further study and application of scaffold or biomaterials with dplts which act as a delivery vehicles and physical support , first of all , structural role of dplts was not proved in the study , where activated epithelial cells through the wound healing had been expected to interact with dplts by emi . a further study , where epithelial component should be injected with dplts ( dermal component ) without wound , was needed to evaluate emi . second , residual dplts and the amount of vegf secreted by dplts were not constant by time according to our observation . the survival rate of cells on the injected site is the major concerns in regenerative medicine . biomaterials which act as a scaffold for extending the survival time and maintaining 3d structure of dplts should be evaluated . although only paracrine effect of dplts was proved in the present study , dplts also have the several positive effect on hair regeneration similar to dps . the dps have a stem cell - like role to differentiate into multi - lineage in itself , as well as the dps contact the bulge and activate the bulge stem cells at the early anagen to form the the new hair follicles after remaining intact during the catagen and the telogen phases . furthermore , dps help neighboring epidermal cells to differentiate into cells of hair follicle lineages [ 5 , 30 ] . other paracrine and signaling pathways , such as wnt/-catenin / bmp , fgf and igf , in dplts also should be evaluated in a further study [ 4 , 16 , 21 ] . in conclusion , this study demonstrates the possibility of dplts made from canine ascs as a substitute for dps in alopecic animals . in addition to the previous in vitro study , the in vivo results indicate that this engineered construct could enhance the hair follicle regeneration in an athymic nude mouse model by increasing neovascularization on the wound bed . to our knowledge , the development of reconstructed dps from mesenchymal stem cells from animals has not been previously reported . despite its imperfections , engineered canine dplts have the feasibility to be a useful treatment option for alopecic animals as an easily obtained source of dpcs as well as a study model to further characterize dpcs .
clay minerals are ubiquitous in nature , and the manner in which they interact with their surroundings has important industrial and environmental implications . consequently , a molecular - level understanding of the adsorption of molecules on clay surfaces is crucial . in this regard computer simulations play an important role , yet the accuracy of widely used empirical force fields ( ff ) and density functional theory ( dft ) exchange - correlation functionals is often unclear in adsorption systems dominated by weak interactions . herein we present results from quantum monte carlo ( qmc ) for water and methanol adsorption on the prototypical clay kaolinite . to the best of our knowledge , this is the first time qmc has been used to investigate adsorption at a complex , natural surface such as a clay . as well as being valuable in their own right , the qmc benchmarks obtained provide reference data against which the performance of cheaper dft methods can be tested . indeed using various dft exchange - correlation functionals yields a very broad range of adsorption energies , and it is unclear a priori which evaluation is better . qmc reveals that in the systems considered here it is essential to account for van der waals ( vdw ) dispersion forces since this alters both the absolute and relative adsorption energies of water and methanol . we show , via ff simulations , that incorrect relative energies can lead to significant changes in the interfacial densities of water and methanol solutions at the kaolinite interface . despite the clear improvements offered by the vdw - corrected and the vdw - inclusive functionals , absolute adsorption energies are often overestimated , suggesting that the treatment of vdw forces in dft is not yet a solved problem .	the accurate treatment of the adsorption of molecules on surfaces is a major challenge of materials modeling , with important applications in nanotechnologies and science : heterogeneous catalysis , sensors , corrosion , lubrication , friction , and coatings , to name but a few . clay minerals are natural aluminosilicates that find use in a wide variety of fields such as medicine , adhesives , paints , and oil drilling . they also act as catalysts to ice nucleation in the atmosphere and help cleanse soils and groundwater through adsorption of pollutants . a clear understanding of how molecules interact with the surfaces of clays is of the utmost importance to understand , improve , and control such processes . reliable reference data from theory and simulation are of intrinsic value and often important as a complement to experiments . computer simulations of water surface interactions , at the molecular level , are often based on force fields ( ff ) and density functional theory ( dft ) approaches . although these techniques are incredibly powerful and useful , there are cases where their accuracy is not satisfactory . ff potentials have parameters that have to be fit in order to reproduce experimental results or higher lever theoretical benchmarks , and this is not always straightforward . unfortunately , dft results are highly sensitive to the choice of the exchange - correlation ( xc ) functional used , and nowadays there are countless xc functionals to choose from . in the field of materials science , the description of weak bonding interactions , and in particular london dispersion forces , is one of the most important challenges . immense progress has been made in this area recently ; however , there is no rigorous way to systematically improve xc functionals , and as a result validation with alternative methods is needed . of the various high level reference methods available , quantum monte carlo ( qmc ) is a powerful approach for obtaining benchmark values for solids , surfaces , and large molecular systems . qmc , within the fixed node diffusion monte carlo ( dmc ) approach , has already been used to tackle interesting materials science problems that have been beyond the reach of dft ( see , e.g. , refs ( 2942 ) ) . this has provided reference data which have exposed shortcomings in existing ff models and dft xc functionals , which in turn aids the development of such approaches . in this paper we will use two qmc approaches to investigate molecular adsorption on a clay surface , namely , dmc and lattice regularized diffusion monte carlo ( lrdmc ) . the particular clay we will examine is kaolinite ( al4si4o10(oh)8 ) , as shown in figure 1 . since the first outline of the kaolinite crystal structure by pauling in 1930 , numerous structural studies using x - ray and neutron powder diffraction , x - ray single crystal , and electron diffraction methods , as well as theoretical studies have been carried out on kaolinite . consequently it is one of the most suitable aluminosilicate clays to assess the performance of various theoretical methods . in addition , when looking at adsorption processes on kaolinite , cleavage along the ( 001 ) basal plane leads to exposure of either aluminate or silicate faces ( figure 1 ) . the aluminate ( aloh ) face is terminated in hydroxyl groups and as a result is regarded as hydrophilic , whereas the silicate ( sio2 ) face which exposes saturated si the distinct chemical nature of these two surfaces means that adsorbates will interact differently with them , making kaolinite an interesting case study for understanding the role of vdw forces on the adsorption at clay mineral surfaces . the hydrogens are sketched in white , the oxygens in red , the silicons by yellow tetrahedra , and the aluminums by pink octahedra . the figures on the right are the hydroxyl - terminated face ( top ) and the silicate - terminated face ( bottom ) . various adsorption sites on the hydroxyl- and silicate - terminated face are labeled . in what follows , we will provide benchmark values for the adsorption of water and methanol molecules at the pristine hydroxyl- and silicate - terminated ( 001 ) faces of kaolinite , by using dmc and lrdmc . then , we probe dft xc functionals by considering a range of generalized gradient approximation ( gga ) functionals , hybrid functionals , and dispersion - corrected density functionals which account for vdw forces . in the case of molecules adsorbed on kaolinite we find that the bare ggas and hybrids are quite unreliable : as expected adsorption energies are underestimated , but more importantly , the relative adsorption energies of water versus methanol do not even agree with qmc . moreover , on the silicate - terminated face the molecules hardly bind at all and move quite far from the surface during geometry optimizations . accounting for vdw forces improves adsorption energies significantly and stabilizes the structures . however , most of the vdw - corrected and vdw - inclusive functionals predict adsorption energies which are slightly too large compared to qmc . this indicated that there remains room for improvement in terms of how vdw forces are handled in dft . since a range of techniques has been used , for brevity the more detailed descriptions of the computational setups are provided in the supporting information . qmc and dft evaluations of the adsorption of water and methanol at both ( 001 ) faces of kaolinite are reported and discussed in section 3 . adsorption was examined on a single layer of kaolinite , a system with 2d periodicity along the a and b axes as indicated in figure 1 . the simulated supercell was 1 2 the conventional unit cell of bulk kaolinite ( ca . 10.38 9.01 ) and is comprised of 8 aluminums , 8 silicons , 36 oxygens , and 16 hydrogens for the kaolinite slab plus the atoms of the adsorbed molecule . note that with ca . 300 electrons the simulations are large for qmc calculations . the adsorption energy , eadsm@x , of the molecule m at the face x of the kaolinite layer , where m can either be the water ( h2o ) or the methanol ( meoh ) and x can be the hydroxyl - terminated ( aloh ) face or the silicate - terminated ( sio2 ) face , can be evaluated in two ways : the first method , hereafter called complex - minus - fragments , is computed as1where eslab+m@x is the total energy for the system with m at the x - face of the kaolinite slab , and eslab and em are the total energies of the isolated slab and the isolated molecule m , respectively . the second method , hereafter called complex - minus - far , is computed as2where eslab m is the total energy of a system where the kaolinite slab and the molecule m are far enough apart that their interaction is negligible . the two methods are equivalent if and only if : ( i ) the size effects due to the periodicity of the system are negligible and ( ii ) the electronic structure calculations are performed with methods that are exactly size consistent . if these conditions are not satisfied in general we have that eslab m em + eslab , meaning that eqs 1 and 2 provide different evaluations of the adsorption energies . in particular , whenever size effects are detected , the complex - minus - far method usually benefits from a larger error cancellation . on the other hand , in cases where size effects are negligible and electronic structure methods are size consistent , there are no residual interactions between the molecule and the periodic partners , and then the complex - minus - fragments method is usually to be preferred . the reason for that is the computational cost : for a system with n electrons the computation is proportional to n , with > 1 ( e.g. , in dft is typically between 2 and 3 and in qmc between 3 and 4 ) , so the cost for calculations of eslab and em is cheaper than eslab moreover , when several adsorption energies need to be evaluated , eslab is calculated only once , whereas a different calculation of eslab m has to be performed for each molecule m. the two qmc approaches used are dmc and lrdmc . they are both projection monte carlo methods : they can access the electronic ground state energy of the system by iteratively projecting an initial trial wave function t into the ground state , with the constraint that the projected wave function has the same nodal surface of an appropriately chosen guiding function g ( fixed node approximation ) . both the trial and the guiding wave functions are parametrized functions , and they have to be the best approximation of the ground state that we can provide ( given the constraint of their ansatz ) thus , usually they are taken such that t = g = vmc , where vmc is the best function obtained within a variational monte carlo approach , with the variational parameters optimized in order to minimize either the variational energy or the variance . whenever g has the exact nodal surface , the approach is exact ; otherwise , it gives the best approximation of the ground state given the fixed node constraint . in projection monte carlo approaches there is a second approximation in how the projection is performed , and it is different in dmc and lrdmc . the projection in dmc comes from the imaginary time schrdinger equation ; it is implemented as an imaginary time evolution , where a time step has to be chosen . the chosen is a trade - off between accuracy and computational cost : exact results are obtained for 0 , but the computational cost is 1/. the finite time - step error can be controlled by performing several calculations with different values of and finally extrapolating to the continuum limit 0 . however , in big systems like those considered here , the extrapolation is impractical and sometimes unfeasible or unreliable , but it is sufficient to consider the results for a small enough that the expected finite - time error is smaller than the required accuracy . here , we have chosen in order to have an expected time - step error smaller than the stochastic error of the evaluations ( see section i in the supporting information ) . on the other hand , lrdmc is based on the spatial discretization of the molecular hamiltonian on a lattice of mesh size a , and it resorts to the projection scheme used also in the green function monte carlo algorithm . the error induced by the finite mesh size a is analogous to the time - step error appearing in standard dmc calculations . lrdmc preserves the variational principle even when used in combination with nonlocal pseudopotentials ( pps ) , and it is size consistent for any value of the mesh a , maintaining its efficiency even for systems with a large number of electrons . both dmc and lrdmc provide excellent benchmark values for weakly interacting systems , as established in a number of studies . we used here a standard setup , described in detail in the supporting information . the stochastic error associated with the qmc evaluations of the adsorption energy is ca . the systems under consideration are too large for a qmc - based structural optimization , even at the variational level , so the reference structures were those obtained from the pbe - d3 functional , as described in section 3.1 . as we will see in section 3 , pbe - d3 configurations are in good agreement with those obtained by all the other vdw - inclusive functionals , thus the bias given by the use of pbe - d3 configurations is expected to be small compared to the stochastic error of the dmc evaluation . there is one aspect of qmc simulations that deserves special care in this specific system , namely , the finite - size errors ( fses ) . qmc is a many - body method , and in contrast to ( effective ) one - particle methods such as dft , qmc can not simply exploit bloch s theorem in calculations for extended periodic systems . fses can be taken into account by performing simulations in larger periodic supercells , through the twist - average method , corrections to the ewald energy , or the kwee , zhang , krakauer ( kzk ) method . in this work we have used the kzk method ( see section 3 in the supporting information ) . there is by now an almost limitless variety of dft xc functionals that we could examine . here we restrict ourselves to the lda functional ; two gga functionals , pbe and rpbe ; two hybrid functionals , pbe0 and b3lyp ; three vdw - corrected pbe functionals pbe - d2 , pbe - d3 , ( both from grimme , d3 correction with zero - dampling ) , and pbe+vdw(ts ) from tkatchenko and scheffler ; two vdw - corrected hybrid functionals , pbe0-d3 and b3lyp - d3 ( both from grimme ) ; and four self - consistent nonlocal functionals ( often called vdw - inclusive functionals ) , the original vdw - df from dion ( also named revpbe - vdw ) , the second generation vdw - df2 , as well as optpbe - vdw and optb86b - vdw from klime et al . we stress that the latter four vdw - inclusive functionals are actually based on ggas and basically differ from the vdw - corrected gga functionals ( e.g. , pbe - d2 , pbe - d3 , and pbe+vdw(ts ) ) only in the way the dispersion energy is approximated . other functionals and vdw corrections have been tested , and results obtained using a comprehensive set of approaches is reported in table s1 of the supporting information . adsorption energies were evaluated using the complex - minus - fragments method ( see eq 1 ) , but the results are the same as those obtained with the complex - minus - far method , as expected . further details about the setup of the dft calculations are reported in section 4 of the supporting information . we also performed a series of molecular dynamics simulations using classical force fields for aqueous water the kaolinite slab was modeled as a single sheet of kaolinite ( approximately 31 36 ) using the clayff force field , and the oh bond lengths were constrained using the p - lincs algorithm . above this slab 538 tip4p/2005 water and 230 opls / ua methanol molecules were randomly placed in order to create a liquid film on the kaolinite surface . the standard lorentz berthelot mixing rules were used to compute cross - interactions , except to adjust the adsorption energies as detailed below . using the gromacs 4.5 simulation package , constant volume and temperature dynamics were propagated using a leapfrog integrator and a nos - hoover chain thermostat , along with replica - exchange among eight replicas with temperatures ranging from 275 to 310 k in 5 k intervals . real - space interactions were truncated at 9 with corrections to the energy applied , and particle - mesh ewald was used to account for long - range electrostatics with the corrections for the slab geometry of the system . a time step of 2 fs was used and molecular dynamics simulations performed for at least 11 ns , with the first nanosecond being disregarded as equilibration . adsorption energies were computed after geometry optimization using the complex - minus - fragments method . this was done in three ways : first , the adsorption energy was computed by applying the standard mixing rules . this yielded adsorption energies of 642 and 640 mev for water and methanol , respectively , and eads = 2 mev . second , the strength of the lennard - jones interaction between the ch3 group of methanol and the oxygen atoms of the kaolinite oh groups was adjusted such that eads matched that of pbe . finally , the same was done to match eads obtained by dmc . water adsorption on the hydroxyl - terminated face of kaolinite has been studied experimentally and theoretically , whereas adsorption on the silicate - terminated face is less well studied . very little is known about methanol adsorption on either face . in the following , the most stable adsorption structures identified for water and methanol on the two kaolinite surfaces are presented . on each surface a range of adsorption sites were considered , as indicated in figure 1 . according to the number of h - bonds formed between the adsorbate and the surface , the adsorption sites can be classified into three categories : 3-fold , 2-fold , and 1-fold sites . the most stable configurations obtained , using dft with the pbe - d3 functional , are shown in figures 2a2h . these structures have been taken as the reference for dmc , lrdmc , and the other dft calculations . in addition , starting from the reference pbe - d3 structures , we have relaxed the geometries for each of the different functionals considered , as shown in figures 3a3d . figure 3e compares the distance of the molecules from the slab as obtained with different functionals . adsorption of water and methanol on the hydroxyl - terminated and the silicate - terminated faces of kaolinite ( side view in first row , top view in second row ) . geometries are relaxed using the pbe - d3 functional and have been taken as reference for the other calculations . the adsorbed molecule on kaolinite is depicted in cyan and gray , and the h - bonds are represented by the blue dashed lines . panels a , b , c , and d show the most stable dft structures for the adsorption of water and methanol on the hydroxyl- and silicate - terminated faces of kaolinite , provided by the different xc functionals considered . the color scheme for the various functionals is blue for pbe , cyan for rpbe , white for pbe - d2 , black for pbe - d3 ( that is also the reference for qmc calculations ) , pink for pbe+vdw(ts ) , violet for vdw - df , green for vdw - df2 , gray for optpbe - vdw , and yellow for optb86b - vdw . panel e shows the height of the center - of - mass of the adsorbed molecules from the average surface plane defined by the surface oxygens for the different xc functionals . the four dashed horizontal lines correspond to the values for the reference pbe - d3 structures . concerning water at the hydroxyl - terminated face ( h2o on aloh ) , all structures initially put in 2-fold and 1-fold sites ( a5a8 ) moved to the 3-fold site a1 . this preference for the a1 site agrees with previous dft studies with local and semilocal xc functionals . in the most stable configuration , shown in figures 2a and 2e , the c2 axis of the water molecule lies almost parallel to the plane of the surface . the water molecule donates one h - bond ( oh distance of 1.69 ) to and accepts two h - bonds ( 2.01 and 2.04 ) from the surface ( pbe - d3 values ) . let us now consider the adsorption of methanol at the hydroxyl - terminated face ( meoh on aloh ) . one way of viewing methanol is as a water molecule with one of its hydrogen atoms replaced by a methyl group . this leads to two possible types of interaction with the surface : ( i ) hydrogen bond formation with the hydroxyl functional group and ( ii ) dispersion interactions arising from the ch3 group . all calculations in which the methanol began parallel to the surface ended with the methanol perpendicular to the surface , maximizing the distance between the ch3 group and the kaolinite . the ch3 group can therefore be considered a spectator group that does not participate directly in the adsorption on the surface . the adsorption of methanol is therefore very similar to that of water , and indeed , we find that a1 is the most favorable site , with the methanol donating one h - bond to and accepting two from the surface . as was the case for the water structure , the h - bond donated by the methanol is much stronger than the two h - bonds it accepts : 1.68 vs 1.97 and 2.03 , respectively , with the pbe - d3 functional . the most stable configuration is shown in figures 2b and 2f . as noted above , adsorption of water at the silicate - terminated face ( h2o on sio2 ) of the six adsorption sites ( s1s6 ) considered here , the 1-fold s5 site turned out to be the most stable at the gga level , and the 2-fold s1 generally is the most stable for the vdw - corrected and vdw - inclusive functionals . the most stable structure found for methanol at the silicate - terminated face ( meoh on sio2 ) using the pbe - d3 functional is shown in figures 2d and 2h . the leading interaction here is dispersion ; there is no h - bond - like interaction because the oh group of the methanol is parallel to the surface of the slab . the dmc and lrdmc results for water and methanol adsorption on the two faces of kaolinite are reported in table 1 . as mentioned in the previous section , dmc and lrdmc evaluations have to be corrected for finite - size effects , and in our lrdmc simulations there is also an unphysical dipole interaction between slabs due to the 3d periodicity employed . the dmc calculations have been performed with 2d periodicity and so do not suffer from the latter problem . the bare and corrected results it can be seen that our best estimates of the adsorption energy of water on the hydroxyl - terminated face are 648 18 mev with dmc and 675 14 mev with lrdmc . for methanol our best estimates of the adsorption energy are 694 18 mev with dmc and 701 13 mev with lrdmc . we notice that we are in the chemisorption regime both for water and for methanol , although the adsorption energy of methanol is slightly larger . note that for both molecules the dmc and lrdmc evaluations are in good agreement , with the differences falling within the stochastic error of the evaluations . this shows that fixed - node projection qmc schemes are robust approaches : they are only slightly affected by the actual computational setup and implementation . nonetheless , two slightly different adsorption energies for each case are obtained , and we should choose only one of them to use as our benchmark . we feel that in the specific case considered here the dmc values are likely to be more reliable since they have been obtained in 2d , as opposed to the lrdmc results which have been corrected for the dipole in the 3d cell . moreover , the reported lrdmc evaluations use eq 1 , which has larger fse than the reported dmc evaluations , which use eq 2 . as discussed in the text , bare dmc and lrdmc results are affected by finite - size errors ( see section 3 and table s2 in the supporting information ) that we have estimated and corrected the adsorption energies for accordingly . dipole interaction between the periodic slabs ( because in this case 2d periodicity was not available , and we had to use 3d periodicity ) , thus we have included a dipole interaction correction . having compared the results of the two qmc approaches on the hydroxyl - terminated face , we have only performed a dmc evaluation on the silicate - terminated face . the dmc adsorption energy at the silicate - terminated face is 184 23 mev for water and 250 18 mev for methanol . the methanol adsorbs more strongly than water , as for the hydroxyl - terminated face ; however , in this case the adsorption is weaker , and we are in the physisorption regime . we now examine how the various dft xc functionals considered in this study perform for water and methanol adsorption on the two faces of kaolinite . in table 2 and figure 4 we summarize the adsorption energies obtained with the different density functionals . at the gga level pbe and rpbe give significantly different adsorption energies , with rpbe providing a value that is roughly 50% that of pbe . in line with the smaller adsorption energy we also see that the bonds the molecule makes with the surface with the rpbe functional are considerably longer than what is obtained with pbe . specifically , with rpbe the two h bonds accepted from the surface are 2.30 and 2.36 , and the one donated is 1.81 , versus 2.03 , 2.06 , and 1.70 with pbe . including dispersion interactions does not drastically change the geometry of the adsorbed water monomer at the hydroxyl - terminated face : the bond lengths at the pbe - d2 , pbe - d3 , pbe+vdw(ts ) , and opt - b86b - vdw level are slightly shortened , but they remain within 0.05 of the pbe structure . pbe - d2 predicts the shortest distance from the surface and the shortest h - bonds . the other functionals give h - bond lengths between the values provided by pbe and rpbe . from the shortest to the longest interaction distance , the functionals are ranked in the following order : pbe - d2 < pbe - d3 optb86b - vdw pbe+vdw(ts ) < pbe < optpbe - vdw < vdw - df2 < vdw - df < rpbe . this trend roughly follows the sequence of adsorption energy predicted by the functionals and is also consistent with previous studies of dft xc functionals for hydrogen - bonded systems . relaxation from the pbe - d3 geometry ( performed for all the gga and gga+vdw functionals ) results in rather small increases in adsorption energies . the maximum difference is observed for vdw - df , with an increase of 36 mev upon relaxation . energies have been obtained on pbe - d3 optimized structures , but in parentheses we also report the adsorption energies when geometries are fully relaxed consistently for each gga and gga+vdw functional . adsorption energies on kaolinite obtained by various xc functionals and dmc , for water on the hydroxyl - face ( h2o on aloh , blue upper triangles ) , methanol on the hydroxyl - face ( meoh on aloh , red squares ) , water on the silicate face ( h2o on sio2 , cyan lower triangles ) , and methanol on the silicate face ( meoh on sio2 , orange diamonds ) . filled points represent the values with the reference structures ( obtained using pbe - d3 ) , and empty points ( reported only for gga and gga+vdw functionals ) correspond to relaxed structures for the specific functional . the solid lines are the reference dmc adsorption energies , and the shaded areas show the stochastic error . a comparison with the qmc adsorption energies shows that vdw - df2 and optpbe - vdw yield the best agreement , with the former providing a slightly underestimated adsorption energy ( by 32 18 mev ) and the latter a slightly overestimated one ( by 41 18 mev ) . it also appears that the two gga functionals ( pbe and rpbe ) , the two hybrid functionals ( pbe0 and b3lyp ) , and vdw - df underestimate the interaction energy , whereas all the other functionals ( pbe - d2 , pbe - d3 , pbe+vdw(ts ) , optb86b - vdw , pbe0-d3 , and b3lyp - d3 ) overestimate the interaction . in particular , evaluations of the adsorption using vdw - corrected hybrid functionals do not seem to improve significantly compared to the gga+vdw approaches . a careful investigation shows that the ordering of the functionals according to the h - bond lengths and to the adsorption energy is almost the same as that observed for water . the only exception is vdw - df , which for methanol gives a larger adsorption energy than that obtained with pbe . the comparison with dmc confirms , as for water , that the best performing functionals are vdw - df2 and optpbe - vdw . again the gga functionals and vdw - df underestimate the adsorption energy , while all the other functionals ( pbe - d2 , pbe - d3 , pbe+vdw(ts ) , optb86b - vdw , pbe0-d3 , and b3lyp - d3 ) overestimate the interaction . as for the previous system , vdw - corrected hybrid functionals do not seem to improve significantly with respect to the gga+vdw approaches . before discussing adsorption on the silicate face of kaolinite an important finding from the results presented in table 2 is that with the gga functionals the adsorption energies of water and methanol are similar ( e.g. , eads , pbeh2o = 608 mev and eads , pbemeoh = 616 mev ) , but upon inclusion of dispersion interactions methanol is stabilized to a greater extent ( e.g. , eads , optpbe vdwh2o = 699 mev and eads , optpbe this is apparent in figure 5 , where the difference in adsorption between water and methanol is plotted . therefore , even though the methyl group is considered a spectator , its vdw interaction with the surface is non - negligible , and it is clearly desirable to properly account for dispersion interactions in these systems . dmc confirms the reliability of the vdw - inclusive functionals on this issue as dmc also finds that methanol binds more strongly than water . difference in adsorption energy , between water and methanol on the hydroxyl - terminated ( left panel ) and silicate - terminated ( right panel ) faces of kaolinite , as obtained from various xc functionals and dmc . filled points represent the values for the configurations optimized using pbe - d3 , and empty points ( reported only for gga and gga+vdw functionals ) correspond to relaxed structures for the specific functional . the solid lines are the reference dmc adsorption energies , and the shaded areas show the stochastic error . in table 3 we present the results from all the functionals for adsorption on the silicate - terminated face . irrespective of which functional is used the adsorption energies obtained are in the physisorption regime . consequently , the inclusion of vdw forces is expected to have a more obvious impact than on the hydroxyl - terminated face . energies have been obtained on pbe - d3 optimized structures , but in parentheses we also report the adsorption energies when geometries are fully relaxed consistently for each gga and gga+vdw functional . as on the hydroxyl - terminated face , rpbe gives an adsorption energy that is noticeably less exothermic than pbe . in the case of the vdw - dfs we see an across - the - board stabilization relative to the gga functionals . like at the hydroxyl - terminated face , vdw - df and vdw - df2 the pbe - d2 and pbe+vdw(ts ) functionals give the strongest overall adsorption energy , with 276 and 273 mev , respectively , followed by optpbe - vdw , pbe - d3 , and optb86b - vdw with values close to 250 mev . overall , the spread of the vdw - based evaluations is much smaller than for the hydroxyl - terminated face . whereas at the hydroxyl - terminated face the adsorption structure was altered only moderately upon inclusion of vdw , at the silicate - terminated face more significant changes are observed . specifically , the gga functionals predict the molecule to be much further away from the surface than the vdw inclusive functionals do . this difference is also reflected in figure 4 , if we consider the difference between the adsorption energies of the gga functionals at the pbe - d3 geometry and when the structures are relaxed . on the other hand , the geometries provided by the vdw - inclusive approaches are in very good agreement with pbe - d3 , so eads evaluated on either the pbe - d3 geometry or on the relaxed structures are similar . comparison with dmc supports the general reliability of the vdw - corrected and vdw - inclusive approaches over the bare gga and hybrid functionals . however , it also shows that almost all the gga+vdw and the two hybrid+vdw functionals overestimate the adsorption energy . similar overestimates have been seen recently for physisorbed water on hexagonal boron - nitride . on this surface the best performance is seen for the vdw - df and the vdw - df2 functionals , both of them being in agreement with dmc , given the dmc stochastic error . it is also interesting to note that even though water still binds preferentially to the hydroxyl - terminated face the relative adsorption strengths are significantly altered : the ratio eadsh2o@aloh / eadsh2o@sio2 is 6.6 for pbe , 9.3 for rpbe , ca . 3 for the vdw - inclusive functionals , and 3.5 0.5 at the dmc level . similar to water , we again expect dispersion interactions to play more of a role at the silicate - terminated than at the hydroxyl - terminated face . indeed , we again observe big differences between functionals including or not the vdw interaction . in the most stable structure found using the pbe - d3 functional there is no hydrogen - bond - like interaction , and methanol is parallel to the surface of the slab ( see figure 2d ) . the geometry at the gga level has the methanol molecule found at a much larger distance from the surface , as depicted in figure 3d . as on the hydroxyl - terminated face , the degree of stabilization due to dispersion interactions is greater for methanol than it is for water . at the gga level , water and methanol bind with similar interaction strengths ( methanol binds more strongly by 27 mev at the pbe level ) , but when vdw is accounted for in the functional we observe that methanol binds more strongly by 64 mev ( for pbe - d2 ) to 121 mev ( for optpbe - vdw ) , as shown in figure 5 . the comparison with dmc shows that in this case the gga functionals underestimate the adsorption energy and that all the gga+vdw and hybrid+vdw functionals overestimate eads . the best agreement is again obtained for the vdw - df and vdw - df2 functionals , the former overestimating the interaction by 47 18 mev and the latter by 42 18 mev . the ratio eadsmeoh@aloh / eadsmeoh@sio2 is 5.2 for pbe , 2.1 for rpbe , between 2.1 and 2.6 for the vdw - corrected and vdw - inclusive functionals , and 2.8 0.2 at the dmc level . in this paper we have used qmc to examine the adsorption of water and methanol on the hydroxyl- and silicate - terminated ( 001 ) faces of kaolinite . the qmc results on the hydroxyl - terminated face have been obtained independently with two different fixed - node projection qmc methods : dmc and lrdmc . the two methods differ in terms of algorithms ( dmc is based on a time - discretization approximation , lrdmc on a space - discretization approximation ) , implementation ( dmc calculations have been performed using the casino code ; lrdmc using the turborvb code ) , and setup ( for instance , different pps , basis sets , and jastrow terms ) . nonetheless , both approaches produce results in good agreement with the small differences between the approaches coming within the stochastic error of the evaluations . qmc results indicate that both water and methanol adsorb on the hydroxyl - terminated face , forming three h - bonds , with an interaction energy larger than 0.6 ev . the adsorption on the silicate - terminated face is much weaker , smaller than 0.3 ev . in both cases as discussed , the qmc results provide a benchmark that can help in further understanding of how other computationally cheaper methods perform for adsorption . specifically , we have compared them with the results provided by a selection of commonly used xc functionals in dft ( covering gga , hybrid , vdw - corrected gga , vdw - corrected hybrid , and vdw - inclusive functionals ) . this shows that the vdw - corrected and vdw - inclusive functionals predict adsorption energies that are considerably larger than those calculated using the bare gga or hybrid functionals , but the degree of stabilization is system dependent . as discussed , in the systems under consideration in this work the qmc references indicate that bare gga and hybrid - based predictions are often underestimated , whereas approaches that account for the vdw interaction yield results in qualitative agreement with qmc , although the absolute value of the adsorption energy can be overestimated , particularly on the silicate - terminated face . overall , the best results are provided by vdw - df2 , and among the vdw - corrected approaches we notice good performance from pbe - d3 . inclusion of exact exchange does not appear to lead to any improvement for the systems considered here ; for instance , results from pbe - d3 and pbe0-d3 are almost identical . the gga+vdw functionals , although based on gga , perform better than the bare ggas also in terms of geometries . indeed , on the silicate - terminated face ( where the interaction is weaker ) structure relaxation performed with the vdw - corrected and vdw - inclusive functionals leads to very similar configurations , whereas with the ggas the adsorbates sometimes strayed away from the surface . looking forward there certainly still seems to be scope for further improvements in the treatment of these systems with dft . of the functional considered vdw - df2 offers the best performance , but it does not convincingly deliver chemical accuracy for all four adsorption scenarios considered . approaches such as hamada s revised vdw - df2 functional or tkatchenko s many - body dispersion would be interesting to explore . the comparison between the adsorption of water and methanol is also interesting . at the gga level there is very little difference in adsorption energies , whereas methanol becomes more strongly bound when vdw interactions are accounted for . as clay minerals can cleanse groundwater through the uptake of pollutants , the relative adsorption energies with respect to water is a highly important quantity . even for methanol , which is one of the simplest organic molecules able to form a hydrogen bond , we see that including vdw interactions can significantly alter the adsorption energy relative to water ; on the hydroxyl - terminated face , water and methanol bind with similar energies , but inclusion of dispersion forces tips the balance in favor of methanol . before closing we note that we have examined the consequences of altering the relative interaction strength of water and methanol with kaolinite through a series of classical molecular dynamics simulations of liquid water specifically in figure 6b we show results obtained with water and methanol interaction parameters that use standard lorentz berthelot mixing rules , values matching pbe , or values matching dmc . as can be seen figure 6b , with the dmc value of eads the adsorption of methanol yields a density profile with a much more pronounced first peak compared to the eads , corresponding to pbe or standard lorentz thus , we see that the standard approach for exploring aqueous solutions at a clay surface with force fields leads to a rather poor description of the interface . this effect is likely to become even more significant as the size of the organic tail of the adsorbate increases and demonstrates the importance of an accurate modeling of dispersion interactions when exploring wet interfaces of environmental relevance . the ability to accurately incorporate nonlocal dispersion interactions is therefore extremely important if one aims to model environmentally relevant adsorption processes on kaolinite and other clays . molecular dynamics simulations of a water methanol mixture on the hydroxyl - terminated face of kaolinite . panel a : snapshot of the simulation system , where the methanol molecules are shown in red and the water molecules as glassy blue circles , and the kaolinite slab is the same as in figure 1 . panel b : density profiles of methanol above the kaolinite surface , for the eight replicas with temperatures ranging from 275 to 310 k , obtained for values of eads ( namely , the difference between adsorption energy of water and methanol ( see text ) ) corresponding to lorentz z is the distance from the average position of the top layer of oxygen atoms in the kaolinite surface . it is clear that an appropriate choice of eads can significantly affect the density of the water
dengue is a vector transmitted viral infection ; tropical and subtropical countries see outbreaks of dengue each year . there is a paucity of literature on effects of dengue infection on pregnancy outcome and this prompted us to undertake a study for better understanding of pregnancy implications with dengue infection . pregnant women admitted during the seasonal outbreak of dengue between september 2015 and october 2015 were studied and maternal and fetal outcomes in sixteen ns1ag positive women were analysed . out of sixteen women diagnosed with dengue fever , three had dengue shock syndrome ( dss ) and eight had dengue haemorrhagic fever ( dhf ) . the most common obstetric complication seen in 43% of the cases was oligohydramnios . bleeding manifestations occurred in seven women and there were three maternal deaths . perinatal complications included three intrauterine deaths , six nursery admissions , and one neonatal death . thus dengue infection was associated with high maternal and perinatal mortality . in view of poor obstetric outcomes , this viral infection warrants early admission and prompt management .	it is caused by viruses of the genus flavivirus , family flaviviridae , and group iv ssrna . dengue is transmitted to humans by the mosquito aedes aegypti and is one of the most rapidly spreading viral infections . there has been a 30-fold increase in its incidence in the last 50 years coupled to increasing migration from rural to urban areas . annually dengue remains a major health concern for southeast asian countries with cyclic epidemics . in india multiple viral serotypes are circulating and some regions have case fatality rates of 35% in general population , which is much higher than other southeast asian regions ( 1% ) . literature suggests an increased risk of maternal hemorrhage , preterm labour , and oligohydramnios [ 35 ] . clinical presentation of dengue may be confused with hellp syndrome , both conditions having elevated liver enzymes , haemolysis , and low platelet counts ; however serology helps in distinguishing the two conditions . as reported in previous studies , dengue is associated with preterm births , low birth weight , fetal deaths , and vertical transmission leading to neonatal thrombocytopenia requiring platelet transfusions [ 610 ] . in a quest to answer vital issues we critically analysed dengue antigen positive cases in our institute and studied maternal and fetal outcomes . this study was conducted in the department of obstetrics and gynaecology of a tertiary level government hospital , delhi , india . during the months of september and october 2015 , 60 pregnant women were seen in the emergency room with complaint of fever and were admitted for evaluation . as per protocol , dengue pcr ( ns1ag ) clinical grading was done according to who classification and case definitions . an acute febrile illness with two or more clinical manifestations like headache , retroorbital pain , myalgia , arthralgia , rash , hemorrhagic manifestation , or leukopenia and positive serology or occurrence at a time of dengue outbreak was taken as the definition of dengue fever ( df ) . dengue hemorrhagic fever ( dhf ) was classified as fever , hemorrhagic tendencies , thrombocytopenia , evidence of plasma leakage , association of hepatomegaly , and circulatory disturbances . increase in serial hematocrit was taken as evidence of plasma leakage and ultrasound showing pleural effusion , ascites , and gall bladder edema were taken as supportive evidence . dengue shock syndrome ( dss ) was classified when dhf symptoms included rapid and weak pulse , narrow pulse pressure of less than 20 mmhg , and hypotension . patients were managed with antipyretics , adequate hydration , and blood product transfusion as necessary . obstetric data , clinical , laboratory parameters , and maternal and fetal outcomes of women are depicted in table 1 . a majority of women were unbooked and presented with classical symptoms of high grade fever , myalgia , headache , and rashes to the emergency room . all women were young , age ranging from 22 to 32 years ; mean age was 25 years . out of 16 cases 13 presented in third trimester and two in second trimester and one was referred after lscs was done in another hospital . she presented with large rectus sheath hematoma following caesarean section needing surgical exploration and drainage . none of the women had any underlying diseases or comorbidity except one ( # case 5 ) who had moderate anemia on admission . in the current study , eight women ( 50% ) had dengue haemorrhagic fever ( dhf ) . ten women required transfusion to maintain platelet count in desired range ; on average each woman required six platelet transfusions . most women had low platelet counts at the time of admission ( mean 81,000/mm ) ; 62% had platelets less than 50,000/mm . platelet count less than 30,000/mm was seen in dengue shock syndrome with increased risk of bleeding manifestations . a sri lankan case series reported 10 women with dhf but only eight required platelet transfusion . three women succumbed to dengue shock syndrome in current study with a mortality rate of 18% compared to 35% case fatality rate in rest of the india in nonpregnant population . in another study done in india by agrawal et al . symptomatic dengue infection may increase the risk of preterm labour and , hence , low birth weight ( lbw ) as suggested by previous studies [ 35 , 11 ] . in the study under discussion most women presented near term ( n = 10 ) and of the four who presented in early gestation two had preterm labour . preterm births reported were as low as 11% in the french guiana study versus 41% in the study by basurko et al . one woman had spontaneous abortion ( 6% ) and seven ( 43% ) pregnancies were complicated with oligohydramnios in present study compared to 52% rate of oligohydramnios from a study in india by agrawal et al . . bleeding manifestations were seen in seven women and three ( 19% ) had postpartum hemorrhage ( pph ) ( # 2 , 11 , and 13 ) . one of them was diagnosed as dss and the other two were diagnosed as df . both the patients did not have any other bleeding manifestations and signs of plasma leakage hence were classified as only df . in retrospective study by basurko et al . on 53 pregnant women , pph was reported in 10% of the cases compared to 19% in our study . there were six neonatal intensive care unit ( nicu ) admissions ( 37% ) , three intrauterine deaths ( iud ) ( 18% ) , and one neonatal death ( 6% ) . none of the neonates in our study showed symptoms of dengue such as thrombocytopenia , anemia , fever , or bleeding manifestations . there are a number of studies on poor fetal outcome following dengue infection in parturient females . they also reported iud rate of 3.8% and neonatal death rate of 1.9% while carles et al . pregnant women with dengue fever should be considered for admission early because of its unpredictable course . oligohydramnios is an ominous sign when seen concomitantly with dengue infection because the high fetal mortality was associated with it . the exact cause of oligohydramnios is not known but dehydration associated with dengue fever may be contributory . hydration should be maintained by encouraging oral intake of oral rehydration solution ( ors ) , fruit juice , and other fluids containing electrolytes and glucose for replacing losses from fever and associated vomiting . another dilemma faced was in differentiating dengue infection from hellp syndrome as both have somewhat similar laboratory parameters ; however serology will help clinch the diagnosis . careful examination and high index of suspicion by the treating obstetrician are required to diagnose and manage such cases especially during epidemics . our knowledge on effect of dengue on pregnancy is somewhat limited . in this study dengue awareness programs and medical education programs on the management of the dengue in pregnancy should be initiated especially during outbreaks to provide quality care .
pauci - immune crescentic glomerulonephritis ( picgn ) is most commonly associated with antineutrophil cytoplasmic antibodies ( anca ) . we report a case of chronic , sclerosing anca - negative picgn discovered when a patient presented with multiple myeloma . a 57-year - old woman presented with complaints of nausea , emesis and weakness . she was found to be in renal failure with a serum creatinine of 9.4 mg / dl , mild hyperkalemia and acidosis . she was noted to have normochromic , normocytic anemia with normal platelet and white cell counts , normal plasma proteins and serum protein electrophoresis . further studies revealed increased concentrations of and light chains in a ratio of 34.89 ; a bone marrow biopsy found 12% plasma cells . serum protein electrophoresis revealed no spike . anca , anti - glomerular basement membrane , antineutrophil antibody , hepatitis panel and serum complements were normal . a kidney biopsy result showed chronic sclerosing picgn plus tubular necrosis , severe tubular atrophy , interstitial fibrosis and severe arteriosclerosis . congo red stains were negative and electron microscopy showed no intraglomerular deposits . the patient was subsequently treated for myeloma with bortezomib and dexamethasone with good hematologic response but never recovered renal function . she remains on outpatient hemodialysis . renal manifestations of myeloma often involve glomerular deposition disease , tubulointerstitial disease , with characteristic proteinaceous casts , or both . in contrast , our patient demonstrated neither of these findings but had chronic sclerosing picgn . crescentic glomerulonephritis occurring in patients with plasma cell dyscrasias has been previously reported , but the association remains extremely rare .	approximately 70% of patients with multiple myeloma have urinary m protein spike and about 2025% have kidney disease at the time of diagnosis . up to half of myeloma patients demonstrate renal involvement during the course of their disease [ 3 , 4 ] . myeloma kidney is implicated in about 41% of cases and is distinctive for multiple myeloma . monoclonal immunoglobulin deposition disease , primary amyloidosis , proximal and distal tubulopathy , renal vein thrombosis , type 1 cryoglobulinemia , proliferative glomerulonephritis , interstitial nephritis , plasma cell infiltration , urate deposition disease , pyelonephritis and nephrocalcinosis represent less common forms of renal involvement [ 6 , 7 , 8 , 9 ] . rapidly progressive glomerulonephritis in multiple myeloma is rare ; cases reported thus far typically demonstrate proliferative glomerulonephritis and/or glomerular deposits on renal biopsy . we here report a case of antineutrophil cytoplasmic antibodies ( anca)-negative , pauci - immune , chronic sclerosing , crescentic glomerulonephritis without deposits on electron microscopy , which was discovered at the time the patient presented with multiple myeloma and which resulted in end - stage kidney disease . a 57-year - old hispanic woman was admitted with nausea , vomiting and weakness of 1 week duration . she described herself to be in good health but had not seen a doctor for 15 years . her history was only significant for pregnancies and visits to the emergency room for epistaxis 3 years prior to admission and after a fall 6 years prior to admission . her father died from malignancy , type unknown . physical examination on admission was normal except for pallor . the patient was started on hemodialysis on admission and was transfused with 2 units of packed red blood cells for severe anemia . urinalysis was positive for microscopic hematuria , and the patient had positive urine m spike with faint light chain on serum immunofixation ( table 1 ) . renal biopsy on day 4 of admission was consistent with chronic sclerosing pauci - immune glomerulonephritis ( fig . initial bone marrow biopsy showed monoclonal plasmacytosis ( 12% ) consistent with plasma cell myeloma . there was no evidence of increased blasts , monoclonal b - cell or aberrant t - cell process . the patient was eventually discharged home with hematology / oncology follow - up and remains on outpatient hemodialysis . she was started on high - dose dexamethasone a few days after discharge and bortezomib 1.3 mg / m twice weekly after hemodialysis approximately 1 month after discharge . she was readmitted 3 months later with fever and symptoms of upper respiratory tract infection and was treated with antibiotics . the immunological investigations were repeated because of the rarity of multiple myeloma presenting with pauci - immune crescentic glomerulonephritis . however , repeat serum protein electrophoresis showed no m spike , and serum immunofixation showed a polyclonal pattern . spot urine protein was 834 mg / dl , the urine creatinine concentration was 24 mg / dl , the urine albumin concentration was 35 mg / dl and the urine protein / creatinine ratio was 35 . a repeat bone survey showed mild generalized osteopenia , but it revealed no focal lesions . a follow - up bone marrow biopsy after 4 months of bortezomib and she continues to require hemodialysis despite reduction of the free light chain burden and disappearance of monoclonal plasma cells in the bone marrow . crescentic glomerulopathy is a rare complication of multiple myeloma first described by kaplan and kaplan in 1970 in a 49-year - old patient with renal failure due to igg myeloma . three cases of extracapillary proliferative glomerulonephritis were reported by meyrier et al . , of which two were due to plasma cell dyscrasias and one was due to waldenstrom 's macroglobulinemia . described a patient with diabetes mellitus who developed rapid progressive renal failure over the course of 5 weeks that led to chronic hemodialysis caused by light chain myeloma . reported a 60-year - old male patient who had a relapse of rapidly progressive glomerulonephritis 10 months after an initial episode that responded to cyclophosphamide and steroid therapy . this time , he had an extensive investigation that revealed light chain monoclonal gammopathy treated with 2 injections of rituximab followed by 2 cycles of bortezomib therapy resulting in clinical remission . perhaps , if she had presented early , she may have recovered her renal function with bortezomib and dexamethasone treatment . the exact mechanism of renal injury in this case remains unclear and needs further investigation since there were no demonstrable immune complexes , monoclonal deposition disease or cast nephropathy . one possible theory could be a form of direct toxic injury to the glomerular basement membrane by the free light chains with subsequent disruption of the basement membrane and formation of fibrocellular crescents . whether this is preceded by extracapillary and/or endocapillary there could also be concurrent direct toxic injury to tubular cells by light chains and interstitial nephritis . this case highlights an atypical renal pathologic manifestation of multiple myeloma and underscores that multiple myeloma should be considered in the differential diagnosis of rapidly progressive glomerulonephritis , especially if there is heavy proteinuria . in such patients , early screening for serum free light chains followed by renal biopsy and bone marrow biopsy can establish the diagnosis and guide therapy the authors declare that no financial or other conflict of interest exists in relation to the content of the article .
the objective of the present research was to determine the appropriate amount of fluoride to use professional topical application and the residual amounts of fluoride in the oral cavity using a 2% neutral sodium fluoride ( naf ) foam with a dedicated tray . using dentition study models , a method for determining the appropriate amount of naf foam was investigated . in eight adult subjects , the appropriate amount of naf foam , the concentration of fluoride in the saliva following professional topical application , and the amount of residual fluoride in the oral cavity following professional topical application was examined . the results indicated that the appropriate amount of naf foam was approximately 0.8 g , the amount of residual fluoride in the oral cavity was approximately 25% of the amount of foam used .	methods of topical fluoride application include professional topical application and home use of fluoride dentifrice and fluoride mouth rinse . professional topical application is a means of preventing dental caries in both deciduous and permanent teeth and is performed by dental specialists ( dentists and dental hygienists ) in dental clinics , health care facilities , and other appropriate settings specializing in dental care . advantages of professional topical fluoride application include the ability to treat patients starting from a very young age , immediately after the deciduous anterior teeth erupted , in addition to the fact that application is necessary only two to four times a year . in addition , the ingestion of fluoride from the professional topical fluoride application is not considered to be a risk factor in dental fluorosis . the fluoride agents in professional topical application are available in liquid , gel , and foam types . because the gel and foam are generally applied using a tray , these have an advantage in that the fluoride can be applied to all of the teeth simultaneously . in the united states and a number of other countries , the tray method using acidulated phosphate fluoride ( apf ) foam is widely used , and the foam application procedure , including the amount of foam used , is standardized . in japan , the ministry of health and welfare has issued guidelines governing professional topical application of fluoride , which describes a method for applying the neutral and acidic naf liquids that were initially introduced in japan in the 1960s . a method for applying apf gel , which was subsequently introduced in the 1980s , has also been established based on fundamental research on factors , such as the amount of gel to be used and the residual amount in the oral cavity [ 59 ] . strongly acidic apf has an advantage in that a large amount of uptake into the tooth substance can be expected , but there are also reports expressing concerns about damage occurring to several types of restorative material [ 1015 ] . these reports underscore the necessity for topical application of neutral naf , and as a result , neutral naf foam is now available on the market in japan . however , no information is currently available on aspects such as methods for applying this neutral naf foam or the amount of foam that remains in the oral cavity after application . first , various amounts of foam ( butler fluodent foam n , sunstar inc . , osaka , japan ) in the deciduous and permanent dentition study model trays ( butler tray , sunstar inc . , osaka , japan ) were prepared to apply to the deciduous and permanent dentition study models ( study model pe - ana004 and study model pe - ana002 , respectively , nissin dental products inc . , initially , the weight of tray without the foam was measured by an electronic balance ( libror aeg-45sm , shimazu co. , kyoto , japan ) , then the foam was placed , and the total weight was measured . the amount of fluoride foam was calculated by subtraction of the weight of tray from the total weight . to sufficiently cover all the dried teeth surfaces of the study models and to minimize leakage outside of the dentition , the appropriate amount of foam ( aaf ) was filled to be adjusted to a level approximately 2 mm below the tray . using the deciduous and permanent dentition study models , after the foam application , the tray was removed from the study model immediately , and the net weight of foam remained in the tray ( trf : tray - remained foam ) and the net weight foam adhering to the study model were measured , respectively . then the foam adhering the area of mucosa of study model was wiped with a wiping paper ( kimwipe s-200 , nippon paper crecia co. , ltd , tokyo , japan ) , and the net weight of foam adhering to the surface of study model ( trsf : tooth - surface retained foam ) was measured . the weight difference between above study models was defined as overflowed foam ( of ) . based on the procedure of experiment 1 , an application experiment was carried out in human subjects . eight healthy participants ( three males and five females ) , ranging in age from 21.3 to 24.8 years , were recruited from the students at kanagawa dental college . each subject signed the informed consent form , which had been approved by the ethics committee of kanagawa dental college ( no . 44 ) . the inclusion criteria in this study were : ( 1 ) with at least 28 natural teeth ; ( 2 ) unstimulated saliva flow rate > 0.3 ml / min ; ( 3 ) no orthodontic appliance in their oral cavity . the exclusion criteria were : ( 1 ) clinically detectable caries ; ( 2 ) periodontitis ; ( 3 ) history of allergies and metabolic diseases such as diabetes ; ( 4 ) other medical condition that could interfere with the study . subjects were instructed to brush their teeth using a nonfluoride toothpaste starting 3 days before the day of the experiment until the end of the experiment , and all other fluoride applications were suspended . the experiment began at 2:00 p.m. before the naf foam was applied to subjects , their teeth were dried with the compressed air . the trays were filled with naf foam to a level approximately 2 mm below the edge of the trays and were placed over the teeth , the subject was instructed to close the jaws with the trays in contact for 4 minutes . the subject was instructed not to swallow but to allow the saliva to dribble into a 500-ml plastic beaker held directly under the mouth . at the end of the topical application , the trays were removed from the mouth and placed in the same beaker , and the subject expectorated the mixture of saliva and foam into the same beaker immediately after removal of the tray and expectorated once again 30 seconds later . whole saliva was collected into a separate 50-ml plastic vessel for 5 minutes a total of eight times ( immediately prior to application , 5 minutes afterwards , 15 minutes afterwards , 30 minutes afterwards , 60 minutes afterwards , 120 minutes afterwards , before the subjects went to bed , and immediately after they awoke the following day ) . the saliva samples obtained up to 30 minutes afterwards were diluted by double - deionized water at the ratio of 1 : 10 , and others were not diluted . total ionic strength adjustment buffer ( tisab ) ( tisab ii , orion research inc . , beverly , mass , usa ) were added to all saliva samples as the ratio of 1 : 1 , and the fluoride concentration of saliva was analyzed as ppm using the ion - specific electrode ( orion 9609bnwp combination electrode , thermo electron corp . the subject was instructed not to swallow but to allow the saliva to dribble into a 500-ml plastic beaker held directly under the mouth . at the end of the topical application , the trays were removed from the mouth and placed in the plastic beaker , and the subject expectorated the mixture of saliva and foam into the same beaker and expectorated once again 30 seconds later . total amount of 500 ml double - deionized water was added to the beaker . the solution was stirred by the magnetic stirrer until no traces of the foam were visible . then the solution was analyzed for fluoride concentration using the ion - specific electrode ( orion 9609bnwp combination electrode , thermo electron corp . the total amount of fluoride recovered to the oral cavity was calculated as the product of the fluoride concentration and volume . the weight of residual fluoride was calculated by subtracting the weight recovered from the weight applied . in addition , the ratio of residual fluoride in the oral cavity was determined from the percentage of fluoride remaining in the oral cavity in relation to the amount of fluoride used . the test of population mean for the fluoride concentrations in saliva following the foam application and the test of coefficient of correlations between with aaf , trf , trsf , and of in the study models were analyzed ( jmp , ver . using deciduous and permanent dentition study models , we found that in order to sufficiently cover the dentition when applying the naf foam and to minimize leakage outside of the dentition , the tray needed to be filled to a level approximately 2 mm below the tray edge . using this method the results showed significant variation in mean appropriate amount of foam ( aaf ) ; that of the deciduous dentition study model was 0.57 0.17 ( 0.360.86 ) g ( table 1 ) , while that of the permanent dentition study model was 0.96 0.24 ( 0.681.39 ) g ( table 2 ) . the mean of the amount of foam retained on the tooth surfaces ( tsrf : tooth - surface - retained foam ) of the deciduous and permanent dentition study models were 0.03 0.01 ( 0.010.05 ) g and 0.12 0.06 ( 0.030.20 ) g , respectively , ( tables 1 and 2 ) . analysis of the coefficient of correlations between the appropriate amount of foam ( aaf ) and the amount of foam adhering to and recovered from the tray ( trf : tray - retained foam ) , aaf and the amount of foam adhering to the mucosa other than the tooth surface on the models ( of : overflowed foam ) , and aaf and the tooth surface retained foam ( tsrf ) showed a strong positive correlation between aaf and trf and between aaf and of in both the deciduous and permanent dentition study models ( table 3 ) . the mean amount of naf foam applied in each adult subjects was 0.81 0.20 ( 0.541.12 ) g. the mean fluoride concentration in saliva following application was 35.95 28.84 ( 7.4885.80 ) ppm , which was the maximum value 5 minutes after application , and by 30 minutes after application had dropped sharply . it continued to drop gradually thereafter , but the concentration of fluoride in saliva measured at the time subjects awoke the following morning was relatively high , at 0.12 ppm ( figure 1 ) . there were significantly differences in salivary fluoride concentrations up to 120 min following the application compared with 0.05 ppm as criterion value ( test of population mean , p < .05 ) . the mean amount of naf foam used was 0.80 0.22 ( 0.561.16 ) g. the mean amount of fluoride used was 7.18 1.94 ( 5.0010.47 ) mg , and the mean amount of fluoride used per kilogram body weight was 0.14 0.04 ( 0.090.19 ) mg f / kg . the amount of fluoride recovered was 5.44 1.75 ( 3.658.70 ) mg , and the amount and ratio of residual fluoride in the oral cavity after foam application were 1.74 0.47 ( 1.322.44 ) mg and 24.9 5.80 ( 16.934.40 ) % , respectively . the amount of residual fluoride in the oral cavity per kilogram body weight was 0.033 0.012 ( 0.0170.053 ) mg f / kg ( table 4 ) . a strong correlation was observed between the amount of fluoride used per kilogram body weight and the amount of residual fluoride in the oral cavity per kilogram body weight ( r = 0.71 , p < .05 ) . the fluoride agents in professional topical application are available in liquid , gel , and foam types . currently , the fluoride agents in professional topical application are available in liquid or gel types in japan . accompanying documentation specifies that the amount of professional topical application to be used is 2 ml or less using the paint - on technique . this applies when a liquid or a gel is used but does not apply to foams . in an experiment conducted by whitford et al . in 46 children between the ages of 8 and 12 years , the appropriate amount of apf foam or apf gel used to cover the dentition without leaking into the oral cavity was one - third the depth of the tray , which was 0.89 0.02 g of foam and 3.86 0.06 g of gel . with these results , the amount of foam needed to sufficiently cover the dental surface was 23.1% that of gel . using apf gel , the amount of gel used with a deciduous dentition study model was 2.13 0.77 g and that for a permanent dentition study model was 3.94 1.38 g , while the amounts of residual gel on the dental surface after wiping away gel that had leaked from the dentition study models were 0.06 0.03 g and 0.18 0.08 g , respectively . in our experiment , we initially prepared enough foam to reach the tray edge , meaning an amount of foam equivalent to the capacity of the tray . when the prepared foam was applied to the study model , a large amount of foam overflowed from the tray and the model . as a result , it was determined that the appropriate amount of foam should be filled to be a level approximately 2 mm below the edge of the tray . the mean amount of foam used with the deciduous dentition study model was 0.57 ( 0.360.86 ) g ( table 1 ) and that with the permanent dentition study model was 0.96 ( 0.681.40 ) g ( table 2 ) . these are 26.7% and 24.9% , respectively , of the amounts of gel reported by arakawa et al . and are largely consistent with the results reported by whitford et al . . for the amounts of foam remaining on tooth surfaces , the results were 0.03 ( 0.010.05 ) g for the deciduous dentition study model and 0.12 ( 0.030.20 ) g for the permanent dentition study model ( table 4 ) , which was approximately half the amount of gel reported by arakawa et al . in the present experiments , the variations in the appropriate amount of foam were comparatively wide , even though the tray was filled with foam to a level approximately 2 mm below the tray edge , using the same study model and using foam from the same container . the reason of this phenomenon is not clear . in experiments 2 and 3 , the tray was filled with foam to a level approximately 2 mm below the tray edge . the mean amount of foam used for the eight adult subjects was approximately the same , 0.80 g. this was largely consistent with the amount of foam used by whitford et al . . this result suggests that with the neutral naf foam used in the present experiments , the amount used can be set at a lower amount than that for gel . in research conducted by sudo et al . , in which apf gel was applied using the toothbrush method in subjects 1.5 years of age , the mean amount of gel applied was 0.66 g , and the mean amount of residual fluoride in the oral cavity per kilogram body weight was 0.19 mg / kg . the mean residual ratio was 25.8% . in our experiments , the mean amount of residual fluoride in the oral cavity following application of naf foam was 1.74 mg , and the amount per kilogram body weight was 0.033 mg / kg , with a mean residual ratio of 24.9% ( table 4 ) . comparing the results of this study , the residual ratios in our experiments were largely the same , but because our experiments targeted adult subjects , the residual amount of fluoride per kilogram body weight was extremely small . the amount of fluoride that causes acute toxicity is 1.35 to 1.8 mg / kg or higher . the results of our experiment indicate that the maximum value would be 0.16 mg / kg , even in a child weighing 15 kg ; therefore , this amount assures adequate safety . where the application of highly acidic dental coatings such as apf is cited as possibly causing corrosion of dental restorative materials such as glass - containing resin composites , glass - ionomer cement , and porcelain , 0.9% neutral naf foam is reported to have little effect on surface hardness of the materials [ 20 , 21 ] . moreover , in patients who have restorative materials , use of a neutral naf coating is recommended in order to avoid corrosion , discoloration , and other problems with the restorative materials . earlier studies have reported that when fluoride products such as dentifrice , rinse , and gels are used , a high fluoride concentration in the saliva was initially obtained , subsequently it dramatically reduced with time [ 23 , 24 ] . the results of this research showed that the concentration of fluoride in saliva immediately following foam application was extremely high and still be elevated at 1.2 ppm f in the next morning ( figure 1 ) . the retention of fluoride in saliva may be very important in the prevention or reversal of caries . / l ) in saliva are sufficient to effectively inhibit demineralisation and/or enhance remineralisation of enamel . it seems that this 2% neutral naf foam is effective in prevention of caries immediately after the application because the salivary fluoride concentration up to 120 min is higher than 0.05 ppm . based on the results of the research described here , using a 2% neutral naf foam enables application using the tray method with smaller amounts of fluoride . foam types of fluoride agents are readily dispersed within the oral cavity and are easier to apply than other forms , even when patients have fixed orthodontic appliances present . furthermore , in vitro research has shown no significant differences between apf gel and foam in the amount of uptake into enamel , and while foam could provide an effect similar to that of gel in terms of preventing dental caries [ 26 , 27 ] , there has not yet been any clinical research showing that the professional topical application of fluoride using 2% neutral naf foam prevents dental caries . in this research , the number of subjects might not be enough large , it caused the large values of standard deviation in the results . further research which included not only adults but also children as subjects is necessary to investigate the appropriate frequency of use and the effects in preventing dental caries . the present results found the following : when carrying out professional topical fluoride application using 2% neutral naf foam with a dedicated tray , filling the tray with foam to a level approximately 2 mm below the tray edge was appropriate , and this amount made it possible to adequately cover all the dentition . the appropriate amount of 2% neutral naf foam used for permanent dentition was approximately 0.8 g on average , which was approximately one - fifth the amount of gel ordinarily used with the tray method . the residual ratio of fluoride in the oral cavity following a 4-minute application of 2% neutral naf foam was approximately 25% of the amount of foam used , and a strong correlation was observed between the amount of fluoride used per kilogram body weight and the amount of residual fluoride in the oral cavity per kilogram body weight .
background : urban family physician program has been launched as a pilot in fars and mazandaran provinces of iran since 2012 . attitudes of policy makers and people toward urban family physician program have become challenging . this study shows what people know and practice toward this program.methods:this cross - sectional population - based study was conducted by a multistage randomized sampling in shiraz , southern iran . knowledge and practice of adults toward urban family physician program were queried through filing the questionnaires . single and multiple variable analyzes of data were performed.results:participation rate was 1257 of 1382 ( 90.9% ) , and the mean age of the respondents was 38.1 13.2 years . of 1257 , 634 ( 50.4% ) were men and 882 ( 70.2% ) were married . peoples total knowledge toward urban family physician program was 5 2.7 of 19 , showed that 1121 ( 89.2% ) had a low level of knowledge . this was correlated positively and in order to being under coverage of this program ( p < 0.001 ) , being under coverage of one of the main insurance systems ( p = 0.04 ) and being married ( p = 0.002 ) . the mean score of people 's practice toward the program was 2.3 0.9 of total score 7 , showed that 942 ( 74% ) had poor performance , and it was correlated positively and in order to being under coverage of this program ( p < 0.001 ) and having higher than 1000 $ monthly income ( p = 0.004 ) . correlation of people 's knowledge and practice toward the program was 24%.conclusions : current evidences show a low level of knowledge , poor practice and weak correlation of knowledge - practice of people toward urban family physician program .	iran by a population of 78 million , 30 provinces , 400 districts and more than 65,000 villages comprises of a 65% of the population that are living in urban areas . according to the 4 5-year national development plan the family physician reform should be extended to the whole country . therefore , family physician program was launched for the first time in 2005 in rural areas and cities with a population below 20,000 . after that and toward extension of this program in urban areas , two provinces of fars , in the south of iran with a population around 4.4 million , and mazandaran , in the north of iran with an approximate population of 3 million , were selected for pilot this program from 8 of july 2012 . similar to any other project , family physician program has its advantage and disadvantages which have to be evaluated in order to get an optimum result . this study as the first population - based study in this issue , aimed to measure the knowledge and practice of people lives in shiraz toward family physician program to present an evidenced - based feedback to national and regional policy makers to improve planning and management of this program . this cross - sectional , questionnaire - based study was conducted from october to december 2014 in shiraz , south of the iran . the sample size was calculated as 1382 , supposed level of knowledge of people toward family physician program as 50% , dropout rate of 20% , design effect of 3 , 5% precision level and a confidence level of 95% . multistage randomized and proportional to size sampling was used . in each address , one person who was at least 18 years and was a resident at shiraz for at least 2 years was asked to fill the questionnaire . the coded anonymous questionnaire comprised of a brief introductory paragraph about title , aims identification and phone call number of the executor of this study , followed by consent form that emphasized on voluntary participation and keeping confidentiality . they asked about their demographic and socioeconomic information including age , gender , level of education , marital status , job status , position in the family ( as breadwinner of family or other family member ) , number of family members and monthly income . being under the coverage of main and supplementary insurance systems and also family physician program was queried . the questionnaire contained questions about knowledge and items about practice of people regarding urban family physician program . in the knowledge section , participants were asked about choosing and changing family physician , family physician addresses , tasks of family physician , work time of family physician in holidays and nonholidays , address of reference and proper action in cases of having complaints or need to more information , electronic record form , referral form and visit - fee . in the practice section , reference to family physician and nonfamily physician , waiting time in the family physician 's waiting room , phone counseling with family physician , average of payment upon each referral to family physician , having problem in obtaining prescribed drugs by family physician , having problem in accessing the specialist family physician and being interviewed and examined completely by family physician in each visit were queried . the questionnaire was validated by two experts in family physician program and its reliability according to cronbach 's alpha was calculated as 0.64 through pilot testing of the questionnaire . all data were entered into spss version 20 software ( spss , chicago , il , usa ) . the accuracy of data entry was ensured by randomly selecting and checking completed questionnaires against their corresponding data in the spss software . chi - squared , t - tests , pearson correlation and stepwise linear regression model were used . voluntary participation in this study , designing of an anonymous questionnaire , possibility of access to executives of this study via two exclusive phone lines and keeping confidentiality in all aspects of research were some ethical aspects that were applied . furthermore , the research protocol as described here was approved by the ethics committee of the health policy research center affiliated with shiraz university of medical sciences . voluntary participation in this study , designing of an anonymous questionnaire , possibility of access to executives of this study via two exclusive phone lines and keeping confidentiality in all aspects of research were some ethical aspects that were applied . furthermore , the research protocol as described here was approved by the ethics committee of the health policy research center affiliated with shiraz university of medical sciences . participation rate of participants was 1257 of 1382 ( 90.9% ) and 997 ( 79.3% ) filled the questionnaire at home addresses [ table 1 ] . mean age of participants was 38.1 13.2 years and of total 1257 , 634 ( 50.4% ) were men , 882 ( 70.2% ) were married and 474 ( 37.7% ) had associate or bachelor degree of education [ table 1 ] . social and demographic characteristics of participants in the population - based study aimed to determine correlates of knowledge and practice toward urban family physician program in shiraz , southern iran ( n=1257 ) six hundred seventeen ( 49.1% ) had job with mean income 1000$/month [ table 1 ] . the mean family member was 3.9 1.5 and 539 ( 42.9% ) were breadwinners of their families . one thousand hundred nineteen ( 89% ) and 479 ( 38.1% ) were under the coverage of one of the main and supplementary insurance systems respectively [ table 1 ] . one thousand fifty - eight ( 84.1% ) of respondents and 1012 ( 80.5% ) of their families members were under the coverage of urban family physician program [ table 1 ] . peoples total knowledge toward urban family physician program was 5 2.7 of 19 , showed that 1121 ( 89.2% ) had a low level of knowledge [ table 2 ] . southern iran ( n=1257 ) of total , 879 ( 69.9% ) of people knew about family physician choosing rules but 880 ( 69.9% ) did not know that , it is possible to change their family physician and 59 ( 4.6% ) stated that family physicians should present both preventive and medical services to their clients . four hundred seventy - fours ( 37.7% ) knew about their family physicians substitutes and 58 ( 4.6% ) were informed about where they should refer in the absence of their family physician . three hundred and fifty - three ( 28% ) and 2 ( 0.1% ) knew correctly about how much they should pay to general and specialist family physician in each visit , respectively . of 1257 , 22 ( 1.8% ) knew that where they should refer if need any information or have any complaint about family physician program while 1173 ( 93.2% ) did not know or could not correctly tell the 4 digits phone number of unit responding to complaints about family physician program . a few of people were informed about referral form ( 233 ; 18.5% ) , about what they should do with filled referral forms ( 154 ; 12.3% ) and about electronic health record ( 11 ; 0.8% ) . family physician office distance to home was less that 1 km in 538 ( 42.8% ) of the responders [ table 2 ] . univariate analysis showed that knowledge toward family physician program was lower in younger than 30 and older than 60 years people , in males , in singles , in whom with < 8 years of education , in whom that were not under coverage of main or supplementary insurance systems and in whom were not under the coverage of family physician program [ table 3 ] . single variable analysis of correlates of knowledge and practice of people toward urban family physician program in shiraz south of iran stepwise linear regression model showed that peoples total knowledge toward their rights in urban family physician program by adjusted r 0.18 and constant = 8.5 ( 95% confidence interval [ ci ] = 7.69.4 , p < 0.001 ) was in order correlated to being under coverage of urban family physician program ( = 2 , 95% ci = 1.42.5 , p < 0.001 ) , being other family member ( s ) under the coverage of urban family physician program ( = 1.1,95% ci = 0.71.6 , p < 0.001 ) , being under the coverage of one of the main insurance systems ( = 0.5 , 95% ci = 0.011 , p = 0.04 ) and being married ( = 0.4 , 95% ci = 0.10.8 , p = 0.002 ) . peoples practice toward urban family physician program has gained mean 2.3 0.9 of total score 7 in this study , showed that 942 ( 74% ) had poor performance while 86 ( 6.8% ) had moderate and 3 ( 0.2% ) had expected level of practice . of total 1257 , 882 ( 70.2% ) stated that they became sick during previous year of this study and 700 ( 55.6% ) referred to their family physician , showing 700 of 882 ( 79.3% ) referral rate to family physicians . eighty ( 6.4% ) had phone counseling with their family physician in the similar period [ table 4 ] . mean number of references to family physicians in whom that were under the coverage of family physician program was 2.4 3.6 in the previous year of this study while mean number of references to nonfamily physicians in whom that were not under the coverage of family physician program was 2.4 4.3 in the same year ( p = 0.3 ) [ table 4 ] . one hundred seventy - eight ( 14.2% ) had changed their family physicians during last year of this study and 152 ( 20.6% ) paid higher than legally approved visit - fee to their family physician . two hundred twelve ( 16.9% ) had problems in providing drugs that were prescribed by their family physicians and 342 ( 27.2% ) had problems in access to specialist family physician [ table 4 ] . practice and problems of people toward urban family physician program in shiraz , southern iran ( n=1257 ) univariate analysis showed that practice toward family physician program was lower in men , in whom which were not under the coverage of main insurance systems and in whom that were not covered by family physician program [ table 3 ] . stepwise linear regression model showed that total practice of people toward their rights in urban family physician program by adjusted r square 0.54 and constant ( = 4.6 , 95% ci = 4.34.8 , p < 0.001 ) was in order correlated to being under coverage of urban family physician program ( = 1.2 , 95% ci = 0.91.4 , p < 0.001 ) , being other family members under coverage of urban family physician program ( = 0.9 , 95% ci = 0.71.1 , p < 0.001 ) and having higher than 1000 $ income monthly ( = 0.2 , 95% ci = 0.050.3 , p = 0.008 ) . after 9 years of establishment and modest achievements in rural family physician program in iran , thought and policy of extension of this system to urban settings has been dominating in recent years as evidenced in health sector reform of this country . therefore , as the pilot , this national project was launched in 2012 in two provinces of iran , including 4.5 million populated fars provinces in the south of the iran . in shiraz , the capital city of fars province , with a population 1.5 million , 650 general family physicians and 300 specialist family physician were included in the family physician program . considering that urban family physician program is a complex and multi - disciplinary structure , it is necessary to monitoring its performance periodically from different aspects . one of the important aspects of the monitoring could be regarded as the assessment of the trend of knowledge and practice of the people toward this program and before and after implemented interventions . therefore , after 2.5 years of starting this program and as the first official report , this study was conducted to evaluate the knowledge and practice of the people toward family physician program . present study demonstrated that the knowledge of the people about their rights in this program is generally low . the results also showed that only few people knew about what to do when they had any question or any complain about the program . this should be considered as an important obstacle toward improvement of the program since the policy makers may hardly get access to the voice of the people . furthermore , most of the people did not know what to do when their family physicians are absent and how to find an alternative one . this leads to ignore or delay to visit by the family physician and gradually results in mistrust to and outgoing from the program . the results of the present study revealed that the lack of knowledge was more common among those who were not under coverage of any health insurance system and also among single people . furthermore , results remarked that the practice of the people toward urban family physician program was so weak . a significant portion of the people had problems with providing drugs that were prescribed by their family physicians and also to access specialist family physicians . furthermore , about one fifth of the people complained that they had paid higher that legally approved visiting fees . this matter could be solved if people get more informed about their rights meanwhile teach them how send their feed backs and complains . however , establishment of an effective and continuous supervision system may also come to help in this regard . another achievement of this survey was that , poor practice is common among those with lower outcome . we found that low knowledge toward this program was not related to level of income , therefore above result pointed that low economics may suffer from weaker infrastructures of family physician system in their areas although other studies are needed to prove such claim . another finding was that practice of people toward urban family physician system had a poor correlation with their knowledge as it was endorsed in previous studies with emphasis on that educating alone may not lead into a better good level of practice . hence , strengthening the software and hardware resources are mandatory for the sake of good performance of this system . present study marked that the reference rate of the patients to family physicians is high in the current system . however , this was not so different from those who were not under coverage of family physician and were referred to out of the family physician system doctors . there are few studies that demonstrate that the number of unnecessary patients referrals to pharmacies , laboratories , and radiology centers has been increased as a result of running family physician program , it is needed to perform other studies about the cost effectiveness of the urban family physician program in our setting , as well as the efficiency of monitoring - evaluation system . saying about limitations in this study , it should be clear that despite our effort to design the questionnaire simple and user friendly and also providing prepaid envelope for resending the filled questionnaires , approximately 10% of the people did not answer the questionnaire . it was half of dropout rate of 20% that we assumed for estimation of sample size and we also noticed that the nonrespondents did not show statistically difference among different postareas , therefore it is unlikely that it could have influence on the results and their representativeness . another point was that this study was conducted in shiraz , the most populated city of the fars province that does not have exactly the same situation as the small cities of this province . however , by choosing participants randomly and from different socioeconomic classes , the possible discrepancy may be faded . this study showed that the knowledge and practice of the people toward family physician program are weak . therefore , continuous education and effective training of people about their rights in this program could lead to a better performance of them and come into play for future outcome of this program . last but not least , lessons from this project could help policymakers at national level before any decision to extension this program to whole the country or even may be , followed by neighboring and regional countries that look to iran as a hub for regional health sector reforms .
diphyllobothrium latum and diphyllobothrium nihonkaiense are the 2 reported main causes of human diphyllobothriasis in the republic of korea . however , the differentiation of these 2 species based on morphologic features alone is difficult . the authors used nucleotide sequencing of the mitochondrial cytochrome c oxidase subunit 1 ( cox1 ) gene to diagnose diphyllobothrium spp . two patients visited the emergency room at kyungpook national university hospital on 3 april and 12 april 2013 , respectively , with fragments of parasites found while defecating . the parasites were identified as diphyllobothrium spp . based on morphologic characteristics , and subsequent cox1 gene sequencing showed 99.9% similarity ( 1,478/1,480 bp ) with d. nihonkaiense . our findings support the hypothesis that d. nihonkaiense is a dominant species in korea .	diphyllobothriasis is caused by the adult tapeworm ( diphyllobothrium spp . ) , and is contracted by consuming raw or undercooked fish in northwest europe and east asia . many patients with diphyllobothriasis are asymptomatic , while other patients develop gastro - intestinal discomfort , diarrhea , or abdominal pain . of the 18 diphyllobothrium spp . known to cause human diphyllobothriasis , diphyllobothrium latum and diphyllobothrium nihonkaiense are usually reported in republic of korea . these 2 diphyllobothrium species have similar morphologic features , and thus , clinicians are not able to differentiate the 2 parasites by naked eyes . as an alternative , genetic studies can be used to identify the species . recently , several nucleotide sequencing analysis studies revealed that previous identifications of d. latum based on morphologic characteristics were incorrect and that the parasite was in fact d. nihonkaiense . although the chemotherapy required for both species is identical , the correct identification of diphyllobothrium species is required from the perspectives of epidemiology and public health promotion , particularly by providing accurate information on intermediate hosts . in this regard , we used nucleotide sequencing analysis of the mitochondrial cytochrome c oxidase subunit i ( cox1 ) gene to identify diphyllobothrium species in 2 korean male patients . on april 3rd and 12th of 2013 , 2 patients visited kyungpook national university hospital with a whitish yellow tapeworm segment . the patient that visited on april 3 was a healthy 50-year - old man ( patient a ) , who visited the emergency room after finding a part of a tapeworm while defecating . he had not experienced any abdominal discomfort or pain , and had not visited any foreign country recently , and reported infrequent consumption of raw salmon and trout in side dishes . he also felt a foreign body while defecating , and visited the same emergency room . no abnormal clinical signs , such as , abdominal discomfort or pain , were observed , and he has taken a vermicide during september 2012 . the specimens obtained from the 2 patients were forwarded to our laboratory with a request to identify the parasite species concerned . the parasite samples from the 2 patients consisted of a number of proglottids ; gravid proglottids showed a rosette - shaped uterus as shown in fig . 1a and e. testes were follicular and ovaries were kidney - shaped in the posterior region . in longitudinal sections of gravid proglottids , uterine and genital pores were separated at midline , and the uterus was filled with eggs ( b , c , f , and g in fig . 1 ; h&e stain ) , which were oval and enclosed by a shell ( d and h in fig . total genomic dna was extracted from each specimen , and oligonucleotide primers were designed to amplify the 1,480-bp product of the mitochondrial cytochrome c oxidase subunit 1 ( cox1 ) gene ( forward primer , 5'-atgactaatcttaaagtttt-3 ' ; reverse primer , 5'-taaagccaacatactataatc-3 ' ) . pcr was performed as follows ; initial denaturation at 96 for 5 min , followed by 30 amplification cycles ( 96 for 1 min , 58 for 1 min , and 72 for 1 min ) , and a final extension step at 72 for 10 min . the purified pcr - amplified fragments obtained were cloned into pgem-t easy vector ( promega , madison , wisconsin , usa ) , and the inserted plasmids ( cox1 gene - pgem-t easy vector ) were sequenced using a big - dye terminator sequencing kit ( applied biosystems , foster city , california , usa ) on an automated dna sequencer ( applied biosystems ) . ( clc bio , aarhus , denmark ) , and similarities in genbank were examined using blastn . the cox1 sequences ( 1,480 bp ) of the 2 parasites showed similarities of 99.9% ( 1,478/1,480 bp ) , 95.1% ( 1,407/1,480 bp ) , 93.1% ( 1,378/1,480 bp ) , and 92.6% ( 1,371/1,480 bp ) with d. nihonkaiense ( genbank no . the patients were treated with a single oral dose of praziquantel ( 15 mg / kg ) and no evidence of recurrence has been noticed . d. nihonkaiense was first identified by yamane et al . in 1986 by comparing its morphologic characteristics , such as egg size , the pit shape of eggshells , and the angle between the long axis of the cirrus sac and seminal vesicle , with those of other diphyllobothrium species . d. nihonkaiense and d. latum had distinct morphologic characteristics . specifically , the average size of d. nihonkaiense eggs was 55.5 ( 1.0 ) 40.5 ( 1.5 ) m , which is smaller than the average size of d. latum eggs ( greater than 60.0 45.0 m ) . the eggshells of d. nihonkaiense had shallower pits distributed on the smooth surface than the eggshells of d. latum did . the cirrus sac is situated obliquely in d. nihonkaiense , and the angle between the long axis of the cirrus sac and seminal vesicle was sharper in d. nihonkaiense than in d. latum . in addition to these distinct morphologies , d. nihonkaiense and d. latum have different intermediate host specificities . the intermediate hosts of d. nihonkaiense are salmon , such as cherry , pink , chum , and sockeye salmon ( onchorhychus masou , o. gorbuscha , o. keta , and o. nerka , respectively ) , which are found in the northern pacific , whereas the intermediate hosts of d. latum are perch , pike , burbot , and walleye . based on his history , patient a likely contracted parasites from these fish . on the other hand , patient b had no history of raw salmon consumption , and similarly , several other cases of d. nihonkaiense infection not associated with salmon consumption have been reported . accordingly , accumulating evidence indicates d. nihonkaiense has another intermediate host and that further research on the intermediate hosts of d. nihonkaiense in korea is needed . previously , most cases of diphyllobothriasis in korea were thought to be caused by d. latum . reported occurrence of d. nihonkaiense in korea ( 35th annual meeting of the korean society for parasitology , kwangju , rok ) . owing to the publication of the complete mitochondrial genome of d. nihonkaiense , d. nihonkaiense can now be accurately identified by dna sequencing of the mitochondrial cox1 gene . in 2009 , jeon et al . reported that 62 cases diagnosed as d. latum infection were actually caused by d. nihonkaiense , which were identified by genetic analysis . moreover , another case of diphyllobothriasis was recently confirmed as d. nihonkaiense infection by cox1 gene analysis . these findings suggest that d. nihonkaiense has been misdiagnosed as d. latum and that d. latum might not exist in korea . the present study , in which 2 korean male patients were diagnosed as d. nihonkaiense by cox1 gene sequencing , supports this suggestion . from the clinical point of view , this specific diagnosis might seem academic because diphyllobothriasis can be easily cured with praziquantel . nevertheless , specific diagnosis at the species level is important in the contexts of epidemiology and public health , and would provide further insight into distribution of different taxa and improve our understanding of imported cases and outbreaks of diphyllobothriasis .
multiple mechanisms underlie the surprising willingness of mothers to tolerate the semi - allogeneic fetal tissues during pregnancy . chief among these is the expression of the hla - g molecules that has been largely demonstrated to be responsible for reprogramming the local maternal immune response towards tolerance . we recently identified a subset of tolerogenic dendritic cells , dc-10 that secrete high amounts of il-10 and express high levels of hla - g and its ligand ilt4 . dc-10 are present in the peripheral blood and are essential in inducing adaptive regulatory t cells . we investigated the presence of dc-10 and hla - g - expressing cd4 + t cells in human decidua in the first trimester of pregnancy . results showed that these cells are highly represented in human decidua as compared to the peripheral blood . this is the first report describing decidual dc-10 and cd4+hla - g+ t cells , strongly suggesting that they may accumulate or be induced at the fetal maternal interface to promote tolerance .	the fetus enjoys special privileges that minimize the risk of being rejected by the maternal immune system during pregnancy . the maternal immune system is alerted and responds actively to the fetal invasion , but the type of inflammation generated is not a milieu in which rejecting t cell responses are favored . at the fetal maternal interface , the decidua serves as an immunologically privileged tissue playing essential functions in pregnancy maintenance . during the first trimester of pregnancy , the majority of leucocyte populations in the human decidua is composed of 70% natural killer ( nk ) cells , and 1020% antigen presenting cells ( apcs ) , whereas t cells are sparse and b cells are virtually absent . dendritic cells ( cd11cdcs ) are the key professional apcs representing 510% of all hematopoietic uterine cells . dcs are not only essential for the induction of primary immune responses but also important for the establishment of immunological tolerance . the local microenvironment influences the functions and differentiation of dcs with tolerogenic activities that play a prominent role in dictating the quantity and quality of immune responses . two different myeloid dc subsets , bdca-1 and bdca-3 , were detected in normal human first trimester decidua . bdca-1 decidual cells express hla - dr , cd80 and cd86 at low levels , consistent with the immature characteristics of myeloid dcs . in addition , kammerer et al . have shown that early human pregnancy decidua harbors c - type lectin - expressing cells ( dc - sign ) that show functional features of immature dcs . during human pregnancy , non - classical hla class i hla - g proteins , specifically expressed in the trophoblasts , contribute to the establishment of immune tolerance . seven different isoforms of hla - g exist , four of which are membrane - bound ( hla - g1 to -g4 ) and three are soluble forms ( hla - g5 to -g7 ) . hla - g locus is low polymorphic in the coding region , but polymorphisms that can regulate its expression are present at both 5 up - stream regulatory region ( urr ) and 3 un - translated region ( utr ) non - coding regions . the immune - regulatory properties of hla - g result from interactions with diverse inhibitory receptors : directly via ig - like transcript ( ilt)2 expressed on myeloid and lymphoid cells , ilt4 specifically expressed on apcs , including dcs , and kir2dl4 on nk cells and cytotoxic t lymphocytes ( ctl ) ; indirectly via cd94/nkg2a on nk cells . myeloid apcs may express hla - g and its expression is greatly enhanced by interferon- , il-10 and maturation stimuli . the expression of membrane - bound hla - g and the secretion of soluble hla - g by myeloid apcs contribute to the generation of a tolerogenic microenvironment that may alter the functions of hla - g - expressing myeloid apcs ( hla - g apcs ) themselves , in a feedback loop . thus , myeloid hla - g apcs may be viewed as suppressor cells capable of inhibiting other effector cells and of generating regulatory cells , such as tolerogenic dcs and regulatory t cells ( tregs ) . recently , a subset of il-10-producing human dc ( dc-10 ) has been characterized in the peripheral blood . these cells secrete high levels of il-10 , express membrane - bound hla - g , ilt2 , ilt3 , ilt4 , and are potent inducers of adaptive il-10-producing type 1 tregs ( tr1 ) in vitro through the il-10-dependent ilt4/hla - g pathway . cd4 t cells constitutively expressing hla - g have been shown to accumulate at sites of inflammation . it has been demonstrated that cd4hla - g cells suppress t cell proliferation via a reversible non - contact il-10- and soluble hla - g5-dependent process that leads to regulation of tissue inflammation at the target organ . in the present study we identify for the first time the presence of dc-10 and cd4hla - g t cells at the fetal maternal interface where they may contribute to the tolerance establishment and maintenance in the first trimester decidua . first trimester decidua ( n = 10 ) at 612 weeks of gestational age were obtained from caucasian women with clinically normal pregnancies which were scheduled for elective abortion due to social or psychological reasons . peripheral blood was collected soon before the aspiration procedure . decidual tissues were taken through the cervix during dilatation and aspiration according to formal clinical procedures . informed consent was obtained from all subjects , and the investigation was approved by the ethical committee of fondazione irccs ca granda , milan , italy . human peripheral blood was obtained from healthy donors upon informed consent in accordance with local ethical committee approval ( tiget periblood ) and with the helsinki declaration . peripheral blood mononuclear cells ( pbmc ) were isolated by centrifugation over lymphoprep ficoll gradients ( fresenius kabi norge as , halden , norway ) . this study was approved and monitored by the internal ethical committee of the san raffaele scientific institute and the ethical committee of fondazione irccs ca granda , milan , italy . decidual tissues were dissected into small pieces and enzymatically dissociated in rpmi 1640 medium containing collagenase type iv ( 1 mg / ml ; sigma aldrich , carlsbad , ca ) , and dnase i ( final 0.01% , invitrogen ) for 90 min at 37 c with gentle shaking . the suspension was filtered through a 40-m nylon mesh ( cell strainer , bd biosciences , san jose , ca ) . decidual mononuclear cells ( dmnc ) were separated by density gradient centrifugation over lymphoprep ficoll gradients ( fresenius kabi norge as , halden , norway ) . dmnc and pbmc were initially incubated for 15 min at room temperature with fcr blocking reagent ( miltenyi biotech , germany ) and stained for additional 30 min at room temperature with the following human monoclonal antibodies ( mabs ) : cd1c ( bdca-1 ) and cd83 ( miltenyi biotech , germany ) , cd14 , cd16 , dc - sign , cd11c , cd4 , cd8 ( bd bioscience , ca ) , cd45 ( biolegend , usa ) , ilt4 and cd56 ( beckman coulter , france ) and hla - g ( mem - g9 , exbio , praha , czech republic ) . samples were acquired using a facs canto ii flow cytometer ( becton dickinson , mountain view , ca ) , and data were analyzed with fcs express ( de novo software ) . significance was defined as p 0.05 , p 0.005 , p 0.0005 , and p < 0.0001 . first trimester decidua ( n = 10 ) at 612 weeks of gestational age were obtained from caucasian women with clinically normal pregnancies which were scheduled for elective abortion due to social or psychological reasons . peripheral blood was collected soon before the aspiration procedure . decidual tissues were taken through the cervix during dilatation and aspiration according to formal clinical procedures . informed consent was obtained from all subjects , and the investigation was approved by the ethical committee of fondazione irccs ca granda , milan , italy . human peripheral blood was obtained from healthy donors upon informed consent in accordance with local ethical committee approval ( tiget periblood ) and with the helsinki declaration . peripheral blood mononuclear cells ( pbmc ) were isolated by centrifugation over lymphoprep ficoll gradients ( fresenius kabi norge as , halden , norway ) . this study was approved and monitored by the internal ethical committee of the san raffaele scientific institute and the ethical committee of fondazione irccs ca granda , milan , italy . decidual tissues were dissected into small pieces and enzymatically dissociated in rpmi 1640 medium containing collagenase type iv ( 1 mg / ml ; sigma aldrich , carlsbad , ca ) , and dnase i ( final 0.01% , invitrogen ) for 90 min at 37 c with gentle shaking . the suspension was filtered through a 40-m nylon mesh ( cell strainer , bd biosciences , san jose , ca ) . decidual mononuclear cells ( dmnc ) were separated by density gradient centrifugation over lymphoprep ficoll gradients ( fresenius kabi norge as , halden , norway ) . dmnc and pbmc were initially incubated for 15 min at room temperature with fcr blocking reagent ( miltenyi biotech , germany ) and stained for additional 30 min at room temperature with the following human monoclonal antibodies ( mabs ) : cd1c ( bdca-1 ) and cd83 ( miltenyi biotech , germany ) , cd14 , cd16 , dc - sign , cd11c , cd4 , cd8 ( bd bioscience , ca ) , cd45 ( biolegend , usa ) , ilt4 and cd56 ( beckman coulter , france ) and hla - g ( mem - g9 , exbio , praha , czech republic ) . samples were acquired using a facs canto ii flow cytometer ( becton dickinson , mountain view , ca ) , and data were analyzed with fcs express ( de novo software ) . significance was defined as p 0.05 , p 0.005 , p 0.0005 , and p < 0.0001 . during pregnancy , subpopulations of leukocytes present at the fetal - maternal interface change participating in a coordinated manner to orchestrate sequential , reproductive events that result in the maintenance of pregnancy . during the first trimester , nk cells represent the most abundant immune cells in the decidua . these cells are cd56cd16 , are distinct from peripheral blood nk subsets , and are critical for the maintenance of pregnancy since their cytotoxicity is inhibited by the expression of hla - e and hla - g on trophoblasts . as expected , 58.3 4.6% , ( mean sem , n = 10 ) of the human decidual mononuclear cells were cd56 nk cells , of which > 90% were cd16 ( fig . the remainder decidual leukocyte population included t and b lymphocytes , cd14 monocytes , cd11b and cd11c myeloid cells . in comparison with peripheral blood lymphocytes , the proportion of cd4 t cells infiltrating human decidua was significantly lower ( 16.01 1.64% , mean sem , n = 10 , vs. 39.12 3.83% , mean sem , n = 9 , p = 0.0009 ) ( fig . no differences in the percentages of cd8 t cells , cd19 b cells , and cd14 monocytes were observed between the human decidua and the peripheral blood . human decidual leukocytes contained also 6.4 1.8% ( mean sem , n = 8) of cd11b cells , 49.95 6.07% ( mean sem , n = 9 ) of cd11c cells , and 3.54 1.2% , ( mean sem , n = 9 ) of bdca-1 ( cd11ccd1c ) cells ( fig . the frequencies of decidual myeloid cd11c and bdca-1 cells were significantly higher than their counterpart in the peripheral blood of pregnant women ( 13.12 1.74% , mean sem n = 9 , p = 0.0027 , and 0.69 0.27 , mean sem , n = 9 , p = 0.0047 , respectively ) ( fig . our data are in line with a previous report demonstrating the presence of several subsets of dcs , including bdca-1 ( 0.2% ) , in the human first trimester decidua ; however , we found a higher percentage of decidual bdca-1 cells ( 3.5% ) . this may be due to the method used for isolating decidua - infiltrating cells : ban et al . used a non - enzymatic method , while we isolated immune cells from decidual tissues by collagenase - mediated digestion . in addition , in the previous study the percentage of bdca-1 cells was calculated based on total decidual cells , whereas we evaluated the frequency of bdca-1 cells by gating on cd45 cells . in our setting , the percentage of cd45 cells ranged from 10% to 70% of the total decidual cells . human decidua harbored a significant population of dc - sign cells that represents 610% of decidual cells . decidual dc - sign cells have been shown to be immature myeloid cells and have been proposed to play a role in normal and pathological pregnancy outcome . in our setting , the percentage of decidual dc - sign cells was 2.9 0.6% ( mean sem , n = 8) , and their frequency was significantly higher than that observed in the peripheral blood of pregnant women ( 1.12 0.23% , mean sem n = 9 , p = 0.01 ) ( fig . dc-10 are tolerogenic dcs characterized by their ability to secrete il-10 and by the high expression of membrane - bound hla - g . since dc-10 are highly potent in inducing tr1 cells , we hypothesized that these cells might represent one of the major subsets of apcs present in the decidua involved in promoting and maintaining tolerance during pregnancy . dc-10 were identified by the concomitant expression of cd14 , cd16 and cd83 on human decidual leucocytes ( cd45 ) . interestingly , a significantly higher percentage of dc-10 was present in the human decidua than in the peripheral blood of pregnant women ( 2.29 0.75% , mean sem , n = 10 vs. 0.25 0.07% , mean sem , n = 8 , p = 0.0014 ) ( fig . human decidual dc-10 expressed similar levels of hla - g , but significantly lower levels of ilt4 ( 80.38 2.45% , mean sem , n = 10 vs. 96.38 1.01% , mean sem , n = 8 ; p = 0.0003 ) as compared to circulating dc-10 ( fig . similarly , inflammatory monocytes ( cd14cd16 ) present in the human decidua expressed similar levels of hla - g but lower amounts of ilt4 as compared to their circulating counterpart ( 56.86 4.89% , mean sem , n = 7 vs. 94.57 1.23% , mean sem , n = 9 ; p = 0.001 , fig . the expression of hla - g and ilt4 on decidual bdca-1 cells was comparable to that observed in their peripheral blood counterpart ( fig . 2c ) . the increased frequency of dc-10 observed in the decidua may be dependent on several mechanisms : dc-10 can be recruited from the peripheral blood , resident decidual dcs can be converted into dc-10 , or the decidual microenvironment can promote the de novo induction of dc-10 . the decidual microenvironment is enriched of several chemokines , including ccl2 and cx3cl1 that have a role in tissue remodeling and in the recruitment of immune cells . peripheral blood dc-10 express ccr2 and cx3cr1 , thus it can be hypothesized that they are attracted and accumulated in the decidua . several cytokines including il-4 , il-10 , and gm - csf as well as growth factors and hormones with anti - inflammatory properties are present at the decidual level ( reviewed in ) . this pro - tolerogenic microenvironment is known to promote alternatively activated macrophages and tolerogenic dcs . in particular , the high levels of il-10 may promote the up - regulation of hla - g , ilt2 and ilt4 on resident decidual immature dcs converting them into dc-10 . alternatively , the presence of gm - csf , il-4 and il-10 can allow , through the il-10 dependent ilt4/hla - g pathway , the de novo induction of tolerogenic dc-10 . further functional studies are required to better define the origin and the role of dc-10 in the decidua . in addition to dcs , cd4cd25foxp3 tregs are enriched at the fetal - maternal interface and are expanded accordingly to gestational stages . we confirmed that cd4cd25foxp3 tregs cells are present in the human decidua , and no differences in the percentages of these cells were observed between human decidua and peripheral blood in our cohort of pregnant women ( data not shown ) . in addition to cd4cd25foxp3 tregs , other tregs either naturally occurring or induced have been described . among them , we focused our attention on a new type of naturally occurring cd4 and cd8 tregs that constitutively express hla - g . cd4hla - g and cd8hla - g tregs are present in the peripheral blood . moreover , cd4hla - g tregs have been shown to accumulate at sites of inflammation , and are potent suppressors of t cell proliferation in vitro . to date , there is no evidence of the presence of hla - g - expressing cd4 or cd8 t cells in the peripheral blood and the decidua during pregnancy . we first investigated the frequency of circulating cd4hla - g t cells in pregnant women as compared to healthy donors . results showed a significantly higher frequency of hla - g expressing cd4 t cells in the peripheral blood of pregnant women ( pbmc pw ) compared to that of healthy donors ( pbmc hd ) ( 5.29 1.3% , mean sem , n = 8 vs. 2.13 0.28% , mean sem , n = 53 , respectively , p = 0.0053 ) ( fig . interestingly , a distinct population of cd4 t cells expressing hla - g was detected in the human decidua ( 18.97 2.98% mean sem , n = 9 ) ( fig . 3a and b ) . the frequency of cd8hla - g t cells in the human decidua was comparable to that observed in the peripheral blood of pregnant women ( data not shown ) . similarly to what has been described in the central nervous system , cd4hla - g t cells can be recruited to the human decidua in the first phases of embryo implantation to control immune responses against the semi - allogeneic fetal antigens . it still remains to be defined whether decidual cd4hla - g t cells express ccr5 or other chemokine receptors , which may allow their recruitment into the decidua , in analogy to what has been described for the same cells during the active inflammatory phase of multiple sclerosis . we can not exclude that cd4hla - g t cells could be directly induced in the decidua by local il-10 as it is known that il-10 promotes hla - g expression in cd4 t cells . moreover , we previously demonstrated that during tr1 cell induction via dc-10 , il-10-derived from dc-10 induces hla - g expression on cd4 t cells that become anergic t cells , and subsequently tr1 cells . in this scenario , it can be speculated that naive cd4 t cells , once recruited in the decidua , can be activated via dc-10 , up - regulate hla - g expression and then , in the continuous presence of il-10 and chronic allo - specific stimulation , they become allo - specific tr1 cells . it still remains to define whether cd4hla - g t cells are precursors of tr1 cells or represent a distinct subset of tregs , which co - operate with other tregs , including cd4cd25foxp3 tregs , in promoting and maintaining fetal - maternal tolerance . in addition to an overall improvement of the knowledge on the biological mechanisms underlying tolerance associated with hla - g expression , our findings may have different potential clinical implications . the ultimate goal of this study is the development of a risk assessment of women with a history of recurrent implantation failure who undergo in vitro fertilization ( ivf ) . from a diagnostic point of view , the tolerance status of women with recurrent embryo implantation failure after ivf can be evaluated . the analysis of dc-10 and hla - g tregs and their potential association with hla - g polymorphisms will allow the identification of specific hla - g genotypes that could be included among the pre - ivf screening tests . from a therapeutic point of view , the study will open new perspectives for the modulation of immune responses in the early phases of pregnancy with the aim of minimizing the negative effects on embryo implantation associated with a reduced maternal tolerance status .
in end - stage renal disease ( esrd ) patients regardless of dialysis modes , i.e. maintenance hemodialysis ( hd ) and continuous ambulatory peritoneal dialysis ( capd ) , potassium ( k ) homeostasis is regulated primarily via dialysis and extrarenal k regulation in the diverse daily k intake . however , k metabolism has been known to differ greatly between the two main methods of dialysis . hyperkalemia is a common complication ( 10 - 24% ) and the most common cause of the death ( 3 - 5% ) among electrolyte disorders in patients on maintenance hd . on the contrary , hypokalemia ( 10 - 36% ) is responsible for a rather common complication and independent prognostic factor on capd . although excessive k intake or inadequate dialysis on maintenance hd and poor nutritional k intake on capd are accused without doubts upto 50% of esrd patients as a primary cause of the k - imbalance , i.e. hyperkalemia on hd and hypokalemia on capd , other contributory factors including certain medications and unknown causes remain still to be resolved . accordingly , the effects of medications as another source of k - imbalance on hd with ras blockades and beta blockers as well as those of conventional and glucose - free dialysates ( icodextrin ) for internal k - redistribution on capd were evaluated with reviewing the literatures and our data . furthermore , new developments in the clinical managements of hyperkalemia on hd following the exclusion of pseudohyperkalemia before the initiation of dialysis were suggested , especially , by the comparison of the effects between mono- and dual - therapy with medications for transcellular k shifting in the emergent situation . also , the intraperitoneal k administration via conventional glucose - containing ( 2.5% ) and glucose - free dialysates ( icodextrin ) as a specific route of k - supplementation for hypokalemia on capd was examined for its efficiency and the degree of intracellular k shift between these two different types of dialysates .	in esrd patients on renal replacement therapy , k homeostasis is mainly maintained on adequate dialytic k removal and adapted extrarenal k regulation1 ) . however , due to the different dialysis modes between hd and capd , i.e. the episodic ( 3 - 4 times / wk ) in hd versus the daily continuous dialysis in capd , hd patients do not achieve a steady blood [ k ] leading to usually highest before dialysis and lowest immediately after dialysis while capd patients approach a steady state for it 's level , so that wide variations in blood [ k ] are not present . excessive dietary k gain before dialysis in hd can temporarily contribute to the development of hyperkalemia but poor dietary k intake in capd may lead to hypokalemia . therefore , hyperkalemia would be more frequent in hemodialysis than in capd , especially predialysis stage , and hypokalemia more frequent in capd than in hd . the k imbalance in dialysis patients seems to be primarily related to poor dietary compliance . in fact , too much k intake with poor compliance to diet in interdialytic interval in hd despite adequacy of hd contributes to the high frequency of hyperkalemia ( 10 - 24% ) responsible for the most common cause of death ( 3 - 5% ) among electrolyte imbalances , whereas malnutrition with poor k intake in capd is a well known factor for the high prevalence of hypokalemia ( 10 - 36% ) leading to the higher cardiovascular mortality than normokalemic capd patients in a recent study2 ) . however , besides dietary factors , there are other important factors to be considered among the causes of k imbalance in dialysis patients regardless of dialysis modes , since they are mostly remediable with identifying and then avoiding or removing them . those are clinical conditions causing the derangement of the external k regulation including abnormal colonic k secretion , i.e. constipation or diarrhea , as well as administration of drugs affecting internal k balance in k homeostatic mechanism related to neurohormones such as insulin , catecholamines , and renin - angiostensin - aldosterone ( ras ) , such as non - selective bet - ablockers , ace - inhibitors and/or arbs , and aldosterone receptor blocker ( spironolactone , eplerenone ) . as an another factor for k homeostasis in the patients on capd , daily continuous infusion of standard glucose containing dialysates might be involved in intracellular k shift related to insulin hormone and showed high intracellular k contents in muscles as shown in a few studies . recently , icodextrin with glucose - polymer solution leading to less glucose load and less insulin secretion has been replaced for the patients on capd with ultrafiltration failure during long - overnight dwell , which might affect k distribution in them3 ) . in this paper , the etiologies responsible for hyperkalemia in hd and for hypokalemia in capd with evaluation of their prevalence and incidence as well as their treatments based on the contributing factors for the k imbalances are reviewed with collected data from the literature and our data . hd patients with esrd are continuously exposed to the risk of hyperkalemia due to episodic removal of body k through dialysis for 3 to 4 hrs with 3 or 4 times per week in frequency rather than continuous k excretion by the intact kidneys . however , the degree of hyperkalemia in hd during the interdialytic perod depends on several factors : 1 ) a dietary k intake , 2 ) the amount of dialysate k removal by hd related to the [ k ] gradient and to the k passive refilling fro icf to ecf , 3 ) increased intestinal excretion of k as an adaptive extrarenal k homeostasis4 ) , and 4 ) the transcellular k shifts as in healthy subjects . hyperkalemia was pointed out as a " potential silent killer " and suggested even one of the leading causes of sudden death in hd patients5 ) . in our survey of hd patients during past 10 yrs , 108 out of 548 patients ( 20% ) expired , and its most common causes are cardiovascular disorder ( 44% ) and infection ( 17% ) same as reported in the world literature , but other notable causes included unknown etiologies ( 22% ) and hyperkalemia ( 6%)6 ) . in the admitted patients with severe hyperkalemia , the most common causes of hyperkalemia were related to inadequate dialysis with noncompliance to hd frequency per week ( 55% ) and excessive k intake with noncompliance to dietary regimen ( 35% ) followed by others including medications7 ) . nonselective beta - blockers have been shown to increase serum [ k ] in patients with esrd due to preventing intracellular k shift by the inhibition of beta-2 adrenergic receptors.8 - 9 however , nonselective beta - adrenergic blocker with a selective alpha-1 blocking activity , carvediolol , did not enhance the hyperkalemic effect of moderate physical exercise on hd , but in our observation predialysis - serum [ k ] with carvediolol was significantly higher than that without it ( 5.131.0 meq / l vs 5.71.0 meq / l)10 ) . though renin - angiotensin - aldosterone system ( raas ) blockers such as acei , arb and spironolactone or eplerenone currently are recommended in hd patients for the cardio - protective effects , it has been reported to be independently associated with an increased risk of developing hyperkalemia11 ) . recently , low - dose spironolactone , 25 mg / d12 ) , and even the combined therapy of ras blockers with ace plus arb failed to show any significant increase in frequency of hyperkalemia and in the higher mean serum k level when compared to basal values without their exposure in our study ( no - exposure , 5.540.67 meq / l ; acei alone , 5.540.75 meq / l ; arb alone , 5.500.66 meq / l ; acei plus arb , 5.420.66 meq / l , p = ns)13 ) . these contradictory data suggest that the cautious trial of ras blockers , either alone or combined , would be justified with the overweighing benefits of the cardioprotective effects for the patients on hd rather than their hyperkalemic risks . before the initiation of hd as the most efficient treatment of hyperkalemia is available , other conservative managements of the emergency treatment of hyperkalemia consists of four basic modalities as monotherapy including calcium gluconate , insulin with glucose , nebulized beta-2 agonists , intravenous nahco3 and oral or rectal cation - resins . however , the effect of nahco3 for the treatment of hyperkalemia is uncertain and its infusion in varying amount failed to lower serum [ k ] in esrd patients14 ) , but when combined with insulin with glucose or beta-2 agonists ( nahco3 alone , -0.130.06 , p = ns ; salbutamol alone , -0.570.03 meq / l , p<0.02 ; nahco3 plus salbutamol , -0.960.08 meq / l , p=0.000 ) , their hypokalemic effect were enhanced probably due to the activation of na - k pump with acute correction of underlying metabolic acidosis in the combined regimen with bicarbonate as dual therapy15 , 16 ) . also , other study introduced the efficacious and safe modalities for the acute treatment of hyperkalemia in esrd patients with the combined regimens as dual therapy , insulin with glucose plus beta 2-agonists17 ) . therefore , once pseudohyperkalemia also present in hd patients as in other general patients is excluded18 ) , the temporizing measures of transcellular k shifting regimens can be initiated alone or combined therapy to buy time until hemodialysis can be initiated with the assessment of ekg at the outset as shown in the flow chart ( fig . 1)19 ) . as a long - term treatment plan of hyperkalemia in hd , the correction of the underlying pathophysiology responsible for hyperkalemia should be started with removal or cautious trials of medications inducing hyperkalemia with balancing the ratio of their benefits and risks in addition to improving the compliance to dialysis and diet . among other therapeutic regimen for hyperkalemia on the long - term basis , the administration of laxative such as bisacodyl increasing colonic k seretion20 ) , and exogenous mineralocorticoid or mineralocorticoid - like substance may provide simple means of reducing interdialytic hyperkalemia in hd21 , 22 ) . in our analysis of serum [ k ] profile in stable capd patients ( n=253)23 ) , normokalemia ( 83% ) and hypokalemia ( 16% ) were much more frequent than hyperkalemia ( 1% ) , being similar to the recent data of chinese capd patients , i.e. normokalemia ( 80% ) , hypokalemia ( 20% ) , and none in hyperkalemia2 ) . the chinese data revealed that serum [ k ] is correlated with serum albumin level ( r=0.17 ; p=0.005 ) suggesting close relation between hypokalemia and malnutrition . also , on analysis of serum [ k ] profile in admitted capd patients for 3 yrs , we observed the significant differences between groups of hypokalemia ( 11/48 , 23% ) and without hypokalemia ( 37/48 , 77% ) in serum albumin , bun , and creatinine levels . furthermore , the history of poor dietary intake was more prominent in hypokalemic patients ( 9/11 vs 6/37 , p<0.01 ) , but daily k loss via dialysates was similar in two groups . these data support strongly that high prevalence of hypokalemia in capd as well as the close correlation of hypokalemia with malnutrition and poor dietary k intake24 ) . however , one study investigated for the pathogenetic mechanism of hypokalemia in capd with measuring the amount of k intake minus that of urinary and peritoneal k excretion and considering stool k losses of around 6 - 50 meq / day . external k imbalance ( i.e. k losses ) alone was not enough to justify the hypokalemia . therefore , the deranged internal k balance , i.e. intracellular k shift by mostly insulin hormone stimulated by the continuous glucose peritoneal infusion in capd , was proposed as a contributory factor for the development of hypokalemia even in the context of a low k excretion25 ) . in our recent study , the mean serum [ k ] was higher in the group of glucose - free dialysates ( icodextrin ) than in the group of standard glucose dialysates for long overnight - dwell ( 4.60.1 meq / l vs 4.40.1 meq / l , p<0.05).13 for k supplementation in the management of hypokalemia furthermore , k mixed with dialysate solution would be administered thru intraperitoneal route upto 40 meq in 2 l dialysate safely . in hypokalemic patient ( < 3.5 meq / l ) , the degree of k absorption via peritoneum and that of intracellular shift of absorbed k were 766% and 7419% , respectively , and the increment of serum [ k ] was 0.80.2 meq / l.26 a recent study of comparison of intraperitoneal k administration ( 40 meq mixed in 2 l dialysate ) after one exchange between standard 2.5% glucose and glucocose - free ( icodextrin ) dialysates revealed the less increase in serum [ k ] ( 0.180.1 meq / l vs 0.560.1 meq / l , p<0.05 ) , despite the less increment of insulin level from basal value ( -199% vs 3712% ) in the latter and similar blood glucose concentrations in two different dialysates13 ) . undoubtedly , acute administration of k in hypokalemic patients in capd regardless its routes would be necessary for its correction to avoid neuromuscular weakness and serious cardic arrhythmia . nevertheless , in the chronic long - term basis , further investigation may be required to investigate the benefits of k supplementation alone , because hypokalemia may simply be surrogate marker of malnutrition and/or other serious comorbid illness . in dialysis patients , i.e. hd or capd , k imbalance should be always considered as a common condition related to either a potentially lethal condition itself or the important contributory factor for their morbidity and mortality . the pathophysiologic factors for hyperkalemia in hd are well understood , and accordingly its managements are somewhat straightforward , while the pathogenetic mechanisms of hypokalemia in capd need further clarification for the role of intracellular k redistribution . for the management of k imbalance in dialysis , the foremost step is to improve compliance to dietary k intake and dialysis regimen besides the reversal or removal of pathophysiologic factors and other potential sources including drugs affecting k imbalance should be always considered . however , cautious administration of certain beneficial drugs such as raas blockers could be justified in esrd on dialysis with tremendous burden with cardiovascular morbidity and mortality . as temporizing acute k - lowering regimens in severe lifethreatening hyperkalemia , dual - therapeutic regimens , i.e. nahco3 plus insulin with glucose or insulin with glucose plus beta-2 agonist , could be recommended as an efficacious modality . in capd patients with hypokalemia , for the long - term management , the correction of malnutrition would be required as one of the essential managements and the selection of glucose - free dialysates such as icodextrin could be considered rather than the conventional glucose dialysates . also , as short - term management , k supplements could be administered safely and efficiently via intraperitoneal route , if required .
pure red cell aplasia ( prca ) may develop in patients with chronic kidney disease receiving erythropoiesis - stimulating agents ( esa ) . we report on a 72-year - old patient who developed hypo - proliferative anaemia unresponsive to esa following the administration of epoetin zeta subcutaneously for 7 months . on the basis of severe isolated hypoplasia of the erythroid line in the bone marrow and high - titre neutralizing anti - erythropoietin antibodies ( ab ) , a diagnosis of ab - mediated prca was made . epoetin zeta was discontinued and the patient was given steroids . this was associated with anaemia recovery . to our knowledge this is the first prca case related to epoetin zeta .	pure red cell aplasia ( prca ) is a rare adverse reaction occurring in patients with chronic kidney disease ( ckd ) who are treated with erythropoiesis - stimulating agents ( esa ) . it is generated by epoetin - induced antibodies ( ab ) that neutralize all the exogenous erythropoietin ( epo ) and cross - react with endogenous epo . in the early 2000s a peak increase in the incidence rate of prca was observed after a change in the formulation of epoetin alpha produced outside the usa . following reinforcement of the cold storage chain , a shift towards the intravenous administration route and the elimination of uncoated rubber stoppers , the incidence of prca has markedly decreased . biosimilars of epoetin alpha have been approved in the european union since 2007 , and following the expiration of the us patent , they will enter the us market soon . we report on a patient who developed prca following the subcutaneous administration of epoetin zeta , which is one of the two biosimilars of epoetin alpha licensed in europe . to our knowledge a 72-year - old caucasian female was regularly followed up in the nephrology outpatient clinic of versilia hospital [ lido di camaiore ( lu ) , italy ] because of a slowly progressive ckd . in 1990 the patient underwent left nephrectomy for severe nephrolithiasis , complicated by acute pyelonephritis and sepsis . in 1991 , she developed new stones in the right kidney and received extracorporeal shock wave lithotripsy . in 1995 since then , she has been under regular cardiologic follow - up . from 2007 until 2014 kidney function slowly deteriorated , reaching stage iv v ckd . in june 2014 , she attended the day hospital of the nephrology unit for severe hip arthrosis . her ( hb ) was 9.7 g / dl with adequate iron stores ; she was prescribed epoetin zeta 4000 iu twice a week subcutaneously ( figure 1 ) and antalgic therapy for hip pain . rbc , red blood cells . in december 2014 , she was hospitalized for 4 days in the nephrology department due to the persistence of severe hip pain . the patient 's serum creatinine was 3.7 mg / dl and hb was 10.2 g / dl . given that occult blood was detected in three stool samples , colonoscopy was suggested . in february 2015 , the patient arrived at the emergency unit because of severe asthenia and mild shortness of breath . she received blood transfusion ( six units of packed red cells ) and was admitted again to the nephrology department . colonoscopy showed diverticulosis , but was not diagnostic for incomplete cleaning . at 2 weeks after admission , at discharge her hb was 9.3 g / dl ; epoetin zeta dose was increased to 4000 iu three times per week . in april 2015 , she was hospitalized again for 8 days because of severe anaemia ( hb 6.2 g / dl , reticulocytes 0.1% , 2500/l ) . the patient underwent bone marrow biopsy showing severe hypoplasia of the erythroid line ( cd34 blasts < 5% ) with hyperplasia of the megakaryocytic and granulocytic lines . table 1.main laboratory parameters at onset of prca25/02/201509/04/2015haemoglobin ( g / dl)3.99.36.27.3reticulocytes ( /l)na2500leucocytes ( n / mmc)68508680platelet count136 000137 000serum iron ( g / dl)227218transferrin saturation ( % ) 80.582.2serum ferritin ( ng / ml)268512serum creatinine ( mg / dl)3.53.7c - reactive protein ( mg / l)na0.3parathyroid hormone ( pg / ml)na155na , not available.post-transfusion . main laboratory parameters at onset of prca in may 2015 , at the time when bone marrow biopsy became available , the patient had become transfusion dependent and epoetin zeta administrations were immediately interrupted . assays for antinuclear ab , anti - dna ab and hepatitis b ab were normal ; parvovirus b19 dna and hepatitis b antigen were not detected . anti - epo ab were tested by ipm biotech gmbh , hamburg , germany and found positive in three different samples by a screening assay , followed by specificity confirmation testing . the patient was prescribed oral prednisone ( 0.5 mg / kg / day ) for 3 months and progressive decrease in the need for blood transfusions was observed . in september 2015 , she received her last blood transfusion and steroid therapy was interrupted . since then , her hb values have stabilized ( last available hb value of 11.5 g / dl in february 2016 ) . no worsening of renal function had occurred over the period ( in february 2016 , serum creatinine was 3.9 mg / dl ) . retesting of anti - epo ab titre has already been planned , in the case the patient needs re - challenging with esa in the future . in the late 1980s , epoetin alfa was developed thanks to the recombinant dna technique in chinese hamster ovary cells . this is a delicate manufacturing process , which invariably leads to heterogeneity of the final product . given that the drug is poorly water - soluble , excipients are required to enhance dispersion or inhibit precipitation when mixed with water . as a result , all esa molecules are delicate and fragile , require cold storage and are sensitive to changes in the stabilizer and/or manufacturing processes . of note , the interruption of the cold chain may be less problematic with long - acting esa , as they have a longer stability at room temperature compared with short - acting esa . the upsurge of prca cases with eprex was preceded by the substitution of human serum albumin with polysorbate 80 . the hypothesis that this stabilizer may elicit the formation of immunogenic epoetin - containing micelles , possibly increasing immunogenicity , has been questioned . the uncoated rubber stoppers in the pre - filled syringes could have possibly increased immunogenicity as well . this may be due to the shift from the subcutaneous to the intravenous route of administration , the reinforcement of the product cold chain or the elimination of uncoated rubber syringe stoppers . in 2008 regulatory authorities readmitted the subcutaneous use of eprex in the absence of a vascular access for intravenous administration . more recently , an unexpected increase in local cases of prca associated with subcutaneous administration of eprex was described in singapore . the introduction of biosimilars in the eu market has brought savings of around 1530% , together with a price reduction of the originator . considering that the manufacturing process of epoetin alfa is owned by the producer of the reference product and can not be precisely duplicated , companies developing biosimilars have to implement a new manufacturing process . this is true also in highly regulated countries such as those of the eu , or the usa , where high - quality processes are used . some years ago , one randomized clinical trial using hx575 was halted for safety reasons following one prca case and one anti - epo ab positivity . the potential cause of immunogenicity was identified in soluble tungsten , most likely derived from the pins used to manufacture the syringes , causing unfolding and aggregation of hx575 in pre - filled syringes . considering that only 160 patients were treated with hx575 in the trial over a follow - up of 52 weeks , the incidence of anti - epo neutralizing ab in this study is particularly high . epoetin theta is an analogue of epoetin beta , which was approved as an originator by european medicine agency in 2009 . data from one clinical trial administering epoetin zeta subcutaneously to 230 ckd patients did not report cases of anti - epoetin ab or prca . similarly , no cases had occurred during a large post - authorization observational study involving more than 1600 ckd patients receiving epoetin zeta intravenously . our patient had been enrolled in a second , ongoing , post - authorization observational study to estimate the incidence of prca and/or neutralizing ab during treatment with epoetin zeta administered subcutaneously in more than 6000 ckd patients [ post - authorisation safety cohort observation of retacrit ( epoetin zeta ) administered subcutaneously for the treatment of renal anaemia , pasco ii ] . to our knowledge , this is the first prca case that has been described for epoetin zeta . our patient suddenly developed severe hypo - proliferative anaemia after 7 months from the start of epoetin zeta administered subcutaneously . this is in line with data in the literature describing a median of 7 months ( range 1 month to 5 years ) from the beginning of therapy until the diagnosis of prca . the severity of anaemia , which had become unresponsive to esa therapy , is testified by the very low reticulocyte count and by the dependence on blood transfusions . prca diagnosis was supported by the findings of the bone marrow biopsy , showing severe hypoplasia of the erythroid line , and by the presence of high - titre anti - epo neutralizing ab . no other causes of prca , such as parvovirus or hepatitis b infection , thymoma , lymphoproliferative disorders , drugs or autoimmune disease were identified . however , the timing from drug exposure and the fact that the patient received uniquely epoetin zeta makes the relationship between the drug and the occurrence of prca extremely likely . considering its rarity , it is difficult to distinguish whether this is a sporadic case or the expression of increased immunogenicity of epoetin zeta . the calculation of prca incidence based on length of exposure to epoetin zeta would be of interest , but is beyond the scope of this case report . however , in the absence of randomized clinical trial , there are insufficient data to provide guidance on the preferred immunosuppressive agents or treatment regimen . this is in line with other reports in the literature [ 14 , 15 ] . interestingly , after therapy her hb stabilized without the need to challenge her with new esa therapy . it is possible that the iron load she received with blood transfusion had contributed to hb stability after the recovery of prca . one possible limitation of this case report is that reticulocytes were not tested at onset and that prca diagnosis was made with a certain delay . while this does not reduce the importance of the relationship between epoetin zeta use and the occurrence of prca , we acknowledge that an earlier testing of reticulocyte values would have prevented needless workup of anaemia with endoscopies and ct scans , and directed us towards bone marrow biopsy sooner . a more timely diagnosis would also have prevented the increase in epoetin zeta dose , which have possibly enhanced anti - epo ab production . to conclude , we report for the first time a patient who developed ab - mediated prca after receiving epoetin zeta subcutaneously . the patient described in this paper was enrolled in the post - marketing observational study epoe-09 - 11 pasco ii , which is sponsored by hospira . hospira has been informed about this case and the intention to publish it . v.p . f.l . was a member of an advisory board and/or speaker at meetings supported by akebia , amgen , astellas , janssen , gsk , hospira , pharmacosmos , roche and sandoz .
objective : to assess whether parkinson disease ( pd ) genes are somatically mutated in cutaneous melanoma ( cm ) tissue , because cm occurs in patients with pd at higher rates than in the general population and pd is more common than expected in cm cohorts.methods:we cross - referenced somatic mutations in metastatic cm detected by whole - exome sequencing with the 15 known pd ( park ) genes . we computed the empirical distribution of the sum of mutations in each gene ( smut ) and of the number of tissue samples in which a given gene was mutated at least once ( ssampl ) for each of the analyzable genes , determined the 90th and 95th percentiles of the empirical distributions of these sums , and verified the location of park genes in these distributions . identical analyses were applied to adenocarcinoma of lung ( adenoca - lung ) and squamous cell carcinoma of lung ( squamca - lung ) . we also analyzed the distribution of the number of mutated park genes in cm samples vs the 2 lung cancers.results:somatic cm mutation analysis ( n = 246 ) detected 315,914 mutations in 18,758 genes . somatic cm mutations were found in 14 of 15 park genes . forty - eight percent of cm samples carried 1 park mutation and 25% carried multiple park mutations . park8 mutations occurred above the 95th percentile of the empirical distribution for smut and ssampl . significantly more cm samples harbored multiple park gene mutations compared with squamca - lung ( p = 0.0026 ) and with adenoca - lung ( p < 0.0001).conclusions : the overrepresentation of somatic park mutations in cm suggests shared dysregulated pathways for cm and pd .	to define pd predisposition genes and loci , omim ( http://www.ncbi.nlm.nih.gov/omim ) was searched with the words parkinson disease and genes and locus / loci . the somatic cm mutation data used in this article merged melanoma exome / genome sequencing from different sources as described elsewhere . all mutational data from 6 different whole - exome / genome sources were collated from 4 published studies and unpublished data . the data were formatted so that all positional data were mapped to the same genome build . in this case , any data that were on hg18 were lifted over to hg19 using the lift genome annotations tool available from ucsc ( http://genome.ucsc.edu/cgi-bin/hgliftover , uc santa cruz software , the regents of the university of california , santa cruz , ca ) . in some cases , as data were merged , it became necessary to eliminate redundant information . for instance , with some samples , both a tumor and a cell line derived from it were sequenced and the overwhelming majority of mutations were shared . this is also true in the case of samples that were sequenced in more than one study and for multiple metastases extracted from the same patient in another study . when removing these duplicates and redundancies , all mutations were retained at a count of one and the sample name was merged into a single entry . this step was taken to ensure that the number of recurrent positions was not inflated in later analysis . once the list of mutations was established , the positional data and changes were formatted to an oncotator input format and annotated using the web - based version of oncotator ( http://www.broadinstitute.org/oncotator , cambridge , ma ) . the next step taken was to remove any samples that were listed as acral , mucosal , or uveal melanoma subtypes , to ensure focusing on cm . in the final step , any samples in the initial publication that did not include a matched normal genotype were also removed . the data were arranged in a table , where the rows represent genes and the columns are cm tissue samples . the entries are the number of somatic mutations per each combination of gene and tissue sample . the somatic cm mutation platform was cross - referenced with the list of the defined park genes and loci ( park1 to park20 ) . to assess cm - related specificity of the findings , identical analyses were performed for adenocarcinoma of lung ( adenoca - lung ) and squamous cell carcinoma of lung ( squamca - lung ) , based on data derived from the cosmic database ( studies cosu417 and cosu418 ) . the data format of the cm mutation data set was compatible with the cosmic data sets . splice variants are annotated in cosmic and are used if a mutation arises in a region that maps to the coding domain of a gene and the predicted protein is different . , we analyzed each gene individually , and in the second step , we analyzed the park genes combined as a group . the question of interest was whether single park genes harbor a relatively high number of somatic mutations , when compared with other genes in cm tissue . to answer this question , 2 measures were defined : ( 1 ) the sum of mutations in each gene ( smut ) and ( 2 ) the number of tissue samples in which a given gene was mutated at least once ( ssampl ) . ( note that the difference between smut and ssampl is that in ssampl , each sample is counted at most once . ) analysis was performed by the following steps : ( 1 ) computation of the empirical distributions for smut and ssampl for all analyzable genes , ( 2 ) determination of the percentiles ( 75th , 90th , and 95th ) of the empirical distribution , and ( 3 ) determination of the location of the known park genes in the above empirical distributions . for comparison , and to ensure cm specificity of the findings , identical analyses were applied for adenoca - lung and squamca - lung in cosmic data sets of studies cosu417 and cosu418 . lung carcinomas were chosen as control tissues because the number of somatic mutations in lung cancer is roughly similar to that of cm ( 1213/megabase in lung vs 1018/megabase in cm ) . because some samples harbored mutations in more than 1 park gene , we analyzed the distribution of the number of mutated park genes in cm using 2 methods : we compared the distribution of the number of mutated park genes in cm samples with the distribution in adenoca - lung and in squamca - lung samples using the kolmogorov - smirnov test.here , the question of interest was how often the 15 park genes are mutated as a group , relative to a randomly chosen group of 15 genes . we compared the distribution of the number of mutated park genes in cm samples with the distribution in adenoca - lung and in squamca - lung samples using the kolmogorov - smirnov test . here , the question of interest was how often the 15 park genes are mutated as a group , relative to a randomly chosen group of 15 genes . using a bootstrap - like procedure , we generated the empirical distributions of the variables of interest ( smut , ssampl ) , aggregated over the 15 park genes . the aggregated variables are denoted by smutgroup , which is the total number of mutations of the 15 park genes as a group in all samples , and ssamplgroup , which is the number of samples in which at least one gene from the group was mutated . the procedure was performed as follows : randomly select a group of 15 genes.compute smutgroup and ssamplgroup for the selected group.repeat steps 1 and 2 , ten thousand times.obtain the empirical distributions of smutgroup and ssamplgroup . we note that the 15 park genes were excluded from the pool of genes ( 18,758 15 = 18,743 genes ) . the same analysis was applied to each of the 3 cancer types . once the empirical distributions of these variables were generated for random gene groups , we verified the locations of the group of 15 park genes in these distributions , for smutgroup and ssamplgroup and for each cancer type . to define pd predisposition genes and loci , omim ( http://www.ncbi.nlm.nih.gov/omim ) was searched with the words parkinson disease and genes and locus / loci . the somatic cm mutation data used in this article merged melanoma exome / genome sequencing from different sources as described elsewhere . all mutational data from 6 different whole - exome / genome sources were collated from 4 published studies and unpublished data . the data were formatted so that all positional data were mapped to the same genome build . in this case , any data that were on hg18 were lifted over to hg19 using the lift genome annotations tool available from ucsc ( http://genome.ucsc.edu/cgi-bin/hgliftover , uc santa cruz software , the regents of the university of california , santa cruz , ca ) . in some cases , as data were merged , it became necessary to eliminate redundant information . for instance , with some samples , both a tumor and a cell line derived from it were sequenced and the overwhelming majority of mutations were shared . this is also true in the case of samples that were sequenced in more than one study and for multiple metastases extracted from the same patient in another study . when removing these duplicates and redundancies , all mutations were retained at a count of one and the sample name was merged into a single entry . this step was taken to ensure that the number of recurrent positions was not inflated in later analysis . once the list of mutations was established , the positional data and changes were formatted to an oncotator input format and annotated using the web - based version of oncotator ( http://www.broadinstitute.org/oncotator , cambridge , ma ) . the next step taken was to remove any samples that were listed as acral , mucosal , or uveal melanoma subtypes , to ensure focusing on cm . in the final step , any samples in the initial publication that did not include a matched normal genotype were also removed . the data were arranged in a table , where the rows represent genes and the columns are cm tissue samples . the entries are the number of somatic mutations per each combination of gene and tissue sample . the somatic cm mutation platform was cross - referenced with the list of the defined park genes and loci ( park1 to park20 ) . to assess cm - related specificity of the findings , identical analyses were performed for adenocarcinoma of lung ( adenoca - lung ) and squamous cell carcinoma of lung ( squamca - lung ) , based on data derived from the cosmic database ( studies cosu417 and cosu418 ) . the data format of the cm mutation data set was compatible with the cosmic data sets . splice variants are annotated in cosmic and are used if a mutation arises in a region that maps to the coding domain of a gene and the predicted protein is different . , we analyzed each gene individually , and in the second step , we analyzed the park genes combined as a group . the question of interest was whether single park genes harbor a relatively high number of somatic mutations , when compared with other genes in cm tissue . to answer this question , 2 measures were defined : ( 1 ) the sum of mutations in each gene ( smut ) and ( 2 ) the number of tissue samples in which a given gene was mutated at least once ( ssampl ) . ( note that the difference between smut and ssampl is that in ssampl , each sample is counted at most once . ) analysis was performed by the following steps : ( 1 ) computation of the empirical distributions for smut and ssampl for all analyzable genes , ( 2 ) determination of the percentiles ( 75th , 90th , and 95th ) of the empirical distribution , and ( 3 ) determination of the location of the known park genes in the above empirical distributions . for comparison , and to ensure cm specificity of the findings , identical analyses were applied for adenoca - lung and squamca - lung in cosmic data sets of studies cosu417 and cosu418 . lung carcinomas were chosen as control tissues because the number of somatic mutations in lung cancer is roughly similar to that of cm ( 1213/megabase in lung vs 1018/megabase in cm ) . because some samples harbored mutations in more than 1 park gene , we analyzed the distribution of the number of mutated park genes in cm using 2 methods : we compared the distribution of the number of mutated park genes in cm samples with the distribution in adenoca - lung and in squamca - lung samples using the kolmogorov - smirnov test.here , the question of interest was how often the 15 park genes are mutated as a group , relative to a randomly chosen group of 15 genes . we compared the distribution of the number of mutated park genes in cm samples with the distribution in adenoca - lung and in squamca - lung samples using the kolmogorov - smirnov test . here , the question of interest was how often the 15 park genes are mutated as a group , relative to a randomly chosen group of 15 genes . using a bootstrap - like procedure , we generated the empirical distributions of the variables of interest ( smut , ssampl ) , aggregated over the 15 park genes . the aggregated variables are denoted by smutgroup , which is the total number of mutations of the 15 park genes as a group in all samples , and ssamplgroup , which is the number of samples in which at least one gene from the group was mutated . the procedure was performed as follows : randomly select a group of 15 genes.compute smutgroup and ssamplgroup for the selected group.repeat steps 1 and 2 , ten thousand times.obtain the empirical distributions of smutgroup and ssamplgroup . we note that the 15 park genes were excluded from the pool of genes ( 18,758 15 = 18,743 genes ) . the same analysis was applied to each of the 3 cancer types . once the empirical distributions of these variables were generated for random gene groups , we verified the locations of the group of 15 park genes in these distributions , for smutgroup and ssamplgroup and for each cancer type . the question of interest was whether single park genes harbor a relatively high number of somatic mutations , when compared with other genes in cm tissue . to answer this question , 2 measures were defined : ( 1 ) the sum of mutations in each gene ( smut ) and ( 2 ) the number of tissue samples in which a given gene was mutated at least once ( ssampl ) . ( note that the difference between smut and ssampl is that in ssampl , each sample is counted at most once . ) analysis was performed by the following steps : ( 1 ) computation of the empirical distributions for smut and ssampl for all analyzable genes , ( 2 ) determination of the percentiles ( 75th , 90th , and 95th ) of the empirical distribution , and ( 3 ) determination of the location of the known park genes in the above empirical distributions . for comparison , and to ensure cm specificity of the findings , identical analyses were applied for adenoca - lung and squamca - lung in cosmic data sets of studies cosu417 and cosu418 . lung carcinomas were chosen as control tissues because the number of somatic mutations in lung cancer is roughly similar to that of cm ( 1213/megabase in lung vs 1018/megabase in cm ) . because some samples harbored mutations in more than 1 park gene , we analyzed the distribution of the number of mutated park genes in cm using 2 methods : we compared the distribution of the number of mutated park genes in cm samples with the distribution in adenoca - lung and in squamca - lung samples using the kolmogorov - smirnov test.here , the question of interest was how often the 15 park genes are mutated as a group , relative to a randomly chosen group of 15 genes . we compared the distribution of the number of mutated park genes in cm samples with the distribution in adenoca - lung and in squamca - lung samples using the kolmogorov - smirnov test . here , the question of interest was how often the 15 park genes are mutated as a group , relative to a randomly chosen group of 15 genes . using a bootstrap - like procedure , we generated the empirical distributions of the variables of interest ( smut , ssampl ) , aggregated over the 15 park genes . the aggregated variables are denoted by smutgroup , which is the total number of mutations of the 15 park genes as a group in all samples , and ssamplgroup , which is the number of samples in which at least one gene from the group was mutated . the procedure was performed as follows : randomly select a group of 15 genes.compute smutgroup and ssamplgroup for the selected group.repeat steps 1 and 2 , ten thousand times.obtain the empirical distributions of smutgroup and ssamplgroup . randomly select a group of 15 genes . compute smutgroup and ssamplgroup for the selected group . we note that the 15 park genes were excluded from the pool of genes ( 18,758 15 = 18,743 genes ) . the same analysis was applied to each of the 3 cancer types . once the empirical distributions of these variables were generated for random gene groups , we verified the locations of the group of 15 park genes in these distributions , for smutgroup and ssamplgroup and for each cancer type . a total of 15 park genes and 20 park - associated loci were identified ( table 1 , first column ) . somatic cm analysis resulted in 315,914 mutations in 18,758 genes by whole - exome sequencing in 246 metastatic cm tissue samples . table 1 summarizes the observed number of mutations and the number of samples harboring park gene mutations and the percentiles of the empirical distribution of mutations . as shown in the table , park8 ( shown as straight bold numbers ) was mutated in cm , as counted by smut , 55 times , thus being above the 95th percentile of the empirical distribution ( which is 49 times ) and in ssampl = 40 cm samples , again above 36 , the 95th percentile of number of samples with mutations . park mutations in cutaneous melanoma , adenocarcinoma , and squamous cell carcinoma of lung three additional park genes ( park2 , park18 , and park20 , shown in table 1 as italic bold numbers ) were mutated above the 90th percentile of the empirical distributions of smut and ssampl . figure 1 summarizes park genes found above the 90th percentile of the empirical distribution of tissue samples ( ssampl ) in cm , adenoca - lung , and squamca - lung , and table 1 depicts the distributions of park gene mutations for the smut and ssampl variables in the 3 cancer types . venn diagram summarizing mutated park genes observed above the 90th percentile of the empirical distribution in melanoma , adenocarcinoma , and squamous cell carcinoma of lung . overall , 48% of cm samples had a mutation in at least 1 park gene and 25% had mutations in multiple park genes ( 28 mutated genes ) ( table 2 ) . samples with mutations in multiple park genes ( 25 mutated genes ) were found in only 6% of adenoca - lung and in 7% of squamca - lung samples . comparison of the distribution of the number of mutated park genes in the 3 cancer types is shown in table 2 . significantly more cm samples harbored multiple park gene mutations compared with squamca - lung ( p = 0.0026 ) and with adenoca - lung ( p < 0.0001 ) ( kolmogorov - smirnov test ) . this finding was significant after bonferroni correction for multiple comparisons ( required p value < 0.025 = 0.05/2 ) . number of samples with mutations in i mutated park genes , i = 1, ,8 , by cancer type the number of samples carrying diverse combinations of multiple park gene mutations in cm samples is depicted in table 3 . the higher frequencies of some of the combinations in cm samples ( in particular , those involving park2 , park8 , park18 , and park20 , table 3 ) vs the paucity of such samples in adenoca - lung ( table e-1 at neurology.org/ng ) and squamca - lung ( table e-2 ) are clearly notable . frequencies of combinations of park gene mutations in cutaneous melanoma ( 246 samples ) to further study the park genes as a group , we used the bootstrap - like permutation method ( see statistical analysis ) . smutgroup and ssamplgroup of park genes shown as the vertical lines appearing to be located in the upper end of the distribution of genes in cm ( figure 2a ) and squamca - lung ( figure 2c ) and not for adenoca - lung ( figure 2b ) . for cm ( figure 2a ) , the location of smutgroup for the 15 park genes as a group was at the 83.5th percentile and that of ssamplgroup was at the 90.8th percentile . for squamca - lung ( figure 2c ) , the location of smutgroup was at the 92.8th percentile and that of ssamplgroup was at the 94.1th percentile . for adenoca - lung ( figure 2b ) , the respective sums were located at the 76.3rd percentile for smutgroup and at the 60.8th percentile for ssamplgroup . histograms of the empirical distributions of the sums of mutations in groups of 15 genes , generated by randomly selecting 10,000 groups of 15 genes in ( a ) cutaneous melanoma , ( b ) adenocarcinoma of lung , and ( c ) squamous cell carcinoma of lung ( smutgroup upper panel , ssamplgroup lower panel ) . the vertical lines depict the locations of the sums of mutations ( smutgroup ) in the park genes . a total of 15 park genes and 20 park - associated loci were identified ( table 1 , first column ) . somatic cm analysis resulted in 315,914 mutations in 18,758 genes by whole - exome sequencing in 246 metastatic cm tissue samples . table 1 summarizes the observed number of mutations and the number of samples harboring park gene mutations and the percentiles of the empirical distribution of mutations . as shown in the table , park8 ( shown as straight bold numbers ) was mutated in cm , as counted by smut , 55 times , thus being above the 95th percentile of the empirical distribution ( which is 49 times ) and in ssampl = 40 cm samples , again above 36 , the 95th percentile of number of samples with mutations . park mutations in cutaneous melanoma , adenocarcinoma , and squamous cell carcinoma of lung three additional park genes ( park2 , park18 , and park20 , shown in table 1 as italic bold numbers ) were mutated above the 90th percentile of the empirical distributions of smut and ssampl . figure 1 summarizes park genes found above the 90th percentile of the empirical distribution of tissue samples ( ssampl ) in cm , adenoca - lung , and squamca - lung , and table 1 depicts the distributions of park gene mutations for the smut and ssampl variables in the 3 cancer types . venn diagram summarizing mutated park genes observed above the 90th percentile of the empirical distribution in melanoma , adenocarcinoma , and squamous cell carcinoma of lung . overall , 48% of cm samples had a mutation in at least 1 park gene and 25% had mutations in multiple park genes ( 28 mutated genes ) ( table 2 ) . samples with mutations in multiple park genes ( 25 mutated genes ) were found in only 6% of adenoca - lung and in 7% of squamca - lung samples . comparison of the distribution of the number of mutated park genes in the 3 cancer types is shown in table 2 . significantly more cm samples harbored multiple park gene mutations compared with squamca - lung ( p = 0.0026 ) and with adenoca - lung ( p < 0.0001 ) ( kolmogorov - smirnov test ) . this finding was significant after bonferroni correction for multiple comparisons ( required p value < 0.025 = 0.05/2 ) . number of samples with mutations in i mutated park genes , i = 1, ,8 , by cancer type the number of samples carrying diverse combinations of multiple park gene mutations in cm samples is depicted in table 3 . the higher frequencies of some of the combinations in cm samples ( in particular , those involving park2 , park8 , park18 , and park20 , table 3 ) vs the paucity of such samples in adenoca - lung ( table e-1 at neurology.org/ng ) and squamca - lung ( table e-2 ) are clearly notable . frequencies of combinations of park gene mutations in cutaneous melanoma ( 246 samples ) to further study the park genes as a group , we used the bootstrap - like permutation method ( see statistical analysis ) . smutgroup and ssamplgroup of park genes shown as the vertical lines appearing to be located in the upper end of the distribution of genes in cm ( figure 2a ) and squamca - lung ( figure 2c ) and not for adenoca - lung ( figure 2b ) . for cm ( figure 2a ) , the location of smutgroup for the 15 park genes as a group was at the 83.5th percentile and that of ssamplgroup was at the 90.8th percentile . for squamca - lung ( figure 2c ) , the location of smutgroup was at the 92.8th percentile and that of ssamplgroup was at the 94.1th percentile . for adenoca - lung ( figure 2b ) , the respective sums were located at the 76.3rd percentile for smutgroup and at the 60.8th percentile for ssamplgroup . histograms of the empirical distributions of the sums of mutations in groups of 15 genes , generated by randomly selecting 10,000 groups of 15 genes in ( a ) cutaneous melanoma , ( b ) adenocarcinoma of lung , and ( c ) squamous cell carcinoma of lung ( smutgroup upper panel , ssamplgroup lower panel ) . the vertical lines depict the locations of the sums of mutations ( smutgroup ) in the park genes . in the present study , we observed that genes associated with pd predisposition ( park genes ) are somatically mutated above random occurrence in cm . it is possible that the co - occurrence of mutations in multiple park genes has a cumulative contribution beyond the influence of a single gene dysfunction . the most commonly mutated park gene observed herein in cm ( 15% of tumors ) was park8 or lrrk2 , a gene that accounts for the most common autosomal dominant form in pd . lrrk2 , leucine - rich repeat kinase 2 , is a large protein displaying dual enzymatic functions , a protein kinase effector domain , and a ras - oncogene like gtpase domain , in addition to multiple protein interaction domains . the g2019s missense mutation , the most common lrrk2 germline mutation in pd , results in gain of function . downregulation of lrrk2 in cultured renal and thyroid cancer cells compromised met activation and reduced downstream signaling , suggesting that lrrk2 and met cooperate in controlling tumor growth . patients with ashkenazi - jewish pd , who carry the germline g2019s*lrrk2 mutation , were reportedly at higher risk for developing mainly hormone - related cancers , breast and prostate , but not for melanoma or any skin cancer compared with pd patients who do not carry this mutation . it is of interest to note that a new bioinformatics tool used for detecting somatic activating mutations identified the lrrk2 locus as a possible oncogene , albeit analyzed only in nonskin cancers , and not cm . the second most commonly mutated gene in our study was park2 encoding for parkin , an ubiquitin ligase 3 . park2-inactivating germline mutations underlie the most common form of autosomal recessive early - onset pd . several studies have shown that park2 is a potential tumor suppressor , inactivated in many cancers including glioblastoma , renal cell carcinoma , colon , lung , and pancreatic cancers . germline park2-inactivating mutations were recently reported to be more frequently encountered in a cm cohort than in controls ( odds ratio = 4.93 ) . furthermore , parkin was underexpressed in melanocytes whereas reexpression of parkin in melanoma cell lines resulted in reduction of cell proliferation rates . in that study , 43% of primary and 66% of metastatic cm cell lines harbored an inactivating park2 mutation . the third most frequently mutated gene in cm in the present study was park18 or eif4g1 . eif4g1 is a member of the translation initiation complex eif4f and plays a central role in eif4f complex formation . the effect of eif4g1 mutations on cap complex assembly and initiation of protein synthesis remains currently unknown . notably , lrrk2 is involved in protein translation and cross - talks with elongations factors , one of which is encoded by eif4g1 . the possible association of this gene with carcinogenesis is indirectly inferred from abnormal expression patterns in cancers such as squamca - lung , inflammatory breast cancer , and nasopharyngeal cancer . the fourth most commonly mutated gene somatically in cm is park20 or synj1 , a gene whose biallelic mutations are associated with an uncommon form of autosomal recessive pd . recently , functional protein - protein interactions of eif4g1 with synj1 were reported . synj1 has not been shown to be involved in cancer pathogenesis , but its homolog , synj2 , was reportedly overexpressed in breast cancer . one caveat to these results and the putative specificity of park genes targeted somatically in cm is the fact that cm displays much higher somatic mutation rates than other cancer types : somatic mutation frequency per megabase is 2 in ovarian cancer , 34 in breast cancer , vs 1018 in cm . to account for these different mutation rates , we chose to compute the relative location of park genes in cm , in comparison with the empirical distributions of the plethora of mutations in cm itself and were able to dissect park genes mutated above these distributions . in addition , we chose lung cancer , which has the closest mutation load ( 1213/megabase ) to cm as control tumor tissue and showed that cm has significantly more mutated park genes compared with adenoca - lung and squamca - lung . the strength of our study is its approach focusing on all park genes and in the use of big data . our study relies exclusively on bioinformatics findings , and there is no assignment of the somatic sequence variants as pathogenic or nonpathogenic and there are no experimental data to prove ( or disprove ) the possible mechanisms of association of genetic variants with phenotypes . additional concerns involve the use of stringent statistical nonparametric analysis that may only be able to detect high odds and omit moderate contributions of single genes or subsets and thus may leave true associations undetected . the present study focused only on park genes that are somatically mutated in cm . yet , the possible functional and genetic intersections between cm and pd are , in all likelihood , more complex . notably , the reported list of somatic cm mutations encompasses several genes that are functionally associated with pd . for example , the alpha - synuclein gene ( snca ) is associated with 2 autosomal dominant inherited forms of pd ( park1 and park4 ) . snca accumulates in neurons and forms the main component of the lewy bodies , the pathologic hallmark of familial and sporadic pd . snca is highly expressed in both primary and metastatic melanoma tissues , but not in nonmelanocytic cells . another example is pla2g6 ( park14 ) , a gene related to nevus count , whose germline mutations cause familial pd and whose variability has been associated with increased risk for developing melanoma . a common denominator between the cells affected in cm and pd is their shared neural crest origin . the initiation of cm malignant transformation process occurs presumably early in life and is associated in a number of cases with sunburn episodes during early childhood . pd genotype may be more prone to uv - induced mutations , increasing the likelihood of acquiring driver this may be suggestive of some genomic instability that ultimately predisposes to pd and cm . the present study highlights an association between somatic pd - related gene mutations and cm . although the functional consequences of the mutations observed in shared genes in both diseases remain to be elucidated , our observation suggests shared pathways and offers one plausible explanation for the observed cm - pd relationship . rivka inzelberg : drafting / revising the manuscript , study concept or design , analysis or interpretation of data . yardena samuels : drafting / revising the manuscript , study concept or design , analysis or interpretation of data , acquisition of data . edna schechtman : drafting / revising the manuscript , analysis or interpretation of data , statistical analysis . eitan friedman : drafting / revising the manuscript , study concept or design , analysis or interpretation of data . y. samuels is supported by the israel science foundation grants 1604/13 and 877/13 , the erc ( stg-335377 ) , the henry chanoch krenter institute for biomedical imaging and genomics , the estate of alice schwarz - gardos , the estate of john hunter , the knell family , the peter and patricia gruber award , and the hamburger family . dr . eytan domany has served on the editorial board of bmc bioinformatics journal of statistical mechanics : theory and experiment , holds a patent for serologic diagnosis of pemphigus vulgaris , has received research support from the leir charitable foundation , has received royalty payments for technology invention ( immunarray co. ) , and holds stocks / stock options in immunarray .
randomized clinical trials ( rcts ) with first- and second - generation drug - eluting stents ( dess ) confirmed the superiority of coronary artery bypass surgery ( cabg ) in patients with multiple vessel disease . in spite of different des designs , investigators in these trials used similar percutaneous coronary intervention ( pci ) strategies hoping to achieve complete revascularization , meaning that all intermediate lesions would be stented . one of these studies also included small vessels in the revascularization policy . on this revision , authors searched for a potential explanation of these intriguing findings and also for solutions to this problem , not seen years ago when other rcts compared cabg with pci in the previous des era . after they revised old and new scientific data , they concluded that improved des design is not itself enough to narrow the gap between pci and cabg and that in the future rcts we should institute more conservative strategies avoiding unnecessary multiple des implantation .	what was different in pci between the two merged analysis described above?14,31 we may argue that patients treated with dess had more three - vessel disease ( 87.3% vs. 36.1% with bms ) and perhaps a higher syntax score than those treated with bms ( in fact , we will never know which was the syntax score for them ) . however , on the other hand , bms pooled data also included diabetic population ( 18.1% vs. 0% with dess ) and more frequently compromise of proximal lad stenosis ( 90% vs. 59% with dess).14,31 the fact that first - generation dess were used in syntax trial could be one of the most attractive explanations ; in fact , syntax patients with definitive set ( 6.8% ) had 35.4% occurrences of cardiac death.24 however , that was not the case with ees used in the best trial , which significantly improved safety compared to the first designs . therefore , perhaps , stent design itself is not solely the reason for the poorer long - term outcome data of these two rcts . if we analyze the trial methods,21,30 in spite of different des designs , both studies share similar pci strategies , meaning that the goal was to achieve complete anatomic revascularization defined by authors as not any residual stenosis 50% in any major coronary artery or their large branches30 ; consequently , we can assume that many intermediate lesions were stented and that concurs with the similarity of stent length in both studies . when we analyze completeness of revascularization with pci,1 we should take into account several different situations such as the amount of myocardium at risk or stenosis severity of the lesions not included in the revascularization strategy . prognosis in patients with incomplete revascularization should be different if the not - attempted vessels had a complete chronic closure with collateral circulation ( fig . 1a ) , high - degree steno - sis in a large vessel ( fig . differences in the amount of completeness of revascularization with both , pci and cabg , was present in all rcts since the first study was performed ; however , in the past , such differences were not associated with poorer outcome4,14 ( 53.2% with pci vs. 82.7% with cabg , p = 0.0003 , see table 1 ) . furthermore , the fact that only 56.7% and 50.9% of syntax and best patients , respectively , in the pci arm achieved the goal of complete revascularization demonstrated how difficult it is for pci to achieve such aim.31 therefore , improved des design is only one face of the problem , while changing pci strategies using a more conservative policy during des implantation would be the other . the eraci iv study,32,33 with a patient population of multiple vessel disease and left main stenosis , used a second - generation des and a conservative pci strategy , defined as stenting only severe lesions ( visually 70% ) in large vessels . intermediate ( > 50 to < 70 ) lesions in small or large vessels or severe lesions in small vessels ( < 2.0 mm ) were not included . this pci strategy of not scoring lesions not included in the revascularization strategy allowed to build a new scoring system where low syntax score was found in 54% of patients and only 17.2% of patients persisted with high syntax score.34 investigators of eraci iv , at more than two years of follow - up , reported remarkable low rates of adverse events including death / mi / stroke of 3.9% , unplanned new revascularization of only 4% , and death / mi / stroke / target vessel revascularization ( tvr ) of 6.7% . it is important to note that there was no major penalty for this conservative policy as it was reflected by the low tvr rate in the not - stented intermediate lesions ( 1.3% ) . we do not know whether these results will remain at a five - year follow - up , although at the present time , the low rate of events in the not - stented lesions supports the pci strategy of this study . in agreement , fame investigators largely demonstrated that nonischemic lesions had better outcome when they were not treated with dess.35,36 we have to take into account that , even in the era of safer dess , neoatherosclerosis as a consequence of des implantation has not disappeared.28 to recap , interventional cardiologists have been doing a lot of work during this 25-year journey trying to close the safety / efficacy gap between pci and cabg in patients with multiple vessel disease and , looking at long - term results from rcts with the old and new dess , we may conclude that improved stent design alone is not enough to narrow the gap between pci and cabg.37 new revascularization strategies during pci and the search for a more functional revascularization avoiding unnecessary des implantation should be the new goal for future randomized comparisons between pci and cabg .
the existing public access policy for our three journals the journal of cell biology , the journal of experimental medicine , and the journal of general physiology is fully compliant with new policies from the research councils uk ( rcuk ) and the wellcome trust . in addition to mandating public access , the new policies specify licensing terms for reuse of content by third parties , in particular for text and data mining . we question the need for these specific terms , and we have added a statement to our licensing policy stipulating that anyone , including commercial entities , is permitted to mine our published text and data .	rup has long championed public access to research articles via a six - month embargo , and we thus support this aspect of the wellcome trust policy and the new rcuk policy . however , we question the value of a provision in their policies that specifies licensing terms for reuse of content by third parties . both organizations now require that the articles for which they have paid an immediate access fee be made available under a creative commons attribution license ( cc - by ) . this means that third parties are allowed to copy , distribute and transmit the work , to adapt the work , and to make commercial use of the work , [ as long as they ] attribute the work in the manner specified by the author or licensor ( 1 ) . cc : creative commons a nonprofit organization that provides standardized copyright licenses , which define the terms of reuse of creative work . by : attribution nc : noncommercial published work can be reused without permission , as long as it is for noncommercial purposes . sa : share alike any subsequent distribution must follow the rules set out in the original license . indeed , we could not release all of our content under a license that permits unfettered commercial reuse of the work . this would allow commercial content aggregators to bundle and sell our content without any recompense to us , thus jeopardizing our subscription revenues . , our content has been released to the public under a cc - by - nc - sa license , which allows third party use of the content for noncommercial purposes but prevents its use for commercial gain without expressed permission ( 2 ) . subscription - based publishers who do take immediate access fees from rcuk and the wellcome trust will , by necessity , have content under two different licenses the one that applies to the bulk of their content and preserves their subscription revenues and the license that applies to immediate access articles funded by rcuk and the wellcome trust . this will add considerable confusion to the research community about what third parties can and can not do with a particular journal s content . there is an initiative by the national information standards organization to tag individual articles with licensing information and to make that metadata accessible to the public ( 3 ) , but those discussions are in their infancy , and implementation is probably several years away . a final issue with the licensing provision is that it is not necessary to achieve one of the stated goals of the wellcome trust to ensure that the content will be fully accessible by anyone the need to differentiate access to content for mining from the right to mine content has been considered in detail elsewhere ( 5 ) . the intellectual property office of the uk government recently issued a clarification of the rights afforded by existing uk copyright law to allow non - commercial researchers to use computers to study published research results and other data without copyright law interfering there is legal precedent in the united states to indicate that the right to mine text and data from published research articles can fall into the category of fair use , and thus any entity noncommercial or commercial may be permitted to do so under any license ( 7 , 8) . the designation of fair use is context specific and is often controversial , confusing , and ambiguous . thus , to clarify the rup s position , we have added a statement to our licensing policy specifying that anyone , including commercial entities , is permitted to mine the text and data : the rup acknowledges that text or data mining by commercial entities for their internal research purposes is allowed without further permission from rup . available to the public for free or for a fee based on text or data mining without further permission from rup , but they may reproduce only snippets of text up to 156 characters in length , or thumbnails of images up to 72 pixels in the long direction , as part of such a service ( 10 ) . the rup s existing license to publish does not restrict mining of the content to advance scientific research by commercial or noncommercial entities ; it only restricts reproduction of the content for commercial gain . this restriction is an essential component of the business models of the vast majority of publishers , which remain subscription based ( 11 ) . cc : creative commons a nonprofit organization that provides standardized copyright licenses , which define the terms of reuse of creative work . by : attribution must be given to the original work . nc : noncommercial published work can be reused without permission , as long as it is for noncommercial purposes . sa : share alike any subsequent distribution must follow the rules set out in the original license . this provides easy access to a machine - readable form of the content for text and data miners . we encourage all publishers to participate in this service . if all participants would agree that text and data mining constitute fair use , the content could be mined by any third party regardless of the copyright / licensing terms attached to it . the name open access subset is actually a misnomer , as the content does not have to be available on an immediate basis ( or under a cc - by license ) to be included . efforts to open up citation data for mining ( 12 ) are commendable if one is trying to break the stranglehold on citation data by large commercial entities , but citations are only a small portion of the textual data in an article . there are no licensing or technical barriers to making all of the text and data published by subscription - based publishers available for mining .
intermittent parathyroid hormone administration can enhance fracture healing in an animal model . despite the success of exogenous parathyroid hormone on fracture healing and spine fusion , few studies have examined the role of parathyroid hormone on cartilage formation . we determined the effects of intermittent parathyroid hormone on cartilage formation in a rabbit microfracture model of cartilage regeneration . twelve rabbits were divided into three equal groups : ( 1 ) microfracture alone , ( 2 ) microfracture + parathyroid hormone daily for 7 days , and ( 3 ) microfracture + parathyroid hormone for 28 days . nonoperated contralateral knees were used as controls . the animals were sacrificed at 3 months and gross and histologic analysis was performed . the microfracture alone group demonstrated the most healing on gross and histologic analysis . treatment with either 1 or 4 weeks of parathyroid hormone inhibited cartilage formation . although discouraging from a cartilage repair point of view , this study suggests that the role parathyroid hormone administration has in clinical fracture healing must be examined carefully . although parathyroid hormone is beneficial to promote healing in spine fusion and midshaft fractures , its deleterious effects on cartilage formation suggests that it may have adverse effects on the outcomes of periarticular fractures such as tibial plateau injuries that require cartilage healing for a successful clinical outcome .	full - thickness cartilage defects remain one of the most common , yet difficult challenges faced by orthopedic surgeons today . curl et al . reported in over 31,000 knee arthroscopies that 63% of patients had a grade iii or iv [ 2 , 3 ] articular cartilage lesion . these lesions demonstrate limited healing potential , with full - thickness cartilage lesions healing with both types i and ii cartilage , and lower than normal concentrations of proteoglycans . the formation of structurally inferior fibrocartilage leads to premature irregularities of the joint surface over time , meniscal tears , and , eventually , degenerative arthritis . microfracture was developed by steadman et al . as a novel technique to treat full - thickness cartilage lesions . the technique of microfracture uses an awl to penetrate the subchondral plate , which leads to migration of mesenchymal stem cells into the cartilage defect and reconstitution of the injured area with a fibrocartilage repair with varying amounts of types i , ii , and iii collagen content . microfracture reportedly results in 70% to 90% of patients having short - term improvement in function [ 59 ] . however , a small but substantial percentage has only moderate to poor fill and worse subjective and objective clinical outcomes . despite the clinical success of microfracture in healing small ( < 2 cm ) cartilage defects in the knee [ 5 , 10 , 11 ] , penetration of the subchondral bone allows for mesenchymal stem cells to enter into a localized defect and produce fibrocartilage . current surgical techniques typically allow for complete fill in 54% , partial fill in 29% , and poor fill in 17% . not surprisingly , there is a correlation between amount of fill and clinical outcome . as the results of microfracture have been variable , there have been many attempts to increase the likelihood of complete lesion fill with hyaline cartilage . previous animal studies have utilized bone morphogenetic proteins ( bmps ) , transforming growth factor- , and insulin - like growth factor-1 to stimulate cartilage healing with promising results [ 1214 ] . in a recent study , morgan et al . found bmp-7 , which induces cartilage formation , worked synergistically with microfracture to heal full - thickness cartilage defects in a rabbit model . despite the early success of these studies in animal models , the role of exogenous parathyroid hormone ( pth ) in fracture healing continues to be elucidated . alkhiary et al . originally reported that intermittent administration of pth enhanced fracture healing in a rat fracture model . the authors found that treatment with 80 g / kg pth increased callus area and strength , with an increase in the amount of new bone formed with histologic analysis . reported increased expression of sox9 and procollagen -1 and increased chondrocyte proliferation in animals receiving intermittent pth . sox9 is a high - mobility group domain transcription factor expressed in chondrocytes during cartilage formation and is essential for cartilage formation in sox9 transgenic mice . expression of sox9 is decreased in cartilage defects that failed to heal , highlighting the importance of this transcription factor in healing of cartilage lesions . increased expression of wnt 4 , 5a , 5b , and 10b in animals treated with intermittent pth to stimulate fracture healing . taken together , these studies suggest that exogenous pth stimulates fracture healing by increasing the activity of genes such as sox9 and the wnt family of proteins to increase chondrogenesis and promote cartilage formation . the purpose of this study was to evaluate the effects of exogenous intermittent pth administration on ( 1 ) the gross and ( 2 ) the microscopic cartilage healing in a rabbit microfracture model . based on the improved chondrogenesis seen with the use of pth in a fracture healing model , we hypothesized that the same mechanisms would stimulate improved cartilage formation in a microfracture setting we specifically aimed to measure the effect that exogenous , intermittent pth administration has following 1- and 4-week treatment periods on the gross and histologic appearance of cartilage defects treated by microfracture compared to animals treated with microfracture alone . female adolescent new zealand white rabbits ( n = 12 ) , weighing from 3.1 to 3.5 kg , were utilized for this study . the animals were divided into three groups : microfracture alone , microfracture + 10 g / kg human recombinant ( 134 ) pth for 7 days , and microfracture + 10 g / kg recombinant ( 134 ) pth for 28 days ( fig . 1 ) . the pth was administered subcutaneously on a daily basis commencing on the day of surgery and continued until the specified day . standard anteroposterior and lateral radiographs of the affected knee were obtained at the time of sacrifice to identify any osseous changes . the research protocol was approved by the animal ethics committee of the hospital for special surgery and animal care and experimental procedures were approved by the animal institutional review board . 1a c diagrams illustrate the experimental design for the pth treatment of 6-mm - diameter full - thickness defects of articular cartilage . a saline was administered as a control , b 10 g / kg pth was administered daily for 1 week , or c 10 g / kg pth was administered daily for 4 weeks . materials and methods section a c diagrams illustrate the experimental design for the pth treatment of 6-mm - diameter full - thickness defects of articular cartilage . a saline was administered as a control , b 10 g / kg pth was administered daily for 1 week , or c 10 g / kg pth was administered daily for 4 weeks . a midline incision was made over the knee and a medial parapatellar arthrotomy was performed to expose the knee . a 6-mm full - thickness defect was made on the weight - bearing surface of the trochlea using a curette . the microfracture holes punctured the subchondral plate and blood was seen to exit each of the holes . at the sacrifice , the soft tissue was removed from the distal femur , and the femur was sectioned approximately 1 cm above the joint . we scored the gross appearance of the lesion based on the fill of the lesion ( 1 = no fill ; 2 = minimal fill ; 3 = near - complete fill ; 4 = complete fill ) , quality of the tissue within the lesion ( 1 = no tissue ; 2 = soft tissue within the lesion ; 3 = normal appearing tissue within the lesion ) , and surrounding changes ( 1 = substantial degenerative cartilage changes surrounding the lesion with loss of cartilage down to bone ; 2 = slight changes in the surrounding cartilage without loss of cartilage to bone ; 3 = no changes ) . this allowed for a calculation of a gross score with a maximum of 10 points . following gross examination , the distal femurs were fixed in 4% paraformaldehyde at room temperature and then transferred to 10% ethylenediaminetetraacetic acid for 3 weeks for decalcification . five - micrometer transverse sections were taken through the center of the defect and stained with hematoxylin and eosin or alcian blue . for semiquantitative analysis of cartilage restoration , we ( bf , zd ) examined the sections in a blinded manner and scored according to the grading scale of pineda et al . with modifications as described by mizuta et al . the maximum score with this scale is 14 , indicating complete fill with reconstitution of the osteochondral junction , normal staining of the matrix , and normal cell morphology . table 1scoring system for the histological appearance of full - thickness cartilage defectscharacteristicscoredefect fill125%3100%475%350%225%10%0reconstitution of osteochondral junctionyes3almost2not close1matrix stainingnormal4reduced3faint2minimal1none0cell morphologynormal4mostly hyaline and fibrocartilage3mostly fibrocartilage2some fibrocartilage1nonchondrocytic cells only0 scoring system for the histological appearance of full - thickness cartilage defects data are presented as the mean standard deviation . differences between groups in both the gross and histologic score were evaluated with a one - way analysis of variance . at the time of sacrifice , there were no complications due to pth administration , and all animals appeared to tolerate the treatment well . one animal developed ossification around the knee and a flexion contracture during the last week of the study . this animal was treated with a microfracture alone , and therefore , this was not a complication of pth administration . administration of pth inhibited or delayed the regeneration of cartilage in the microfracture sites compared to the untreated animals . grossly , all animals in the microfracture alone group appeared to have completely healed their defects with normal - appearing cartilage in three of four animals . the overall gross score was 9.1 0.8 . in the 1-week pth group , there was minimal or no new cartilage present and the defects remained present in all the animals . the overall gross score was 2.3 1.4 ( p < 0.001 versus control ) . in the 4-week pth group , there was minimal cartilage formed in two of the four animals although the cartilage felt soft . the overall gross score was 3.6 2.5 ( p < 0.001 versus control ; p = ns versus 1-week pth ; fig . 2 ) . 2a c photographs show the gross appearance of representative specimens at the time of sacrifice . a in the microfracture alone specimen , there is reconstitution of the lesion with near - complete fill and normal - appearing cartilage . b in the microfracture + 1-week pth specimen , the lesion is still clearly evident with minimal fill . c in the microfracture + 4-week pth specimen , similar to the 1-week treatment group , the lesion remains evident with minimal fill within the lesion . a in the microfracture alone specimen , there is reconstitution of the lesion with near - complete fill and normal - appearing cartilage . b in the microfracture + 1-week pth specimen , the lesion is still clearly evident with minimal fill . c in the microfracture + 4-week pth specimen , similar to the 1-week treatment group , the lesion remains evident with minimal fill within the lesion . there appears to be some mild erosive as well histologic analysis confirmed the findings of the gross analysis further suggesting that pth administration inhibited cartilage regeneration in the microfracture sites . there was near - complete fill of the defect in the microfracture alone group , although there were fewer cells and reduced matrix staining within the microfracture lesion compared to control contralateral femurs ( fig . , there was no fill of the lesion and there appeared to be thinning of the surrounding cartilage in all the samples ( fig . similar results were found in the 4-week pth group , with minimal fill of the defect and thinning of the surrounding cartilage and decreased matrix staining ( fig . 3c ) . the average histologic score in the microfracture alone group was 10.3 3.4 , compared to 2.7 2.5 in the 1-week pth group ( p < 0.001 versus microfracture alone ) and 3.4 2.5 in the 4-week pth group ( p < 0.001 versus microfracture alone ; fig . 4 ) . 3a c photomicrographs show the histologic appearance of representative specimens at the time of sacrifice ( stain , hematoxylin and eosin , alcian blue ; original magnification , 10 ) . a in the microfracture alone specimen , there is reconstitution of the lesion with near - complete fill , although there are fewer than normal cells and the matrix staining is less than normal . b in the microfracture + 1-week pth specimen , the lesion is still clearly evident with minimal fill . c in the microfracture + 4-weeks pth specimen , no cartilage formation is evident within the lesion , and the surrounding cartilage appears to be thinned as wellfig . p < 0.001 versus control animals . mfx microfracture a c photomicrographs show the histologic appearance of representative specimens at the time of sacrifice ( stain , hematoxylin and eosin , alcian blue ; original magnification , 10 ) . a in the microfracture alone specimen , there is reconstitution of the lesion with near - complete fill , although there are fewer than normal cells and the matrix staining is less than normal . b in the microfracture + 1-week pth specimen , the lesion is still clearly evident with minimal fill . c in the microfracture + 4-weeks pth specimen , no cartilage formation is evident within the lesion , and the surrounding cartilage appears to be thinned as well a graph shows the histologic score as described by pineda et al . intermittent pth administration can enhance fracture healing in an animal model . despite the success of exogenous pth on fracture healing and spine fusion , we investigated the role of intermittent administration of exogenous recombinant pth on healing of a microfracture model . based on the improved chondrogenesis seen with the use of pth in a fracture healing model , we hypothesized that the same mechanisms would stimulate improved cartilage healing in a microfracture setting . contrary to our hypothesis , we found that intermittent pth given for 1 or 4 weeks was effective at inhibiting cartilage production in this model . although disappointing from a cartilage repair standpoint , our results are interesting as it increases our understanding of the role of pth in bone and cartilage formation . first , since we designed a preliminary study , we used no alternate doses or administration time courses . a dose of 10 g / kg was chosen based on its success in previous studies that it found successfully improved bone formation via chondrogenesis [ 17 , 18 ] . likewise , a 1- and 4-week time course of pth administration was chosen based on its success in previous studies [ 17 , 18 ] . it is possible that a shorter period of time ( i.e. , 25 days ) would be sufficient to stimulate cartilage formation without leading to inhibition of osteochondral healing . second , the underlying mechanism of inhibition of cartilage formation with intermittent pth is not clearly understood . with continuous pth administration , there is downregulation of the pth / pth - related protein ( pthrp ) receptors , but it is unknown if intermittent pth functions by the same mechanism to inhibit cartilage repair . clearly , these factors should be clarified in subsequent studies . based on the promising results with other growth factors and the enhanced chondrogenesis seen using recombinant intermittent pth in a fracture healing model , we presumed that administration of 10 g / kg intermittent pth would enhance cartilage formation in a microfracture model . however , we found that intermittent pth actually nearly completely inhibited the formation of new cartilage within the defect in our model . with 1 week of pth , there was no cartilage formed within the defect , a finding that was clearly evident on both gross and histologic examination . the findings were similar in animals treated for 4 weeks of intermittent pth . on gross examination , there was minimal or no fill in almost all the animals treated with intermittent pth , suggesting an inhibition of chondrocyte proliferation . a smaller proportion of the animals also demonstrated adjacent cartilage breakdown after treatment with pth . interestingly , there appeared to be decreased staining of the surrounding cartilage matrix in those animals treated with pth , suggesting an alteration of collagen or proteoglycan concentration in these animals . these results clearly demonstrate that intermittent pth administration is not an effective means to improve the ability of microfracture to heal chondral defects . two previous studies utilizing continuous pth administration in a chondral defect model have demonstrated results similar to ours . reported that pth , when given continuously via an osmotic pump into the knee , completely inhibited chondrogenesis . the authors found that , although proliferation of the chondral precursors was normal , pth administration effectively inhibited chondrogenic differentiation . in a subsequent study , the authors found that continuous pth administration for 4 weeks was able to irreversibly block expression of pth / pthrp receptor , suggesting cells that migrate into the chondral defect maintain their chondrogenic potential for no more than 2 weeks . our data suggest that even intermittent pth administration results in a similar outcome in this model . this is surprising as it is traditionally thought that continuous and intermittent pth function in separate ways in a fracture healing model . these results demonstrate that it is vital to more completely understand the mechanisms that govern the outcome of pth administration in osteochondral injury models . although it may be alluring to use pth to improve fracture healing in these difficult to treat fracture models , it is possible that the pth would have a deleterious effects on the cartilage injury and result in a much poorer overall outcome . we found that intermittent pth did not improve healing of full - thickness chondral defects in a rabbit microfracture model and in fact inhibited cartilage formation within the defect . although these results did not support our hypothesis , they do highlight the importance of understanding the molecular mechanisms that govern the relationship between pth and chondrogenesis . future studies focusing on the molecular mechanisms of pth - mediated chondrogenesis and the time course of gene expression may help elucidate why pth is effective in stimulating cartilage formation in a fracture healing model yet is unable to stimulate cartilage in a full - thickness chondral injury model .
backgroundthe effects of antihypertensive agents on endothelial function have not been fully evaluated in human hypertension and data on the forearm circulation of humans are controversial . the aim of this study was ( 1 ) to evaluate the endothelial function in hypertensive patients ( 2 ) to investigate whether vitamin c administration has any benefit on the endothelial function and ( 3 ) to determine whether treatment with calcium antagonist improves endothelial dysfunction in hypertensive patients.methodsthe endothelial function was estimated using venous occlusion plethysmography ( vop ) in 8 hypertensive patients and 8 healthy volunteers . the patients in the hypertension group were treated with amlodipine , then examined again . the change of forearm blood flow ( fbf ) was measured with acetylcholine infusion through brachial artery and also with intra - arterial vitamin c.resultsforearm blood flow response to acetylcholine was significantly enhanced with intra - arterial infusion of vitamin c in hypertensive group before antihypertensive treatment . co - infusion of l - nmma , an inhibitor of nitric oxide synthase , blunted forearm blood flow response to acetylcholine . after treatment with amlodipine for 2 months in hypertensive group , endothelium - dependent vasorelaxation to acetylcholine was significantly improved compared to pretreatment , and vitamin c did not affect the improved endothelial function by amlodipine treatment.conclusionvitamin c ( acutely ) and amlodipine ( chronically ) improved endothelial function in hypertensive patients . these results suggest that increased oxidative stress , at least in part , may be involved in the decreased endothelial function in hypertension .	the main endothelium derived factor is nitric oxide ( no ) which is not only a potent vasodilator but also inhibits platelet aggregation , smooth muscle cell migration and proliferation , monocyte adhesion and adhesion molecule expression , thus protecting the vessel wall against the development of atherosclerosis and thrombosis . endothelium - dependent vasodilation in response to substances , such as acetylcholine , bradykinin and substance p and reactive hyperemia , was reduced in brachial , coronary , renal arteries and femoral areteries in patients with essential hypertension . impairment of endothelial function has been shown to play an important role in the development and maintenance of hypertension . therefore , an important aim of antihypertensive therapy would be not only to normalize blood pressure values but also to reverse endothelial dysfunction by restoring no availability . several studies have demonstrated the restoration of endothelial function in essential hypertensive patients through the administration of antihypertensive agents , while others have shown that effective antihypertensive therapy did not restore impaired endothelium - dependent vasodilation in the forearm circulation of hypertensive patients . the effects of antihypertensive agents on endothelial function have not been fully evaluated in human hypertension and data on the forearm circulation of humans are controversial . it may be clinically important to select an appropriate antihypertensive agent that is effective in improving endothelial dysfunction in patients with established essential hypertension . recently , the role of superoxide anion and its interaction with nitric oxide has been investigated . under physiological conditions , these oxygen - free radicals are potent chemical inactivators of no and the balance between no and superoxide is more important than the absolute levels of either alone . vitamin c is an important antioxidant in human plasma , capable of scavenging oxygen - free radicals and sparing other endogenous antioxidants from consumption . therefore , the aim of this study was ( 1 ) to evaluate the endothelial function in hypertensive patients ( 2 ) to investigate whether vitamin c administration has any benefit on the endothelial function and ( 3 ) to determine whether treatment with calcium antagonist improves endothelial dysfunction in hypertensive patients . eight hypertensive patients ( age range 35 to 73 years ) were recruited . they had a clinical blood pressure reading ( the average of 3 different sphygmomanometric measurements , each performed on 3 separate days ) of > 140/90 mmhg . the possibility of secondary causes of hypertension was excluded by standard clinical and laboratory tests . exclusion criteria were 1 ) evidence of overt atherosclerotic disease , i.e. , coronary artery disease , peripheral vascular disease , stroke , etc . 2 ) having other risk factors of atherosclerosis , i.e. , current smoking and smoking within 1year , severe hypercholesterolemia ( > 240 mg / dl ) and diabetes mellitus 3 ) advanced organ failure 4 ) malignancy . in all patients , subjects were required to refrain from drinking alcohol or caffeine - containing beverage for 12 hours before the study . the protocol of the study was approved by the ethics committee of our institution and informed consent was obtained from each participant . the procedures followed were in accordance with institutional guidelines . the examination was done at supine position . a mercury - filled silastic strain gauge was placed around the thickest part of the forearm . the size of the strain gauge was selected to be about 2 cm less than the maximal forearm circumference . the strain gauge was connected to plethysmograph ( hokanson ec - r5 , issaquah , washington ) to record the forearm volume change . a rapid cuff inflator ( hokanson e-10 ) was used to inflate the arm cuff to 40 mmhg instantaneously , thus occluding venous return from the forearm . a wrist cuff was inflated to 20 mmhg above the systolic pressure to cut off the arterial flow to hand . intra - arterial pressure was measured continuously ( transpac ; abbot laboratories ) throughout the study . the measurement of forearm volume change was repeated 7 times for each stage . between infusions , the cuffs were deflated , allowing at least 15 min for forearm blood flow to recover from the preceding infusion and before further baseline measures were recorded . all solutions were prepared aseptically from sterile stock solutions or ampoules immediately before infusion into brachial artery . acetylcholine and l - nmma had been diluted in distilled water and filtered through 0.22 m filter . an intra - arterial infusion of 5% dextrose solution was begun at 1 ml / min and continued throughout drug infusion . basal measurement was obtained during the infusion of dextrose solution . acetylcholine ( sigma chemical ) acetylcholine was infused for 5 minutes for each dose level and forearm blood flow was measured during the last 2 minutes of each dose . after 15 minutes of wash - out period , intra - arterial infusion of vitamin c at 24 mg / min was done for 10 minutes . with continued infusion of vitamin c at the same rate , acetylcholine was infused at the same incremental doses as the previous stage and measurement of forearm blood flow was made during the last 2 minutes of each dose . after another 15 minutes of rest period , intra - arterial infusion of vitamin c at the same rate , plus infusion of l - nmma ( ng - monomethyl - l - arginine , sigma chemical ) , an inhibitor of nitric oxide synthesis , was begun at 100 g / min and continued for 5 minutes . then infusion of acetylcholine at incremental dose and measurement of forearm blood flow were done as in previous stages . forearm blood flow was measured and forearm blood flow changes during acetylcholine infusion were expressed as percentage changes from the baseline immediately preceding each drug administration . forearm blood flow changes were compared between normal and hypertensive groups with and without vitamin c infusion . comparison was also made between before and after antihypertensive treatment in hypertensive group and forearm blood flow was compared in hypertensive group with and without vitamin c infusion . 6.12 all values were expressed in mean standard error of mean and p<0.05 was considered significant . eight hypertensive patients ( age range 35 to 73 years ) were recruited . they had a clinical blood pressure reading ( the average of 3 different sphygmomanometric measurements , each performed on 3 separate days ) of > 140/90 mmhg . the possibility of secondary causes of hypertension was excluded by standard clinical and laboratory tests . exclusion criteria were 1 ) evidence of overt atherosclerotic disease , i.e. , coronary artery disease , peripheral vascular disease , stroke , etc . 2 ) having other risk factors of atherosclerosis , i.e. , current smoking and smoking within 1year , severe hypercholesterolemia ( > 240 mg / dl ) and diabetes mellitus 3 ) advanced organ failure 4 ) malignancy . in all patients , subjects were required to refrain from drinking alcohol or caffeine - containing beverage for 12 hours before the study . the protocol of the study was approved by the ethics committee of our institution and informed consent was obtained from each participant . a mercury - filled silastic strain gauge was placed around the thickest part of the forearm . the size of the strain gauge was selected to be about 2 cm less than the maximal forearm circumference . the strain gauge was connected to plethysmograph ( hokanson ec - r5 , issaquah , washington ) to record the forearm volume change . a rapid cuff inflator ( hokanson e-10 ) was used to inflate the arm cuff to 40 mmhg instantaneously , thus occluding venous return from the forearm . a wrist cuff was inflated to 20 mmhg above the systolic pressure to cut off the arterial flow to hand . intra - arterial pressure was measured continuously ( transpac ; abbot laboratories ) throughout the study . the measurement of forearm volume change was repeated 7 times for each stage . between infusions , the cuffs were deflated , allowing at least 15 min for forearm blood flow to recover from the preceding infusion and before further baseline measures were recorded . all solutions were prepared aseptically from sterile stock solutions or ampoules immediately before infusion into brachial artery . acetylcholine and l - nmma had been diluted in distilled water and filtered through 0.22 m filter . an intra - arterial infusion of 5% dextrose solution was begun at 1 ml / min and continued throughout drug infusion . acetylcholine ( sigma chemical ) was infused intra - arterially at 7.5 , 15 and 30 g / min . acetylcholine was infused for 5 minutes for each dose level and forearm blood flow was measured during the last 2 minutes of each dose . after 15 minutes of wash - out period , intra - arterial infusion of vitamin c at 24 mg / min was done for 10 minutes . with continued infusion of vitamin c at the same rate , acetylcholine was infused at the same incremental doses as the previous stage and measurement of forearm blood flow was made during the last 2 minutes of each dose . after another 15 minutes of rest period , intra - arterial infusion of vitamin c at the same rate , plus infusion of l - nmma ( ng - monomethyl - l - arginine , sigma chemical ) , an inhibitor of nitric oxide synthesis , was begun at 100 g / min and continued for 5 minutes . then infusion of acetylcholine at incremental dose and measurement of forearm blood flow were done as in previous stages . forearm blood flow was measured and forearm blood flow changes during acetylcholine infusion were expressed as percentage changes from the baseline immediately preceding each drug administration . forearm blood flow changes were compared between normal and hypertensive groups with and without vitamin c infusion . comparison was also made between before and after antihypertensive treatment in hypertensive group and forearm blood flow was compared in hypertensive group with and without vitamin c infusion . 6.12 all values were expressed in mean standard error of mean and p<0.05 was considered significant . eight hypertensive patients and eight normotensive controls ( age range 35 to 73 years ) were recruited . there were not any significant differences in cholesterol levels between normotensive controls and hypertensive patients . only systolic and diastolic blood pressure differed in the two groups ( table 1 ) . after 2-month antihypertensive treatment , there were significant decreases in systolic and diastolic blood pressure ( mean blood pressure ; 169/98 mmhg 131/74 mmhg , p<0.05 ) . absolute fbf data recorded in the infused limbs at baseline and during the infusion of acetylcholine at three dose levels before and after antihypertensive treatment are presented in table 2 . endothelium - dependent vasorelaxation ( vasodilatory response to acetylcholine ) was significantly greater in normal control group compared to hypertensive group before antihypertensive treatment ( maximum forearm blood flow in nc : 44863% , ht : 30258% , p<0.05 , figure 2 ) . forearm blood flow response to acetylcholine was significantly enhanced with intra - arterial infusion of vitamin c in hypertensive group before antihypertensive treatment ( maximum forearm blood flow in vit c ( ) : 30258% , vit c ( + ) : 44643% , p<0.05 , figure 3a ) . such an enhanced response was not observed in normal control group ( maximum forearm blood flow in vit c ( ) : 44863% , vit c ( + ) : 38351% , p=0.11 , figure 3b ) . co - infusion of l - nmma , an inhibitor of nitric oxide synthase , blunted forearm blood flow response to acetylcholine ( maximum forearm blood flow in vit c ( + ) : 44643% , vit c + l - nmma ( + ) : 22923% , p<0.05 , figure 3c ) . after antihypertensive treatment with amlodipine for 2 months in hypertensive group , endothelium - dependent vasorelaxation ( vasodilatory response to acetylcholine ) was significantly improved in amlodipine treated group compared to before treatment ( maximum forearm blood flow in before treatment : 30258% , after treatment : 53981% , p<0.05 , figure 4a ) . intra - arterial infusion of vitamin c in amlodipine treated hypertensive group did not change the forearm blood flow response to acetylcholine ( maximum forearm blood flow in vit c ( ) : 53981% , vit c ( + ) : 44878% , p=0.21 , figure 4b ) . co - infusion of l - nmma , an inhibitor of nitric oxide synthase , blunted forearm blood flow response to acetylcholine in amlodipine treated hypertensive group ( maximum forearm blood flow in l - nmma ( ) : 53981% , l - nmma ( + ) : 24022% , p<0.05 , figure 4c ) . endothelium - dependent vasorelaxation ( vasodilatory response to acetylcholine ) was significantly greater in normal control group compared to hypertensive group before antihypertensive treatment ( maximum forearm blood flow in nc : 44863% , ht : 30258% , p<0.05 , figure 2 ) . forearm blood flow response to acetylcholine was significantly enhanced with intra - arterial infusion of vitamin c in hypertensive group before antihypertensive treatment ( maximum forearm blood flow in vit c ( ) : 30258% , vit c ( + ) : 44643% , p<0.05 , figure 3a ) . such an enhanced response was not observed in normal control group ( maximum forearm blood flow in vit c ( ) : 44863% , vit c ( + ) : 38351% , p=0.11 , figure 3b ) . co - infusion of l - nmma , an inhibitor of nitric oxide synthase , blunted forearm blood flow response to acetylcholine ( maximum forearm blood flow in vit c ( + ) : 44643% , vit c + l - nmma ( + ) : 22923% , p<0.05 , figure 3c ) . after antihypertensive treatment with amlodipine for 2 months in hypertensive group , endothelium - dependent vasorelaxation ( vasodilatory response to acetylcholine ) was significantly improved in amlodipine treated group compared to before treatment ( maximum forearm blood flow in before treatment : 30258% , after treatment : 53981% , p<0.05 , figure 4a ) . intra - arterial infusion of vitamin c in amlodipine treated hypertensive group did not change the forearm blood flow response to acetylcholine ( maximum forearm blood flow in vit c ( ) : 53981% , vit c ( + ) : 44878% , p=0.21 , figure 4b ) . co - infusion of l - nmma , an inhibitor of nitric oxide synthase , blunted forearm blood flow response to acetylcholine in amlodipine treated hypertensive group ( maximum forearm blood flow in l - nmma ( ) : 53981% , l - nmma ( + ) : 24022% , p<0.05 , figure 4c ) . the present study demonstrates that the endothelium - dependent vasodilation is impaired in essential hypertensive patients as compared with normotensive control subjects . in addition , short - term , intra - arterial administration of the antioxidant vitamin c restores endothelium - dependent vasodilation in patients with essential hypertension . a nitric oxide ( no ) synthase inhibitor , l - nmma , blunted the improvement of endothelium - dependent vasodilation . these findings suggest that oxygen - derived free radicals may decrease the bioavailability of endothelium - derived nitric oxide and impair endothelium - dependent vasodilation in patients with essential hypertension . these results were consistent with previous observations that , in essential hypertensive patients , impaired endothelium - dependent vasodilation of forearm circulation could be improved by the antioxidant vitamin c. solzbach et al demonstrated that impaired endothelium - dependent vasodilatory function of human coronary arteries in hypertensive patients could be improved by the administration of the antioxidant vitamin c. assessment of the clinical relevance of the present results should take into account the fact that superoxide anion production has been found to cause endothelial dysfunction in the presence of several cardiovascular risk factors . in patients with coronary artery disease , both single dose ( 2 g po ) and long - term ( 500 mg / d ) administration of vitamin c reverse endothelial vasomotor dysfunction in the brachial circulation . with regard to mechanism , it has been proposed that ascorbic acid improves no action by scavenging superoxide anion and preventing inactivation of no . this explanation is attractive because atherosclerosis and hypercholesterolemia are linked to excess generation of superoxide , and superoxide reacts with no and eliminates its biological activity . in addition to scavenging superoxide anion or inhibiting ldl oxidation , vitamin c could alernatively improve no action by sparing intracellular glutathione which , together with vitamin c , is the primary regulator of intracellular redox state . in essential hypertension , impaired endothelium - dependent vasodilation seems to be a primary phenomenon and the endothelial vasomotor dysfunction is not normalized by the mere reduction of blood pressure . several investigators have addressed the possibility that antihypertensive treatment could restore or at least improve endothelial function . calcium antagonists have been shown to be effective in reversing endothelial dysfunction of angiographically normal and stenotic epicardial coronary vessels in essential hypertension . in agreement with these results , but several other studies have demonstrated that the treatment with long - term calcium antagonist did not improve forearm vasodilator response to reactive hyperemia . hirooka et al also reported that single dose administration of nifedipine did not alter forearm vasodilation with acetylcholine in hypertensive patients . in our findings , prolonged ( 8 weeks of oral treatment ) amlodipine administration could improve endothelium -dependent vasodilation in essential hypertensive patients . intra - arterial infusion of vitamin c to the treated patients did not increase forearm vasodilation in response to acetylcholine . these data indicate that amlodipine is effective in improving endothelial dysfunction in essential hypertension and amlodipine appears to act specifically on the no pathway by a mechanism that is probably related to antioxidant activity . experimental data indicate that calcium antagonists exert an antioxidant effect and therefore , could protect endothelial cells against free radical injury and diminish oxidative breakdown of no . an insufficient blood pressure reduction with an antihypertensive drug with antioxidant activity would not improve the endothelial function . so , a sufficient blood pressure reduction with an antihypertensive drug with antioxidant activity would be required . all the hypertensive patients in this study have normalized their systolic and diastolic blood pressure . even though the relative importance of the various possible mechanisms leading to depressed endothelial function in essential hypertension remains to be elucidated , our study shows that vitamin c or amlodipine result in demonstrable improvement by a mechanism that is probably related to antioxidant activity .
study design survey . objective sports - related spinal cord injury ( sci ) represents a growing proportion of total scis but lacks evidence or guidelines to guide clinical decision - making on return to play ( rtp ) . our objective is to offer the treating physician a consensus analysis of expert opinion regarding rtp that can be incorporated with the unique factors of a case for clinical decision - making . methods ten common clinical scenarios involving neurapraxia and stenosis , atlantoaxial injury , subaxial injury , and general cervical spine injury were presented to 25 spine surgeons from level 1 trauma centers for whom spine trauma is a significant component of their practice . we evaluated responses to questions about patient rtp , level of contact , imaging required for a clinical decision , and time to return for each scenario . the chi - square test was used for statistical analysis , with p < 0.05 considered significant . results evaluation of the surgeons ' responses to these cases showed significant consensus regarding return to high - contact sports in cases of cervical cord neurapraxia without symptoms or stenosis , surgically repaired herniated disks , and nonoperatively healed c1 ring or c2 hangman 's fractures . greater variability was found in recommendations for patients showing persistent clinical symptomatology . conclusion this survey suggests a consensus among surgeons for allowing patients with relatively normal imaging and resolution of symptoms to return to high - contact activities ; however , patients with cervical stenosis or clinical symptoms continue to be a challenge for management . this survey may serve as a basis for future clinical trials and consensus guidelines .	an estimated 1,000 cases of spinal cord injury ( sci ) related to athletics are treated yearly in the united states , representing 8.9% of total sci cases,1 2 and although much has been written about both surgical and nonsurgical treatment methods , the literature guiding return to function is scant . transient neurapraxia and cervical cord neurapraxia ( ccn ) have been written about extensively , but still only very low - level medical evidence exists regarding management.3 4 5 6 the risk of recurrence after return to sports has been debated in the literature , and there is no definitive universally accepted guideline.1 7 various criteria based on numerous retrospective studies for return to play ( rtp ) have been suggested by torg,8 watkins,9 cantu et al,10 bailes et al,11 and torg and ramsey - emrhein6 ; however , the decision regarding rtp is individualized to the specific athlete , the level of function , and the expectations of the athlete.7 12 13 14 15 moreover , these return criteria are subjective , difficult to apply clinically , and poorly validated on prospective studies . completion of a randomized trial regarding rtp may not ever become possible , and instead information on medical decision - making must be found in other sources . a recent meta - analysis of literature evaluating evidence for rtp with various injury patterns concluded that only grade c or d practice recommendations supported by level iii evidence at best were available for rtp after sci.16 that is , evidence supporting interventions is compiled from limited cohort studies and clinical trials , resulting in clinicians using significant judgment of the current evidence to make patient treatment decisions . in patients with sci , the data are clear that improved function is dependent upon early initiation of rehabilitation17 ; however , in patients with minimal or no deficits , the expectations are greater than in patients with a severe or complete sci . once the patient has fully recovered from the injury , decisions must be made to release the patient to a specific level of activity , with return to sports being one of the more demanding levels of function . consensus opinion regarding rtp may provide better evidence than individual case reports or small case series regarding opinions about return to sporting activity . consensus regarding expert opinion can be especially useful in guiding surgical decision - making where other types of evidence are absent or interpretation of available evidence is subjective.18 19 this approach has been widely used in the spine literature for interpreting guidelines and assessing interobserver reliability in clinical decision - making.20 21 22 23 24 this study was designed to identify the maximum level of sporting activity that a group of experienced spine trauma surgeons would allow patients to participate in after recovery as a guide to making general recommendations , which from a medical perspective is essentially a subjective determination of the likelihood that an individual who returns to sports will suffer an additional significant injury directly related to the original trauma . whether a patient can function at the preinjury performance level would depend on many factors and can not be addressed in this article . a treating physician is frequently faced with the decision to release a patient to a given level of contact and currently must make a decision based on his or her own anecdotal experience with little supportive literature . the aim of this study is to offer the treating physician a consensus analysis of expert opinion regarding rtp that can be incorporated with the unique factors of the case for the final individual decision . twenty - five spine surgeons in the spine trauma study group ( stsg ) who consider spine trauma to be a significant component of their practice , all from separate level 1 trauma centers , were surveyed.20 21 22 23 24 the survey was administered at a national meeting discussing guidelines for cervical neurapraxia . the stsg was founded in 2004 and consists of 50 surgeons from 12 countries specifically focused on the study of traumatic spine injuries . each surgeon was presented 10 common case scenarios involving cervical injuries and asked to identify the level of sports participation they would allow following recovery ( tables 1 and 2 ) . the levels of sports were defined as high - contact , intermediate - contact , noncontact , or no sports , representing the frequency and severity of expected stress on the spine . cases were categorized into those involving cervical neurapraxia and stenosis , atlantoaxial cervical injury , subaxial cervical injury , and general cervical injury . participants were also asked how soon after recovery they would allow a return to activity and what would be the minimum imaging studies necessary to make the decision . the surveys were completed simultaneously after an explanation of each scenario , the definition of level of play , the use of time to return as that of return to maximum participation , and the use of imaging tools . abbreviations : acdf , anterior cervical diskectomy and fusion ; ncaa , national collegiate athletic association ; mri , magnetic resonance imaging . each brief case scenario ( table 3 ) represented increased levels of injury and assumed that the patient has fully recovered subjectively and objectively . full clinical recovery was defined as minimal to no neck pain , return of full or near - full range of motion , and return of normal motor and sensory function . after the recommendations for full recovery were collected , the surgeons were presented with the same scenarios but instructed that the patient had persistent symptoms , such as moderate neck pain , upper extremity paresthesias , or single - level radicular weakness ( table 4 ) . because this study focused on cervical injury , the levels of contact were stratified by the frequency and severity of head / neck impact ( table 1 ) . the surveyed surgeons were given seven specific time frames within which they would allow the patient to return to the maximum level of play : same game , 1 to 2 days , 1 week , 2 to 4 weeks , 2 to 3 months , 3 to 6 months , or greater than 6 months ( table 4 ) . finally , the surgeons were asked what imaging studies were the minimum necessary to make the final rtp decision after a history and physical exam plain radiographs , computed tomography ( ct ) scanning , and/or magnetic resonance imaging ( mri ) scanning . no difference between orthopedic surgeons and neurosurgeons recommendations seen , mann - whitney u test . no difference between orthopedic surgeons and neurosurgeons recommendations seen , mann - whitney u test . after the surveys were completed , the data was tabulated for analysis of each case scenario . the responses from the orthopedic surgeons were compared with those from the neurosurgeons to determine whether any significant differences in recommendations existed using the kruskal - wallis and mann - whitney u test . a p value < 0.05 was considered significant . fourteen orthopedic surgeons and 11 neurosurgeons who were members of the former stsg completed the survey . a summary of the level of play recommendations for each scenario with full recovery is shown in table 3 . there were no differences between orthopedic and neurosurgeons ( results not shown ) , so the data was analyzed as one group , which is consistent with a previous finding from this group of fellowship - trained spine surgeons regarding evaluation of cervical dislocation injuries.20 the scenarios in which the surgeons most consistently allowed for return to high - contact sports were the episodes of ccn with early resolution of symptoms and no stenosis on mri ( case 1 , 88% , p < 0.0001 and case 3 , 64% , p = 0.03 ) . in the setting of diffuse stenosis and early resolution of ccn , the percentage of surgeons allowing return to high - contact sports dropped to 35% ( case 2 , p = 0.6 ) and 27% ( case 4 , p = 0.6 ) , with greater heterogeneity of opinions . cases in which some surgeons favored allowing return to high - contact sports included c1 ring or c2 hangman 's fractures that healed nonoperatively ( 67% , case 5 , p = 0.005 ) , c5c6 unilateral facet dislocation presenting neurologically intact with subsequent posterior fusion ( 56% , case 8 , p = 0.02 ) , and herniated disks repaired operatively ( 71% , case 9 , p = 0.003 ) . the surgeons generally favored a return to a less vigorous level of activity such as intermediate or noncontact sports when the case involved ccn with os odontoideum and c1c2 fusion ( 82% , case 6 , p = 0.04 ) , c5 flexion compression of american spinal injury association grade d with c4c6 anterior cervical corpectomy and fusion ( 80% , case 10 , p = 0.06 ) , and c5 burst fracture treated nonoperatively ( 52% , case 7 , p = 0.03 ) . good consensus was seen for these cases in favoring return to a degree of activity less than high contact . changes in the clinical scenario to indicate a lack of patient symptom resolution resulted in greater variation and less consensus of recommendations and a lower number of physicians recommending return to high - contact sports ( results not shown ) . with a lack of resolution of symptoms , only case 2 ( p = 0.03 ) and case 4 ( p = 0.02 ) showed significant consensus in recommending noncontact or no sports activity . table 4 outlines the time frame within which surgeons would allow rtp at the level they recommended . a relative consensus in recommending return after 2 to 4 weeks was seen in cases 1 to 4 involving cervical neurapraxia and stenosis . all surgeons recommended waiting 2 to > 6 months for cases 5 to 10 , involving atlantoaxial , subaxial , or general cervical spine injury patterns . there was no difference in the recommendations of orthopedic surgeons and neurosurgeons for level of activity or time to rtp ( results not shown ) . recommendations for x - rays ( 64 to 92% ) , cts ( 20 to 84% ) , and mris ( 50 to 100% ) varied among different cases , indicating a high consensus for obtaining imaging in making recommendations ( results not shown ) . there were also no differences in recommendations between orthopedic surgeons and neurosurgeons for imaging prior to making rtp decisions . most surgeons favored extensive imaging with radiographs , ct , and mri before allowing rtp . decisions about safe rtp after cervical injury can be difficult because there are opposing forces to be considered , such as the patient 's desires and the medicolegal implications . other than for ccn , there is almost no literature to determine an appropriate strategy for return to sports after cervical injury . torg et al reported that cervical stenosis was predictive of another episode of ccn ( 53% ) but not predictive of catastrophic injury25 ; however , repeated episodes of neurapraxia and cord contusions were thought to be a relative contraindication for rtp . more recently , brigham and capo followed four professional athletes who had cord contusions from presumed hypermobility at c3c4 or a disk herniation elsewhere in the spine.7 despite the positive findings on radiographic images , the players were completely asymptomatic after stabilization and subsequently returned to professional sports . the authors emphasize close observation , careful assessment , and thorough counseling of the patients . the results of studies of return to professional sports after treatment of cervical injury by hsu and colleagues supported rtp after treatment of cervical disk herniations , with a greater rate of return for operatively managed cases12 26 ; however , the authors discussed the unclear consensus in management of patients with herniated disks and concomitant cervical stenosis . despite evidence to suggest that athletes with spinal stenosis may return to high - contact sports if they are asymptomatic,3 6 the current study reveals the prevailing caution regarding rtp when an athlete has lost the space available for the spinal cord . morganti et al completed a similar rtp survey based on case scenarios of cervical trauma administered to members of the cervical spine research society , the herodicus sports medicine society , and members of the authors ' own department.27 an evaluation of 113 responses ( 32.7% response rate ) demonstrated that the consensus on rtp was poor and that most of the differences were based on type of subspecialty interest ( i.e. , spine or sports ) and seniority . although 49% of respondents reported using guidelines at the time for decision - making , only 1 of 10 survey cases was evaluated as appropriate in this manner . the results of the study presented a question of how recommendations for rtp could be made in the face of limited data . in contrast , our study was directed specifically at spine surgeons at level 1 trauma centers who include trauma as a significant component of their practice . the surgeons surveyed for this study had a thorough understanding of the available literature on transient neurapraxia , including a recent issue of spine that was focused on spine trauma and included an evidence - based review of rtp after transient neurapraxia.28 the conclusions of that review were based on a consensus of members of the stsg , as well as available literature.5 because there is so little literature available on the other scenarios , most of those recommendations were based on expert opinion from clinical experience . some important differences can be observed in the rtp recommendations after transient neurapraxia . if a patient had congenital stenosis after an episode of neurapraxia , then experienced trauma spine surgeons were distinctly less willing to allow that patient to return to a high - contact sport . if the neurapraxia was brief , 88% would allow return to high - contact sports if the athlete did not have congenital stenosis whereas only 35% would allow the return if congenital stenosis existed . if the deficit lasted for a few hours rather than resolving quickly , only 64% would allow return to high - contact sports even if the patient did not have congenital stenosis and just 27% would allow return to high - contact sports if congenital stenosis existed on mri . thus , it appears that duration of neurapraxia , perhaps indicating a more significant injury , was important in the rtp decision , as was the presence of congenital stenosis . our study did not address the issue of repetitive neurapraxia , which could also influence decision - making just as repeated cerebral concussions do.29 the case scenarios set forth do not cover the entire gamut of potential cervical injuries but they were thought to include a broad representation of the common injury patterns so that some extrapolation to other injury patterns may be made . for example , a single - level posterior fusion for facet fracture without dislocation could be assessed in a similar fashion to a single - level posterior fusion for a unilateral facet dislocation in scenario 6 . the only other injury patterns in which a majority of those surveyed would allow return to high - contact sports were in the scenarios that resulted in a stable single - level subaxial fusion , either anterior or posterior , and nondisplaced , healed upper cervical fractures . in the scenarios of a healed posterior c1c2 fusion ( even with normal neurology ) , those with two - level fusions , and those in which patients had an incomplete sci ( excluding transient neurapraxia ) , only a small minority of less than 20% would consider return to high - contact sports . absolute contraindications to high - contact sports have generally included patients with odontoid abnormalities , atlanto - occipital fusion , klippel - feil fusions above c3 , and acute fracture with instability.16 limitations of this study include the reliance on expert opinion for surgical recommendations because of the difficulty of studying these injury patterns in the active athlete . the findings are highly dependent on individual interpretation of data and personal experience ; however , in the face of convincing data , consensus opinion can be a source of guidance on patient management as well as starting point for further exploration . other limitations of the study include an inability to totally account for all mechanisms and types of cervical sport injury in making practice recommendations . clinicians applying this survey may not find every scenario fits their specific patient , although the principles evaluated in each case may be widely applied . although this study will not completely answer the questions about rtp , it establishes a reasonable consensus of expert surgeon opinion with substantial experience to guide the treating physician involved in similar case scenarios . it can also serve as a basis upon which future prospective , multicenter studies can be designed to confirm or disprove current dogma .
until 2004 , identification of nipah virus ( nv)-like outbreaks in bangladesh was based on serology . we describe the genetic characterization of a new strain of nv isolated during outbreaks in bangladesh ( nv - b ) in 2004 , which confirms that nv was the etiologic agent responsible for these outbreaks .	virus isolation was performed in the bsl-4 laboratory at the centers for disease control and prevention in atlanta . vero e6 cells were inoculated and observed for characteristic cytopathic effect , syncytium formation ( 1,5 ) . nv was isolated from 2 oropharyngeal swabs ( spb200401066 , spb200406506 ) , 1 cerebrospinal fluid ( spb200401617 ) , and 1 urine specimen ( spb200405758 ) from human patients , and isolation was confirmed by reverse transcription polymerase chain reaction ( rt - pcr ) . two isolates were from rajbari , and 1 was from faridpur ; the fourth isolate , from the rajshahi district ( 100 km from rajbari ) , was not linked to the other 2 outbreaks . the complete genomic sequence of the first viral isolate ( spb200401066 ) from rajbari was derived and submitted to genbank ( accession no . the sequences of the open reading frame ( orf ) coding for nucleoprotein ( n ) were obtained for the other 3 isolates . the methods used for rt - pcr , sequencing , cdna cloning , rapid amplification of cdna ends ( race ) , and sequence analysis were previously described ( 2,3 ) . the genome of nv - b is 18,252 nt in length , 6 nt longer than nv - malaysia ( nv - m ) , the prototype strain of nv ( spb199901924 ) . the additional 6 nt map to the 5 nontranslated region of the fusion protein ( f ) gene . the length of the nv - b genome is evenly divisible by 6 , suggesting that nv - b follows the " rule of six " ( 3 ) . the gene order and sizes of all the orfs except v are conserved between nv - b and nv - m ( table , figure , a ) . the overall nucleotide homology between the genomes of nv - b and nv - m is 91.8% , but the changes are not uniformly distributed throughout the genome . nucleotide homologies are higher in the protein coding regions than in the noncoding regions , although the sizes of the nontranslated regions remain highly conserved ( table ) . the predicted amino acid homologies between the proteins expressed by nv - m and nv - b are all > 92% ( table ) . * nv , nipah virus . . percentage amino acid identity or nucleotide homology after sequences were aligned by using gap from gcg . nv - malaysia gene lengths and 5 and 3 nontranslated sequences and gene lengths were obtained from genbank accession no . open reading frames ( orfs ) are indicated by shaded boxes : n , nucleoprotein ; p , phosphoprotein ; m , matrix protein ; f , fusion protein ; g , attachment protein ; l , polymerase protein . a phenogram of the n orfs of members of this subfamily was created by using maximum parsimony analysis with paup 4.02 ( sinauer associates , sunderland , ma , usa ) . abbreviations and accession numbers : hpiv-1 , human parainfluenza virus , d01070 ; sendai , x00087 ; hpiv-3 , d10025 ; cdv , canine distemper virus , af014953 ; pdv , phocine distemper virus , x75717 ; rpv , rinderpest virus , x68311 ; mv , k01711 ; dmv , dolphin morbillivirus , x75961 ; ndv , newcastle disease virus , af064091 ; gp , goose paramyxovirus , af473851 ; hpiv-4b , m32983 ; hpiv-4a , m32982 ; tioman , af298895 ; menangle , af326114 ; hpiv-2 , m55320 ; simian virus 5 ( sv5 ) , m81442 ; mumps , d86172 ; nv - umcc1 , ay029767 ; nv - malaysia , af212302 ; nv - p . hypomelanus , * af376747 ; nv - bangladesh ; hendra virus , af017149 ; tupaia paramyxovirus , af079780 ; mossman virus , ay286409 ; and salem virus , af237881 . c ) the phylogenetic relationship between the n gene sequences of the 4 human nv isolates from the bangladesh outbreak in 2004 and the n gene sequences from pig and human nv isolates from malaysia . accession numbers for the pig isolates of nv ( 13 ) are aj627196 , aj564622 , and aj564621 . hypomelanus is sequence from a virus isolated from pteropus hypomelanus , the island flying fox ( 8) . overall , the predicted amino acid homologies of the surface glycoproteins , f and g , of nv - b and nv - m are high ( table ) . in the f protein , the predicted cleavage site , f1 amino - terminal domain , transmembrane domain , and predicted n - glycosylation sites are identical in nv - b and nv - m ( 2 ) . four of the 9 predicted amino acid changes occur in the first 11 amino acids ( aa ) of the precursor of the f protein , f0 , which fall within the predicted signal peptide and would be cleaved from the mature protein . within the g proteins , the predicted transmembrane domains , and the positions of all 17 cysteine residues are conserved between nv - b and nv - m . of 8 predicted n - linked glycosylation sites in the g protein of nv - m , 6 are conserved in nv - b and in hv ( 2 ) . the coding strategy of the p gene is identical in nv - b and nv - m . in these viruses , the p gene contains the c , v , and w orfs in addition to the p orf . like most other paramyxoviruses , the henipaviruses have a conserved ag - rich region that acts as an editing site to facilitate the addition of nontemplated g residues into the transcripts of the p gene . the edited transcripts encode 2 proteins , v and w , which are co - amino - terminal with p but have unique carboxy termini ( 2 ) . the addition of 1 g residue generates the mrna for the v protein , and the addition of 2 g residues produces the mrna for the w protein . sequence analysis of multiple cdna clones containing the editing site of nv - b identified edited transcripts that encoded both the v and w proteins ( data not shown ) . the conserved 20-nt region encompassing the editing site is identical in nv - m , hv , and measles virus ( mv ) ( 2 ) ; however , the editing of site of nv - m ( uggguaauuuuucccguguc ) differs from nv - b ( gggauaauuuuucccguguc ) at 2 nt positions ( underlined ) all of the cysteine residues are conserved in the v proteins of nv - b and nv - m ; however , the unique portion of the v protein of nv - b is predicted to be 55 aa , 3 aa longer than the v protein of nv - m . the predicted w protein of nv - b is identical in size and sequence to the w protein of nv - m . recently , aa 100160 and 230237 of the v protein of nv - m have been identified as necessary for inhibition of interferon signaling ( 14 ) . these regions are highly conserved in the v protein of nv - b , which has 4 predicted amino acids substitutions ( 1 conservative ) between positions 100160 and no predicted substitutions between positions 230237 . in addition , the v protein of nv - b has 3 predicted amino acid substitutions in the crm1-dependent nuclear export signal that was identified between aa 174193 in the v protein of nv - m ( 14 ) . the l proteins of nv - b and nv - m had a high level of predicted amino acid conservation ( table ) . the 6 highly conserved domains of viral polymerases , originally described by poch et al . ( 3 ) , remain largely unchanged between nv - b and nv - m . domains 1 , 2 , and 5 have 1 conservative amino acid change each and domain 3 , which is considered the most conserved domain within the l proteins of paramyxoviruses , has 2 aa changes . the 4 motifs identified in domain 3 , including the qgdne motif , which is assumed to be the active site of the polymerase , are identical between the nv - b and nv - m , as is the predicted nucleotide - binding motif in domain 6 . the cis - acting control sequences are highly conserved in the genomes of nv - b and nv - m . as in nv - m , the intergenic sequences in nv - b are gaa , with the exception of the sequence between the g and l genes , uaa , which is unique among the henipaviruses . however , the intergenic sequence between the g and l genes is gaa in the second isolate from bangladesh . the transcriptional start and stop signals of each gene of nv - b are highly conserved in relation to the other henipaviruses . the 3 leader sequence of nv - b is identical in length to those of all other paramyxoviruses and has nucleotide changes at positions 14 and 47 compared to nv - m . the 5 trailer of nv - b is identical in length and sequence to nv - m . phylogenetic analysis was used to compare the sequence of the n orf of nv - b to the sequences of the n orfs from other members of the subfamily paramyxovirinae . the results confirmed the results of the sequence comparisons , which show that nv - b is most closely related to the henipavirus nv - m , and support the conclusion that nv - b should be regarded as new strain of nv ( figure , b ) . phylogenetic analyses conducted with the sequences of the other genes produced similar results ( data not shown ) . the sequences of the n orfs of 4 nv isolates from bangladesh share 99.1% nt homology ( figure , c ) but exhibited more interstrain nucleotide heterogeneity than the sequences of the human isolates in malaysia , which were nearly identical ( 1,8,13 ) . these varying amounts of genetic variability may reflect differences in the mode of transmission of nv in the 2 countries . in malaysia , molecular evidence suggests that at least 2 introductions of nv into pigs occurred ( figure , c ) . however , the nearly identical sequences of human and pig isolates from the later phase of the outbreak suggest that only 1 of the variants spread rapidly in pigs and was associated with most human cases ( 13 ) . in contrast , the sequence heterogeneity observed in bangladesh may be the result of multiple introductions of nv into humans from different colonies of fruit bats . this first look at strain variation in nv indicates that viruses circulating in different areas have unique genetic signatures and suggests that these strains may have coevolved within the local natural reservoirs . until 2004 , the isolation and genetic characterization of nv - b confirm that nv was the etiologic agent responsible for these outbreaks . note : after this article was accepted for publication , nipah virus was isolated from pteropus lylei in cambodia ( 16 ) . phylogenetic analysis of the n gene sequences demonstrated that this virus is more closely related to nipah - malaysia than to nipah - bangladesh and represented another lineage of nipah virus .
to achieve safe airway management , it is essential first to predict whether there will be difficulties in intubating or ventilating the patient 's airway . an enlarged thyroid mass can produce a tracheal obstruction by compression or intraluminal invasion or both . we report two patients with thyroid cancer that obstructed the trachea by compression or invasion . there was no difficulty in endotracheal intubation of the patients with marked thyroid enlargement or in securing passage of the endotracheal tube through the compressed or narrowed portion of the trachea .	a 72 year - old , 150 cm , 57 kg , female patient presented with a neck mass that developed a few years earlier . she had no specific medical records except for taking medication for hypertension for 2 years . a physical examination revealed a palpable , broad , and firm tumor around both thyroid glands , but she did not complain of any symptoms . neck ct detected a thyroid neoplasm on both sides ( about 6 3 cm on the right thyroid gland ; about 7 7 cm on the left thyroid ) , which revealed the presence of tracheal stenosis . the region of tracheal constriction narrowed from 3.6 mm below the glottis to the sternal notch , in which the narrowest lumen of the trachea was approximately 3.9 21 mm in diameter and appeared to be compressed by the tumors of both sides ( fig . a thyroid papillary carcinoma was diagnosed by an aspiration biopsy , and on thoracic ct , a concern of metastasis into the lung urged immediate treatment . arterial blood gas analysis ( abga ) showed a ph of 7.424 , paco2 of 43.1 mmhg , pao2 of 74.8 mmhg , and base excess of 2.9 . the distance between the glottis and constriction site was so short that an endotracheal tube could not to be placed above the constriction region . however , there were no episodes of respiratory disturbance despite the severe tracheal obstruction , nor was there any increase in the paco2 . the partial pressure of oxygen ( pao2 ) also was in the normal range for her age . upon auscultation , normal breathing sounds were heard , which made us suspect that the tracheal obstruction was mobile . after explaining the potential difficulty of endotracheal intubation due to the tracheal stenosis one day before surgery , the patient provided informed consent for the procedure . a flexible fiberoptic bronchoscopy and laryngeal mask airway ( lma ) were prepared , and uncuffed endotracheal tubes , 4.0 , 4.5 , 5.0 in size , and cuffed endotracheal tubes , 4.0 to 7.0 in size , were ready for use . for pre - anesthesia medication , 0.5 mg of atropine was administrated 30 minutes before the onset of anesthesia . an electrocardiogram ( ecg ) , non - invasive blood pressure ( nibp ) monitor , and pulse oximetry were placed after arriving in the operating room . her vital signs indicated a bp , heart rate and oxygen saturation of 145/85 mmhg , 92/min and 99% , respectively , and she did not complain of any discomfort in the supine position . before the onset of anesthesia , denitrogenation was induced with 100% oxygenation at 6.0 l / min for 5 minutes . because she had no dyspnea during sleep midazolam 3 mg was administered and the patient fell asleep . however , when a bronchoscope intubation was attempted , her spontaneous respiration stopped and support ventilation was provided . when assisted ventilation with 100% oxygen was confirmed to function well , thiopental 100 mg was administered and the ventilation function was reconfirmed . succinylcholine 50 mg was then administrated and endotracheal intubation was attempted . after inserting an uncuffed endotracheal tube of 4.0 without resistance , the tube was withdrawn while keeping the bronchoscope in place . when a cuffed endotracheal tube ( size 5.0 ) was inserted without difficulty , rocuronium 30 mg was then injected into the vein and anesthesia was maintained with both oxygen and nitrous oxide of 1.5 l / min , and sevoflurane 1.0 - 2.5 vol% , while remifentanil was administrated continuously as a supplement . the tidal volume ( tv ) , respiratory rate ( rr ) and peak inspiratory pressure ( pip ) was 500 ml , 10/min , and 18 cmh2o , respectively . the operation was started after placing a catheter into the left dorsalis pedis artery for continuously monitoring of the arterial blood pressure and abga . extubation was performed after confirming that the patient 's spontaneous breathing was restored and she had reached consciousness . she was transferred to the ward after sufficient monitoring in the post - anesthesia recovery unit . a 66 year - old , 175 cm , 73 kg , male patient was referred to our institution from a clinic due to an episode of dyspnea over the last 3 - 4 months . he was diagnosed with bronchial asthma and received medical treatment , but there were no favorable changes and he was admitted to our hospital . he had no other abnormalities in his medical records other than taking medication due to a 20-year history of hypertension . he often experienced respiratory disturbances in the left lateral decubitus or supine position , whereas there were no complaints of dyspnea in the right lateral decubitus or semisupine position . a physical examination revealed a firm tumor , approximately 3 cm in size , below the right thyroid , but a simple chest x - ray did not show any abnormalities . the abga results were ph 7.413 , paco2 44.0 mmhg , pao2 106.4 mmhg , and base excess 2.4 . the pulmonary function test showed mild lung impairment with a mild obstructive pattern ( fvc 3.8 l / min [ 89.4% of predicted value ] , fev1 2.53 l / min [ 93 % of predicted value ] ) . cervical magnetic resonance imaging ( mri ) detected an ill - defined tumor in the lower part of the right thyroid and showed that the tumor had invaded the trachea and formed a polypoid tumor in the postro - internal direction . the tumor was distributed from approximately 2 - 3 cm below the glottis to the sternal notch with the narrowest region being 2.79 mm in diameter at approximately 4 cm below the glottis ( fig . a partial resection of the thyroid and trachea was determined after diagnosing thyroid papillary carcinoma by an aspiration biopsy . the distance from the glottis was so short that a tracheal tube was not placed above the region of the tracheal obstruction . extracorporeal circulation was planned under the suspicion that a metastasis of the tumor into the trachea would cause a high airway pressure , and extracorporeal circulation would be safer than laryngeal mask airway due to the symptoms of dyspnea . after consultation regarding our plan , his attending surgeon strongly recommended endotracheal intubation on the grounds that the base of the polypoid legion was so broad that the tissue would be far from being damaged or cut off and that there would be no bleeding or necrosis . therefore , it was decided to attempt endotracheal intubation through a bronchoscope under the sleep state . in case of failure of endotracheal intubation , a lma was provided and endotracheal tubes ( size 4.0 and 4.5 without a cuff and sizes 4.0 to 7.5 with a cuff ) were set . one day before the operation , the patient was given an explanation of the potential difficulty in endotracheal intubation due to the presence of the tumor inside the trachea , the process of intubation under conscious sedation or in the awakened state , and the possibility of extracorporeal circulation . there was no pre - operative medication and the upper body was elevated to approximately 30 after arriving in the operating room . the bp measured immediately after entering the or was 180/112 mmhg , which necessitated the administration of hydralazine 10 mg . a catheter was placed into the right dorsalis pedis artery to monitor the arterial bp and perform abga . propofol and remifentanil were administered continuously for sedation to maintain normal breathing , and oxygenation was provided by a mask . when respiration became shallow , assisted ventilation was performed and the ventilation rate was increased until the patient 's respiration had returned to normal , and reached a proper sleep state and did not respond to hearing stimulus . a size 5.0 cuffed endotracheal tube was threaded over a 4.0 mm flexible bronchoscope and the bronchoscope was inserted carefully into the region of the tumor . although there was a cough attack when the bronchoscope was passed over the glottis , the bronchoscope was advanced uneventfully up to the tumor location in a single attempt . after confirming the tumor , the bronchoscope was advanced further to confirm the carina . to prevent damage to the tumor , the tracheal tube threaded over the bronchoscope was advanced with a 180 turn in order for its bevel to be directed toward the right . the length of the 5.0 tracheal tube was 22 cm , and it was pushed up as much as possible to allow the adapter part of the tube to be attached to the patient 's incisor . the tip of the tube was confirmed to be placed above the carina . before removing the bronchoscope , we checked for any damaged tissue , and after removing it , suction was performed twice to remove the unfound tissue debris . mannual ventilation was provided with 100% oxygen at 4 l / min and sevoflurane , using the anesthesia machine . a cuffed 6.0 endotracheal tube was inserted through a tube exchanger because the pip increased sharply to 35 cmh2o and the length of the tube was too short to be advanced further on resection of a part of the trachea . a bronchoscope was inserted to again confirm the removal of fragments due to tumor damage , followed by further suction . no fragments of damaged tissue were found , and the administration of protofol was ceased . anesthesia was maintained with both 500% oxygen and nitrous oxide 1.5 l / min , respectively and sevoflurane supplemented with the continuous administration of remifentanil . a 72 year - old , 150 cm , 57 kg , female patient presented with a neck mass that developed a few years earlier . she had no specific medical records except for taking medication for hypertension for 2 years . a physical examination revealed a palpable , broad , and firm tumor around both thyroid glands , but she did not complain of any symptoms . neck ct detected a thyroid neoplasm on both sides ( about 6 3 cm on the right thyroid gland ; about 7 7 cm on the left thyroid ) , which revealed the presence of tracheal stenosis . the region of tracheal constriction narrowed from 3.6 mm below the glottis to the sternal notch , in which the narrowest lumen of the trachea was approximately 3.9 21 mm in diameter and appeared to be compressed by the tumors of both sides ( fig . a thyroid papillary carcinoma was diagnosed by an aspiration biopsy , and on thoracic ct , a concern of metastasis into the lung urged immediate treatment . arterial blood gas analysis ( abga ) showed a ph of 7.424 , paco2 of 43.1 mmhg , pao2 of 74.8 mmhg , and base excess of 2.9 . the distance between the glottis and constriction site was so short that an endotracheal tube could not to be placed above the constriction region . however , there were no episodes of respiratory disturbance despite the severe tracheal obstruction , nor was there any increase in the paco2 . the partial pressure of oxygen ( pao2 ) also was in the normal range for her age . upon auscultation , normal breathing sounds were heard , which made us suspect that the tracheal obstruction was mobile . after explaining the potential difficulty of endotracheal intubation due to the tracheal stenosis one day before surgery , the patient provided informed consent for the procedure . a flexible fiberoptic bronchoscopy and laryngeal mask airway ( lma ) were prepared , and uncuffed endotracheal tubes , 4.0 , 4.5 , 5.0 in size , and cuffed endotracheal tubes , 4.0 to 7.0 in size , were ready for use . for pre - anesthesia medication , 0.5 mg of atropine was administrated 30 minutes before the onset of anesthesia . an electrocardiogram ( ecg ) , non - invasive blood pressure ( nibp ) monitor , and pulse oximetry were placed after arriving in the operating room . her vital signs indicated a bp , heart rate and oxygen saturation of 145/85 mmhg , 92/min and 99% , respectively , and she did not complain of any discomfort in the supine position . before the onset of anesthesia , denitrogenation was induced with 100% oxygenation at 6.0 l / min for 5 minutes . because she had no dyspnea during sleep midazolam 3 mg was administered and the patient fell asleep . however , when a bronchoscope intubation was attempted , her spontaneous respiration stopped and support ventilation was provided . when assisted ventilation with 100% oxygen was confirmed to function well , thiopental 100 mg was administered and the ventilation function was reconfirmed . succinylcholine 50 mg was then administrated and endotracheal intubation was attempted . after inserting an uncuffed endotracheal tube of 4.0 without resistance , the tube was withdrawn while keeping the bronchoscope in place . when a cuffed endotracheal tube ( size 5.0 ) was inserted without difficulty , rocuronium 30 mg was then injected into the vein and anesthesia was maintained with both oxygen and nitrous oxide of 1.5 l / min , and sevoflurane 1.0 - 2.5 vol% , while remifentanil was administrated continuously as a supplement . the tidal volume ( tv ) , respiratory rate ( rr ) and peak inspiratory pressure ( pip ) was 500 ml , 10/min , and 18 cmh2o , respectively . the operation was started after placing a catheter into the left dorsalis pedis artery for continuously monitoring of the arterial blood pressure and abga . extubation was performed after confirming that the patient 's spontaneous breathing was restored and she had reached consciousness . she was transferred to the ward after sufficient monitoring in the post - anesthesia recovery unit . a 66 year - old , 175 cm , 73 kg , male patient was referred to our institution from a clinic due to an episode of dyspnea over the last 3 - 4 months . he was diagnosed with bronchial asthma and received medical treatment , but there were no favorable changes and he was admitted to our hospital . he had no other abnormalities in his medical records other than taking medication due to a 20-year history of hypertension . he often experienced respiratory disturbances in the left lateral decubitus or supine position , whereas there were no complaints of dyspnea in the right lateral decubitus or semisupine position . a physical examination revealed a firm tumor , approximately 3 cm in size , below the right thyroid , but a simple chest x - ray did not show any abnormalities . the abga results were ph 7.413 , paco2 44.0 mmhg , pao2 106.4 mmhg , and base excess 2.4 . the pulmonary function test showed mild lung impairment with a mild obstructive pattern ( fvc 3.8 l / min [ 89.4% of predicted value ] , fev1 2.53 l / min [ 93 % of predicted value ] ) . cervical magnetic resonance imaging ( mri ) detected an ill - defined tumor in the lower part of the right thyroid and showed that the tumor had invaded the trachea and formed a polypoid tumor in the postro - internal direction . the tumor was distributed from approximately 2 - 3 cm below the glottis to the sternal notch with the narrowest region being 2.79 mm in diameter at approximately 4 cm below the glottis ( fig . a partial resection of the thyroid and trachea was determined after diagnosing thyroid papillary carcinoma by an aspiration biopsy . the distance from the glottis was so short that a tracheal tube was not placed above the region of the tracheal obstruction . extracorporeal circulation was planned under the suspicion that a metastasis of the tumor into the trachea would cause a high airway pressure , and extracorporeal circulation would be safer than laryngeal mask airway due to the symptoms of dyspnea . after consultation regarding our plan , his attending surgeon strongly recommended endotracheal intubation on the grounds that the base of the polypoid legion was so broad that the tissue would be far from being damaged or cut off and that there would be no bleeding or necrosis . therefore , it was decided to attempt endotracheal intubation through a bronchoscope under the sleep state . in case of failure of endotracheal intubation , a lma was provided and endotracheal tubes ( size 4.0 and 4.5 without a cuff and sizes 4.0 to 7.5 with a cuff ) were set . one day before the operation , the patient was given an explanation of the potential difficulty in endotracheal intubation due to the presence of the tumor inside the trachea , the process of intubation under conscious sedation or in the awakened state , and the possibility of extracorporeal circulation . there was no pre - operative medication and the upper body was elevated to approximately 30 after arriving in the operating room . the bp measured immediately after entering the or was 180/112 mmhg , which necessitated the administration of hydralazine 10 mg . a catheter was placed into the right dorsalis pedis artery to monitor the arterial bp and perform abga . propofol and remifentanil were administered continuously for sedation to maintain normal breathing , and oxygenation was provided by a mask . when respiration became shallow , assisted ventilation was performed and the ventilation rate was increased until the patient 's respiration had returned to normal , and reached a proper sleep state and did not respond to hearing stimulus . a size 5.0 cuffed endotracheal tube was threaded over a 4.0 mm flexible bronchoscope and the bronchoscope was inserted carefully into the region of the tumor . although there was a cough attack when the bronchoscope was passed over the glottis , the bronchoscope was advanced uneventfully up to the tumor location in a single attempt . after confirming the tumor , the bronchoscope was advanced further to confirm the carina . to prevent damage to the tumor , the tracheal tube threaded over the bronchoscope was advanced with a 180 turn in order for its bevel to be directed toward the right . the length of the 5.0 tracheal tube was 22 cm , and it was pushed up as much as possible to allow the adapter part of the tube to be attached to the patient 's incisor . the tip of the tube was confirmed to be placed above the carina . before removing the bronchoscope , we checked for any damaged tissue , and after removing it , suction was performed twice to remove the unfound tissue debris . mannual ventilation was provided with 100% oxygen at 4 l / min and sevoflurane , using the anesthesia machine . a cuffed 6.0 endotracheal tube was inserted through a tube exchanger because the pip increased sharply to 35 cmh2o and the length of the tube was too short to be advanced further on resection of a part of the trachea . a bronchoscope was inserted to again confirm the removal of fragments due to tumor damage , followed by further suction . no fragments of damaged tissue were found , and the administration of protofol was ceased . anesthesia was maintained with both 500% oxygen and nitrous oxide 1.5 l / min , respectively and sevoflurane supplemented with the continuous administration of remifentanil . a tracheal obstruction has many symptoms according to the location , degree and cause , and is generally treated according to the symptoms . the airway obstruction is caused by primary tumors in the trachea , or secondarily by compression on the trachea or metastasis into the trachea by a neighboring tumor or by granuloma growth from a previous history of tracheal intubation , injury , or inflammation [ 11 - 13 ] . from the location , tracheal obstructions are divided into two groups : obstructions inside the thorax and obstructions outside the thorax . in the case of obstructions inside the thorax , it is essential to determine if the obstruction occurs in the trachea or in the bronchus . regardless of the cause , a bronchial obstruction is less life - threatening than a tracheal obstruction . a tracheal obstruction in the posterior mediastinum caused by the compression of a tumor inside the thorax does not carry a large risk . on the other hand , an obstruction in the anterior mediastinum may impede the maintenance of proper ventilation by increasing the compression on the trachea by the tumor when the patient 's spontaneous respiration disappears or muscle relaxation is achieved . tumors in the anterior mediastinum require a prompt warning of severe post - general anesthesia complications if measurements of the peak expiratory flow rate and tracheal area are < 50% of the expected one . in the case of a tracheal obstruction outside the thorax , it is essential to confirm whether it is caused by neighboring tumors , intratracheal tumors , or scars resulting from trauma or inflammation . a tracheal obstruction caused by trauma or inflammation , regardless of its position , allows only endotracheal tubes with a smaller size than the measured diameter . therefore , it is essential to make an accurate estimate of the extent of tracheal obstruction . in cases of difficult endotracheal intubation due to a severe obstruction , the anesthetic method should be decided according to whether the obstruction site is above or below a possible site of the tracheostomy . a tracheal obstruction can also be caused by tissue fragments from a primary intratracheal tumor during or after tracheal intubation . although such obstructions are rare , it is important to understand the features and characteristics of tumors even in smaller ones . reported that a huge thyroid tumor did not raise the issue of difficult endotracheal intubation ( dei ) , and some cases with dei did not show any difficulty when the endotracheal tube was passed over the compressed region [ 6 - 8 ] . by contrast , the degree of thyroid tumor progression is a more dangerous factor for dei because fibroblastic proliferation caused by the progression of thyroid cancer decreases the mobility of the larynx . regarding the anesthetic methods for patients with tracheal obstruction , there are several options worth considering . these include placement of a tracheal tube above the tracheal obstruction site , placement of a tube with a smaller diameter being passed over the obstruction site , tracheostomy , use of a lma , high frequency jet ventilation , high frequency positive pressure ventilation , extracorporeal circulation , etc . all these methods can secure the airway , but each method is vulnerable to certain complications . in any patient in whom a tracheal obstruction is suspected , anesthesia should be induced with the preparation of all necessary tools and equipment ready in case of emergency . in the present cases , thyroid cancer obstructed the trachea by compression or invasion . in case 1 , the tumor grew in both thyroid glands and compressed the trachea on both sides ; the narrowest diameter of the trachea was approximately 3.9 21.2 mm . abga revealed normal findings , whereas the trachea with an anterioposterior diameter of approximately 21.2 mm was compressed by the tumor on both sides but the thyroid tumor was believed not to be fixed to the surrounding tissues and was mobile . her surgeon stated that a tracheotomy under local anesthesia would be difficult because the tumor was large enough to cover the tracheotomy site . taking the use of lma or extracorporeal circulation into account , the relevant clinical reports were reviewed , which showed that endotracheal tubes with diameter a larger than the tracheal diameter could inserted in cases of a tracheal obstruction caused by the compression of thyroid cancer [ 2 - 8 ] . therefore , a flexible fiberoptic bronchoscope , lma , and a variety of endotracheal tubes of different sizes were made ready in case of emergency , and the induction of general anesthesia was scheduled after making preparations for an otolaryngologist to perform an emergency tracheostomy . although tracheal intubation under consciousness is safer , the patient did not have any symptoms of dyspnea during sleep , so a sleep state was induced with midazolam , and we tried to attempt tracheal intubation during sleep under a bronchoscope . manual ventilation was needed due to the occurrence of apnea during the induction of sleep , and tracheal intubation was performed under the direct laryngoscope . the tracheal obstruction was so severe that the endotracheal tubes were changed gradually to a larger size from 4.0 , 5.0 , to 6.5 , while checking the airway resistance to reduce the potential damage caused by intubation . in retrospect , it may have been worth attempting to insert a 6.5 size tube next to a 4.0 size one to save time and trouble . as expected , an endotracheal tube with a diameter larger than the measured inside diameter ( i d ) was easily inserted . in this case , a partial tracheostomy was not scheduled because there were no signs of endotracheal metastasis , so lma was not employed because it was believed that it would not be able to maintain anesthesia for more than 6 hours until the thyroid tumor had been removed completely . however , a plan for lma and tracheostomy was made as a contingency plan for difficult endotracheal intubation ( dei ) . extracorporeal circulation also was not attempted due to the risk of blood loss because this method was rather invasive and required heparin despite its advantage of enabling anesthesia without touching the trachea when it is difficult to secure the airway . in case 2 , to avoid damage to the tumor , a 4.0 mm flexible fiberoptic bronchoscope was inserted through a cuffed 5.0 size endotracheal tube to reduce the space between the bronchoscope and tube while a 6.5 size endotracheal tube was inserted for replacement with its bevel being directed toward the tumor lesion . 3 ) , but the anesthetic method in case 2 did not appear to be the proper choice in retrospect . this is because naked eye observation does not ensure that there is no tumor tissue debris remaining and the presence of such fragments may be another issue of bronchial obstruction . the reason why the method in this case did not present any problems is that the endotracheal tube with a diameter larger than the measured i d could be inserted because the trachea compressed by the thyroid tumor was not constricted by foreign surrounding tissue . in conclusion , endotracheal tubes with a diameter larger than the measured i d were used despite the severe tracheal obstruction due to the absence of dyspnea or very mild symptoms in patients showing normal abga and pulmonary function test results . in addition , the normal results of these two tests demonstrated that the trachea compressed by the foreign tumor outside the thorax was mobile . as suggested in our cases , anesthesia should be initiated only after making all preparations for securing the trachea , even when easy tracheal intubation is anticipated . as in case 2 ,
an increase in dentin roughness , associated with surface composition , contributes to bacterial adherence in recontaminations . surface roughness is also important for micromechanical interlocking of dental materials to dentin , and understanding the characteristics of the surface is essential to obtain the adhesion of root canal sealers that have different physico - chemical characteristics.objectivesto evaluate the effects of sodium hypochlorite ( naocl ) , ethylenediaminetetraacetic ( edta ) , etidronic ( hebp ) , and citric acid ( ca ) associated with different irrigation regimens on root dentin roughness.material and methodsforty - five root halves of anterior teeth were used . the root parts were sectioned in thirds , embedded in acrylic resin and polished to a standard surface roughness . initially , the samples of each third were randomly assigned into 3 groups and treated as follows : g1 - saline solution ( control ) ; g2 - 5% naocl+18% hebp mixed in equal parts ; and g3 - 2.5% naocl . after initial measuments , the g3 samples were distributed into subgroups g4 , g5 and g6 , which were subjected to 17% edta , 10% ca and 9% hebp , respectively . following the new measuments , these groups received a final flush with 2.5% naocl , producing g7 , g8 and g9 . the dentin surface roughness ( ra ) was determined before and after treatments using a profilometer . the wilcoxon test ( <0.05 ) was used to compare the values before and after treatments , and the friedman test ( <0.05 ) to detect any differences among root thirds . results(i ) naocl did not affect the surface roughness ; ( ii ) there was a significant increase in roughness after the use of chelating agents ( p<0.01 ) ; and ( iii ) only the g3 group showed a difference in surface roughness between apical third and other thirds of the teeth ( p<0.0043).conclusiononly the irrigation regimens that used chelating agents altered the roughness of root dentin .	adhesion involves interactions at the interface between materials and depends of several factors as cleanliness , composition , and roughness of adherent surface . mechanical bonding is the most effective means of creating strong joints . in this type of adhesion , the material penetrates into the adherent , becoming mechanically interlocked at some level . to attain effective adhesive bonding irrigating solutions used in endodontics clean the dentin surface , and may interfere with the chemical structure of dentin , changing the calcium / phosphorus ( ca / p ) ratio of the surface . these alterations can increase the surface roughness , which may affect the sealing ability and adhesion of dental materials - such as resin - based cements and root canal sealers - to dentin and alter the nature of adhesion and the adhesion strength of various bacteria . sodium hypochlorite solutions ( naocl ) are the most common irrigating agents used in biomechanical preparation based on their excellent microbicidal activity and tissue - dissolving capabilities . however , despite these properties , this solution only removes the organic structure of the smear layer produced during mechanical instrumentation , and combining it with chelating agents is necessary to remove the inorganic phase of this layer . various chelating agents can be used for this purpose , such as ethylenediaminetetraacetic acid ( edta ) , citric acid ( ca ) , mtad ( mixture of doxycycline , citric acid and tween 80 ) , and chitosan . recently , etidronate ( hebp ) , a substance that prevents bone resorption has been used in medicine for patients suffering from osteoporosis or pagets disease , and was suggested as substitute for traditional chelators due to fewer effects observed on dentin structure . it is considered the unique chelator that can be mixed with naocl without interfering with its antimicrobial property . however , chelating solutions also remove calcium ions from the dentin surface , exposing the collagen matrix , which may contribute to bacterial adherence in recontaminations , as with enterococcus faecalis . the use of naocl solutions has been suggested as a strategy to remove this exposed collagen matrix in a process called deproteination , which restores to the surface characteristics of untreated dentin . the importance of roughness studies is supported due to the strong relationship between surface topography and its influence on dentin wettability , a property that directly influences the bonding of the dental materials and microorganism adhesion . the increase in roughness could be potentiated by the combination of the irrigation solutions and , to date , there have been no studies evaluating the effect of hebp and different irrigation regimens on the roughness of root canal dentin . therefore , the purpose of this in vitro study was to evaluate the effects of naocl , edta , hebp and ca associated with different irrigation regimens on root dentin roughness . solutions of 2.5% ( wt / vol ) and 5% naocl , 10% ca ( sigma - aldrich , st louis , missouri , usa ) , 9 and 18% hebp ( zschimmer & schwarz mohsdorf gmbh & co kg , burgstdt , sn , germany ) were prepared using pure chemicals dissolved in deionized water . the 17% edta solution ( sigma aldrich ) was prepared as previously described in other study . all solutions were stored at 5c in airtight dark containers between experiments ; prior to being used , the solutions were removed from the refrigerator and stored for 60 min at room temperature . a fresh 1:1 mixture of 5% naocl and 18% hebp was prepared immediately before the experiments , producing a solution that contained 2.5% naocl and 9% hebp . remnants of debris and soft tissue on the tooth surfaces were removed , and all teeth stored in 0.1% thymol at 9c until use . tooth crowns were removed at the cement - enamel junction using a low - speed diamond disk ( kg sorensen ind . the canals were explored with a size 15 k - type file ( dentsply maillefer , ballaigues , switzerland ) until the tip of the instrument was adjusted to the apical foramen . at this point , another sectioning was performed to standardize the root apical limit and ensure that only the canal dentin was analyzed . subsequently , each root was sectioned longitudinally in the buccolingual direction to expose the entire canal extension , and the pulp tissue was removed . the tooth halves thirds were transversely marked with the aid of a digital caliper pd-150 ( vonder , curitiba , pr , brazil ) , and then the halves were horizontally sectioned into apical , middle and cervical thirds . each segment was identified and horizontally mounted in autopolymerizing acrylic resin ( dentbras ind . the specimens were wet polished on a circular grinding machine with a series of ascending grades ( 400 , 600 , 1200 , and 2000 ) of silicon carbide abrasive papers ( 3 m do brasil ltda . , sumar , sp , brazil ) under water coolant to achieve a standard surface roughness . then , the samples were thoroughly washed and sonicated in distilled water to remove residual particles . the surface roughness of each sample was determined in ra ( arithmetic average roughness - m ) with a portable digital roughness tester sj 301 ( mitutoyo , tokyo , japan ) within a pre - established cut - off ( distance traveled in each reading ) of 0.8 mm . specimens with similar surface roughness were included in the study ; the other samples were again subjected to polishing until they reached the roughness standard mean ( 0.10 - 0.14 ra ) determined by the pilot test . the 45 specimens of each third were randomly divided into groups as shown in figure 1 . the specimens were immersed in irrigating solutions for 30 minutes to simulate the biomechanical preparation . in stage 2 , the inorganic phase of the smear layer was removed through the application of chelating agents , and in step 3 , deproteination was performed with the use of 2.5% naocl solution for 3 min . the irrigation solutions were renewed every 5 min to ensure their chemical effectiveness . after each step , the specimens were washed for 1 min with distilled water in an ultrasonic tub to avoid residual effects from the solutions . the measurements were performed after each stage in the same manner as the initial measurements , and the roughness values were recorded . twenty - seven specimens of each root third were subjected to naocl and then distributed according to the irrigation regimens used . to avoid false - positive inflation , the roughness values of only 9 specimens the nonparametric wilcoxon test ( <0.05 ) was used to compare the dentin surface roughnesses before and after treatments , and the friedman test ( <0.05 ) was used to detect differences among the root thirds . solutions of 2.5% ( wt / vol ) and 5% naocl , 10% ca ( sigma - aldrich , st louis , missouri , usa ) , 9 and 18% hebp ( zschimmer & schwarz mohsdorf gmbh & co kg , burgstdt , sn , germany ) were prepared using pure chemicals dissolved in deionized water . the 17% edta solution ( sigma aldrich ) was prepared as previously described in other study . all solutions were stored at 5c in airtight dark containers between experiments ; prior to being used , the solutions were removed from the refrigerator and stored for 60 min at room temperature . a fresh 1:1 mixture of 5% naocl and 18% hebp was prepared immediately before the experiments , producing a solution that contained 2.5% naocl and 9% hebp . remnants of debris and soft tissue on the tooth surfaces were removed , and all teeth stored in 0.1% thymol at 9c until use . tooth crowns were removed at the cement - enamel junction using a low - speed diamond disk ( kg sorensen ind . the canals were explored with a size 15 k - type file ( dentsply maillefer , ballaigues , switzerland ) until the tip of the instrument was adjusted to the apical foramen . at this point , another sectioning was performed to standardize the root apical limit and ensure that only the canal dentin was analyzed . subsequently , each root was sectioned longitudinally in the buccolingual direction to expose the entire canal extension , and the pulp tissue was removed . the tooth halves thirds were transversely marked with the aid of a digital caliper pd-150 ( vonder , curitiba , pr , brazil ) , and then the halves were horizontally sectioned into apical , middle and cervical thirds . each segment was identified and horizontally mounted in autopolymerizing acrylic resin ( dentbras ind . the specimens were wet polished on a circular grinding machine with a series of ascending grades ( 400 , 600 , 1200 , and 2000 ) of silicon carbide abrasive papers ( 3 m do brasil ltda . , sumar , sp , brazil ) under water coolant to achieve a standard surface roughness . then , the samples were thoroughly washed and sonicated in distilled water to remove residual particles . the surface roughness of each sample was determined in ra ( arithmetic average roughness - m ) with a portable digital roughness tester sj 301 ( mitutoyo , tokyo , japan ) within a pre - established cut - off ( distance traveled in each reading ) of 0.8 mm . specimens with similar surface roughness were included in the study ; the other samples were again subjected to polishing until they reached the roughness standard mean ( 0.10 - 0.14 ra ) determined by the pilot test . the 45 specimens of each third were randomly divided into groups as shown in figure 1 . the specimens were immersed in irrigating solutions for 30 minutes to simulate the biomechanical preparation . in stage 2 , the inorganic phase of the smear layer was removed through the application of chelating agents , and in step 3 , deproteination was performed with the use of 2.5% naocl solution for 3 min . the irrigation solutions were renewed every 5 min to ensure their chemical effectiveness . after each step , the specimens were washed for 1 min with distilled water in an ultrasonic tub to avoid residual effects from the solutions . the measurements were performed after each stage in the same manner as the initial measurements , and the roughness values were recorded . twenty - seven specimens of each root third were subjected to naocl and then distributed according to the irrigation regimens used . to avoid false - positive inflation , the roughness values of only 9 specimens the nonparametric wilcoxon test ( <0.05 ) was used to compare the dentin surface roughnesses before and after treatments , and the friedman test ( <0.05 ) was used to detect differences among the root thirds . the median and interquartile range of roughness values of root canal dentin before and after treatment with irrigation regimens in cervical , middle , and apical thirds are summarized in tables 1 , 2 and 3 , respectively . a significant increase in dentin roughness was observed following the treatment with the different irrigation regimens , except when saline ( g1 ) or naocl ( g3 ) were used alone . median ( med ) and interquartile range ( iqr ) and p values ( wilcoxon test ) for roughness analysis in the cervical third before ( t0 ) and after ( t1 ) the application of the irrigation regimens median ( med ) and interquartile range ( iqr ) and p values ( wilcoxon test ) for roughness analysis in the middle third before ( t0 ) and after ( t1 ) the application of the irrigation regimens median ( med ) and interquartile range ( iqr ) and p values ( wilcoxon test ) for roughness analysis in the apical third before ( t0 ) and after ( t1 ) the application of the irrigation regimens the data for initial roughness revealed no statistically significant difference among the different thirds ( table 4 ) . after treatments , the different thirds behaved the same way , except in g2 in which the apical third had a lower increase in surface roughness than did the other thirds ( p=0.0043 ) . the p values ( friedman test ) of the roughnesses before ( t0 ) and after ( t1 ) the same treatment on different thirds ct = cervical third ; mt = middle third ; at = apical third ; p=0.0043 : ct x mt p = not significant ; ct x at p<0.05 ; and mt x at p<0.05 the irrigation solutions might influence the physicochemical properties of human root canal dentin , including microhardness , permeability , solubility , wettability and roughness . in endodontics , an increase in surface roughness could be clinically beneficial because it may enhance the micromechanical bonding of root canal sealers , which requires irregularities on the surface of the adherent for penetration . however , too much roughness can facilitate bacterial adhesion , which might lead to plaque formation . in the present study , naocl treatment did not modify the roughness of the dentin surface when used before or after the chelating agents , consistent with previous findings that naocl does not cause decalcification or changes in dentin wettability , a factor that has been correlated with dentin roughness . however , these results are in contrast to those of other studies , most likely due to the standardization of the initial polishing used here ; this procedure was not reported in the other studies . initial roughness standardization is recommended in studies of chemical conditioning because it offers a controlled reference point for the correct and unambiguous assessment of the morphological effects induced by subsequent chemical treatments . the regimens that employed ca ( g5 and g8 ) and hebp mixed with naocl ( g2 ) demonstrated greater increases in roughness values than did other groups . the strong activity of ca is most likely due to its greater capacity for demineralization that , in addition to removing the smear layer , can also cause extensive demineralization in dentinal tubules and peritubular dentin . when mixed , the hebp causes some reduction in the activity of naocl after 1 h , but the two substances remain active in fresh mixture . the results observed in g2 are most likely due to the ability of naocl to create deproteination canals in the dentin , which may increase the area of action available for hebp . associated with this factor , the longer use of this weak chelating agent for 30 min also may have potentiated its effect . the smallest changes in surface roughness after the use of a chelating agent were observed in regimens that employed 9% hebp for 5 min after naocl treatment ( g6 and g9 ) . these findings confirm that hebp is a weak chelating agent that attacks less dentin surface than other commonly used chelators , such as edta , but the hebp solutions need 300 seconds to completely remove the smear layer . in terms of the behavior of different root thirds in each of the irrigation regimens , it was observed that , despite the heterogeneous structure of these regions , they exhibit similar behaviors in response to direct contact between the irrigation agent and the root surface ( table 4 ) . one exception was the apical third in g2 , possibly because the greater percentage of sclerotic dentin in this area reduced the number of deproteination canals created by naocl , decreasing the area of action available for hebp . the results of this study showed the effects of direct contact from different irrigation regimens using different auxiliary chemical substances on dentin surface roughness . however , these results can not be extrapolated to clinical practice , because it is difficult to introduce irrigation solution to the apical region , which , associated to the use of endodontic instruments , may change these values . however , the analysis of the behavior of different root thirds into direct contact with the irrigating solutions is relevant to assess if , besides the difficulty of the irrigators to reach the apex , the dentin composition also influences the increase in roughness . it was possible to observe that only the irrigation regimens that employed chelating solutions increased the surface roughness and the use of naocl before the chelating agents or as a final flush did not modify the dentin roughness . further studies should be performed to evaluate not only the effect of different irrigation regimes on the dentin structure but also the effect of these protocols on the adhesion of root canal sealers and bacteria in recontaminations . the findings of this study indicated that only the irrigation regimens that used chelating agents were capable of increasing the roughness of root canal dentin . this study was supported in part by capes - coordination of higher education and graduate training - and by cnpq - national council for scientific and technological development .
atypical hemolytic uremic syndrome ( ahus ) is a very rare , life - threatening , progressive disease that frequently has a genetic component and in most cases is triggered by an uncontrolled activation of the complement system . successful treatment of ahus with plasma infusions and therapeutic plasma exchange ( tpe ) is well reported . tpe has been the treatment of choice in most adult patients with ahus . however , due to severe hemolysis , which is common among ahus patients , there are some technical challenges that can affect tpe treatment such as the continuous activation of the blood leak alarm due to hemolysis . our experience shows that such patients can be managed better on a centrifuge based tpe machine compared to a membrane based tpe machine .	hemolytic uremic syndrome ( hus ) is a hematological disease characterized mainly by the triad of microangiopathic hemolytic anemia , thrombocytopenia , and acute renal failure . it is commonly caused by infections of shiga - like toxin producing bacteria , such as escherichia coli strain o157:h7 , o111:h8 , o103:h2 , o123 , and o26.1 diarrhea is often present in such cases which can also be referred to as typical hus . on the other hand , atypical hus ( ahus ) is characterized by the absence of diarrheal illness and can be acquired , genetic , or idiopathic . ahus represents approximately 10% of all hus cases.2 currently , plasma exchange and/or plasma infusions is the recommended first - line management for ahus.3 this paper presents the technical challenges faced while treating a patient who had ahus with therapeutic plasma exchange ( tpe ) . a 21-year - old female with a known history of ahus presented to our emergency department in september 2012 . she had three relapses before when she was aged 8 , 10 , and 20 . she presented with 3 days of fever and sore throat followed by dark urine . on examination , she was found to be afebrile , alert , oriented , and she was talking in full sentences . her sitting blood pressure was 140/90 mmhg and her heart rate was 70 beats per minute with an oxygen saturation of 99% on room air . hematological investigations revealed a hemoglobin ( hb ) count of 96 g / l , platelet count 16 10/l , red cell count 3.01 10/l , and a hematocrit of 0.27 there was also marked thrombocytopenia and microangiopathic anemia on the film , features suggestive of relapse of known hus . biochemistry results showed a lactate dehydrogenase ( ldh ) level of 1,201 u / l , haptoglobin < 0.06 g / l , potassium 3.7 mmol / l , urea 20.7 mmol / l , creatinine 202 umol / l , estimated glomerular filtration rate 27 ml / minute , and c - reactive protein ( crp ) of 27 mg / l . high levels of ldh and low haptoglobin levels pointed to severe hemolysis while the renal markers , such as creatinine , urea , and estimated glomerular filtration rate showed a deteriorating renal function . earlier attempts to perform the biochemical tests had been futile due to severe hemolysis . after a multidisciplinary team collaboration that involved the intensive care unit , hematology , nephrology , and the emergency department , a left femoral vascath was inserted with the view of commencing tpe using 3 l of fresh frozen plasma ( ffp ) . the procedure was scheduled to be done in the emergency department using a membrane based tpe machine ( mtpe ) . plasma flux psu 2s plasma exchange filters manufactured by fresenius se & co ( bad homberg , germany ) were used . these filters have a surface area of 0.6 m , blood priming volume of 70 ml , and a plasma sulphone membrane . filtration is primarily based on pressure gradients allowing filtration of molecules of up to 1,000 kda including immunoglobulins , complement factors , and albumin . soon after commencing tpe , the machine showed a blood leak alarm and it was evident that the membrane had ruptured gauging by the color of the effluent ( figure 1 ) . the filter was replaced with another one which also did not last long before the machine showed a blood leak alarm . an attempt was made to resume treatment with a centrifuge based tpe machine ( ctpe ) . the centrifugal device we used was a spectra optia apheresis system , a product of terumo bct ( lakewood , co , usa ) . this machine operates by separating blood products according to their specific gravity using centrifugal force . the spill over alarm persisted on ctpe and red blood cell detected was shown on the machine . at this point , it was agreed to stop tpe due to the nature of the technical problems which were attributed to severe hemolysis . on day 2 , it was agreed that the patient could be treated with eculizumab ( complement c5 blocker ) , but the drug was not available for compassionate access . disable the red blood cell detector and ctpe was initiated successfully using a very low inlet flow rate ranging from 2070 ml / minute with an anticoagulant infusion rate of 1.0 ml / minute . the patient continued to receive daily ctpe until day 16 when a decision was made that she had reached clinical remission , and she was discharged home . her hematological ( table 1 ) and biochemical ( figure 3 ) results continued to improve . a follow up of this patient after 6 weeks revealed that she was clinically well and had resumed her normal daily routines . however , plans had been made for her to have a permanent vascular access in the form of an arterio - venous fistula ( avf ) in the setting of these recurrent ahus episodes which seem to resolve after aggressive tpe treatment . both machines ( mtpe and ctpe ) were fitted with safety mechanisms to prevent loss of red blood cells during treatment and if there was a blood leak , an alarm was triggered and the machine would stop automatically . interestingly , in our case , we knew that the effluent was very rich in hemolysis products and the machine that won the day was one that we could manipulate easily to tailor our treatment plan ; this was the ctpe . furthermore , another dilemma with using the mtpe is that plasma has different optical properties from ultrafiltrate , which can set off the blood leak alarm and stop the pump even if there is no actual blood leak . in this respect , nursing staff were not comfortable overriding the mtpe blood leak alarm since it is challenging to determine whether there is an actual blood leak or not . the ctpe also gave us an opportunity to assess the color changes of the patient s effluent and this boosted our confidence as we visually noticed some positive results of the treatment . we believe that our patient received the best tpe treatment we could offer since some clinical cross over trials have reinforced the notion that ctpe allows a higher plasma exchange rate compared to mtpe without adverse effects on treatment quality and tolerability.4 this sentiment has been shared by another researcher who has found that plasma removal is more efficient with ctpe compared to mtpe systems , which have a lower plasma extraction ratio and therefore require longer procedure times.5 the ctpe treatment also ensured that our patient received the 3 l of ffp that was ordered and that an equal volume of patient plasma was removed . from our experience with dialysis , in patients whose post dialysis weight does not correlate with the reported ultrafiltration , the preciseness of the built - in ultrafiltration controller in the mtpe machine is questionable . ffp was used as a substitution solution during tpe despite the high rate of side effects , such as anaphylactic reactions6,7 that may actually require cessation of plasmatherapy.8 we used ffp because it provides normal amounts of complement factors ( cfh , cfi , cfb , and c3 ) and functional proteins9 which are vital for treating ahus . it was unfortunate that our patient could not access eculizumab , which is an anti - complement factor 5 ( c5 ) monoclonal antibody that binds to c5 thereby preventing activation of the terminal complement cascade . recent impressive improvement in the management of ahus has been reported with the use of eculizumab which is becoming the new breakthrough treatment option for patients with primary ahus , providing improved control of the disease over plasma exchange , with a good safety profile.10 however , it is still not quite clear whether life - long or recurrence - specific treatment is necessary and how genetics may or may not impact care of persons on eculizumab.11 what is known , however , is that defects in more than one complement regulator in ahus cases may pose significant therapeutic challenges.12 recent reports indicate that mutations in dgke ( which encodes diacylglycerol kinase ) were found in several ahus patients and this may have an impact on the successful treatment of individuals with eculizumab.13 nevertheless , we are delighted that we successfully delivered first line treatment8 for ahus through ctpe . traditionally , ctpe is the method preferred by hematology or blood bank based physicians for plasma exchange while , on the other hand , nephrology based physicians prefer mtpe . from our experience , it may be better to individualize these treatment options since patient needs vary . for a patient with ahus , treating with centrifuge devices such as the spectra optia apheresis system ( terumo bct ) may result in better outcomes . clinicians also seem to be more comfortable managing the technical challenges , such as the continuous activation of blood leak alarms caused by severe hemolysis for patients on ctpe compared to mtpe .
here , we investigate the effect of the structure ( generation ) and nature of the surface groups of different polyamidoamine ( pamam ) dendrimers on electron - mediated dissociation , either electron capture dissociation ( ecd ) or electron detachment dissociation ( edd ) , and compare the fragmentation with that observed in collision - induced dissociation ( cid ) . ecd and edd of the pamam dendrimers resulted in simple mass spectra , which are straightforward to interpret , whereas cid produced complex mass spectra . the results show that electron - mediated dissociation ( ecd and edd ) of pamam dendrimers does not depend on the nature of the surface group but tends to occur within the innermost generations . cid of the pamam dendrimers showed a strong dependence on the nature of the surface group and occurred mostly in the outer generation . the results demonstrate the potential utility of ecd and edd as a tool for the structural analysis of pamam dendrimers .	polyamidoamine ( pamam ) dendrimers were purchased from sigma aldrich ( poole , dorset , uk ) and used without further purification . the pamam dendrimers with amino and amidoethanol surface groups were diluted to a concentration of 10 pmol/l in solutions of methanol ( fisherscientific , loughborough , uk ) to water ( j. t. baker , middlesex , uk ) to acetic acid ( fisherscientific ) ( 49:49:2 , vol / vol ) . the 0.5-generation pamam dendrimer with sodium carboxylate surface groups was prepared ( 10 pmol/l ) in methanol : acetic acid ( 98:2 ) solution . mass spectrometry analysis was performed on a thermo finnigan ltq ft mass spectrometer ( thermo fisher scientific , bremen , germany ) . samples were injected by use of an advion biosciences triversa nanomate electrospray source ( advion biosciences , ithaca , ny , usa ) . data acquisition and analysis were conducted using the xcalibur 2.0 ( thermo fisher scientific ) software . precursor ions were selected and isolated for ecd in the linear ion trap before transfer to the icr cell . electrons were generated on the surface of an indirectly heated barium tungsten cylindrical dispenser cathode ( 5.1 mm diameter ; heat wave labs , inc . , watsonville , ca , usa ) , situated 154 mm from the cell , 1 mm off - axis . the current across the electrode was about 1.1 a. ions were irradiated with electrons for 70 ms . cid experiments were performed in the front - end linear ion trap and the fragments transferred to the icr cell for detection . cid experiments were performed with helium gas at a normalized collision energy of 35% . each cid scan consisted of five coadded microscans . all tandem mass spectrometry ( ms / ms ) spectra were averaged over 30 scans and analyzed manually . ecd fragments were assigned based on the nomenclature devised by oh and colleagues ( see scheme 2 ) . assignments are given in the form gn(m ) , where subscript n refers to the generation in which fragmentation takes place and m describes the type of fragmentation ; a / x , b / y , c / z . for example , fragment g1(y ) was derived from cleavage of the amide bond in generation 1 . gn(in ) and gn(out ) notations concern fragmentation that takes place core - side of the tertiary amines . the second - generation pamam dendrimer with amidoethanol surface groups and ethylenediamine core is a symmetrical molecule that contains three tertiary amine branches ( generations 0 , 1 , and 2 ) and 16 neutral alcohol surface groups . electrospray ionization of this pamam dendrimer leads to the formation of multiply protonated molecular ions [ m + 4h ] through [ m + 7h ] . the most abundant multiply protonated molecular ions of the pamam dendrimer[m + 4h ] , [ m + 5h ] , and [ m + 6h ] ions were isolated and subjected to ecd . the ecd ms / ms spectrum of [ m + 6h ] precursor ions is shown in figure 1 ( top ) . all fragments detected are described in supplementary table s-1 , which can be found in the electronic version of this article . the mass spectrum shows peaks corresponding to triply charged fragments g1(in ) , g0(in ) singly charged gcore(in ) , and quadruply charged g1(in ) . in addition , g(out ) fragments , which also result from cleavage at tertiary amines but with the charge retained toward the surface of the dendrimer , were observed . these fragments also undergo secondary fragmentations , resulting in singly charged fragment ions [ g1(out)g2(a ) ] at m / z 160.1205 , [ g0(out)g1(a ) ] at m / z 389.2624 , and [ g0(out)g1(out ) ] at m / z 459.2915 . the c / z cleavages that are the most abundant in the ecd of peptides / proteins were found here only as minor channels : g1(z ) at m / z 274.1757 and g0(z ) at m / z 732.4592 . as mentioned earlier , similar fragmentation patterns were seen for each charge state of pamamg2oh . in each case , fragmentation within these cations occurred in the innermost generations , i.e. , the core , g0 or g1 . electrospray ionization of the generation 1 pamam dendrimer , containing ethylenediamine core , two tertiary amine branches ( generations 0 and 1 ) , and eight primary amino surface groups , results in the formation of multiply protonated molecular ions [ m + 2h ] through [ m + 4h ] . mass spectra shown here were obtained from the most abundant charge state , i.e. , [ m + 4h ] ions . the ecd mass spectrum of the [ m + 4h ] ions of pamamg1nh2 is shown in figure 2 ( top ) and the fragments observed are detailed in supplementary table s-2 . the peaks at m / z 592.9225 and 289.2342 can be assigned to g0(in ) and g0(y ) fragments , respectively . other abundant fragment ions at m / z 388.3021 and 159.1366 are attributed to secondary fragmentation and can be assigned to [ gcore(out)g0(a ) ] and [ g0(out)g1(a ) ] , respectively . these fragments are presumably the result of cleavage of the amide bond followed by loss of co , together with cleavage at the tertiary amine . comparison of the ecd mass spectra obtained from pamamg1nh2 and pamamg2oh dendrimers leads to the conclusion that , despite the different chemical properties , the nature of the surface groups does not affect the ecd fragmentation behavior of these polymers . in both cases the mechanism of ecd is bound up with protonation of the tertiary amines and the presence of amide functionalities in the polymer backbone and can be explained on the basis of a charge - solvation model ( proton sharing ) . in both cases the ecd mass spectra are dominated by fragments that come from the inner generation(s ) . the most abundant fragments are of the same type for each dendrimer ; gn(y ) , gn(in ) and gn(out ) , where n stands for the inner generation(s ) ( n = 0 , 1 , in the case of pamamg2oh ; n = 0 in the case of pamamg1nh2 ) . assignments are given in form agn , bgn , and cgn , where subscript gn refers to the generation in which cleavage takes place . note that bgn is equivalent to ecd assignment gn(kout ) , but the multiplicity of the cid fragmentation ( see following text ) demands simpler nomenclature . the collision - induced dissociation of [ m + 6h ] ions of pamamg2oh ( figure 1 , bottom ) , is dominated by the loss of two neutral fragments , ag2 ( m = 103.0633 ) and bg2 ( m = 115.0633 ) and combined losses of the two in various stoichiometries . the precise connectivity of this fragment is unknown : the proton localized on the tertiary amine can be bound to the oxygen atom from carbonyl group . as a result of proton transfer and rearrangement , the fragment ch2=c(oh)n(h)ch2ch2oh may be formed . neutral fragment bg2 is the result of cleavage at the tertiary amine in the second generation and has the formula ch2=ch c(o)n(h)ch2ch2oh . fragment ions are observed following cid that result from loss of the same neutral , but differ by charge . for example , the most abundant peak ( m / z 511.6547 ) can be assigned to the loss of two ag2 neutrals ; i.e. , [ m 2ag2 + 6h ] and the peak at m / z 613.7838 can be assigned to [ m 2ag2 + 5h ] . in general , we observe series of peaks that can be assigned to combined losses of various stoichiometries of ag2 and bg2 for charge states + 6 through + 3 . for example , for the + 5 charge state , the following ions were detected : [ m ag2 + 5h ] , [ m bg2 + 5h ] , [ m 2ag2 + 5h ] , [ m ag2 bg2 + 5h ] , [ m 2bg2 + 5h ] , [ m 3bg2 + 5h ] , [ m 2ag2 bg2 + 5h ] , [ m ag2 2bg2 + 5h ] , [ m 4ag2 + 5h ] , [ m 3ag2 bg2 + 5h ] , [ m 2ag2 2bg2 + 5h ] , and [ m ag2 3bg2 + 5h ] . in all dissociation reactions , hydrogen migration followed by cleavage at tertiary amine ( fragments bg2 , bg1 , bg0 ) or between two carbon atoms ( fragments ag2 comparison of our results for [ m + 6h ] ions with those obtained for both singly and doubly charged precursors by brodbelt et al . suggests that cid of pamam dendrimers does not depend on the number of mobile protons present . the collision - induced dissociation of [ m + 4h ] ions of pamamg1nh2 ( figure 2 , bottom ) , is dominated by loss of water ; loss of two neutral fragments ag1 [ ch2=c(oh)n(h)ch2ch2nh2 , m = 102.0793 , equivalent to ag2 in pamamg2oh ] and bg1 [ ch2=ch c(o)n(h)ch2ch2nh2 , m = 114.0793 , equivalent to bg2 in pamamg2oh ] ; and combined losses of water and ag1 and bg1 in various stoichiometries ( see supplementary table s-4 ) . such fragmentation behavior is quite surprising , particularly with regard to the cid of pamamg2oh , in which no loss of water was observed . we speculate that the unusual cid behavior of pamamg1nh2 dendrimer proceeds as shown in scheme 4 . the loss of water is pronounced probably because p is particularly stable as a result of charge delocalization between the two nitrogen atoms . this idea is supported by a recent study of deamination and dehydration processes of n - terminal glutamine in cid of protonated peptides . when mobile protons are present the predominant neutral loss process from n - terminal glutamine is elimination of water because of formation of a protonated five - member aminopyrroline ring . when no mobile protons are present , deamination is observed as a result of formation of a neutral pyrrolidinone ring . both reactions depend on the charge state and stability . in the present case of quapruply protonated pamamg1nh2 ions , four protons are present and a maximum of four molecules of water are eliminated during cid . in contrast , cid of both singly and doubly charged pamamg1nh2 ions resulted in loss of ammonia but not loss of water . neutral losses observed in the cid of pamamg1nh2 depend on the charge state ; for higher charge states , the dominant process is loss of water and , for lower charge states , elimination of nh3 is observed . unlike ecd , the results of the cid experiments performed for pamamg2oh and pamamg1nh2 suggest a strong dependence of the dissociation processes on the nature of the surface groups . for pamamg1nh2 , the pamam dendrimer , generation 0.5 , with eight sodium carboxylate surface groups and ethylenediamine core ( pamamg0.5coona ) , forms multiply charged anions via negative electrospray . the dendrimer ions have mixtures of sodium and proton counterions because protons present in the meoh / nh4oh solution compete with sodium ions associated with the surface groups . within each charge state , mixtures of counterions were found according to the formula [ m ( n + m)na + mh ] , where n is charge state ( n = 24 ) , and m is the number of protons replacing sodium in the surface groups . figure 3 shows the edd and cid mass spectra obtained from [ m 5na + 2h ] ions . the edd mass spectrum ( figure 3 , top ) is dominated by peaks corresponding to the loss of one , two , and three t fragments [ ch2=ch c(o)o ] ( m = 71.01333 ) , which results from cleavage at an outer tertiary amine . loss of t fragments is also observed following cid ( figure 3 , bottom ) . the only peak present in the edd mass spectrum , but not present in the cid mass spectrum , occurs at m / z 497.7054 and can be assigned to the [ g1/2(in ) ] fragment ( m / zcalc 497.7080 ) . this fragment results from cleavage at the tertiary amine , which was also a major fragmentation channel in the ecd of pamam cations . no fragments corresponding to cleavage of the amide bond or c / z - type cleavages were observed following edd . we have investigated the electron - mediated dissociation ( ecd and edd ) of three pamam dendrimers with the aim of determining the effect of the macromolecular properties ( number of generations ) and the nature of the surface group . in all cases , fragmentation was dominated by cleavage at the tertiary amines with some amide bond cleavages ( ecd ) . the c / z - type dissociation , prevalent in ecd of peptides and proteins , is observed only as a minor channel . the results suggest that ecd ( and edd ) are independent of the nature of the surface group , but tend to occur within the innermost generations . in contrast , cid of the pamam dendrimers tends to occur in the outermost generation and is strongly dependent on the nature of the surface group . in comparison with cid the results demonstrate the potential utility of ecd as a tool for the structural analysis of pamam dendrimers .
purposeto determine the ocular biometric characteristics during the menstrual cycle using the optical low - coherence reflectometry ( olcr ) biometry.methodstwenty-two healthy women between the ages of 19 and 36 years with regular menstrual cycles were enrolled in this prospective study . subjects with irregular menstrual cycles , those taking contraceptive pills , those with a history of ocular surgery or trauma , and women unable to cooperate with the ocular biometry device were excluded from this study . a complete ophthalmic examination was performed between 8.30 and 10.30 am for all participants . also , central corneal thickness , axial length , anterior chamber depth , lens thickness , and keratometric measurements were made at the same time using the olcr device . measurements were taken at the beginning of the cycle ( 13 days ) , at ovulation ( 1216 days ) , and at the end of the cycle ( 2632 days).resultsthe mean age of the participants was 22.864.22 ( range : 1836 ) years . the difference in central corneal thickness , axial length , anterior chamber depth , lens thickness , and keratometry values were not statistically significant during the menstrual cycle.conclusionthe ocular biometric parameters did not significantly vary during the menstrual cycle according to the olcr biometry .	sex steroid hormone receptors are located in various human ocular tissues , such as cornea , iris , ciliary body , lens , conjunctiva , retina , lacrimal and meibomian gland , in both males and females.1,2 epidemiological studies show that sex hormones act differently according to sex and increase the incidence of age - related cataract , glaucoma , dry eye , neovascular age - related macular degeneration , central serous chorioretinopathy , etc.35 there are variations in the levels of the sex steroid hormones during the menstrual cycle . thus , some researchers have shown that these hormone fluctuations show correlation with the ocular tissue variables.6,7 on the other hand , some researchers advocate that these fluctuations do not affect ocular variability significantly.8 the ocular biometric parameters may change during the different phases of the menstrual cycle . they found that ophthalmic artery perfusion is increased after administration of intranasal 17-beta - estradiol in postmenopausal women . the lenstar ls 900 optical biometer is a device that is based on optical low- coherence reflectometry ( olcr ) technology . the lenstar biometer measures central corneal thickness ( cct ) , anterior chamber depth ( acd ) , lens thickness ( lt ) , axial length ( al ) , keratometry , pupil diameter , and white - to - white distance.10 the aim of our study was to evaluate the ocular biometric characteristics during the menstrual cycle using the lenstar ls 900 biometer . twenty - two healthy women between the ages of 19 and 36 years with regular menstrual cycles were enrolled in this prospective study . the study protocol was approved by adnan menderes university s ethics committee and complied with the tenets of the declaration of helsinki . subjects with irregular menstrual cycles , those taking contraceptive pills , those with a history of ocular surgery or trauma , and those unable to cooperate with the biometry device were excluded from this study . subjects were asked to define the phases by counting forward the days from the start of menses . also , biometric measurements ( cct , al , acd , lt , and keratometric values ) were made at the same time using the olcr device . both eyes were measured , but only the right eye was taken to the study . two - way analysis of variance for intergroup comparisons was used for normally distributed variables and descriptive statistics are shown as mean standard deviation . for variables which were not in accordance with normal distribution , the friedman test was used for intergroup comparisons , and descriptive statistics are shown in median ( 25th to 75th percentile ) format . two - way analysis of variance for intergroup comparisons was used for normally distributed variables and descriptive statistics are shown as mean standard deviation . for variables which were not in accordance with normal distribution , the friedman test was used for intergroup comparisons , and descriptive statistics are shown in median ( 25th to 75th percentile ) format . the mean cct , al , acd , lt , and kerato - metric measurements ( steep and flat keratometry readings ) of the right eye are summarized in table 1 ( two - way analysis of variance ) . measurements were made at the beginning of the cycle ( 13 days ) , at ovulation ( 1216 days ) , and at the end of the cycle ( 2632 days ) , on different menstrual cycle days , for 1 month ( table 2 ) ( two - way analysis of variance ) . the cct and al were thinnest at the beginning of the cycle and increased steadily until the end of the cycle , but these differences were not statistically significant ( p=0.498 and p=0.421 , two - way analysis of variance ) . conversely , the lt was the thickest , and steep and flat keratometry readings were the highest , at the beginning of the cycle and decreased regularly at the end of the cycle ( p=0.179 , p=0.892 , p=0.434 , two - way analysis of variance ) . the acd was reached the thickness value at the middle of the cycle ( table 2 ) ( friedman test ) . changes in the anterior segment parameters during the menstrual cycle are referred to in the literature . some researchers claim that these variations are significant , while others advocate that these differences are inconsiderable . they reported the cornea was thickest at the end of the cycle and thinnest at the beginning.11 on the other hand , hashemi et al investigated corneal thickness , corneal curvature , and acd during the menstrual cycle using the scheimp - flug imaging technique.12 they found no significant difference in measurements during the menstrual cycle period . corneal biomechanical parameters like corneal resistance factor and corneal hysteresis are determined by the ocular response analyzer . goldich et al reported that corneal resistance factor and corneal hysteresis decreased at the ovulation phase of the menstrual cycle.13 the cct was thickest at the end of the cycle and thinnest at the beginning.13 seymenolu et al determined corneal biometric properties during the menstrual cycle by using the ocular response analyzer.14 in contrast to goldich et al s study , they could not find differences in corneal biomechanical properties and intraocular pressure during the menstrual cycle . the lenstar is an optical biometer that provides cct , acd , lt , al , and keratometric measurements . the lenstar uses the olcr measurement principle and allows fast , comfortable , noncontact , less user - dependent , highly reliable , and reproducible measurements.15 recently , uakhan et al compared the corneal curvature and acd measurements using the lenstar ls 900 , pentacam ( oculus , wetzlar , germany ) , and a manual keratometer in healthy eyes.16 the authors reported good correlation between the lenstar and pentacam for measuring the acd and corneal curvature . another study , by cruysberg et al evaluated the reproducibility of the lenstar ls 900.17 the cct and acd measurements were compared between the lenstar ls 900 and visante as - oct . acd and al measurements , keratometric readings , and chamber depth measurements were compared between the lenstar ls 900 and iol master . they found differences between the lenstar ls 900 , iol master , and visante as - oct , and they did not recommend the use of these three optical devices interchangeably . on the other hand , they showed that the lenstar ls 900 is an excellent reproducible optical biometric device for all reported measurements . in our study , cct , al , acd , lt , and keratometric values were found to change during the menstrual cycle . some measurements were highest at the beginning of the cycle ( lt and keratometric values ) , some measurements were highest at the middle of the cycle ( acd ) , and other measurements were highest at the end of the cycle ( cct and al ) . secondly , different phases of the menstrual cycle should be taken into consideration to avoid inconsistent results of changes in ocular variables . lastly , we could not perform a multivariate regression model in order to evaluate the role of confounding factors ( cardiovascular risk factors , the pharmacological history , etc ) on results . biometric measurement is important in refractive surgery calculation , in intraocular lens selection , prior to cataract surgery , for eyeglass or contact lens prescription , etc . the lenstar ls 900 biometric measurement device is not affected by the variations of hormonal changes . therefore , measurement with the lenstar ls 900 can be used reliably in healthy women without reckoning with variations of hormonal changes during the menstrual cycle .
trigeminal meningoceles , lateral to the maxillary nerve ( v2 ) , have seldom been reported as underlying pathology for spontaneous rhinoliquorrhea . in contrast to sphenoid meningoceles arising from a persistent lateral craniopharyngeal canal ( sternberg cruveilhier , medial to v2 ) , their occurrence seems to be generated by addition of erosive processes to the constitutively thin bony shell underneath the semilunar ganglion , lateral to the round foramen ( and v2).the developmental and anatomical relationships of trigeminal meningoceles to the sphenoid bone are depicted , and in a review of the literature we present the different surgical approaches employed for sealing the dura leak . in view of these techniques we discuss an unusual case of therapy - resistant rhinoliquorrhea with left - sided trigeminal meningocele involving the meckel cave at the lateral sphenoid and reaching the superior orbital fissure and the medial orbital space.in contrast to patients who have lateral sphenoidal meningoceles with a persistent lateral craniopharyngeal canal ( sternberg cruveilhier ) , who can be treated successfully using an endoscopic transsphenoidal approach ( recurrence rate 13.7% ) , the recurrence rate of cerebrospinal fluid ( csf ) efflux for trigeminal meningoceles lies much higher ( endoscopically 66% , open craniotomy 33% ) . the surgical strategy thus has to be chosen individually , taking into account specific anatomical situations and eventually preceding operations .	among the different entities causing rhinoliquorrhea in closest proximity to the sphenoid bone , trigeminal meningoceles ( tms ) , lateral sphenoidal meningoceles ( lsms ) , and the persistence of the lateral craniopharyngeal canal ( sternberg cruveilhier canal , sc ) have been described . tms have only been reported several times in the literature , and nomenclature is still heterogeneous . in general , reports cover various mass lesions in the meckel cave , ranging from lipoma and meningioma to schwannoma . only a few deal with meningoceles in the pterygopalatine region , such as in patients with typical stigmata of neurofibromatosis type 1 ( nf 1).1 2 tms are localized in the lateral sphenoid wing lateral to v2 and the foramen rotundum underneath the semilunar ganglion , reaching to the superior orbital fissure , pterygopalatine fossa , and medial orbital space , mostly emerging from an enlarged meckel cave and giving rise to osseous erosion of the sphenoid wing . as a different and more frequent reason for rhinoliquorrhea , lsms have been described . they lie medially to v2 near the base of the sphenoidal bone3 4 5 6 7 8 and can be distinguished from tms by the absence of orbital or pterygopalatine fossa involvement . the third dura leak associated pathology of this region is the developmental anomaly of a persistent sc embryonal canal ( lateral craniopharyngeal canal ) . still controversially discussed in literature , it has been found in some cases as an underlying condition for lsms.9 10 11 the canal lies medially to the foramen rotundum and extends from the maxillary nerve root ( v2 ) to a recess of the lateral sphenoid wall , opening into the sphenoid sinus . from an anatomical point of view , there are several weak spots in the embryonic development of the sphenoidal bone , which can explain the occurrence of meningoceles or spontaneous rhinoliquorrhea , especially in the area of the trigeminal ganglion and the carotid artery . the cartilage precursor of the skull base has to form around the cranial nerves , arteries , and veins while respecting their lumen . this can lead to thinned cranial base structures that might be eroded during a lifetime by inflammatory processes or increased intracranial pressure.12 the bone formation itself is inhomogeneous . the collision zone between endochondral ossification ( lesser wing ) and intramembranous ossification ( greater wing ) lies laterally to the foramen rotundum , extending to the region of the foramen ovale ( fig . study , 25 patients with lateral sphenoidal cerebrospinal fluid ( csf ) leaks were all shown to have the bony defects in this area , lateral to the foramen rotundum.8 cranial base in a 6-month - old embryo shown from above ( modified from fenart and landouzy ) depicting the four main parts forming the sphenoid bone . cranial nerves : ii , v1 , v2 , v3 , vii , ix , x , xi , xii . alisph , alisphenoid ; boc , basioccipitale ; bsph , basisphenoid ; fm , foramen magnum ; fz , fusion zone between ( lateral ) and endochondral ( medial ) ossification ; orbsph , orbitosphenoid ; petr , petrous bone ; prsph , presphenoid ; scc , site of the sternberg cruveilhier canal . the sutures between the ossification centers can leave small basal clefts , such as the lateral craniopharyngeal canal ( sc ) . the canal represents a remnant of the fusion zone between the alisphenoid ( greater wing ) and the basisphenoid ( fig . 1).13 development of the sphenoid bone is crucial for the whole cranial base.14 15 16 distorted midline structure development can result in encephaloceles and palate cleft malformations,17 18 19 and midline basal encephaloceles are known as very rare malformations.11 20 lateral sphenoid malformations give rise to lateral sphenoid meningoceles , as well as to meningoceles of the trigeminal nerve . the meningoceles seem to be associated with sphenoid bone malformations , increased intracranial csf pressure , and accompanying erosive processes . as shown in the following case description , tms can result in rhinoliquorrhea and csf leakage to the pterygopalatine fossa and periorbital fatty tissue , followed by exophthalmos and conjunctivitis together with conjunctival edema . in literature , many different terms have been employed to describe the pathology of a tm . arachnoid cyst of meckel 's cavity appears to be imprecise , because lsms also arise from it . the terms transalar sphenoid meningocele and transsphenoidal and transethmoidal meningoceles have been used synonymously in literature.19 20 we suggest the term lateral sphenoidal meningocele to describe pathologies medial to v2 and the term trigeminal meningoceles as anatomically more precise for lesions lateral to the foramen rotundum and v2 . she also reported a left - sided orbital swelling , especially when bending forward or in prone position . her first meningitis had been at the age of 17 years , a second occurred a few years later , and both had been treated successfully with antibiotics . a former traumatic head injury was denied and basal cranial fractures had been ruled out with multiple imaging techniques . extended neuroradiological imaging included cisternographic magnetic resonance ( mr ) scan after intrathecal gadolinium - application in prone position . it revealed a left - sided csf leak along a csf - containing enlargement of the temporal fossa that extended into the pterygopalatine fossa . origin of the fistula was suspected in the temporopolar parasellar region in close proximity to the cavernous sinus and to the meckel cavity . the clivus , sellar area , and ethmoidal cells appeared to be anatomically altered in the ct - scan ( fig . ( a ) preoperative magnetic resonance ( mr ) t1-weighted axial images enhanced by cisternography . ( b ) oblique reconstruction parallel to the optic canal , ( c ) axial and ( d ) coronal reconstruction . the surgical strategy was discussed in the multidisciplinary skull - base board and under suspicion of a temporomedial meningocele of the maxillary nerve , a pterional craniotomy with transsylvian approach and closure of the parasellar entry point of the meningocele was advised . in a first operation , the suspected entry point of the fistula the patient then decided to be treated in another neurosurgical department , where a second pterional operation was performed without relieving the symptoms . about 2 years after first surgery , the patient decided to restart treatment in our institution . intrathecal contrast - enhanced ct revealed the refilled fistula and an enlarged , csf - containing space in the paraclival region , close to the maxillar nerve and the meckel cave . a third pterional exploration was proposed , but the patient opted for conservative therapy . only after increasing orbital swelling and reappearance of rhinoliquorrhea fusion of the ct dataset with neuronavigation pictures allowed identification of the entrance of the meckel cavity . the wall of the cavity showed a cisternlike arachnoid covering in which the nerve fibers crossing the cavity were partly adhering to the basal arachnoid layer of the cyst and were spread apart . two walls of the cavity were found , corresponding to arachnoid cystic structures and dural tissue , thus displaying typical criteria for meningoceles . to close the fistula , abdominal fat and muscle tissue were harvested and the periarachnoidal space of the meckel cave was filled in proximal and distal direction . hereafter , fat tissue was positioned in the remaining arachnoid space rostrally . between fat and muscle tissue , liquid dura glue ( duraseal xact , covidien , mansfield , massachusetts , usa ) the opening of the cavity was sealed with tachosil ( takeda pharmaceuticals ; zurich , switzerland ) and surgicel ( ethicon , somerville , new jersey , usa ) . after this intervention , rhinoliquorrhea and orbital swelling disappeared and the third nerve palsy nearly completely recovered within 3 months . however , about 4 months postoperatively the patient experienced an intermittent csf leak . shortly thereafter , orbital swelling and conjunctivitis reoccurred . t2- and ciss 3d t2-scans revealed a partly occluded , yet csf - containing , meningocele . the patient refused any further interventions , especially implantation of a ventriculoperitoneal ( vp ) shunt to reduce the intracranial csf pressure , and she was lost to follow - up . review of the literature was performed in the medline database using the search terms trigeminal , encephalocele , meckel 's cave , sphenoid , pterygopalatine , csf fistula , and sternberg in all combinations of two keywords . search resulted in 38 relevant cases from 23 reports , ranging from 1888 to 2011 ( table 1 ) . within these 38 reports , patients presented with rhinoliquorrhea in 92.1% ( 35 patients ) , headaches in 34.2% ( 13 patients ) , and meningitis in 15.7% ( 6 patients ) . abbreviations : csf , cerebrospinal fluid ; lsm , lateral sphenoid meningocele ; sc , sternberg cruveilhier canal ; sof , superior orbital fissure ; tm , trigeminal meningocele . out of 38 patients , 6 had a tm , whereas a persistent sc or lsm was found in 32 patients . a total of 29 patients underwent endoscopic surgery for lsms , whereas in 3 cases the transcranial approach was preferred . in two cases4 transcranial reoperation was performed after endoscopic surgery due to recurrence of csf leaks.4 recurrence rate was 13.7% ( 4 of 29 cases ) . from the six tms , three were treated by an endoscopic transsphenoidal approach , with recurrence of csf leaks in two of them ( 66% ) . one of three patients undergoing craniotomy and open repair experienced recurrent therapy - resistant csf leakage ( present case , 33% ) . apart from persistent rhinoliquorrhea , the most common complications in the endoscopic group were meningitis ( 6.3% ) and maxillary nerve irritation ( 3.1% ) . in the craniotomy group , the case presented here illustrates a rare tm extending from an enlarged meckel cave into the medial cranial and pterygopalatine fossa , causing therapy - resistant csf leakage and orbital affection with an unusual collection of csf in the temporal muscle ( fig . although the predominant symptoms of csf leaks such as rhinoliquorrhea , headache , and meningitis do not help in localizing the dural defect , clinical appearance may vary with the localization of the specific arachnoidal cyst , meningocele , or encephalocele . for example , arachnoid cysts of the region of the meckel cavity often become symptomatic with facial numbness or pain due to their relation to the trigeminal nerve.21 the therapeutic difficulties of that unusual entity have not been solved . in contrast to the far lateral tms , lsms and persistent sc can be treated successfully with a transsphenoidal approach . this may be caused by their more medial localization , medially to the foramen rotundum and v2 at the sphenoid base . patients with tms are likely to have a greater benefit from a transcranial approach , which provides better access to the lateral aspects of the meckel cave and the semilunar ganglion . this is underlined by the higher recurrence rate when using the transsphenoidal approach ( table 1 ) . alternatively , transmaxillar transpterygoid approaches may be employed for tms with a limited pterygoid fossa leakage.1 csf leaks associated with tms can be assumed to be of idiopathic origin with an elevated intracranial pressure ( icp ) as an additional factor . thus , icp recording and icp normalization should be taken into account , and routine use of lumbar drainage as a diagnostic and therapeutic measure has been proposed in lateral sphenoid csf leaks.22 in light of the reviewed literature , together with the presented therapy - resistant case , alternative strategies such as implantation of a vp shunt or temporary lumbar drainage to reduce csf pressure have to be discussed . the occurrence of the spontaneous csf leak as part of an idiopathic hydrocephalus syndrome should be considered , with acetazolamide or furosemide being a treatment option.22 however , the one case treated conservatively experienced persistence of symptoms , similar to our patient , who experienced recurrence after deciding for nonsurgical follow - up ( table 1 ) . midfacial degloving has been suggested as approach to the pterygopalatine fossa meningoceles and remains an option for recurrence.21 a staged treatment algorithm using lumbar csf drainage , icp recording , and invasive location of the skull base defect with intrathecal administration of contrast medium seems to be most promising . depending on the location and anatomical shape of the tm , open craniotomy or endoscopic transpterygoid approach can be selected.6 7 8 22 whereas for medial pathologies ( sc , lsm ) endoscopic transsphenoidal approaches appear to be advantageous , tms should be primarily considered for an open craniotomy . alternatively to transcranial approaches , transfacial or transmaxillary / transpterygoid approaches have to be considered as treatment for recurrent , extended tms involving the pterygopalatine fossa , in which transsphenoidal endoscopic techniques have limited success rates . in case of therapy - refractory csf leaks and marked elevation of icp , vp shunt placement and/or medical treatment should be considered before reintervention.22 compared with the spontaneous rhinoliquorrhea caused by a persistent sc or lsms , the point of leakage in tms lies laterally to v2 and can not always clearly be visualized , even by sophisticated neuroradiological techniques . this can result in considerable technical difficulties and therefore therapeutic decisions should follow the advice of a multidisciplinary skull base team after taking into account the individual anatomic situation of a patient , preceding operations , and specific risk factors for elevated icp .
diagnosis of palatal swellings is a challenge . benign and malignant tumors may be misinterpreted as lesions of inflammatory origin . we present a case of b - cell non - hodgkin lymphoma on the palate of a 40-year - old male . a number of factors can make the diagnosis of oral lymphoma difficult . many lymphomas are extranodal , there is usually a prominent superimposed nonspecific inflammatory response and thus they mimic benign lymphoid hyperplasia . it is important for the pathologist to be familiar with features that distinguish benign from malignant lymphoid proliferations .	the palate is a complex area of the mouth , with a variety of native tissue types that give rise to a plethora of pathological conditions , both benign and malignant [ table 1 ] . they are generally classified as either hodgkin lymphoma or non - hodgkin lymphoma ( nhl ) and may be of either b - lymphocyte or t - lymphocyte origin . lymphoma is the second most common neoplasm of the head and neck region after squamous cell carcinoma . nearly 24%48% of nhl can arise in extranodal locations , and 3%5% are primarily located in the oral cavity . differential diagnosis of palatal swellings oral lymphomas are relatively rare and are often difficult to diagnose as they may mimic other pathologies such as periodontal diseases , osteomyelitis , or some other malignancy . lymphoid lesions of the palate can be divided into three categories , the management and prognosis of each category being different : primary lymphoma of the palate , with no other lymphomatous lesion detected elsewhere in the bodylymphoma of the palate occurring as one of the lesions in a case of disseminated lymphomabenign lymphoid hyperplasia ( blh ) of the palate primary lymphoma of the palate , with no other lymphomatous lesion detected elsewhere in the body lymphoma of the palate occurring as one of the lesions in a case of disseminated lymphoma benign lymphoid hyperplasia ( blh ) of the palate in addition , histologically , lymphoid lesions of the palate may be misinterpreted as inflammatory in nature . the purpose of our report is to present a case of b - cell lymphoma on the palate and distinguish it from benign lymphoid hyperplasia ( blh ) . a 40-year - old man with a swelling in the right palatal region was referred to the department of oral pathology for evaluation and diagnosis . intraoral examination exhibited a firm , exophytic , oval mass with an intact overlying mucosa in the region of the right hard palate measuring 3 4.5 cm in size [ figure 1 ] . there were no signs of ulceration , bleeding , discharge , or numbness in the area . the patient did not have the habit of chewing tobacco or betel nut . on general examination , he was found to be afebrile , with no palpable lymph nodes in the head and neck region . he did not mention any sudden weight loss in the recent past and the medical history was noncontributory . clinical photograph showing swelling on the palate ct scan revealed a mass on the right side of the hard palate , with no involvement of the maxillary sinus [ figure 2 ] . computed tomography scan of patient an excisional biopsy [ figure 3 ] was performed under local anesthesia . and a bony crater - like defect was seen on the palatal bone after soft tissue removal . histopathological examination of sections of the resected specimen revealed an intact stratified squamous epithelium with underlying vaguely follicular and diffuse proliferation of lymphoid cells [ figure 4a and b ] . the follicle - like structures were composed of central large cells ( giving a washed - out appearance ) surrounded by a thin rim of small , round lymphocytes . the central large cells had abundant pinkish cytoplasm and convoluted nuclei with inconspicuous nucleoli [ figure 5 ] . epimyoepithelial islands were also observed . on microscopic examination of hematoxylin and eosin ( h and e)stained sections , there was a conflict of opinion over the distinction between benign ( reactive ) lymphoid hyperplasia ( pseudolymphoma ) and non - hodgkin lymphoma . the large lymphoid cells showed immunoreactivity for cd20 and the rim of small lymphocytes were positive for cd5 [ figure 6 ] . the large lymphoid cells were negative for cd5 , bcl-2 , and cd10 [ figures 79 ] . photograph of gross excisional tissue ( a ) the overlying epithelium is seen separated from follicles . the histological picture in this view gives the impression of reactive follicular hyperplasia of the lymphoid tissue ( h and e , stain ; original magnification , 2.5 ) . ( b ) high - power view of the follicular pattern gives the impression of a reactive lesion ( h and e , stain ; original magnification , 10 ) magnified view showing central larger cells with mitotic figures , convoluted nuclei , and inconspicuous nucleoli . peripherally , small round lymphocytes are seen ( h and e , stain ; original magnification , 20 ) the large lymphoid cells show positive immunoreactivity for cd20 , whereas the peripheral small lymphocytes are negative . inset shows control stain ( original magnification , 10 ) the peripheral small lymphocytes show positive immunoreactivity for cd5 , whereas the central large cells are negative . inset shows control stain ( original magnification , 10 ) the peripheral small lymphocytes show positive immunoreactivity for bcl2 , whereas the central large cells are negative . inset shows control stain ( original magnification , 10 ) negative immunoreactivity for cd10 ( original magnification , 10 ) immunohistochemistry profile showing very low ki67 proliferative index ( original magnification , 20 ) the patient was referred to a general physician for systemic evaluation and was found to be free of other systemic manifestations . a final diagnosis of low - grade b - cell lymphoma of mucosa - associated lymphoid tissue ( malt ) was accorded based on the clinical , radiographic , histopathologic , and immunohistochemical investigations . lymphoma constitutes a diverse and complex group of malignancies of lymphoid histogenesis . according to the revised european american classification of lymphoid neoplasms ( real ) , classification the category of marginal zone b - cell lymphoma includes lymphomas that were originally designated as monocytoid b - cell lymphoma and low - grade b - cell lymphoma of malt . both these neoplasms were thought to represent different clinical presentations of a b - cell lymphoma believed to arise from normal marginal zone b - cells in the lymph node or their extranodal counterparts , respectively . the most frequent location of extranodal lymphoma in the head and neck is the palate . many salivary gland lymphocytic infiltrates of the palate are usually non - hodgkin b - cell lymphomas of malt . extranodal marginal b - cell lymphomas of malt ( b - malt ) are known to show histologic features similar to benign lymphoid hyperplasias ( blh ) . in the past , many low - grade lymphomas were probably misdiagnosed and inappropriately categorized as blh . extranodal infiltration composed of small round or slightly irregular lymphoid cells often admixed with plasma cells , histiocytes and lymphoid follicle were classified as pseudolymphomas since clinical studies showed that in patients with these lesions , the disease pursued an indolent clinical course . the term pseudolymphoma / benign lymphoid hyperplasia should be restricted to tumefactive lesions with prominent lymphoid infiltrate that by routine morphology appears to be reactive and that on ancillary immunophenotypic and molecular genetic analysis lacks evidence of clonality . the subtle distinctions in interpretation of the histological characteristics between benign and malignant lymphoid proliferations makes the diagnosis of these lesions difficult . several papers have dealt with the histologic features of blh and nodular lymphoma.[1214 ] fortunately , with the advent of advanced immunophenotyping and molecular genetic techniques , it is now possible to differentiate the two lesions . table 2 lists out the essential features that help us distinguish blh from b - cell malt lymphomas . lymphoid hyperplasia vs b.cell malt lymphoma blh is characterized by numerous well - demarcated germinal centers of varying size and shape often rimmed by a mantle of small lymphocytes . while many b - cell lymphomas have a nodular growth pattern , these nodules are neoplastic and should not be considered germinal centers . in addition , in lymphomas , these neoplastic nodules tend to be ovoid to round and show little variation in size . in blh , the cells demonstrate all stages of follicular center cell transformation , including plasma cells . mitosis may be abundant , although never atypical and never seen outside the germinal centers . the cells comprising malignant lymphomas , on the other hand , are neoplastic and do not show the wide range of cell types seen in reactive lesions . nuclear atypia as well as mitosis ( occasionally atypical ) may be seen , but the mitotic rate rarely approaches that seen in benign lesions . in our case , the follicle - like structures showed variation in size and were composed of central large cells surrounded by a thin rim of small , round lymphocytes . mitotic activity was found to be quite low . hence , it was quite difficult to give a confirmative diagnosis solely on the basis of routine histopathologic examination . therefore we performed immunohistochemical analysis on the lesional tissue for the following markers : cd20 , cd5 , cd10 , bcl2 , and ki-67 . the result was positive for cd20 and negative for cd5 , cd10 , and bcl2 , while ki-67 showed positivity of 1% . immunophenotypic studies have shown that low - grade b - cell lymphomas of malt typically do not express the b cell - associated antigens cd10 , cd21 , or cd23 , or the t - cell antigens ( including cd5 ) . these findings were consistent with the immunohistochemistry profile of our case and thus confirmed our diagnosis of low - grade b - cell lymphoma of malt . patients with localized low - grade b - cell lymphomas follow a very indolent clinical course . therapeutic strategies for this kind of disease are not standardized yet due to the small number of malt lymphomas described in the head and neck region . remedial measures include combined radiochemotherapy for advanced cases and either radiation therapy or surgery for small lesions . our patient underwent surgical excision of the lesion and continues to be well after 6 months of follow - up . a number of factors can make the diagnosis of oral lymphoma difficult , despite the fact that the histological features are the same as at other sites . many lymphomas are extranodal and there is almost always a prominent superimposed , nonspecific , inflammatory response . clinically obvious lymphomas present primarily to the medical rather than the dental practitioner or oral surgeons . hence to see a spectrum of lymphomas in oral pathology practice is unusual . early recognition and biopsy are extremely important because the lymphoma may be confined entirely to the palate in the early stages and such localized palatal lymphomas respond well to irradiation , whereas disseminated disease necessitates chemotherapy . malignant b - cell lymphoma mimics blh , both clinically and histologically . the pathologist must be familiar with the features that distinguish between these two diseases .
clear cell odontogenic carcinoma ( ccoc ) is a rare jaw tumor that was classified as a malignant tumor of odontogenic origin in 2005 by the world health organization because of its aggressive and destructive growth capacity and metastasis to the lungs and lymph nodes . we report a case of a 66-year - old female who had swelling , incision and drainage history and a well - defined unicystic radiolucent lesion that was comparable to a cystic lesion . at first , the patient received decompression , and the lesion size decreased . three months after decompression , cyst enucleation was performed . the pathologic result indicated that the lesion was ccoc . in this report we emphasize that patients with painful cystic lesions in addition to jaw enlargement and loosening teeth should be considered for the possibility of malignancy .	clear cell odontogenic carcinoma ( ccoc ) is a rare jaw tumor that was first described by hansen et al.1 in 1985 , in which patients presented with local bony invasions without metastasis . at first , ccoc was called a clear cell odontogenic tumor or clear cell ameloblastoma , and was considered more aggressive than ameloblastoma . the potential for ccoc to metastasize was initially unclear ; however , ccoc was defined as a benign tumor in the 1992 world health organization ( who ) classification2 . the who reclassified ccoc as a malignant tumor of odontogenic origin in 20053 because of its aggressive and destructive growth capacity and metastasis to the lungs and lymph nodes4,5,6,7,8 . about onethird of ccoc cases were initially misdiagnosed , and the primary diagnosis was ameloblastoma which lead to inadequate treatment for some patients . in this case report , we describe a case of a ccoc , which presented in a similar manner to a cystic lesion ; the patient was misdiagnosed and received insufficient treatment . we introduce this case to discuss differential patient presentations to reduce the rate of malignancy misdiagnosis . a 66-year - old female visited the department of oral and maxillofacial surgery at yonsei dental hospital due to swelling and pain on the right premolar maxillary area . clinical examination showed fluctuating swelling on the right maxillary vestibule with tenderness to palpation , severe tooth mobility and loss of vitality on the first and second premolars and no appreciable cervical lymphadenopathy . a panoramic radiograph revealed an approximately 2729 mm well - defined radiolucency in the apex of the maxillary right premolars with root resorption.(fig . 1 ) a computed tomography ( ct ) scan showed a low attenuated cystic lesion with a volume of 7,419 mm ( simplant software ; materialise , leuven , belgium).(fig . 2 ) at first , the lesion was diagnosed as infected odontogenic cyst ( radicular cyst ) , the patient received decompression in order to separate the cyst from maxillary sinus wall . two months after decompression , a panoramic radiograph showed that the lesion size was reduced to approximately 2526 mm.(fig . 3 ) the lesion was reduced to a volume of 4,797 mm ( simplant software ) on the 3-month follow - up conebeam ct.(fig . 4 ) the measurement and comparison may not be precise because of the differences in ct imaging ; however , there was definite shrinkage of the lesion.(fig . 5 ) because there was not a change in the lesion size , a cyst enucleation was performed and specimens were sent to oral pathology . b ) magnetic resonance imaging of the neck and positron emission tomography ct , were taken to examine the lymph nodes and distant metastasis.(fig . an obturator was placed at the time of surgery and a 2.42.0 cmsized ccoc with a positive basal resection margin , massive bone marrow infiltration and lymphovascular permeation was reported.(fig . nearly 100 cases of ccoc have been reported in the englishlanguage literature to date , and these tumors have a higher reported incidence in females with an male / female ratio of 1 : 1.8 . most cases of ccoc have been diagnosed in patients over the age of 40 , with an average age of 54 years . the mandible is more frequently involved than the maxilla , and the posterior of the jaw is a more frequent site than the anterior site9,10,11 . radiographic findings show mainly radiolucent lesions with or without regular margins , although some cases exhibited a mixed radiolucent - radiopaque lesion . the likelihood of the recurrence depended on the method of initial therapy and the extent of the tumor invasion . three - quarters of the patients who were treated with conservative care ( curettage or enucleation ) had recurrence and onethird of the patients who were treated with resection experienced a recurrence . conservative care ( curettage or enucleation ) and presence of soft tissue involvement were associated with a higher recurrence rate10,12 . the ideal treatment approach for ccoc a wide resection with at least 1 cm of a tumor - free margin is recommended and when there is evidence broad soft tissue invasion , palpable neck lymph node , perineural invasion or tumor removal without free margins , adjuvant neck dissection and/or radiotherapy should be considered10,12,13 . however , except for squamous cell carcinoma and high - grade central mucoepidermoid carcinoma , neck dissection is rarely required for maxillary carcinomas without nodal metastases12,13,14 . in our case , the patient had swelling , incision and drainage history and a well - defined unicystic radiolucent lesion , that was comparable with a cystic lesion . at first we misdiagnosed the lesion as an infected cyst , performed decompression and were unable to send specimen for pathologic examination . our treatment was limited because we did not make a correct diagnosis when the patient first presented at our clinic . when decompression was performed , the clinical findings were similar to a cystic lesion and during follow the up period we found that lesion size decreased . currently , mass properties have not been described in previous reports , and our case study presented with both radiographic and clinical similarities to cystic lesion symptoms ; for these reasons our diagnosis was delayed . additional reports have indicated that many cases of ccoc are misdiagnosed because of its rarity and similarity to cystic lesions11,13 . in this report , we discuss a patient that presented with a painful cystic lesion , jaw enlargement jaw and loosening teeth . similar cases should be considered for the possibility of malignant ccoc to identify and treat patients with ccoc .
currently , the most promising strategy to improve the prognosis of advanced esophageal cancer is neoadjuvant chemoradiation ( crt ) followed by surgery . however , patients who achieved pathological complete response can experience more survival benefit . therefore , it is critical to identify the responders early in the course of treatment . published data demonstrate that clinic - histopathological factors , molecular biomarkers , and functional imaging are predictive of neoadjuvant therapy . the existing biomarkers , including epidermal growth factor receptors , angiogenetic factors , transcription factors , tumor suppressor genes , cell cycle regulators , nucleotide excision repair pathway , cytokines , and chemotherapy associated genes , need to be validated and novel biomarkers warrant further exploration . positron emission tomography ( pet ) is useful for differentiating the responders of neoadjuvant crt . the most valuable parameters and the time point of performing pet in the course of treatment remains to be elucidated . furthermore , predictive models incorporating the multiple categories of factors need to be established with a large , prospective , and homogeneous patient cohort in the future . standardization of staging , biomarker detection method , and image acquisition protocol will be critical for the generalization of this model . prospective , multi - center controlled trials , which stratified patients according to these predictive factors , will help guide individualized treatment strategies for patients with esophageal cancer .	according to the international agency for research on cancer , there were an estimated 482,000 incident cases of esophageal cancer ( ec ) with high mortality ( 84% ) around the world in 2008 1 . ec often infiltrates neighboring organs and easily metastasizes to lymph nodes , and as a result , the prognosis of locally advanced patients is extremely poor , with a 5-year survival of 15 - 34% 2 . more recently , preoperative chemoradiotherapy ( crt ) has gained popularity with physicians , as its tolerance is better than postoperative crt , permits downstaging and higher respectability in the subsequent surgery , and may eradicate occult distant disease . several randomized clinical trials have testified a significant survival benefit for neoadjuvant crt in patients with squamous - cell carcinoma or adenocarcinoma of the esophagus 2 - 4 . 5 recently conducted a meta - analysis , which included 24 clinical trials and 4188 patients with resectable esophageal carcinoma . the results showed that neoadjuvant crt provided an 8.7% absolute survival benefit at 2 years after surgery alone and 5.1% survival benefit after neoadjuvant chemotherapy . a proportion of patients show minor or no response to crt and are merely exposed to its toxicity . furthermore , neoadjuvant treatment produces a pathological complete response , and outcomes are better 6 . one retrospective study from stahl et al . 7 showed that the overall survival was significantly hampered in patients with residual tumor in their resected specimen compared with patients who showed a pathological complete tumor remission ( overall survival rate at 3 years 25.2% versus 65.6% ; hazard ratio [ hr ] = 3.50 ; 95% confidence interval [ ci ] 1.91 - 6.44 ; p < 0.0001 ) it is vital to select patients who will experience survival benefit from receiving neoadjuvant crt before treatment . furthermore , several studies have reported that surgery can be omitted in patients that achieved pathological complete response ( pcr ) after neoadjuvant crt 8 , 9 . in this article , we summarize the recent literature with respect to the predictive factors of neoadjuvant crt response and provide a review the progress of the field and future challenges to be expected . predictive biomarkers and functional imaging have become a hot technique for the individualized treatment of cancer . however , traditional clinical factors , such as tumor stage , patient age , and performance status , are still used to select the best therapy for a particular patient . 10 initially found that tumor invasion depth was the only clinical factor significantly correlated with response to preoperative chemotherapy for thoracic esophageal squamous cell carcinoma ( escc ) . 12 identified that age , baseline hemoglobin level , smoking habit , and tumor length were important pcr predictors in escc . in patients with esophageal adenocarcinoma , patel et al . 13 found that signet ring cell histology on pretreatment biopsy predicts a decreased likelihood of pcr and survival . 14 created a regression classification based on two parameters ( histomorphologic tumor regression and postoperative pathological node stage ) to predict the complete resections following neoadjuvant crt for ec patients . the following parameters were incorporated into this model : post - chemoradiation positron emission tomography ( pet ) standardized unit value ( suv ) , post - chemoradiation biopsy , sex , histologic tumor grade , and baseline endoscopic ultrasonography tumor stage . the area under the receiver - operating characteristic curve was 0.72 ( 95% ci : 0.662 - 0.787 ) . this model needs to be prospectively validated before it can be used in clinical practice . serum c - reactive protein ( crp ) as an inflammatory factor has also been evaluated in the treatment response prediction of ec patients . 16 was the first to demonstrate that serum crp levels during crt were closely associated with the pathological response , particularly in patients with elevated crp prior to crt , a decrease in crp within normal ranges 2 - 3 weeks following crt initiation predicted a favorable pathological response with the highest accuracy . a large spectrum of biomarkers at the level of alterations of genomic dna , gene expression of messenger rna ( mrna ) , micro - rna ( mirna ) , and protein expression have been identified and analyzed to predict the response of neoadjuvant therapy . cisplatin ( cddp ) and 5-fluorourcil ( 5-fu ) based neoadjuvant chemotherapy have been widely used in clinical practice . the excision repair cross - complementing 1 ( ercc1 ) gene codes for a nucleotide excision repair protein involved in the repair of radiation- and chemotherapy - induced dna damage . 18 testified that ercc1 mrna expression and ercc1 ( rs11615 ) gene polymorphisms correlated with treatment response to cddp - based chemotherapy . furthermore , rna expression levels of 5-fu metabolism - associated genes , thymidylate synthase ( ts ) , dihydropyrimidine dehydrogenase ( dpd ) , thymidine phosphorylase ( tp ) , methylenetetrahydrofolate reductase ( mthfr ) , as well as of cddp and taxane - related genes gluthatione s - transferase ( gstp-1 ) , caldesmon , and multi - drug resistance gene ( mrp1 ) have been testified to be predictors of response to neoadjuvant therapy 19 - 22 . mirnas are small noncoding rnas , which are involved in the regulation of gene expression by inhibiting messenger rna translation 23 . 24 conducted a comprehensive mirna profiling in 16 specimens with pre - neoadjuvant and post - neoadjuvant therapy , and the selected mirnas were verified in 80 ec patients . the results showed that mir-192 and mir-194 in pre - therapeutic biopsies are considered indicators of major histopathologic regression . moreover , in vitro assays showed that mir-296 and mir-200c expression correlated with chemotherapy resistance 25 , 26 . furthermore , mir-148a has been reported to improve response to chemotherapy in sensitive and resistant esophageal carcinoma cells 27 . long non - coding rnas ( lncrnas ) are a new class of non - protein - coding rnas , which are longer than 200 bases 28 . 29 initially explored the relationship between lncrna loc285194 and the response to neoadjuvant crt in escc and showed that the decreased expression of loc285194 indicated crt resistance and poor prognosis . other biomarkers , such as epidermal growth factor receptor ( egfr ) , vascular endothelial growth factor ( vegf ) , proliferating cell nuclear antigen ( pcna ) , p53 status , p21 status , bcl-2 , ki-67 , transcription factor nuclear factor kb ( nf - kb ) , and rad51 have been shown to correlate with response to neoadjuvant therapy in ec 30 - 39 ( table 1 ) . 30 adopted the proximity ligation assay ( pla ) followed by enzyme - linked immunosorbent assay ( elisa ) to identify serum biomarkers that predict treatment response of neoadjuvant crt in 79 escc patients . both methods testified that low pretreatment serum vascular endothelial growth factor - a ( vegf - a ) significantly correlated with pcr . however , the predictive value of transforming growth factor ( tgf)-1 was not validated by elisa . 22 also revealed that ts and dpd rna expression in the peripheral blood of ec patients could be highly specific predictors to identify a subset of patients who do not benefit from neoadjuvant chemoradiotherapy . it has been reported that chemoradiotherapy induces cancer cell death through tumor antigen - specific t cell response 40 . 41 conducted serum profiling of 84 cytokines in escc patients who received neoadjuvant crt plus surgery and revealed that increased serum soluble interleukin-6 receptor was correlated with a poor response to preoperative therapy . it is recognized that multiple gene alterations are involved in the development and progression of ec 42 . markers originating from different molecular levels , such as gene expression , mrna expression , protein expression , epigenetic modification , and mutation , have always been validated independently in separate studies . given that tumor cells interact at different levels in the organism , which interferes with angiogenesis , dna repair and apoptosis , cell cycle control pathways , or cell - to - cell communication pathways , analysis of one pathway alone can not cope with the complexity of the interacting tumor cells . whole genome microarray technology allows for high - throughput identification of gene expression profiles in cancers 43 . this approach had already been used to identify genes that could serve as biomarkers of neoadjuvant crt response prediction . table 2 summarizes the whole genome profile - related studies regarding the neoadjuvant crt response prediction . 44 initially identified a combination of three differentially expressed genes ( perp , s100a2 , and sprr3 ) that allowed for the discrimination between pcr and < pcr with sensitivity and specificity of 85% after profiling pretreatment cancer biopsies from 19 ec patients that received neoadjuvant crt . 45 performed cdna microarrays of 46 pretreatment endoscopic biopsy samples and identified a 32-gene classifier that can be used to predict the response to crt in escc . 46 performed gene expression profiling on pretreatment samples of escc patients who received chemotherapy , and constructed a diagnostic system with 199 most informative genes that showed 82% accuracy . 47 proved that the ephrin b3 receptor , a differentially expressed gene via microarray , is related to the neoadjuvant chemotherapy response . 48 identified two novel markers , cul2 and stk11 using human genome microarrays , for response prediction in ec . 49 established a five gene based model that predicted the response to neoadjuvant crt with 95% accuracy in 74% of ec patients . 50 also performed gene expression profiling on pretreatment biopsies from 28 esccs who received neoadjuvant crt in a phase iii clinical trial and developed a prediction model based on three genes ( mmp , limch1 , clorf226 ) with 81% accuracy in the validation cohort ( table 2 ) . first , most markers presented within this review were mainly generated by focusing on relatively small cohorts within retrospective analyses . the results are mostly preliminary and require further validation . for popularization and application of these biomarkers , large prospective trials are warranted . finally , the crt or chemotherapy response must be evaluated pathologically . conventional imaging modalities ( endoscopy , endoscopic ultrasonography , computed tomography , and magnetic resonance imaging ) can not reliably differentiate between viable tumor and inflammatory reactions , edema , and scar tissue 51 , 52 . positron - emission - tomography with the glucose analog fluorodeoxyglucose ( fdg - pet ) is a functional imaging modality that can detect changes in tissue metabolism . current evidence has shown that tumor metabolic activity has been proven to correlate with histopathologic response in ec 53 . however , pet - based parameters to stratify prognosis in the literature has varied from different studies , including pre - radiation standard unit value ( suv ) , post - radiation suv , a percentage decrease of suv , pet - tumor length , and pet - tumor volume based parameters . the pretreatment pet evaluation is quite important in developing a strategy to identify the value of therapy in its early stage . 54 showed that pretreatment suv is a reliable predictor of response to definitive crt in escc . 55 also demonstrated that an initial suv higher than the median ( 10.1 ) was associated with a better pathologic response . more attention has been paid to the dynamic changes of suv and the timing of post - radiation fdg - pet imaging . several studies testified that the decrease of suv post - neoadjuvant therapy or preoperatively can be useful for predicting pathologic response 58 - 61 . however , swisher et al . 56 found that post - crt suv was predictive of pathologic response with a relatively low specificity . this high false positive rate may be due to the inflammatory changes following radiotherapy , which leads to falsely elevated suv values because of the presence of metabolically active leukocytes and macrophages . therefore , the timing of rechecking fdg - pet in the course of therapy may be critical because the false positive rate appears to decrease with time 57 . to address this question , wieder et al . 58 studied the time course of changes in tumor fdg - uptake in patients with escc patients treated with preoperative therapy . the results showed that metabolic changes within the first 2 weeks of therapy are slightly better predictors compared with later changes . this observation is most likely related to the complex proinflammatory and anti - inflammatory effects of radiation , which are strongly dependent on time and dose . the authors conceived that the cytotoxic effects of radiotherapy on radiosensitive cells , such as lymphocytes , limit the intensity of inflammatory reactions in the tumor tissue during and early after completion of therapy . additionally , in consideration of the application of suv in clinical practice , relative changes are better predictors of crt response than absolute suvs , as absolute suvs are much more sensitive to differences in data acquisition , image reconstruction , and data analysis than relative changes 59 . except for the suv value , other pet image - derived parameters , such as tumor longitudinal length ( tl ) and volume ( tv ) and total lesion glycolysis ( tlg = tv suv mean ) , 62 investigated the predictive value of baseline fdg - pet image - derived parameters regarding therapy response in ec patients . for study purposes , the tumor was automatically delineated on the baseline pet image using an adaptive threshold and the automatic fuzzy locally adaptive bayesian ( flab ) methodologies to attract the tl , tv , suv , and the derived tlg values . the results showed that commonly used parameters , such as suvs , were not significant predictive factors of the response ; parameters related to tumor functional spatial extent ( tl , tv , tlg ) could significantly differentiate histological response with sensitivity above 75% and specificity above 85% , regardless of the functional volume delineation strategy . 63 , who revealed that baseline mtv and tlg were not found to be predictors of response to neoadjuvant therapy in ec patients , although a trend towards a correlation between response to crt and smaller mtv was observed . this discrepancy may be related to the limited accuracy and reproducibility of the available tumor delineation methods , small sample size , and different response evaluation criteria in both studies . more recently , the spatial - temporal fdg - pet has gained popularity with physicians , which offers more information , including intensity features , texture features ( spatial patterns ) , geometry features , and geometry - intensity features ( total glycolytic volume ) , compared to the conventional pet measures with suv 64 . 65 initially built a predictive model using multiple , comprehensive tumor response measures , including conventional fdg - pet measures , clinical parameters , and demographics , and spatial - temporal fdg - pet features . this model achieved very high accuracy ( 100% sensitivity and 100% specificity ) for prediction of pathologic tumor response to crt in 20 patients with ec . this model needs to be validated with a large and prospective patient cohort . in conclusion , conventional fdg - pet image - derived parameters , especially the relative changes of suv values , have been proven a significant predictor of treatment response . furthermore , spatial - temporal fdg - pet offers more information about the intensity , texture , geometry , and geometry - intensity features compared with the conventional pet measures with suvs and will be useful for differentiating the responders to neoadjuvant crt . nevertheless , there are significant issues to be resolved with regard to the standardization of pet imaging protocols , image - processing methods and the time point for repeat imaging . diffusion - weighted magnetic resonance imaging ( dwmri ) is also a functional imaging that is based on the microscopic random translational motion of water molecules in biological tissues . the magnitude of this motion is characterized by its apparent diffusion coefficient ( adc ) values . recently , a few studies have been published to evaluate the efficacy of adc values in predicting neoadjuvant crt response . 66 analyzed the pretreatment adc values of 80 patients with escc and found that an adc value of 1.10 10 mm /s can differentiate crt responders from non - responders ( a high adc group responded better to crt than did a low adc group ) with a sensitivity , specificity , and accuracy of 73.8% , 86.8% and 80.0% , respectively . similarly , imanishi et al . 67 reported that the adc at the time of 20 gy and the increased rate of the adc at the time of 20 gy were significant predictors of treatment response in locally advanced escc . computed tomography perfusion ( ctp ) images can quantify tumor vascularity by measuring the temporal changes in tissue attenuation following intravenous contrast administration , which has been reported to associate with tumor characterization , survival , and therapy response in ec 68 - 70 . the tumor blood flow was closely related to tissue oxygen status and tumor microcirculation , which was demonstrated to be an important factor for determining chemoradio - sensivity . thus , ctp parameters will provide an important insight into the individualized treatment of ec . published data demonstrate that clinic - histopathological factors , molecular biomarkers , and functional imaging are predictive of neoadjuvant therapy . these clinical factors and biomarkers need to be further validated and novel biomarkers warrant additional exploration . fdg - pet image - derived parameters , especially the relative changes of suv values , have been proven a significant predictor of treatment response . nevertheless , there are significant issues to be resolved with regard to the standardization of pet imaging protocols , image - processing methods and the time point for repeat imaging . in addition , it is noteworthy that there is currently no comprehensive clinical study that incorporates clinical factors , biomarkers , and functional imaging to identify patients that may benefit from receiving neoadjuvant therapy . therefore , a predictive model based on these factors needs to be established with a large , prospective , and homogeneous patient cohort in the near future . standardization of staging , biomarker detection method , and image acquisition protocol will be critical for the generalization of this model . aside from these outstanding discoveries , prospective , multi - center controlled trials , which stratify patients according to these predictive factors , will help guide individualized treatment strategies for patients with ec .
spontaneously diabetic torii leprfa ( sdt fatty ) rat , established by introducing the fa allele of the zucker fatty rat into sdt rat genome , is a new model of obese type 2 diabetes . both male and female sdt fatty rats show overt obesity , and hyperglycemia and hyperlipidemia are observed at a young age as compared with sdt rats . with early incidence of diabetes mellitus , diabetic complications , such as nephropathy , retinopathy , and neuropathy , in sdt fatty rats were seen at younger ages compared to those in the sdt rats . in this paper , we overview pathophysiological features in sdt fatty rats and also describe new insights regarding the hematology , blood pressure , renal complications , and sexual dysfunction . the sdt fatty rats showed an increase of leukocytes , especially the monocyte count , prominent hypertension associated with salt drinking , end - stage renal disease with aging , and hypogonadism . unlike other diabetic models , the characteristic of sdt fatty rat is to present an incidence of diabetes in females , hypertension , and retinopathy . sdt fatty rat is a useful model for analysis of various metabolic disorders and the evaluation of drugs related to metabolic disease .	type 2 diabetes mellitus is a polygenic disorder that is caused by a metabolic and/or hormonal imbalance between insulin secretion from cells and insulin sensitivity in peripheral tissues , both of which might be modified by genetic and environmental factors . the decreased sensitivity to insulin leads to an increased requirement for insulin and is often associated with obesity in which metabolic disturbances are marked in insulin - target organs , such as the liver , muscle , and adipose tissues . obesity plays key roles in the pathophysiology of several metabolic diseases and is a risk factor for diabetes mellitus and for dyslipidemia . diabetic animal models play critical roles in the elucidation of the mechanisms of diabetes mellitus and the complications and in the development of novel drugs as treatments [ 5 , 6 ] . based on the previously mentioned concept , a novel model of obesity - related diabetes they established a congenic line of the spontaneously diabetic torii ( sdt ) rat by introducing the fa allele of the zucker fatty rat into the sdt rat genome via the speed congenic method using a pcr technique with dna markers . they have normal body weights , blood glucose levels , insulin levels , and lipid levels until about 16 weeks of age and , thereafter , develop hyperglycemia associated with hypoinsulinemia , which results from the degeneration of pancreatic beta cells [ 1113 ] . as a result of chronic hyperglycemia , the sdt rats develop profound complications in eyes , peripheral nerves , kidneys , and bones [ 8 , 9 ] . the fa / fa ( sdt fatty ) rats of both sexes became overtly obese and showed a significant hyperphagia . also , the bmi ( body mass index ) was greater in sdt fatty rats ( mean value , 0.91 and 0.87 g / cm in males and females , resp . ) than that in lean rats ( 0.75 and 0.58 g / cm in males and females , resp . ) at 14 weeks of age . both visceral and subcutaneous fat weights were significantly higher in sdt fatty rats , but , especially , the visceral fat weight was markedly elevated with aging ( table 1 and figure 1 ) . serum glucose levels in sdt fatty rats of both sexes were elevated from 6 weeks , and lipid parameters such as serum triglyceride and total cholesterol levels in the rats were elevated from 4 weeks of age . the hyperglycemia and hyperlipidemia were sustained for a long time afterwards [ 15 , 16 ] . the male sdt fatty rats showed hyperinsulinemia from 4 to 8 weeks of age , but after 16 weeks their insulin levels decreased to levels similar to those in sdt rats . in the female rats , hyperinsulinemia was shown from 4 to 12 weeks of age , and the insulin levels decreased gradually . also , a remarkable rise in renal parameters such as urine volume and urine protein was shown in sdt fatty rats of both sexes . effect of food restriction in sdt fatty rats was investigated [ 14 , 17 ] . body weights of the pair - fed rats were similar with those of sdt rats . improvement of hyperglycemia or hypertriglyceridemia was observed , but hypercholesterolemia was not entirely improved ( figure 2 ) . the visceral / subcutaneous ( v / s ) fat ratio decreased in the pair - fed rats ( mean standard deviation : control rats , 2.04 0.66 ; pair - fed rats , 1.28 0.19 ) , although the total fat ( visceral fat and subcutaneous fat ) weight did not change ( mean standard deviation : control rats , 137.5 52.5 g ; pair - fed rats , 135.2 10.4 g ) . cell size of the epididymal fat in the pair - fed rats tended to decrease , and glucose oxidation level in epididymal fat in the pair - fed rats was recovered to a similar level with that in sdt rats . in the glucose tolerance test conducted at 9 weeks of age , sdt fatty rats showed higher serum glucose levels after glucose loading without any response of plasma insulin . those impaired glucose tolerance and insulin secretion were deteriorated with aging . in pancreatic islets of female sdt fatty rats , pathological findings such as vacuolation , hypertrophy , and hemorrhage were observed from 8 weeks of age , and findings such as atrophy and fibrosis in the islets were observed from 24 weeks of age ( figure 3 ) . in sdt rats , glucose intolerance was observed in prediabetic stage [ 12 , 13 ] . the histological features in the pancreas of sdt rats were as follows : in 1020 weeks of age , slight changes such as hemorrhage , hemosiderin deposition , inflammatory cell infiltration , and fibrosis in and around the islets ; in 25 weeks of age , hemosiderin deposition , cellular infiltration with lymphocytes and macrophages , and fibrous tissue proliferation in and around the islets . nonfasted serum parameters , such as leukocyte count ( wbc ) , erythrocyte count ( rbc ) , hemoglobin ( hb ) , and hematocrit ( ht ) level , were examined at 6 and 12 weeks of age . also , differential counts of leukocytes , such as neutrophils , eosinophils , basophils , monocytes , and lymphocytes , were determined , respectively . the levels were measured using an automatic analyzer ( advia 120 hematology system ( siemens ag ) , erlangen , germany ) . wbc and ht levels in sdt fatty rats were significantly higher as compared with those in sd rats at both 6 and 12 weeks of age . in differential counts of leukocytes , the monocyte count was significantly higher at 12 weeks of age in sdt fatty rats . it is reported that type 2 diabetes mellitus or obesity is a chronic inflammatory state aggravated by factors that promote inflammation at the level of vasculature and adipose tissue [ 1921 ] . since the monocyte count in wbc is elevated , in the future , it is necessary to investigate the inflammatory state in sdt fatty rats . rbc and hb levels in sdt fatty rats were comparable to those in sd rats at 6 and 12 weeks of age . we examined blood pressure , known to be a risk factor for metabolic syndrome , in sdt fatty rats . in male sdt fatty rats , blood pressure was significantly higher from 8 to 24 weeks of age , as compared with age - matched sd rats . furthermore , we confirmed a new insight regarding blood pressure in sdt fatty rats : a high sensitivity to sodium . a 1% nacl solution was given to male sdt fatty rats for 8 weeks , from 4 to 12 weeks of age . the systolic blood pressure in sdt fatty rats was significantly higher as compared with that in control - sdt fatty rats ( mean standard deviation : nacl - sdt fatty rats , 200.2 22.0 mmhg ; control - sdt fatty rats , 137.2 13.0 mmhg ) . in other words , an elevation of blood pressure in the rats was more prominent after sodium loading . with early incidence of diabetes mellitus , diabetes - associated complications in sdt fatty rats were seen at younger ages compared to those in the sdt rats . in male sdt fatty rats , histopathological examination of the kidneys revealed changes in the glomeruli from 16 weeks and in the renal tubules from 8 weeks of age . in the glomeruli , glomerulosclerosis was observed from 16 weeks of age , and the sclerosis progressed with aging . nodular lesions were observed at 40 weeks of age . in the renal tubules , glycogen deposition in the tubular epithelium ( armanni - ebstein lesions ) and tubular dilation were noted from 8 weeks of age , and the change progressed from 8 to 16 weeks of age . in female sdt fatty rats , the female rats revealed changes in the glomeruli from 32 weeks of age , and in the renal tubules from 16 weeks , and the changes progressed with aging . furthermore , we investigated histopathological characteristics of the kidneys in male and female sdt fatty rats at 60 weeks of age . diffuse glomerulosclerosis , including increased mesangial matrix and glomerular hypertrophy , was severely progressed in the sdt fatty rats . moreover , tubular and interstitial lesions , including fibrosis and inflammatory cell filtration , were progressed in the sdt fatty rats ( figure 4 ) . histopathological findings in lens , including hyperplasia of epithelium , vacuolation of fiber , and occurrence of morgagnian globules , were observed from 8 weeks of age in male sdt fatty rats , and these changes progressed with aging . also , retinal lesions , such as folding and thickening , were observed with aging in male and female sdt fatty rats . diabetic peripheral neuropathy was evaluated at 8 , 24 , and 40 weeks of age in male sdt fatty rats . tail motor nerve conduction velocity ( mncv ) in the sdt fatty rat was delayed at 24 weeks of age and was further decreased at 40 weeks of age ( figure 5 ) . histopathologically , at 40 weeks of age , the fiber number was significantly decreased , and sdt fatty rats revealed significant atrophy in myelinated nerve . six - week treatment of pioglitazone , a peroxisome proliferator - activated receptor ( ppar)- agonist , lowered blood glucose level and prevented delay of sciatic mncv in sdt fatty rats . we investigated the effects of obese type 2 diabetes on bone turnover , bone mass , and bone strength in sdt fatty rats [ 24 , 25 ] . both serum osteocalcin , a bone formation marker , and urine deoxypyridinoline , a bone resorption marker , levels were lower in male sdt fatty rats compared to age - matched sd rats from 8 to 40 weeks of age ( figures 6(a ) and 6(b ) ) . the early onset of diabetes and obesity in male sdt fatty rats induced decreases in both serum osteocalcin and urine deoxypyridinoline and resulted in low bone turnover at a young age , 8 weeks of age . male sdt fatty rats showed lower bone mineral density ( bmd ) and bone mineral content ( bmc ) of the whole tibia ( figures 7(a ) and 7(b ) ) and shortening of the tibia and femur compared to age - matched sd rats . deterioration in bone geometrical properties of the femur midshaft , such as cortical thickness and minimum moment of inertia , was observed in male sdt fatty rats . furthermore , trabecular bone volume of the distal femur was lower in the sdt fatty rats . these negative effects on bone in the sdt fatty rats caused severe decreases in maximum load , stiffness , and energy absorption of the femur . in addition , serum levels of homocysteine , a candidate bone fragility marker , were elevated in male sdt fatty rats compared to age - matched sd rats ( mean standard deviation : sdt fatty rats , 14.4 3.4 nmol / ml ; sd rats , 6.6 1.7 nmol / ml ) . we also investigated changes in bone metabolism and bone quantity at 10 , 15 , 25 , and 40 weeks of age in female sdt fatty rats . serum osteocalcin and urine deoxypyridinoline levels were lower at 10 weeks of age in the sdt fatty rats compared to those in sd rats , but only the urine deoxypyridinoline levels were elevated in the sdt fatty rats from 25 weeks of age ( figures 6(c ) and 6(d ) ) . female sdt fatty rats showed lower bmc and bmd of the whole tibia from 8 to 25 weeks of age ( figures 7(c ) and 7(d ) ) . sdt fatty rat is a useful model to investigate bone abnormalities in obese type 2 diabetes . since sdt fatty rats ( fa - homozygous ) are infertile in both males and females , we used the sdt - fa/+ rats ( fa - heterozygous ) for reproduction . the reason the rats show infertility is unknown , but some functional disorders related to reproduction are observed . we monitored the estrus cycle by vaginal smear cytology between 12 and 17 weeks of age and compared them with normal sd rats used as control . sd rats showed a regular 4-day estrus cycle , whereas sdt fatty rats showed an irregular 4- to 5-day estrus cycle with persistent estrus stage and metestrus extension . weight of reproductive organs ( ovary , uterus , and vagina ) in each estrus cycle stage was measured at 12 weeks of age , and the organs were examined by histopathological analysis . relative weights of ovary , uterus , and vagina of sdt fatty rats were significantly lower than those of sd rats . in sdt fatty rats , histopathological changes , such as atrophy in uterus and inflammation in vagina , were observed ( figures 8 and 9 ) . irregular estrus cycle , increased leukocytes in vagina , or dysgenesis of reproductive organs might cause the infertility in female sdt fatty rats . moreover , testosterone levels in male sdt fatty rats tended to be lower as compared with those in sd rats at 8 and 24 weeks of age ( mean standard deviation : sdt fatty rats , 212.2 84.4 pg / ml ; sd rats , 960.4 802.7 pg / ml ; at 8 weeks of age , resp . ) . since the testosterone levels in male sdt fatty rats are low , the reproductive function is considered to be decreased . in histological analysis of testis in the sdt fatty rats in further study , it is necessary to elucidate the mechanism of hypogonadism in sdt fatty rats . in 2004 , dr . masuyama and dr . shinohara ( research laboratories of torii pharmaceutical co. , ltd . , japan ) established the congenic type 2 diabetes model spontaneously diabetic torii fatty ( sdt fatty ) rat by introducing the fa allele of the zucker fatty rat into the genome of the original sdt rat . clea japan has received right of production and sales from japan tobacco inc . and has distributed the animals as sdt fatty rats since 2012 . diabetes mellitus and diabetic complications in sdt fatty rats were found at a younger age than those in sdt rats . the early onset of diabetes or diabetic complication has advantages for the use of sdt fatty rats in diabetes research . furthermore , not only the male rats but also the female rats developed diabetes mellitus at a young age . female sdt fatty rat has the potential to become an important animal model of type 2 diabetes mellitus with obesity , especially in women , where few models currently exist . since sdt fatty rats showed a hypertension with obesity , hyperglycemia , and hyperlipidemia , the rat might have potency to be established as a metabolic syndrome model . also , it is interesting that sdt fatty rats showed various diabetic complications , such as osteoporosis and hypogonadism , with microangiopathy . use of sdt fatty rats will assist in the further elucidation of the pathogenesis of human diseases related to metabolic disorder and in discovery of new drugs .
this article reviews a new concept in magnetic resonance as applied to cellular and biological systems . diffusion weighted magnetic resonance imaging can be used to infer information about restriction sizes of samples being measured . the measurements rely on the apparent diffusion coefficient changing with diffusion times as measurements move from restricted to free diffusion regimes . pulsed gradient spin echo ( pgse ) measurements are limited in the ability to shorten diffusion times and thus are limited in restriction sizes which can be probed . oscillating gradient spin echo ( ogse ) measurements could provide shorter diffusion times so smaller restriction sizes could be probed .	this short invited article reviews diffusion techniques used to infer sizes of structures in samples that lead to restricted diffusion . then it explains how the time dependence is used to infer sizes of structures , such as axon diameters , in the sample . it then discusses the limitations of current methods inferring small sizes and briefly explains one possible alternative to overcome these limitations . their mean square displacement depends on the diffusion time , , as described by einstein s relation < r > = 2d ( in one dimension ) where d is the diffusion coefficient.1 molecules diffusing in a uniform medium with no barriers experience unrestricted diffusion . in non - uniform media ( eg , porous samples and cellular tissues ) barriers restrict molecular displacements so that the diffusion depends on the time scale of the study and the permeability of the barriers . assuming einstein s relation , < r > during the time can be used to find an apparent diffusion coefficient ( adc ) that is less than d because of restrictions or hindrances to motion . understanding the influence of the restrictions or hindrances on the adc gives information about the geometry of the boundaries of the surrounding medium which , for mri , could be tissues , rocks , concrete , cement , polymers , gases , etc.2 as an example , the adc in a simple system consisting of molecules with diffusion coefficient d that are entirely restricted to a single pore of diameter a will depend on . for a/(2d ) , all molecules , regardless of their starting position , will be found anywhere in the pore.3 measurements as a function of thus provide information about the structure in which the molecules are diffusing . specifically , there will be a change in the adc when measuring through the time = a/(2d ) which can be used to determine the pore size . traditional mr measurements of the adc in different samples use the pulsed gradient spin echo ( pgse ) sequence,4 see figure 1 . after excitation , a magnetic field gradient pulse is applied to the sample for a short time . this causes the spins to obtain a phase based on their position at the time of the pulse . a rf pulse is then applied to the system which reverses the phase of the spins . another identical gradient pulse is applied to the sample which changes the phase of the spins based on their position at the time of the second pulse ( ) . if no diffusion has occurred , the phase acquired from the second pulse will be equal and opposite to the phase of the spin just before the pulse , resulting in a net phase of zero . if diffusion occurs , the mean squared phase of all spins will be nonzero and cause a loss in mr signal which can be used to calculate the adc.4 alternatively , callaghan proposed q - space imaging using pgse with short gradient pulses to measure the conditional probability , p(rr , ) , that a spin initially at r has migrated to r over the time . by measuring the signal as a function of gradient strength , one can calculate the probability distribution of displacements of spins.5 using this probability distribution , one can infer from mr images tissue geometric information , such as axon diameter distributions and axonal and cellular volume fractions or densities.59 it is difficult , however , to find p in white matter because of the variability of axon diameter.2 in vivo measurements using echo planar imaging ( epi ) sequences are used to acquire images more rapidly.2 this allows full diffusion tensor imaging ( dti ) in vivo although the resolution is often not as good as standard imaging sequences.10 perfusion can also be measured using mri and the effects of perfusion on diffusion measurements must be taken into account.1114 many changing cellular structures , due to disease or injury , have already been shown to affect adc measurements . specifically for white matter imaging , some have measured changes in dti metrics with changing myelin content or myelin damage.2,1522 there are several methods for estimating axon diameter distributions and densities using mri and most use pgse with adc( ) or q - space . one method , axcaliber , uses a framework that combines composite hindered and restricted models of water diffusion ( charmed ) and pgse measurements.23 this method uses one fixed gradient direction and multiple diffusion times and gradient strengths to make the charmed model more sensitive to axon diameter.8 it fits the mr signal to an equation which has components due to restricted diffusion , hindered diffusion , and later free diffusion.24 water in each axon size will experience restricted diffusion at different . for example , water in an axon with a small diameter will experience restriction for much smaller than water in a larger axon.2 thus by shortening , smaller and smaller axons move from the restricted component to the hindered component allowing the inference of axon diameter of smaller and smaller axons . the framework used in axcaliber for dividing the models into different types of diffusion was based on another model , which describes bovine optic nerve tissue as a three - compartment system , ( axons , glial cells , and extracellular space ) . each compartment has its own diffusion coefficient , size , volume fraction , membrane permeability , and nmr relaxation times . it uses multiple s and gradient strengths in a pulse sequence similar to pgse ( stimulated echo ) which allows for longer diffusion times and measures the mean axon diameter , but not the distribution.25 another method , activeax , extended and optimized these methods to determine the accuracy and precision with which this important new biomarker , axon diameter , can be estimated in live human subjects.9 the work from this group combined a simplified version of charmed with high - angular - resolution diffusion imaging ( hardi ) and a model with a single axon diameter.9 it has been modified to be robust in the presence of orientation dispersion.26 the same group has also combined two - shell hardi with a three - compartment tissue model to create neurite orientation dispersion and density imaging ( noddi).27 table 1 summarizes the literature for some of the axon diameter measurements made with mri . diffusion times used ranged from 7 to 305 ms measuring diameters from 0.414 m . measuring similar restriction sizes in porous objects at room temperature would likely need longer imaging times because of the slower diffusion at the lower room temperature compared to body temperature . measuring axon diameter distributions with mri , even with intact ex vivo brains , has advantages over traditional ex vivo histological techniques . ex vivo histological techniques are cumbersome , require tissue sectioning and are subject to inaccuracies such as cell shrinkage.9 with mri , the brain remains intact and images can provide measurements over large regions of the brain . because of the difficulties with ex vivo histological measurements of axon diameter distributions , the variation of fiber composition over the population and during development is largely unstudied.9 although fixation can affect samples , studies have been done to compare adc and other dti metrics between in vivo and fixed ex vivo samples.22,2830 while absolute values of adc change , partially due to the temperature of the sample , fractional anisotropy is similar.31 thus ex vivo measurements of axon diameters on intact fixed brains can still offer important information . the pgse sequence requires , however , that be large with respect to the restriction sizes in biological tissues , thus limiting the information that could be obtained from the measurements as will now be explained . the signal from a pgse sequence is given by ln ( s / s0 ) = g ( /3 ) d = bd , where is the gyromagnetic ratio of the hydrogen nucleus , is the duration of the gradient pulse , and g is the amplitude of the gradient pulse . in order to measure the diffusion coefficient , the difference in signal with , s , and without , s0 , given that small s are desired , and < for pgse , the only remaining factor that can be increased is the gradient strength . with q - space imaging , the smallest displacements which can be probed depend on the largest q which can be used.8 again , to make a large q , large gradient strengths need to be used . moreover , for small duration , large amplitude gradient pulses , there will be a large change in magnetic field experienced by the subject in a short amount of time . this induces an electric field in the subject , which could cause twitching of the skin , pain , or interference with the function of the heart or brain.32 thus , in practice , it is difficult to make measurements at short diffusion times using the pgse sequence . pgse signals from water molecules in small axons do not change with diffusion time in the typical range used . thus methods to determine axon diameter from these pgse measurements are insensitive to these small axons . shortening would allow the inference of restriction sizes , for example rodent sized axon diameters , and surface - to - volume ratios in samples . in 1969 , an oscillating gradient spin echo ( ogse ) sequence was proposed to make measurements at short diffusion times,34 see figure 2 . in this sequence , the trapezoidal gradient pulses of the pgse sequence are replaced with sinusoidally varying gradient pulses . each period of the sine wave acts as a diffusion weighting so that the magnetic moments are dephased by the first lobe of the sine wave and rephased by the second lobe . these sine waves are repeated multiple times so that sufficient diffusion weighting can be obtained . the first measurements were made of surface - to - volume ratios from packed beads in water33 and in vegetables.36 however , only in the case of unrestricted brownian motion can these multiple diffusion - weighting periods be considered independent.37,38 for this reason , the signal attenuation should be described in terms of a frequency spectrum rather than simply a scalar value ( ) . more complicated gradient modulations that selectively sample a narrow frequency domain of the diffusion spectrum have been proposed.39 these provide a straightforward means of characterizing the diffusion spectrum , but are difficult to implement accurately.35 thus double - sine- and apodized cosine - modulated gradient waveforms , which are modified forms of ogse , have been used and are still termed ogse sequences.35 in this so - called temporal diffusion spectroscopy , the effective diffusion times are changed by varying diffusion gradient frequencies , and thus a spectrum of diffusion rates , which describe the biological tissue microenvironment , can be measured . these measurements are different from q - space imaging where the propagator is usually defined at a given long diffusion time and has spatial and directional dependences over a large range of q - space.5,40 the contrast between tissues might be greater at a discrete , moderately high frequency than at low frequencies , as suggested by theoretical consideration of simple geometries and preliminary studies in tumors41 and rat brain.42 using ogse sequences might improve axon measurements . as alexander notes : more significant improvements may come from replacing the standard pgse sequence with other diffusion - sensitive sequences such as oscillating gradient combination of the experiment design with these other pulse sequences should allow the a priori range of axon diameters to extend to include smaller diameters . this should provide protocols with sensitivity to wider ranges and provide more discriminative axon diameter indices.9 this same group performed experiments with optimized gradient waveforms ( gen ) and were able to make axons with smaller radii more distinguishable with gen than with pgse.43,44 they still suggest that oscillating waveforms would provide valuable information for in vivo studies.43 developing dti techniques to visualize better smaller fibers is considered an active area of research45 and creating new techniques using short diffusion times will cause water in the larger fibers to become less restricted allowing for easier visualization of the restricted diffusion of water in the smaller fibers . extending the measurements made with ogse from studying the variation of adc with diffusion time or frequency to create a method which uses this variation to infer restriction sizes using the shortest possible diffusion times could allow for the probing of smaller axon diameters . in theory , this method overcomes problems with high gradient strengths necessary in current mr diffusion methods ( pgse and q - space ) to allow smaller restriction sizes to be distinguished.35 the ogse and pgse methods could be used together to distinguish a large range of restriction sizes from very small ( ogse ) to fairly large ( pgse ) . combining the two methods might allow for a more complete understanding of the geometry of the sample .
schizophrenia and bipolar disorder are chronic psychiatric disorders , both considered as major psychosis ; they are thought to share some pathogenetic factors involving a dysfunctional gene x environment interaction . alterations in the glutamatergic transmission have been suggested to be involved in the pathogenesis of psychosis . our group developed an epigenetic model of schizophrenia originated by prenatal restraint stress ( prs ) paradigm in mice . prs mice developed some behavioral alterations observed in schizophrenic patients and classic animal models of schizophrenia , i.e. deficits in social interaction , locomotor activity and prepulse inhibition . they also showed specific changes in promoter dna methylation activity of genes related to schizophrenia such as reelin , bdnf and gad67 , and altered expression and function of mglu2/3 receptors in the frontal cortex . interestingly , behavioral and molecular alterations were reversed by treatment with mglu2/3 agonists . based on these findings , we speculate that pharmacological modulation of these receptors could have a great impact on early phase treatment of psychosis together with the possibility to modulate specific epigenetic key protein involved in the development of psychosis.in this review , we will discuss in more details the specific features of the prs mice as a suitable epigenetic model for major psychosis . we will then focus on key proteins of chromatin remodeling machinery as potential target for new pharmacological treatment through the activation of metabotropic glutamate receptors .	schizophrenia and bipolar disorder are severe and chronic psychiatric disorders , both considered as major psychosis that are thought to share several pathogenetic factors involving a dysfunctional gene x environment interaction . epigenetic events in telencephalic gabaergic and glutamatergic neurons contribute to the pathogenesis of psychotic symptoms in patients affected by schizophrenia [ 1 , 2 ] . atypical antipsychotics and valproic acid , drugs commonly used to treat psychosis , have been demonstrated to exert a potential effect on epigenetic mechanisms and to modulate chromatin structure remodeling by inducing dna demethylation and histone modifications [ 3 , 4 ] . refers to stable alterations in chromatin structure and genes expression that occur without modifying the underlying dna sequence . these alterations strongly depend on gene - environment interactions , can be transmitted through generations of cell divisions and may induce phenotypic modifications in the organisms . epigenetic modifications of the genome may occur via different pathways : ( i ) cytosine dna methylation at specific clusters of dinucleotyde sequence ( cpg ) by a family of enzymes called dna methyltransferases ( dnmts ) ; ( ii ) covalent modifications of histones at their amino ( n)-terminal tails , the most important of which being acetylation and methylation ; ( iii ) rna interference [ 7 - 10 ] ( fig . , an increasing interest arose among the scientific community on this topic : epigenetic mechanisms have been identified in the pathogenesis of many diseases , including psychiatric diseases [ 11 - 16 ] , and the development of drugs with specific ability to modulate the epigenetic machinery is at the moment a very intriguing and promising therapeutic strategy . evidence from neuroimaging , neuropathology , and epidemiological studies has led to the conclusion that schizophrenia ( sz ) and bipolar ( bp ) disorders are likely to be neurodevelopmental disorders that originate before birth [ 17 - 19 ] . moreover , an association between prenatal stress and risk for developing sz and associated psychiatric disorders including anxiety and affective disorders has been established [ 20 - 22 ] . adult offspring of mice exposed to repeated episodes of restraint stress during pregnancy , here defined as prenatally restraint stressed mice or prs mice , exhibit a sz - like behavioral phenotype characterized by hyperactivity , stereotyped and compulsive behaviors , deficits in social interaction , pre - pulse inhibition ( ppi ) , fear conditioning , and object recognition , and hypersensitivity to n - methyl d - aspartate ( nmda ) receptor blockers . this behavioral phenotype recapitulates positive and negative symptoms , as well as cognitive dysfunction displayed by patients affected by sz or bp disorder . prs mice also show a deficit in cortical gabaergic innervation [ 23 - 25 ] , which is expected to cause abnormal synchronization of the firing rate of pyramidal neurons , a putative electrophysiological substrate of cognitive dysfunction in psychotic patients [ 1 , 26 , 27 ] and neurodevelopmental animal models of sz . from a molecular standpoint , prs mice show a disrupted signature of chromatin remodeling at genes typically expressed in gabaergic and glutamatergic neurons , i.e. , genes encoding for glutamate decarboxylase-67 ( gad1 ) , reelin ( reln ) and brain derived neurotrophic factor ( bdnf ) respectively [ 23 , 29 , 30 ] . these molecular changes in prs mice are similar to those observed in the brain of sz and bp patients , [ 2 , 31 - 35 ] strongly suggesting that aberrant epigenetic gabaergic / glutamatergic mechanisms may underlie psychotic symptoms . the epigenetic endophenotypes common to the frontal cortex ( fc ) and hippocampus of prs mice and the brain of patients affected by sz and bp disorder are the following : ( i ) increased mrna and protein levels of the dna methylating enzymes , dna methyltransferase 1 ( dnmt1 ) and ten - eleven metylcytosine dioxygenase -1 ( tet1 ) ; ( ii ) enhanced dnmt1 binding to gad1 , reln , and bdnf - ix promoters ; ( iii ) increased levels of 5-methylcytosine ( 5mc ) and 5-hydroxymethylcytosine ( 5hmc ) at gad1 , reln , and bdnf promoters ; ( iv ) increased binding of methyl cpg binding protein ( mecp2 ) to gad1 and reln promoters ; and ( v ) a reduced expression of gad1 , reln and several bdnf - splice variants [ 2 , 23 , 30 , 31 , 36 - 38 ] ( fig . different studies [ 23 , 39 , 40 ] have shown that the sz - like behavioral alterations present in neurodevelopmental rodent models of sz are corrected by systemic administration of valproate and clozapine . in the present review , we address the question of whether prs mice may provide a behavioural and molecular epigenetic animal model of sz valuable for the study of the antipsychotic - like activity of types-2/3 metabotropic glutamate ( mglu2/3 ) receptor agonists and their interaction with typical or atypical antipsychotic drugs . metabotropic glutamate receptors are gtp - binding - protein ( g - protein ) coupled receptors that modulate both excitatory and inhibitory synaptic transmission in the cns , and are considered as candidate drug targets for neurological and psychiatric disorders . in the last decade , a role for mglu2/3 receptors in schizophrenia has been suggested by preclinical and clinical studies . pharmacological activation of mglu2/3 receptors decreases glutamate release in the cns , thereby reducing synaptic firing . this effect is shared with atypical antipsychotic drugs such as clozapine , which suppress serotonin - induced post - synaptic excitation in different brain regions ( fig . 3 ) . both orthosteric mglu2/3 receptor agonists and selective mglu2 receptor enhancers reverse the effect of nmda receptor antagonists on working memory , sensorimotor gating , locomotor activity , and cortical glutamate efflux in rodents [ 43 - 47 ] . in addition , drugs that activate mglu2/3 receptors restrain the electrophysiological and behavioral effects of agents acting on 5-ht2a serotonin receptors , i.e. hallucinogens [ 48 - 52 ] . in patients affected by schizophrenia , systemic treatment with pomeglumetad methionyl , an oral prodrug of the mglu2/3 receptor agonist , ly404039 , displayed significant antipsychotic activity in a phase-2 clinical trial , but not in subsequent trials [ 54 , 55 ] . perhaps a reason for these contrasting findings was the recruitment of patients that had been treated with atypical antipsychotic drugs , which are known to epigenetically down - regulate mglu2 receptors in mice [ 56 , 57 ] . thus , mglu2/3 agonists might still be promising for the treatment of targeted subpopulations of patients affected by sz . as outlined above , prs mice exhibit a sz - like phenotype characterized by alterations in in social interaction , prepulse inhibition , and locomotor activity , and , by an epigenetic down - regulation of reelin , bdnf , and gad67 proteins in the frontal cortex and hippocampus . interestingly , prs mice also showed significant reductions in mglu2 and mglu3 receptor mrna and protein levels in the frontal cortex , which was manifest at birth and , at least for mglu2 receptors , persisted in adult life . this reduction was associated with an increased binding of dnmt1 to cpg - rich regions of the grm2 and grm3 gene promoters and an increased binding of mecp2 to the grm2 receptor promoter . more recently , we have found a significant increase of tet1 ( the enzyme that catalyzes the transformation of 5-methylcytosine ( 5-mc ) into 5-hydroxymethylcytosine ( 5-hmc ) in the frontal cortex of prs mice . all epigenetic and behavioral changes of prs mice were reversed by treatment with the mglu2/3 receptor agonist , ly379268 . a reduced expression of dnmt1 and dnmt3a was also observed in response to ly354740 ( another potent mglu2/3 receptor agonist ) ( authors unpublished observation ) . in addition ly379268 increased both mrna and protein levels of growth arrest and dna damage 45- ( gadd45- ) , a protein involved in the regulation of dna demethylation , in mice frontal cortex and hippocampus . the binding of gadd45- to specific promoter regions of reln , bdnf , and gad1 genes was also increased after treatment with ly379268 . the effects of mglu2/3 receptor agonists in prs mice were similar to those produced by the atypical antipsychotic clozapine and by valproate , a drug that has multiple mechanisms of action including the inhibition of hdacs . in contrast , the classical antipsychotic , haloperidol , had no effect on prs mice . all together , these findings suggest that treatment with mglu2/3 receptor agonists or mglu2 receptor pams might correct epigenetic alterations typical of patients affected by sz . a large body of evidence suggests that 5-ht2a and mglu2 receptors tightly interact in regulating the response of cortical pyramidal neurons to thalamic inputs in the prefrontal cortex , and that this interaction is relevant to the pathophysiology of schizophrenia . activation of 5-ht2a receptors enhances glutamate release in the apical dendritic region of cortical layer v pyramidal cells , and this effect is blocked by pharmacological activation of presynaptic mglu2/3 receptors . gonzales - maeso and his associates have shown that mglu2 physically associates with 5-ht2a receptors via specific amino acid residues located at the c - terminal end of the 4 transmembrane ( tm ) domain , and that activation of mglu2 receptors restrains specific signaling pathways activated by hallucinogens acting at 5-ht2a receptors [ 61 - 63 ] . the finding that expression of mglu2 receptors is reduced , and expression of 5-ht2a receptors is increased , in post - mortem cortical tissue of patients affected by sz suggests that an imbalance between mglu2 and 5-ht2a receptors contributes to the pathophysiology of sz and can be targeted by therapeutic intervention . this imbalance was also shown in prenatally stressed mice , and in the adult progeny of dams exposed to viral infection during pregnancy . in both models , early life stress caused a down - regulation of mglu2 receptors and an up - regulation of 5-ht2a receptors in the cerebral cortex in the adult life [ 64 , 65 ] . these changes in receptor expression might not be interrelated because it is the blockade or genetic deletion of 5-ht2a receptors that epigenetically down - regulate mglu2 receptors [ 56 , 57 ] . 5-ht2a receptor knockout mice showed a reduced expression of mglu2 receptors in the frontal cortex , which was associated with a reduced h3 and h4 histone acetylation and an increased h3-k27 tri - methylation at the grm2 gene promoter , two epigenetic changes that cause gene repression . similar changes were found in normal mice chronically treated with atypical antipsychotic drugs inhibiting 5-ht2a receptors . all these findings suggest that mglu2 and 5-ht2a receptors are specifically targeted by a pathological epigenetic programming that is triggered by adverse events occurring early in life and that ultimately results in a long - lasting dysfunction of thalamic - cortical transmission , which is a key event in the pathophysiology of sz . pharmacological activation of mglu2 receptors seems to have a favorable impact on epigenetic changes associated with sz , but the effect of mglu2/3 receptor agonists or mglu2 receptor pams might be limited by the down - regulation of mglu2 receptors , a process associated with the disorders and even amplified by the use of atypical antipsychotics . thus , mglu2 receptors should be targeted in drug - nave patients , preferentially at the first episode of psychosis , or at least in the early phase of the disease . one can easily predict that epigenetic drugs acting at the grm2 gene promoter to enhance the expression of mglu2 receptors may have a permissive effect with mglu2 receptor agonists / pams in the treatment of sz . accordingly , pharmacological inhibition of hdacs has been shown to reverse the epigenetic down - regulation of mglu2 receptors caused by atypical antipsychotics . l - acetylcarnitine enhances mglu2 receptor expression via a mechanism mediated by acetylation of histones or transcription factors [ 66 - 68 ] . l - acetylcarnitine is currently marketed for the treatment of neuropathic pain and shows an excellent profile of safety and tolerability . it will be interesting to examine whether l - acetylcarnitine is able to reverse the epigenetic programming triggered by early life stress by enhancing the expression of mglu2 receptors . if so , a combination of mglu2/3 receptor agonists ( or mglu2 receptor pams ) and l - acetylcarnitine might be proposed as a valuable strategy in the treatment of sz .