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Evaluation on the Hub 34

aegypti was maintained in the laboratory of chemical ecology of vector insects of the federal university of minas gerais from 2007 . the breeding room was kept at a temperature of 27 1c , 75 - 80% relative humidity ( rh ) and a 12l:12d photoperiod ( eiras & jepson 1991 ) . the larvae were kept in plastic tubs containing water ( approximately 5 cm deep ) and fed reptile food ( reptolife , alcon , brazil ) until they reached the pupa stage . the pupae were collected daily and transferred to adult screened cages ( bugdorm-1 , mega view science education services , taiwan ) ( 30 30 30 cm ) and fed 10% sucrose solution . a selection cage was used to separate the gravid females that were used in the assays . blood meal was prepared using chicken blood ( gallus domesticus ) , which was obtained from a slaughterhouse , including the anticoagulant heparin ( gomes et al . . aegyptifemales ( f6 and f7 ) of five-10 days of chronological age and three days after blood meal ( gomes et al . experimental area - the tests were performed in laboratory and semi - field conditions . in laboratory conditions , four transparent acrylic boxes ( 150 50 41 cm ) were kept in a room with monitored temperature and humidity conditions ( 27 3c , 60 - 90% rh ) and a photoperiod of 12l:12d ( eiras & jepson 1991 ) . the semi - field tests were performed in an experimental area of 14.0 7.0 3.5 m , where eight cages ( 2.5 2.5 2.0 m each ) were installed ( roque & eiras 2008 ) . the temperature , rh and the photoperiod ranged according to the external environment and were monitored with a thermo - hygrometer . the tests were considered valid only when the temperature reached a minimum of 25c ( roque & eiras 2008 ) . bioassays - the tests evaluating the pattern of distribution of eggs by ae . the females were evaluated in laboratory conditions in experiment 1 and in semi - field conditions in experiment 2 . experiment 1 ( laboratory ) was performed in the four acrylic boxes in which four different densities of breeding sites ( 2 , 4 , 8 and 16 ) were simulated . in each box , a single gravid female of ae . aegypti was released and allowed to remain for 96 h in the interior of the box . the breeding sites were ovitraps that were set with 300 ml of tap water and a rectangular wooden paddle ( 12 cm 3 cm ) ( fay & perry 1965 , fay & eliason 1966 ) . for each density level , 12 repetitions of the experiment were performed . 1 ) . sugar solution ( 10% ) absorbed into cotton was provided as food to the insects during the experimental processes . 1:representation of the distribution of the different densities ( 2 , 4 , 8 and 16 breeding sites ) and the positions of the breeding sites in the interior of the acrylic boxes . in the centre , after 96 h , the paddle and the water that were present in each trap were collected , labelled and taken to the laboratory . to verify the distribution of eggs at each breeding site , the number of eggs that were present in the water and in the paddle of each ovitrap was counted using a manual counter and a stereoscopic microscope ( 20 ) . experiment 2 ( semi - field ) was conducted in the interior of the four fabric cages ( 2.5 2.5 2.0 m ) ( roque & eiras 2008 ) . the same densities ( 2 , 4 , 8 and 16 ovitraps ) that were evaluated in the laboratory conditions were evaluated in this environment . for each density the positions of the breeding sites were kept constant and the same methodology as used in experiment 1 was followed . however , to avoid visual effects of the treatment itself , each cage received all 16 of the ovitraps , but only the test traps ( densities 2 , 4 , 8 or 16 ) received water ( fig . 2 ) . fig . 2:diagram of the distribution of the ovitrap with and without water of each treatment / density of breeding sites . statistical analyses - for statistical analyses , the specific locations of egg deposition ( water and paddle ) were called habitats and the local of performance of the experiment ( laboratory or semi - field ) was called environment . to compare the number of eggs that were deposited in each environment / density , data were submitted to a normality test at a significance level of 95% . when the distributions were not in accordance with the criteria of normality , medians were compared using the kruskal - wallis test . the proportions of eggs that were deposited on water by each single female ( n eggs on the water / n total eggs ) at the different densities of breeding sites both in laboratory and semi - field conditions were compared by two - factors anova . before analyses , the data were transformed in arcsin(sqrt ) to normalise the distribution and stabilise the variances . the number of colonised breeding sites in relation to the amount of available ones , both in laboratory and semi - field conditions , was compared by the kruskal - wallis test . by investigating the female groups above and below the regression lines , we obtained two groups : those that colonise various breeding sites and those that colonise few sites . we compared these two groups by the mann - whitney u test to determine whether there was any difference between the groups with higher and lower frequencies of colonised breeding sites and the proportion of eggs that were deposited in the preferential breeding site . the r program ( available from : r-project.org ) environment ( laboratory or semi - field ) of the experiment and egg deposition - the oviposition behaviour of ae . aegypti was maintained in both environments . under laboratory conditions , the mean number [ standard deviation ( sd ) ] of eggs that were laid by one ae . aegyptiper repetition was 73.9 ( 25.64 ) , ranging between 40 - 142 eggs . similarly , under semi - field conditions , the mean number ( sd ) of eggs that were laid by oneae . aegypti per repetition was 72.2 ( 20.57 ) , ranging between 41 - 123 eggs . on average , females in both the experimental environments distributed the same number of eggs and colonised the same number of breeding sites ( anova p > 0.05 ) . therefore , these oviposition behaviours do not seem to be influenced by changes in the tested environments . oviposition on the water - the tested females exhibited a strong tendency to deposit their eggs on the water of the ovitrap . the mean proportion of eggs that were laid on the water surface tended to be higher than that on the paddle when the number of available breeding sites was greater ( fig . habitat received significantly more eggs in semi - field than in laboratory conditions ( anova p < 0.05 ) ( fig . 3:box plot of the proportion of eggs on the water in different density of oviposition breeding sites . 4:box plot of the number of eggs laid on water and paddle in semi - field and laboratory conditions . breeding sites colonised in relation to available ones - the availability of breeding sites directly influenced the dispersion of eggs by females . there was a significant difference ( p < 0.05 ) between the densities 2 * 4 , 2 * 8 , 2 * 16 and 4 * 16 both in laboratory and semi - field conditions ( fig . 5 ) . one female dispersed the eggs among 11 breeding sites , the highest number observed in the study . skip oviposition behaviour , although widely used , may not occur , as observed in eight ( 7.4% ) females . 5:mean and standard deviation of breeding sites colonised on the basis of available ones . different letters mean statistical difference ( kruskal - wallis p < 0.05 ) . one - by counting the number of eggs at each breeding site , we noticed that one of the sites usually received most of the eggs ( 40% or more of the total deposited eggs ) . although all the breeding sites were identical and in semi - field and were not statistically different from each other , it one site generally received more eggs than the others . the amount of eggs that were deposited at the favourite site was similar in both laboratory and semi - field conditions . the average percentage of eggs that were deposited in the favourite site was significantly higher with a density of two breeding sites and did not vary among the other densities ( fig . 6 ) , showing that females tend to aggregate more eggs when there are only two available breeding sites . this number can be reduced by an increase in the dispersion among several breeding sites . there was a negative relationship between the number of colonised breeding sites and the percentages of eggs that were laid at the favourite breeding site ( simple linear regression y = -0.0682x + 0.9256 , f1,106 = 65.9 , r = 0.38 , p < 0.001 ) ( fig . 7 ) . however , even those females that used many breeding sites seemed to deposit at least 40% of their eggs at the favourite breeding site , as shown by the dashed line ( fig . 7 ) . nevertheless , comparing the percentages of eggs that were laid by the ae . aegypti females of the two groups ( above and below the regression lines , fig . 8) , a significant difference was observed in both semi - field ( t = -6.62 , p < 0.001 ) and laboratory ( t = 2.87 , p = 0.001 ) ( fig . 7:percentage of eggs laid on favourite breeding site in relation to the number of colonised breeding sites under laboratory and semi - field conditions . 8:percentage of eggs laid in the favourite breeding site in laboratory by aedes aegypti female s who had higher frequency of breeding site colonisation ( a ) and smaller frequency ( b ) . percentage of eggs laid in the favourite breeding site in semi - field by ae . aegypti female s who had higher frequency of breeding site colonisation ( a ) and smaller frequency ( b ) . in other words , both environments ( laboratory and semi - field ) showed higher percentages of eggs laid at favourite breeding sites by females those that distributed their eggs in small numbers of containers . although all the breeding sites were identical and in semi - field they were not statistically different from each other , one site generally received more eggs than the others . the results that were obtained under laboratory and semi - field conditions were similar , showing that the general aspects of the oviposition behaviour were maintained despite the individual variations that were observed between the tested females ( wong et al . the variation in the number of eggs per female is common in experiments that assess a single gravidae . these variations are probably due to the blood - feeding efficiency ( xue et al . 2008 ) and the size of the adults , which may interfere with the reproductive capacity of males and females ( blackmore & lord 2000 , ponlawat & harrington 2009 ) . the average number of eggs was similar to that found by other authors ( christophers 1960 , madeira et al . as expected , the average number of eggs that were laid did not significantly differ between densities ( 2 , 4 , 8 and 16 breeding sites ) or between environments ( laboratory or semi - field ) . this result is probably due to the duration of the tests , which were performed between the third - seventh day after the consumption of blood meal , during which the females laid almost all of the eggs that were produced ( gomes 2006 , reiter 2007 , chadee 2010 ) . it was demonstrated for the first time that the behavioural response of females depended on the resources ( number of breeding sites ) that were provided to them . in an experiment with eight available containers , chadee ( 2010 ) noted that 87% of females used one to four breeding sites , with a maximum of seven . in our study , the number of containers that were used by the females increased following the increased availability of breeding sites under both laboratory and semi - field conditions . however , the number of breeding sites that were colonised seemed to stabilise at around five , even when there were 16 breeding sites available and reached a maximum of 11 under semi - field conditions . this result has epidemiological importance , as the search for breeding sites seems to be a crucial factor in the dispersal of the females and hence the diseases that they transmit ( edman et al . the determination of the average and maximum number of breeding sites that were used by each female can aid in the development of methodologies for monitoring and controlling the vector . therefore , further studies should be conducted to verify this behaviour in other environments and with different densities of breeding sites , given that few females colonised more than eight ovitraps . the results of the present study support the existence of the skip oviposition aegypti females , as previously observed ( christophers 1960 , fay & perry 1965 , corbet & chadee 1993 , apostol et al . 2003 , reiter 2007 , chadee 2010 ) . the ability of ae . aegypti females to distinguish potential breeding sites that will sustain the survival of their offspring during their development is a crucial factor in the life cycle of mosquitoes ( zahiri & rau 1998 ) . the selective pressure in favour of the females that make choices that may maximise the survival of their offspring ( reiter 2007 , harrington et al . nevertheless , this behaviour does not occur sometimes , as observed in a few females in this study ( 7.4% of females ) and by other authors ( harrington & edman 2001 , chadee 2010 ) . the large proportion of eggs that were laid on water contradicts the findings of most authors , who reported that the number of eggs that were deposited on water is negligible compared to that deposited on the walls , filter paper or paddles of the breeding site ( chadee & corbet 1987,chadee et al . however , a study in brazil demonstrated a large number of eggs that were deposited on water by females of two populations and at different humidities . the obtained figures were 42.9% and 57.3% ( 80% rh ) and 61.4% and 63.2% ( 51% rh ) for populations l and b , respectively ( madeira et al . 2002 ) . these findings were similar to the observations of the present work and almost 10 times higher than those reported by other authors ( chadee & cobert 1987 , chadee et al . aegypti because females of the same population distributed their eggs in different available proportions in oviposition substrates , as observed in our study . considering the behavioural plasticity of ae . aegypti , it is possible that the oviposition in both environments ( water or paddle / side walls of the breeding site ) can be advantageous depending on the circumstance . in anthropic environments , females can lay eggs in a large range of ephemeral containers that are very susceptible to disturbance ( reiter 2007 ) . this behaviour can make the choice of laying most of their eggs out of water relevant in places that can only be achieved by increasing the level of the water and can offer more chances of the larvae hatching and reaching adulthood . furthermore , deposition on the walls of the breeding sites may be an example of germ banking for the future of the ae . aegyptipopulation ( tsunoda et al . 2010 ) . in unfavourable conditions , the maintenance of germ banking until the return of favourable periods may be more advantageous to the offspring than the immediate eclosion of the larvae . on the other hand , the behaviour of laying eggs directly on the water is also relevant to the vector from an epidemiological perspective , as it helps to maintain their populations when there is no rainwater to supply breeding sites , thus allowing the existence of floating populations of mosquitoes during the dry season ( gomes et al . the deposition of eggs on water can also prevent predation by ants and cockroaches and the reduction of viability due to delays in hatching ( madeira et al . this result could perhaps explain why the number of eggs on the water was higher in semi - field conditions , where the eggs would be more exposed to these attacks . the increase in the number of eggs on water as observed in this study could also be explained by the abundance of resources ( breeding sites ) . the quick hatching of the larvae would increase the chances of the offspring finding the same available resources . however , to be considered a viable strategy , the eggs that were laid on the water surface must have hatching rates similar to those that were laid outside of the water . the literature is controversial on this issue because some authors have reported an extremely low hatching rate ( 2% ) ( silva et al . 2003 ) , while others have found higher rates , such as from 47 - 53% ( madeira et al . 2002 ) and from 73 - 80% ( rey & oconnell 2014 ) . therefore , we believe that the deposition of eggs on the water may be , at least in certain cases , a good choice for the survival of ae . aegypti . our results are also relevant in the context of the strategies of monitoring vector populations . in brazil , ovitraps can be used to obtain the indices of infestation ( ovitrap positivity index = number of ovitraps positive 100/number of ovitraps inspected and egg density index = number of eggs / number of positive ovitraps ) by counting the number of eggs on the paddle of the installed ovitraps ( gomes 1998 , ms / svs 2009 ) . in these surveys , the liquids ( water or infusion grass ) that are present in the ovitraps are discarded without performing any type of analysis . however , madeira et al . ( 2002 ) and the present study , both conducted in brazil , found high amounts of eggs in the ovitrap water . additional studies should be performed to determine if this trend is maintained in other populations of the country and under field conditions . the present work demonstrated for the first time the existence of a favourite breeding site and showed that egg distribution was not equal . the term favourite breeding site was used to denote the breeding site that received the highest percentage of eggs . this site was observed in semi - field and in laboratory conditions and with different amounts of available breeding sites . the ae . aegypti females usually choose the most productive containers to deposit most of their eggs . these containers are usually large , dark and unmanaged ( maciel - de - freitas & loureno - de - oliveira 2011 , wong et al . 2012 ) . in the present study , this behaviour was maintained even when all of the breeding sites were identical . either the females were able to recognise clues that were insignificant to us or this was such an intrinsic behaviour that it was maintained even when there was no advantage among the breeding sites . it is not known if the females lay their eggs in a single visit to the breeding site or if the breeding site receives a portion of the eggs and then the female returns to the same place and deposits eggs again . the observations on the proportion of eggs demonstrated the existence of two distinct patterns : ( i ) females lay many eggs at one favourite breeding site ( 40% or more ) and spread the remainder at other breeding sites and ( ii ) females lay few eggs ( less than 40% ) at the favourite breeding site and display a greater potential for spreading the eggs remaining over other breeding sites . ( 2012 ) , who observed an increase in the distribution of eggs in semi - field conditions when the highly productive containers were removed . the first pattern was more frequently observed in our study , although these two strategies can be important , depending on the condition of the breeding site and the environment in which the mosquitoes live . the behaviour of females laying most of their eggs at the favourite breeding site may favour the species in periods during which temporary breeding sites are scarce . this behaviour can also be advantageous when a female finds high - productivity breeding sites containing ideal conditions , such as large volumes and diameters , dark colouring and the presence of co - specific larvae . this behaviour would indicate the capacity to support large quantities of immature forms ( harrington et al . 2008 , maciel - de - freitas & loureno - de - oliveira 2011 , wong et al . females that choose other strategies , spreading the higher proportion of their eggs and depositing some at the favourite breeding site or spreading their eggs without considering the favourite breeding site , can favour their offspring during the rainy season , when there is a wide variety of breeding sites that are constantly supplied by rain water . thus , the plasticity of behaviour that was observed in this study and in previous studies ( madeira et al . the choice of a favourite breeding site and the deposition of large percentage of eggs in water were remarkable behaviours in the assessed population . aegypti ( madeira et al . 2002 , paduan et al . 2006 , hiragi et al . 2009 ) because they inhabit a variety of environmental conditions and present short life cycles . aegyptimosquitoes contain populations of individuals of various origins that have experienced different selective pressures . mosquito populations show great adaptability , primarily because their larval stages develop in different seasons and under different environmental conditions ( becker 1989 ) and because different selective pressures of the environment can lead to populations presenting distinct characteristics and great genetic and behavioural plasticity ( begon et al . 2011 ) . to determine whether the oviposition behaviour is the result of phenotypic plasticity or intraspecific differences between different populations , studies are required . however , regardless of the origin of behavioural plasticity in the oviposition ofae . . therefore , studies that elucidate the vector behaviour are important to the definition of control measures and despite efforts , little is known about the oviposition behaviour . thereby , it is extremely important that further studies investigate these aspects , especially under field and different conditions . finally , it is also important to understand the behaviour of mosquitoes that are used in the most modern and promising control techniques , such as those infected with the bacteria wolbachia and genetically modified mosquitoes .

despite the importance of the mosquito aedes aegypti in the transmission of arboviruses , such as yellow fever , chikungunya fever and dengue fever , some aspects of their behaviour remain unknown . in the present study , the oviposition behaviour of ae . aegypti females that were exposed to different densities of breeding sites ( 2 , 4 , 8 and 16 ) was evaluated in laboratory and semi - field conditions . the number of breeding sites that were used was proportional to the number available , but tended towards stabilisation . females used four - six breeding sites on average , with a maximum of 11 . a high percentage of eggs was observed in the water , along with the presence of a breeding site termed favourite , which received at least 40% of the eggs . the results are discussed in ecological , evolutionary and epidemiological approaches .

point - of - care testing ( poct ) is an increasingly popular means of delivering laboratory tests close to the patient . poct allows for rapid diagnostics and turnaround of test results to provide for faster medical decision - making and improved patient outcomes ( 1 ) . however , if inappropriate samples are collected , specimen is mislabeled , analysis is performed incorrectly , or test is misinterpreted , wrong results may be reported and acted on by the clinician . studies have indicated that for central laboratory testing , most errors occur in the preanalytical phase , prior to the sample arriving in the lab ( 2 ) . for poct , the majority of errors occur in the analytic phase of testing ( 3 ) . in fact , errors can occur in any phase of laboratory testing whether performed in a central laboratory or at the point - of - care . as laboratory directors , we should know our processes and take steps to detect and prevent errors before those mistakes reach the clinician and affect patient care . risk management is a way to reduce errors with poct . risk management is defined as the systematic application of management policies , procedures , and practices to the tasks of analyzing , evaluating , controlling , and monitoring risk ( 4 ) . risk is generally assumed from the patient s perspective , but risk can also apply to the operator of the poct device , the laboratory administration and even the hospital and its reputation . risk is the chance of suffering harm or loss , and risk can be estimated through a combination of the probability of occurrence of harm and the severity of that harm(5 ) . errors that occur more frequently have greater risk , and errors that lead to greater harm also present greater risk . staff troubleshoots control failures and followsup on complaints from clinicians . when errors are detected , the harm to patients is estimated and actions are taken to prevent recurrence in the future . so , risk management is simply a formal term for many of the activities that laboratories are already doing . quality control is a means of detecting and preventing errors . besides frequency and severity , detectability is a third factor in the risk estimation equation . as products are constructed on a factory line , the quality of the product is inspected to ensure that it meets manufacturer specifications . if problems are noted , the line can be stopped and production corrected to ensure the quality of the final product . these industrial risk management principles have application for reducing errors in laboratory testing as well . in the central laboratory , laboratories analyze liquid quality control , a stable sample with predefined acceptability ranges , in order to detect reagent problems before they affect the test result . traditionally , liquid controls are analyzed at two concentration levels each day of testing or more frequently as required by the stability of the test system . these are errors that affect the patient sample in the same manner as the quality control sample . reagent degradation , calibration errors , dilution and pipetting errors are examples of systematic errors that quality control can effectively detect and prevent before the errors affect a patient result . quality control , however , does a poor job at detecting random errors which uniquely affect individual samples . bubbles , clots , drugs , hemolysis and other sample specific errors are not detected by liquid quality control . other mechanisms , like bubble and clot detection , or analyzer hemolysis indices must be utilized to detect random errors . so , analyzing two levels of liquid quality control each day of testing does not entirely eliminate risk , and laboratories have still produced bad results despite analyzing liquid quality control . analysis of liquid quality control consumes the entire test in the process , and there is no guarantee that the next test will perform identically . alternative control processes must be used for these devices in addition to liquid controls in order to optimize the quality of these tests . many poct kits have built - in biologic and chemical controls to ensure the performance of individual tests . fecal guaiac occult blood cards have a positive and negative control area on each card to ensure the reactivity of the card and developer . urine pregnancy tests have a control line on each test to verify test storage and viability of the antibodies on the test . drug , rapid strep , hiv and other poct unitized tests have similar control lines or regions that guarantee the quality of the test kit and result with each test . these control lines are control processes that act as an alternative to traditional liquid controls in order to detect the risk of specific errors when using those tests . some tests , like bilirubinometers , can not even accept a liquid sample , so how does a laboratory effectively control the quality of these tests ? it is neither economical nor possible to analyze two levels of liquid controls for every reaction on this test each day . the effective way to ensure quality would consider risk of those errors that are most likely to occur or cause greatest severity of harm from an incorrect result . the amount and quality of specimen , the reactivity of the replicating enzyme , and the thermocycling of the device are key failure points , and those are the steps that should be monitored by the quality processes . although the practice of analyzing two levels of liquid quality control have given laboratories some degree of assurance that results are valid , newer devices have built - in electronic controls , and on - board chemical and biological controls , no single quality control procedure can cover all devices , since devices may differ in design , technology , function , and intended use ( 6 ) . quality control information from the manufacturer increases the user s understanding of device overall quality assurance requirements , so that informed decisions can be made regarding suitable control procedures . manufacturers understand their devices and the limitations of those devices , while laboratories know how the device will be utilized and test results applied for patient care . a quality control plan identifies the weaknesses in the testing process and defines the roles of the manufacturer built - in control processes and laboratory actions required to maintain risk to an acceptable level . the clinical and laboratory standards institute ( clsi ) document ep23-a introduces the industrial risk management principles to the clinical laboratory ( 7 ) . ep23 describes good laboratory practice for developing a quality control plan based on manufacturer s information , applicable regulatory and accreditation requirements , and the individual healthcare and laboratory setting . this guideline recommends collecting information about a test system and processing that information through a risk assessment to develop a quality control plan . weaknesses in the testing process are identified and for each hazard identified , the laboratory defines a control process which will detect and prevent that error , controlling risk to a clinically acceptable level . some hazards , like use of expired reagent , may be effectively controlled through a manufacturer built - in process such as barcoding which prevents the operator from utilizing expired reagents . other hazards may require the laboratory to take an action , like instrument maintenance or operator training / competency . a quality control plan is essentially a summary of all the hazards considered and laboratory actions required to minimize risk . if errors continue to occur , the laboratory is encouraged to troubleshoot , reassess their risk and modify the quality control plan as required . a unit - use blood gas device may be used as an example of the risk management process . the first step let s consider a generic poct blood gas and electrolyte analyzer intended for use in a same - day surgical center . the need for testing is low , only 1 2 tests per day . at a cost of $ 10 20 per test , the requirement to perform two levels of liquid control each day of testing will increase the cost of testing significantly and add to the turnaround time of results since control results will need to be evaluated before patient testing can be conducted . the use of alternative control processes provided by the manufacturer will improve cost , test and labor efficiency . review of the package insert allows the laboratory to determine intended use , test system operation and test limitations . the system is a portable clinical analyzer for the in vitro quantification of various analytes in whole blood . the test system consists of the portable clinical analyzer , test cartridges sealed in a foil pouch for protection during storage , quality assurance materials ( liquid control and calibration verification solutions ) , and a data management system with a server class computer , data management software , wireless connectivity , and laboratory and hospital information system interfaces . the unit - use cartridge contains all the components to perform testing including ; a calibrant solution , reagents , sample handling system , and sensors . the analyzer automatically controls all steps of the testing process such as fluid movement , calibration , fluid mixing , and thermal control . the cartridges are standardized to plasma core - laboratory methods using multi - point calibration curves stored in the device memory that are stable over many lots . upon insertion , signals produced by the sensor responses to the calibrant solution are measured , and a one - point calibration adjusts the sensor offset to the stored multi - point calibration curve . the analyzer then moves the sample over the sensors and the signal of the sensor responses to the sample are measured from the adjusted calibration curve . examination of the manufacturer , internal control processes allows an understanding of how the process functions and what errors can be detected and prevented with that process . the blood gas and electrolyte analyzer contains simulated internal control processes that check the edge connector , internal electronics and analyte circuitry . an isolated region of the internal circuit board sends a range of simulated sensor signals through the cartridge measurement channels . next , conductivity out of the connector pins is measured , insuring no contamination is present on the edge connector which would interfere with the test . the internal simulated control is performed automatically every 8 hours or if there has been a significant change in analyzer temperature , from cold to hot , since this can cause condensation on the connectors . the internal control can also be performed manually whenever the performance of the device is in question . internal simulated controls are never intended to entirely replace liquid quality control , and the manufacturer recommends analyzing liquid controls with each shipment of cartridges , new lots of cartridges , whenever cartridges experience a temperature shift > 8c , or as required by the laboratory . temperature is monitored continuously during each test , but a temperature verification cartridge is recommended at least annually . the information about the test system and the function of the internal control processes can now be processed through a risk assessment . risk assessment is best started by mapping the testing process to look for weaknesses and steps that could lead to error . follow the sample from order to specimen collection , analysis , and reporting of results . areas of focus should include the sample , the reagents , the operator , the analyzer , and the environment . examine those hazards of greatest risk first including errors that occur frequently or lead to greater severity . for compliance with federal and state regulations , testing should only be conducted based on a physician order . with poct so , operators must be trained to only conduct a blood gas or electrolyte test with this system based on an existing physician order . with appropriate training and demonstration of ongoing operator competency , the laboratory can conclude that risk of this error is reduced to a clinically acceptable level . inappropriate collection or use of the wrong specimen additive can affect blood gas and ionized calcium results . failure to adequately mix or over - mixing the sample can further lead to clots or hemolysis of the sample . whole blood samples continue to metabolize after collection , so prompt analysis , no more than 15 30 minutes after collection , is important . these are additional elements that should be added to the operator training and competency program to reduce risk of these errors . operator technique can impact poct results , so the effect of operator technique is critical to assessing risk with poct . operator lock - out features on poct devices require a personal identification number to unlock the device and perform patient testing . this feature ensures that only those trained and competent operators are conducting testing . adding too much or too little sample can affect test results by flooding the cartridge or contaminating the connector pins , and insufficient sample failing to adequately contact the sensors in the cartridge . this analyzer has volume detection and will not allow overfilling or start a test until an adequate amount of sample has been added . the analyzer also automates all steps of the testing process , preventing incorrect timing , misinterpretation , or other procedural steps common for poct . the analyzer also detects the expiration date of the cartridge through barcoding , preventing use of expired reagents . documentation of results into the patient s medical record presents an additional step for operators , so there is a risk of manual test results not being documented . the test system wireless connectivity and data management system ensure documentation of results without need for operator intervention or requiring additional operator actions . poct devices can transmit nosocomial infections between patients , so cleaning and disinfection between patients is important . training and reminders for staff on proper cleaning will effectively reduce risk of this error . exposure of cartridges to temperatures outside of manufacturer specifications during shipping and lot - to - lot variation can affect test results . analysis of liquid quality control upon receipt of new shipments and lots of cartridges can prove the viability of the cartridge prior to use for patient samples . however , cartridges can also degrade during storage , so temperature monitoring of storage conditions is required to ensure continued viability through the life of the cartridges . temperature monitoring of liquid control sample storage is also important to ensure control viability . periodic analysis of liquid quality control will further ensure cartridge and control stability . at what frequency should control samples the manufacturer recommends testing liquid control samples upon receipt of each shipment , with new lots of cartridges , and periodically to verify cartridge stability during storage . to determine the frequency of liquid control testing during storage , laboratories can perform side - by - side testing of daily liquid controls with internal control processes to document shelf stability for a period of several weeks . once stability is documented for several weeks , the laboratory will have data to decrease the frequency of liquid control to every few days , and eventually weekly or monthly , depending on the life - span of cartridges after receipt . the analyzer does not require water , works on battery power and internally detects the electrical circuitry and sensor connector pins . so , these risks are not a consideration with this device . once the testing process has been mapped , hazards recognized and control processes identified , the third step of the risk management process is summarizing the quality control plan . the quality control plan summarizes all of the hazards recognized during the risk assessment and the error mitigations selected , both those internal control processes from the manufacturer and the actions from the laboratory . if risk is not reduced to an acceptable level , then the laboratory must take additional mitigation steps to control the risk . the final step of the risk management process is implementing the quality control plan and monitoring the effectiveness of the plan . benchmarks for this blood gas and electrolyte analyzer could include trends in quality control , internal controls as well as liquid quality controls , analyzer error codes , physician complaints , or any other unexpected trends . when errors do arise , the laboratory should troubleshoot to determine the source , correct the process and reassess risk in light of the new information , modifying the quality control plan as required . the us centers for medicare and medicaid services ( cms ) recently implemented new clinical and laboratory improvement amendments interpretive guidelines in january 2014 ( 8) . risk management principles have been incorporated into the new interpretive guidelines in the form of individualized quality control plans ( iqcp ) . cms will begin inspecting for laboratory iqcps beginning in 2016 . at that time , laboratories will have two quality control options : 1 ) perform two levels of liquid quality control each day of testing or 2 ) develop an iqcp in order to reduce the frequency of liquid quality control . the laboratory can not reduce frequency below manufacturer recommendations , and the laboratory must perform liquid quality control at some frequency ( i.e. , performing no liquid quality control is not an option . ) . although iqcp will initially only apply to clia moderate complexity devices , any laboratory will benefit from mapping their processes and assessing weaknesses in their tests . since the chemistry of the test reaction is in the unit - use test cartridge , facilities with dozens of the same device can select a subset of devices and rotate the analysis of liquid controls , since all devices share the same lot and supply of unit - use cartridges . for laboratory - developed tests , most importantly , by developing an iqcp the laboratory will embrace industrial risk management principles and learn how to better detect and control risks with their test systems .

point - of - care testing ( poct ) is growing in popularity , and with this growth comes an increased chance of errors . risk management is a way to reduce errors . originally developed for the manufacturing industry , risk management principles have application for improving the quality of test results in the clinical laboratory . the clinical and laboratory standards institute ( clsi ) , ep23-a laboratory quality control based on risk management guideline , introduces risk management to the clinical laboratory and describes how to build and implement a quality control plan for a laboratory test . a simple , unit - use blood gas analyzer is utilized as an example for developing a laboratory quality control plan . the us centers for medicare and medicaid services ( cms ) has revised the clinical and laboratory improvement amendments ( clia ) interpretive guidelines to provide a new quality control option , individualized quality control plans ( iqcp ) , for decreasing the frequency of analyzing liquid controls from two levels each day of testing to manufacturer recommended frequencies in conjunction with a device s built - in internal control processes and the risk of error when testing with that device . iqcps have the advantage of allowing laboratories the flexibility to adopt alternative control processes in concert with traditional liquid controls to improve efficiency and cost effectiveness while providing optimal quality poct results for patient care .

the societal burden of seasonal influenza epidemics is quite heavy , with approximately 3 - 5 million cases and 250,000 - 500,000 estimated deaths worldwide every year and a large economic impact , which includes both direct and indirect costs [ 2 , 3 ] . conventionally , attention has been directed toward influenza a , which accounts for the majority of influenza cases in most seasons [ 4 - 7 ] . since the 1980s , influenza b viruses have belonged to two antigenically distinct lineages , called the victoria and yamagata lineages ; this has constituted a challenge for seasonal influenza vaccines , as only one influenza b strain is included in the trivalent vaccine . studies in the united states have shown that the frequent influenza b vaccine mismatches of recent years have been associated both with substantial increases in cases , hospitalizations and deaths ( up to 970,000 cases , with 8200 hospitalizations and 485 deaths annually , in the usa ) , and with high influenza - related medical costs and costs due to productivity loss . despite the important role of influenza b , much of the published scientific literature regarding the epidemiology of influenza has focused on influenza a , and we still have a relatively poor understanding of the global epidemiology and disease burden of influenza b. several studies have reported on the burden of disease attributable to influenza b in a single season , or during consecutive seasons in a single country [ 12 , 13 ] . in order to improve our understanding of the burden and epidemiology of influenza b , we reviewed the influenza b viruses circulating in italy from 2000 to 2015 . in italy , the influenza sentinel surveillance system ( influnet ) was implemented nationwide in the 1999 - 2000 season by the influnet working group . influnet is based on the voluntary participation of an average of 830 ( range 648 - 902 ) general practitioners ( including paediatricians ) per year , covering about 1.5 - 2% of the national population in all italian regions . the system aims to monitor the incidence of influenza - like illness ( ili ) and to determine the extent , timing and severity of seasonal epidemics . gps are asked to report ili cases ( defined as acute onset of fever + respiratory symptoms + one of the following symptoms : headache , general discomfort , asthenia ) weekly ( from week 42 to week 17 ) using standardized forms . specific information regarding age ( 0 - 14 , 15 - 64 , > 64 years ) and influenza vaccine status are also collected . we excluded the first years of data collection and focused the analysis on influnet data collected from the 2005/2006 to 2014/2015 seasons . influenza virological surveillance in italy is routinely carried out , between week 46 and week 17 of the following year , by the national who ( world health organization ) influenza centre at the istituto superiore di sanit ( nic - iss ) , in collaboration with a network of 15 peripheral laboratories located in 14 of the 21 italian regions . the main objective of these activities is to rapidly characterize the influenza viruses circulating in the country and to identify antigenic variants emerging in human populations during the winter season , in order to update the vaccine composition , in collaboration with the who and ecdc ( european centre for disease prevention and control ) . during the virological surveillance period , sampling kits are sent out to a random sample of gps participating in the influnet surveillance system , who collect throat swabs from the first ili patients seen each week . collected swabs are then sent to the regional reference laboratories for influenza diagnosis , and the isolated strains are characterized at the regional laboratory or directly sent to the nic - iss for further molecular and antigenic analyses . overall results obtained throughout the country are reported to the niciss weekly by means of web - based electronic forms . every year , approximately 2000 samples are collected , with a proportion of positive specimens of about 34% . a web - based data collection form for the surveillance of severe confirmed hospitalised cases and deaths due to pandemic influenza was drawn up in mid - september 2009 . since then , regional and local authorities have filled in forms during the influenza season ( october - april ) ; the data are analysed weekly at the national level ( by the iss and the ministry of health ) . our analysis included virological surveillance data collected from the 2010/2011 to 2014/2015 seasons , as all confirmed cases during the 2009/2010 season were due to a / h1n1pdm09 virus . in italy , the influenza sentinel surveillance system ( influnet ) was implemented nationwide in the 1999 - 2000 season by the influnet working group . influnet is based on the voluntary participation of an average of 830 ( range 648 - 902 ) general practitioners ( including paediatricians ) per year , covering about 1.5 - 2% of the national population in all italian regions . the system aims to monitor the incidence of influenza - like illness ( ili ) and to determine the extent , timing and severity of seasonal epidemics . gps are asked to report ili cases ( defined as acute onset of fever + respiratory symptoms + one of the following symptoms : headache , general discomfort , asthenia ) weekly ( from week 42 to week 17 ) using standardized forms . specific information regarding age ( 0 - 14 , 15 - 64 , > 64 years ) and influenza vaccine status are also collected . we excluded the first years of data collection and focused the analysis on influnet data collected from the 2005/2006 to 2014/2015 seasons . influenza virological surveillance in italy is routinely carried out , between week 46 and week 17 of the following year , by the national who ( world health organization ) influenza centre at the istituto superiore di sanit ( nic - iss ) , in collaboration with a network of 15 peripheral laboratories located in 14 of the 21 italian regions . the main objective of these activities is to rapidly characterize the influenza viruses circulating in the country and to identify antigenic variants emerging in human populations during the winter season , in order to update the vaccine composition , in collaboration with the who and ecdc ( european centre for disease prevention and control ) . during the virological surveillance period , sampling kits are sent out to a random sample of gps participating in the influnet surveillance system , who collect throat swabs from the first ili patients seen each week . collected swabs are then sent to the regional reference laboratories for influenza diagnosis , and the isolated strains are characterized at the regional laboratory or directly sent to the nic - iss for further molecular and antigenic analyses . overall results obtained throughout the country are reported to the niciss weekly by means of web - based electronic forms . every year , approximately 2000 samples are collected , with a proportion of positive specimens of about 34% . a web - based data collection form for the surveillance of severe confirmed hospitalised cases and deaths due to pandemic influenza was drawn up in mid - september 2009 . since then , regional and local authorities have filled in forms during the influenza season ( october - april ) ; the data are analysed weekly at the national level ( by the iss and the ministry of health ) . our analysis included virological surveillance data collected from the 2010/2011 to 2014/2015 seasons , as all confirmed cases during the 2009/2010 season were due to a / h1n1pdm09 virus . from the 2005/2006 to 2014/2015 seasons , an estimated average number of approximately 4,800.000 ili cases were reported to the surveillance system ( tab . the national database that was used for the analysis included 40,000 ili cases , with testing of samples from cases that occurred between 2000/2001 and 2011/2012 . during the study period , several waves of infections by influenza a and b viruses were observed in italy ( fig . 1 ) . in detail , 11,488 influenza cases were confirmed in the study period : 9,842 influenza a cases and 1,646 influenza b cases . influenza a and b viruses co - circulated during most influenza seasons , with numbers of influenza b infections approaching or exceeding those of influenza a virus during three seasons in the study period considered ( 2001/02 , 2007/08 and 2012/13 ) . the number of samples tested for influenza viruses by pcr in italy increased by a factor of 1.5 over this period , from an average of 2,774 per season in the period 2000 - 2007 to an average of 4,312 per season in 2008 - 2012 . influenza b appeared to be relatively more frequent among older children ; a(h1n1)pdm2009 among young and older adults , and a(h3n2 ) among the elderly . on average , the proportion of influenza b cases on the total of tested samples was 23% ( range < 1 - 78% ) ( fig . 2 ) . distribution of estimated influenza - like illness cases and cumulative incidence by season , italy , 2005/2006 2014/2015 . distribution of confirmed severe cases , by influenza virus , italy , 2010/2011 2014/2015 . number of influenza a and b virus infections , italy , 2000/2001 2011/2012 . influenza b circulation as a proportion of the total percentage of all circulating influenza strains , italy 2001/2002 2010/2011 seasons . from 2010/2011 to 2014/15 , on a total of 1,545 severe confirmed influenza cases reported to the surveillance system ( tab . i ) , 102 were confirmed influenza b virus - infected individuals admitted to icu ; 2 were pregnant ( both in the third trimester ) ; 7 needed extracorporeal membrane oxygenation ( ecmo ) treatment , and 24 died . the median age was 63 years ( range 0 - 92 ) , 58 were male , and 16 were vaccinated almost 15 days before symptom onset . the median age of the individuals who died was 76 ( range 39 - 85 ) . of the 102 severe influenza b patients , 73 belonged to groups recommended for vaccination ( 65 years and older or a clinical risk group ) , but only 16 had actually received the seasonal influenza vaccination . most of the influenza b cases were reported during the 2012/2013 season , when the b virus co - circulated with the a / h1n1pdm09 . from 2010/2011 to 2014/15 , on a total of 1,545 severe confirmed influenza cases reported to the surveillance system ( tab . i ) , 102 were confirmed influenza b virus - infected individuals admitted to icu ; 2 were pregnant ( both in the third trimester ) ; 7 needed extracorporeal membrane oxygenation ( ecmo ) treatment , and 24 died . the median age was 63 years ( range 0 - 92 ) , 58 were male , and 16 were vaccinated almost 15 days before symptom onset . the median age of the individuals who died was 76 ( range 39 - 85 ) . of the 102 severe influenza b patients , 73 belonged to groups recommended for vaccination ( 65 years and older or a clinical risk group ) , but only 16 had actually received the seasonal influenza vaccination . most of the influenza b cases were reported during the 2012/2013 season , when the b virus co - circulated with the a / h1n1pdm09 . our results on influenza b virus infections in italy are timely , in view of the recent introduction of a tetravalent influenza vaccine containing influenza b viruses of both the victoria and yamagata lineages . our study reveals that influenza b virus was the predominant overall cause of influenza in two of the 13 influenza seasons from 2000/01 through 2012/13 . however , circulation of the b virus during the inter - pandemic season was always demonstrated . similar patterns of influenza b virus circulation have been described in the us , europe and hong kong [ 12 - 13 ] . in italy , the proportion of influenza b cases in the study period averaged 23% ( range < 1 - 78% ) , a value similar to the european ( 23% ( 1 - 60% ) ) and us ( 24% ( < 1 - 44% ) ) averages . in our study , < 1% of all ili cases are tested for influenza each season , and very few b viruses were antigenically and genetically characterized , thus reducing the opportunity to identify the co - circulation of the two lineages in italy . however , evidence from two northern italian regions clearly demonstrated a complete or partial mismatch in the 2001/2002 , 2004/2005 , 2005/2006 , 2007/2008 , 2008/2009 , 2010/2011 , and 2011/2012 seasons ; this was almost always due to co - circulation of the two lineages [ 15 - 17 ] . influenza is generally recognised as an important disease which causes high excess mortality among the elderly , although children have been shown to play an important role in its transmission [ 18 - 19 ] . unlike the influenza a(h3n2 ) virus , the b virus predominantly infects children and young adults and is generally recognised as a mild influenza virus . a large study conducted in the us reported that 25% of all influenza - related mortality could be attributed to influenza b virus . this is higher than the percentage seen in the present study , in which influenza b infections accounted for 6% of all influenza virus - related deaths ( 24/428 ) during the period considered , but is similar to figures reported for scotland . among those admitted to icu with influenza b infections , influenza immunisation is recommended in italy for specific groups with an increased risk of complications following influenza infection ( e.g. > 65 years old , > 6 months with chronic conditions , healthcare personnel , pregnant women in their second or third trimester etc . ) . only 16% of those treated in icu who should have been vaccinated had received the seasonal influenza vaccine , and 70% of those treated in icu were included in the target categories of national recommendations . the magnitude of the problem created by mismatching between circulating influenza b strains and the influenza b lineage contained in the vaccine varies by season . the most striking recent examples occurred during the 2005 - 2006 and 2007 - 2008 seasons . in 2005 - 2006 , the influenza b component of the northern hemisphere influenza vaccine was of the b / yamagata lineage , but 81- 91% of the circulating influenza b viruses antigenically characterized in the us and europe were of the b / victoria lineage , and influenza b was found in 34 - 60% of all samples . similarly , in 2007 - 2008 , the influenza b component of the vaccine was of the b / victoria lineage , but 98 - 99% of the circulating influenza b viruses characterized in the us and europe belonged to the b/ yamagata lineage . unfortunately , as very few b influenza viruses in italy were genotyped , information on the antigenic characteristics of circulating b viruses was not available at the national level . our study demonstrates that influenza b virus infections are associated with substantial morbidity and that influenza surveillance and interventions including vaccination and treatment are still suboptimal . incorporating viral and epidemiological data will help obtain more accurate estimates of influenza disease burden and result in a better selection of strategies for influenza prevention and control .

summarywe describe the burden of influenza b infections in italy over a 12-year study period . influenza a and b viruses co - circulated throughout the period , with numbers of influenza b cases approaching or exceeding those of influenza a during three influenza seasons . influenza b virus infections led to fewer admissions to an intensive care unit ( icu ) and a lower mortality rate than influenza a from 2010 to 2015 . however , only 16% of those admitted to icu with influenza b had been immunized . this highlights the need for consistent influenza vaccination for identified risk groups . our study demonstrates that influenza b virus infections are associated with substantial morbidity and that influenza surveillance and interventions including vaccination and treatment are still suboptimal . our findings have important public health implications . incorporating virus and epidemiological data will help obtain more accurate estimates of influenza disease burden and result in a better selection of influenza prevention and control strategies .

obesity in humans has been established as a risk factor for a multitude of maladies including cardiovascular disease ( cvd ) , type 2 diabetes mellitus ( t2 dm ) , hypertension , renal disease , and neurologic dysfunction . furthermore , obesity has been causally linked to a variety of cancers either as a risk factor or as a negative factor for prognosis [ 13 ] . recent studies have suggested that the link to these disorders or diseases is due to a chronic low - grade inflammation that is associated with obesity [ 47 ] . debate exists whether the inflammation is a product of obesity or rather inflammation results in an obese state . nevertheless , once excess adipose tissue is established , the functions of the tissue appear to be comparable with a dynamic endocrine organ [ 8 , 9 ] . in the obese individual , macrophages , adipocytes , and epithelial cells communicate via obesity - associated hormones , inflammatory cytokines , and other mediators . for example , adipose tissue is known to produce and secrete various adipokines , such as leptin and adiponectin , and pro - inflammatory factors , tnf , il-6 , il-1 , and c - reactive protein ( crp ) [ 4 , 5 ] . all of these factors not only are important in adipogenesis but have been strongly linked to the onset of cvd , t2 dm [ 13 ] , and metabolic syndrome [ 6 , 7 ] . in addition , recent reports have associated this chronic inflammation seen in obesity with cancer promotion and development [ 1012 ] . a longitudinal community - based cardiovascular risk factors prevalence study ( crisps ) in human subjects from hong kong shows that il-6 , soluble tumor necrosis factor receptor 2 ( stnfr2 ; as a surrogate marker of tumor necrosis factor- activity ) , leptin , lipocalin 2 , adiponectin , and adipocyte - fatty acid binding protein ( a - fabp ) are predictors for cancer development [ 1012 ] . in the last decade , several studies have shown elevated il-6 , tnf , and il-1 levels in obese patients [ 1317 ] ; however , data regarding the relevance of these cytokines are controversial [ 18 , 19 ] . this could be partially attributable to the complex etiology of obesity , which consists of an interaction between genetics , diet , and physical activity levels , and is additionally influenced by environmental , socioeconomic , and behavioral factors . chimpanzees have the closest homology to humans and also share a multitude of similar diseases related to obesity including cvd , t2 dm , hypertension , and renal disease . although understood in humans , it is unknown if obese chimpanzees share a chronic inflammatory state . the study of obese chimpanzees could both help in the management of this species and also lend clues to the human obesity epidemic . in the present study , we examined cytokine , chemokine , and metabolic hormones levels in plasma of three chimpanzee weight categories ; lean , overweight , and obese . we measured plasma concentrations of the following cytokines and chemokines : interferon gamma ( ifn- ) , interleukin-6 ( il-6 ) , interleukin-12p40 ( il-12p40 ) , tumor necrosis factor ( tnf ) , soluble cd40l ( scd40l ) , interleukin-1 ( il-1 ) , interleukin-4 ( il-4 ) , interleukin - ra ( il - ra ) , interleukin-10 ( il-10 ) , interleukin-13 ( il-13 ) , eotaxin , fractalkine , monocyte chemoattractant protein-1(mcp-1 ) and chemokine ( cxc motif ) ligand8 ( cxcl8 or formally known as il-8 ) ) , and metabolic hormones such as connecting peptide ( c - peptide ) , glucagon - like - peptide-1 ( glp-1 ) , glucagon , insulin , peptide tyrosine or pancreatic peptide yy336 ( pyy ) , and leptin . the purpose of this study is to investigate the relationship between obesity and these cytokine / chemokine / metabolic hormones in chimpanzees . the hypothesis is that chimpanzees will be similar to humans and will demonstrate an inflammatory profile with increasing adiposity . the study population was grouped according to the body condition scoring and defined as lean , overweight , and obese . chimpanzees in lean group were defined as muscular body , normal body condition , some abdominal tuck , and neither concave nor convex abdomen . overweight chimpanzees were defined as round convex abdomen , big thighs , and the presence of fat around gluteal muscles . obese chimpanzees were defined as having a very large abdomen that extends outside of body frame , pectoral fat , the presence of fat around gluteal muscles , and fatty deposits in axillary regions and/or below biceps . all subjects were considered healthy and in their normal social groups at the time they were sampled . the study population consisting of chimpanzees used in the study is shown in table 1 . all chimpanzees are group housed ( multimale , multifemale ) at the michale e. keeling center for comparative medicine and research at the university of texas md anderson cancer center and maintained in accordance with the guide for the care and use of laboratory animals of the institute of laboratory animal resources , national research council . the facility is fully accredited by the association for the assessment and accreditation of laboratory animal care international . the primary source of nutrition is harlan teklad chimpanzee diet ( # 7775 ) , a high fiber , 20% protein - containing diet that is fed twice a day . environmental enrichment is provided daily using various food puzzle devices , forage such as popcorn or sunflower seeds scattered in the grass of outdoor enclosures , manipulable items such cardboard boxes and through treats given as rewards for positive reinforcement training . blood sample ( 10 ml ) was collected in heparin coated collection tubes and immediately plasma was separated by centrifugation and stored at 80c until further use . peripheral blood mononuclear cells ( pbmcs ) were separated from heparinized blood by centrifugation through a histopaque ( density , 1.077 g / ml ; sigma , st louis , mo ) . peripheral blood mononuclear cells were removed from the interface and washed twice with complete rpmi 1640 ( hyclone , logan , ut ) supplemented with 100 000 u / l penicillin ( sigma ) , 100 mg / l streptomycin ( sigma ) , 2 mmol / l l - glutamine ( sigma ) , and 25 mmol / l hepes ( sigma ) . cells were resuspended in appropriate concentrations in complete rpmi for cultures in various immune assays . plasma concentration of cytokines / chemokines , ifn- , il-6 , il-12p40 , tnf , scd40l , il-1 , il-4 , il-10 , il-13 , il - ra , mcp-1 , fractakine , eotaxin , il-15 , il-17a , tgf- , macrophage inflammatory proteins-1 , ( mip-1 or ccl3 ) , macrophage inflammatory proteins-1 ( mip-1 or ccl4 ) , and rantes and metabolic hormone panel containing leptin , c - peptide , glp-1 ( active ) glucagon , insulin , pancreatic peptide , and pyy ( total ) were measured using the multiplex map magnetic bead - based immunoassay kits ( millipore corp . blood was collected from lean ( n # 28 ) , overweight ( n # 10 ) , and obese ( n # 10 ) chimpanzees . following centrifugation , plasma was aliquoted immediately and frozen at 80c until assayed . on the day of assay , frozen plasma was thawed , mixed by vortexing , and then centrifuged at 10,000 rpm for 5 min to isolate debris prior to use in the assay . all assays were conducted according to the manufacturer 's instructions using handheld magnetic separator block for 96-well flat bottom plates ( millipore , millipore corp . ) and analyzed using the luminex 200 system ( bio - rad corp . ) . all samples were run in duplicate and cytokine standards supplied by the manufacturer were run on each plate . acquisition gates were set at 8,00015,000 , sample volume was 25 l , and 50 events per bead were acquired . mean fluorescence intensity was analyzed using the bioplex manager software version 5.0 ( bio - rad ) and compared to a standard curve to generate concentration values . values below the range of the standard curve were set to the lower limit of detection . the assay sensitivities ( minimum detectable concentrations , pg / ml ) were for ifn- and tnf ( 0.1 pg / ml ) , il-6 and il-10 ( 0.3 pg / ml ) , for il-1 and il-13 ( 0.4 pg / ml ) , for il-12p40 ( 10.5 pg / ml ) , for scd40l ( 4.9 pg / ml ) , for il-4 ( 0.6 pg / ml ) , for il - ra ( 2.9 pg / ml ) , for eotaxin ( 1.2 pg / ml ) , for fractakine ( 6.0 pg / ml ) , for mcp-1 ( 0.9 pg / ml ) , for cxcl8 ( 0.2 pg / ml ) , for c - peptide ( 24 pg / ml ) , for glp-1 ( 7.0 pg / ml ) , for glucagon ( 6.0 pg / ml ) , for insulin ( 58 pg / ml ) , for leptin ( 27.0 pg / ml ) , and for pyy ( 8.0 mg / ml ) . the proliferation of pbmcs samples from the chimpanzees was determined by the standard [ h ] thymidine incorporation as described previously [ 20 , 21 ] . briefly , aliquots of the pbmcs ( 10/well ) were seeded in triplicate wells of 96-well plates and stimulated for 6 days individually with the mitogens concanavalin - a ( con a ) , phytohemagglutinin ( pha ) , and pokeweed mitogen ( pwm ) ( each at 5 g / ml final concentration ) ( sigma , st louis . the culture medium without added mitogens served as negative control . after culturing for 5 day at 37c in 5% co2 , each well was pulsed for 18 h with 0.1 ci of methyl - h - thymidine ( icn , irvine , ca ) . these mitogen concentrations , pbmcs numbers , and incubation times were found to be optimal conditions for stimulation of pbmcs from healthy animals in our laboratory . the contents of the wells were then harvested onto glass fiber discs using a skatron cell harvester ( skatron laboratories , va , and usa ) . the amount of radioactivity was determined in a wallac liquid scintillation counter ( wallac1409 , mustionkatu , tarku , finland ) . the results were reported as corrected counts per minute ( cpm ) , which is the average count per minute ( cpm ) of mitogen - stimulated cultures minus the average cpm of cultures without mitogen . freshly - isolated pbmcs , as described above , were stimulated with the mitogens pha , con a , and pwm ( each at 5 g / ml final concentration ) to determine the numbers of ifn--producing cells by the enzyme linked immuno spot ( elispot ) assay using the methodology reported earlier [ 20 , 22 ] . briefly , aliquots of pbmcs ( 10/well ) were seeded in triplicate wells of 96-well plates ( polyvinylidene difluoride backed plates , maip s 45 , millipore , bedford , ma ) precoated with the primary ifn- antibody and the lymphocytes were stimulated with the different mitogens . after incubation for 30 hr at 37c , the cells were removed and the wells were thoroughly washed with pbs and developed as per protocol provided by the manufacturer . purple colored spots representing individual cells secreting ifn- were counted by an independent agency ( zellnet consulting , new jersey , nj ) using the ks - elispot automatic system ( carl zeiss inc . , thornwood , ny ) for the quantitative analysis of the number of ifn- spot forming cells ( sfc ) for 10 input pbmcs . responses were considered positive when the numbers of sfc with the test antigen were at least five and also were five above the background control values from cells cultured in the medium alone . aliquots of 1 10 cells resuspended in culture medium rpmi-1640 ( hyclone laboratories ) were dispensed in each well of a 96-well plate . the culture medium used was free of detectable endotoxin ( < 0.1 eu / ml ) and all other solutions were prepared using pyrogen - free water and sterile polypropylene plastic ware . the cells were then incubated with or without tlr ligand peptidoglycan ( pgn , tlr-2 ligand ) , ultrapurified lipopolysaccharide ( lps , tlr-4 ligand ) , polyinosinic - polycytidylic acid ( poly ic , tlr-3 ligand ) , and cytosine - phosphate - guanine ( cpg ) dna ( tlr-9 ligand ) all from invivogen ( invivogen corp . , san diego , ca , usa ) at 1 g / ml each for 24 hr at 37c in a 5% co2 atmosphere . the cell - free supernatant was harvested and stored at 70c for subsequent assays of cytokines and chemokines with the human inflammation cytometric bead array ( cba ) kit as described above . natural killer cells activity was assessed using the standard 4 h radioactive chromium ( cr-)release assay as described previously . peripheral blood mononuclear cells were isolated from heparinized blood and incubated with chromium 51 ( cr- ) labeled k562 target cells to yield 100 : 1 , 50 : 1 , 25 : 1 , and 12.5 : 1 effector - to - target ratios . the microtiter plates were then incubated for 4 hr at 37c in a 5% carbon dioxide incubator . at the end of the incubation , 100 l of supernatant was collected from each well and the amount of cr released was determined using the -counter . to account for the maximum release the % of specific lysis was calculated using the mean counts per minute ( cpm ) by the following formula : % specific lysis = ( experimental release spontaneous release)/(maximum release spontaneous release ) 100 . for statistical analysis , samples were grouped according to body condition of the animals from which samples were obtained . comparison between groups of chimpanzees was done by one - way analysis of variance with the kruskal - wallis test and gaussian approximation with dunn 's multiple comparison tests . data are presented as group means sd . only differences with a probability less than 0.05 all statistical analyses were conducted using graphpad prism 6.00 ( graphpad software , san diego , california , usa ) . in the present study , we compared the circulating plasma concentration of proinflammatory cytokines , anti - inflammatory cytokines , growth factors , and metabolic hormones in lean , overweight , and obese chimpanzees . plasma ifn- , il-6 , il-12p40 , tnf , scd40l , and il-1 levels were found to be significantly higher ( p < 0.0005 ) in overweight and obese chimpanzees than in lean controls ( figure 1(a ) ) . anti - inflammatory cytokines il-4 , il-10 , il-13 , and il - ra were also significantly higher in overweight and obese chimpanzees ( figure 1(b ) ) . plasma mcp-1 and fractakine levels were significantly higher in the lean group relative to the obese group while eotaxin levels were higher in the overweight group compared to the obese group ; however , no difference was found between obese and lean group ( figure 1(c ) ) . cytokine cxcl8 was significantly higher in the obese group compared to lean and overweight group ( figure 1(c ) ) . moreover , no significant differences were observed in il-12 , il-15 , il-17a , tgf- , mip-1a , mip-1 , and rantes plasma concentrations between the 3 groups of chimpanzees ( data not shown ) . obesity was also associated with increased levels of leptin , insulin , glucagon , glp-1 , pyy , and c - peptide in overweight and obese chimpanzees ( figure 2 ) . we also studied the immune responses of lean , overweight , and obese chimpanzees to test the hypothesis that obesity is associated with an impaired immune function and deregulated inflammatory response . in vitro proliferative responses of pbmcs and the production of ifn- we observed cona , pha , and pwm , 3 different mitogens inducing proliferative response of lymphocytes significantly lower in the overweight and obese groups compared to the lean group ( figure 3 ) . in ifn- producing t cells in ifn- elispot assay , pha , con a , and pwm stimulated pbmcs from the obese and overweight groups produced significantly higher levels of ifn- ( p < 0.005 ) than those from the lean group ( figure 4 ) . we also studied natural killer ( nk ) cells activity in lean , overweight , and obese chimpanzees using standard cr release assay . we observed significantly lower nk activity ( 47% compared to 51% ) in obese chimpanzees compared to lean chimpanzees ( figure 5 ) . tlrs play a crucial role in host defense against invading pathogens by mediating innate and adaptive immunities . recent studies suggest that adipocytes may play an important role in the physiological regulation of immune responses in fat deposits via toll - like receptor ( tlr ) signaling cascades . therefor we decided to study effect of various tlr ligands on pbmcs isolated from different groups of chimpanzees . pbmcs isolated from lean and obese chimpanzee blood were stimulated with tlr ligands for 20 hr and supernatants were measured for cytokines . obese chimpanzees showed increased capability to produce il-6 and cxcl8 after tlr-2 , tlr-3 , tlr-5 , and tlr-9 stimulation ( figure 6 ) . in contrast , when pbmcs were stimulated with tlr-4 , the obese chimpanzees had decreased il-6 and cxcl8 production compared to lean chimpanzees . however , no statistical difference was found between lean and obese chimpanzees for production of inflammatory cytokines by pbmcs stimulated with lipopolysaccharide ( tlrs ) . epidemiologic evidence suggests an important link between obesity and inflammation , although these findings were not appreciated in terms of the pathophysiologic conditions associated with obesity . for example , the levels of circulating fibrinogen and other acute phase reactants , including tnf , il-6 , and c - reactive protein ( crp ) were found to be elevated in obesity [ 24 , 25 ] . in the present study we investigated the relationship between cytokines and obesity by measuring plasma concentrations of different cytokines and metabolic hormones in lean , overweight , and obese chimpanzees . furthermore , we also examined the influence of obesity on immune function . the important finding from the present study is that obesity is associated with simultaneous induction of several cytokines and metabolic hormones in plasma of overweight and obese chimpanzees compared to lean counterparts . we demonstrated that obesity is associated with increased levels of c - peptide , leptin , insulin , glp-1 , pyy , and glucagon . our findings are in agreement with the studies demonstrating positive relationships between levels of adipose tissue and circulating plasma c - peptide , insulin , leptin , il-6 , and c - reactive protein levels and release of adipokines by adipose tissue matrix of obese humans [ 2628 ] . the plasma c - peptide has proved to be extremely valuable in the study of the natural history of type 1 diabetes to monitor insulin secretion in patients with insulin antibodies and as an adjunct in the investigation of patients with hypoglycemic disorders . have shown in a human study that serum leptin concentrations rise in proportion to the body adiposity and therefore , obese individuals with metabolic syndrome generally have higher circulating leptin concentrations . in animal models , increased expressions of leptin are associated with release of proinflammatory cytokines such as tnf and il-6 in monocytes and macrophages . leptin also has a significant effect on adaptive immunity , such as inducing a switch toward th1-cell mediated immune responses by increasing ifn- , tnf secretion , and the suppression of th2-cell responses in adipose tissue [ 30 , 31 ] . glucagon - like peptide 1 ( glp-1 ) , an insulinotropic hormone , also increased with obesity in our study . glp-1 has been shown to inhibit food intake and induce weight loss in humans independent of type 2 diabetes status . the anorectic effect of glp-1 could be attributable to both its effect on gastric emptying and a direct effect on neurons in the central nervous system involved in appetite regulation . overweight and obese chimps also had elevated levels of ifn- , tnf , il-6 , cxcl8 , il-12p40 , scd40l , il-1 , il-4 , il - ra , il-10 , il-13 , and mcp-1 compared to the lean counterparts . these findings are consistent with the recent reports in a human study by jung et al . , demonstrating the effect of weight loss on serum cytokines in human obesity . other studies have also revealed that proinflammatory cytokines il-6 and tnf were among the first to be implicated as a predictor or pathogenic mediator of insulin resistance , cvd , and in patients with type 2 diabetes [ 36 , 37 ] . . found that plasma levels of cxcl8 were higher in obese subjects compared with lean subjects . this finding could be related to the fact that obesity is known to be associated with a chronic state of low - grade inflammation . our study showed il- , il-6 , and il-10 were higher in obese chimpanzees . il-1 together with il-6 concentrations reportedly predicts risk for type 2 diabetes in humans better than either cytokine alone . il-10 is an anti - inflammatory cytokine produced by immune cells in adipose tissue that acts on adipocytes to improve insulin signaling , potentially decreasing further macrophage recruitment [ 41 , 42 ] . in 2003 , esposito et al . showed that circulating levels of il-10 were elevated in obese women and speculated that the higher il-10 levels represented the body 's attempt to inhibit continued proinflammatory cytokine production . in our study , we also found significantly increased levels of il-10 in overweight and obese chimpanzees . esposito et al . also reported that changes in life style aimed at reducing body weight and increasing physical activity over 1 year significantly reduced high il-10 levels in obese women , while manigrasso et al . reported no significant change in il-10 levels after an observed reduction in body weight in another obesity study . in the present study owed experimental evidence from high - fat - diet - fed mice suggesting that il-12 could have an additional role in the systemic low - grade inflammation and the ensuing development of obesity - related insulin resistance . schernthaner et al . demonstrated circulating scd40l levels in obese patients were closely related to insulin levels , triglycerides , and to the inflammatory biomarkers ( mcp-1 ) . two independent studies convincingly showed that the adipocyte - specific overexpression of mcp-1 in mice was sufficient to increase macrophage recruitment to adipose tissue and cause systemic insulin resistance , hepatic steatosis , and insulin resistance in liver and muscle . data from this study indicate obesity is related to lower t- and b - cell proliferation function and activity of natural killer cells and higher ifn- secretion in obese compared to lean chimpanzees . obesity has been linked to impaired t- and b - cell function , suggesting that metabolic alterations can be induced by or are a consequence of an altered self - immune tolerance and/or modulation of immune responses as reported in obese men and women who were compared with nonobese control subjects . obese children and adolescents have been reported to have impaired cutaneous delayed - type hypersensitivity responses , mitogen - stimulated lymphocyte proliferation , and bactericidal capacity [ 49 , 50 ] . few reports exist , comparing activity of natural killer cells in obese and nonobese human beings . moriguchi et al . showed that obesity is related to reduced activity of natural killer cells in older men and women . similar results were reported in obese zucker rats ; activity of natural killer cells has been reported to be suppressed , an effect found to be reversible through exercise training via improved lymphocyte glucose uptake and enhanced glut-1 expression . we have observed decreased cytokines ( il-6 and cxcl8 ) in obese chimpanzees when pbmcs were stimulated with specific ligands for tlr3 , tlr4 , and tlr9 but differences were not significant compared to lean chimpanzees . recently , kopp et al . demonstrated that il-6 release is stimulated in murine adipocytes by specific ligands for tlr1/2 , tlr2/tlr6 , tlr3 , and tlr4 , whereas monocyte chemoattractant protein-1 ( mcp-1 ) release is increased upon stimulation with specific ligands for tlr1/2 , tlr3 , and tlr4 . an advantage of the chimpanzee model for obesity and inflammatory studies , besides a genomic similarity greater than 98% in comparison to humans , is that we can control obesity - related exogenous factors , such as nutrition and lifestyle , thus studying the molecular events leading to metabolic syndrome , inflammatory , and/or autoimmune conditions . one of these limitations is that we are reporting systemic circulating cytokines / chemokines and metabolic hormones concentrations . for example , leptin function may be modulated by local leptin concentration , the ratio between free and bound leptin , the expression of different forms of the receptors , the ratio between signaling and nonsignaling receptors , and/or the presence of specific inhibitors . a second limitation is that we have a limited number of overweight and obese animals in our chimpanzee colony from which to acquire samples . the most important finding of the present study is that plasma cytokines / chemokines and metabolic hormones concentrations are significantly increased in obese chimpanzees relative to normal - weight healthy chimpanzees . also , in the present study , we observed an influence of obesity on immune function . in fact , the current study provides evidence that a proinflammatory state could be regarded as a significant prognostic indicator of the risk of obesity , cvd , and metabolic syndrome . although obesity is a complex , multifactorial trait that can not be explained by one factor , the findings of the present study represent important directions for the future planning of programs designed to prevent obesity - related diseases and in the identification of novel antiobesity targets .

obesity is characterized by chronic low - grade inflammation and serves as a major risk factor for hypertension , coronary artery disease , dyslipidemias , and type-2 diabetes . the purpose of this study was to examine changes in metabolic hormones , inflammatory cytokines , and immune function , in lean , overweight , and obese chimpanzees in a controlled environment . we observed increased plasma circulating levels of proinflammatory th-1 cytokines , interferon gamma , interleukin-6 , interleukin-12p40 , tumor necrosis factor , soluble cd40 ligand , and interleukin-1 and anti - inflammatory th-2 cytokines , interleukin-4 , interleukin - ra , interleukin-10 , and interleukin-13 in overweight and obese chimpanzees . we also observed increased levels of metabolic hormones glucagon - like - peptide-1 , glucagon , connecting peptide , insulin , pancreatic peptide yy336 , and leptin in the plasma of overweight and obese chimpanzees . chemokine , eotaxin , fractalkine , and monocyte chemoattractant protein-1 were higher in lean compared to obese chimpanzees , while chemokine ligand 8 increased in plasma of obese chimpanzees . we also observed an obesity - related effect on immune function as demonstrated by lower mitogen induced proliferation , and natural killer activity and higher production of ifn- by pbmc in elispot assay , these findings suggest that lean , overweight , and obese chimpanzees share circulating inflammatory cytokines and metabolic hormone levels with humans and that chimpanzees can serve as a useful animal model for human studies .

pott 's disease accounts for 50% of the cases of skeletal tuberculosis ( tb ) , 15% of the cases of extrapulmonary tuberculosis ( eptb ) and 2% of all cases of tb.1 the diagnosis of pott 's disease is mostly based on clinicoradiological observations substantiated by staining and culture methods to detect the causative organism . the typical tubercular lesions of the spine can be diagnosed by radiological methods as their sensitivity is increased by the advent of newer imaging techniques like computed tomography and magnetic resonance imaging ( ct and mri ) . these techniques give sufficient information about lesions in the bone and tissues ; although the definite diagnosis is based on tissue / pus examination.2 the conventional bacteriological examination includes ziehl - neelsen ( zn ) microscopy and culture for acid fast bacilli ( afb ) . zn microscopy method is a popular technique routinely used in the clinical laboratories worldwide , due to its simplicity , cost effectiveness and rapidity , but it suffers with low sensitivity and requires 10 to 10 bacilli / ml in the clinical specimens to be positive.34 the culture of mycobacterium tuberculosis ( m. tuberculosis ) is a gold standard method for diagnosis but it also has limitations like that required 6 to 8 weeks due to the slow growth of m. tuberculosis and is often negative , it still needs 10 -10 bacilli / ml ( live bacilli ) in clinical specimens for culture recovery and also stringent test conditions that is difficult to implement at primary or secondary clinical laboratories.56 moreover , histopathological examination plays a valuable role in the diagnosis of pott 's disease but sometime it may be inconclusive and in addition need high expertise and the final reporting also takes more than 1 week.6 recently , the molecular biology technique , polymerase chain reaction ( pcr ) represents a major advance in the diagnosis of tb.6 with the use of amplification systems , nucleic acid sequences unique to m. tuberculosis can be detected directly in clinical specimens , offering better accuracy than zn microscopy and greater speed than culture . the pcr has shown very promising results for early and rapid diagnosis of the disease due to its detection limit of one to 10 bacilli in various clinical specimens.6 the present study was undertaken to evaluate the efficiency and effectiveness of different laboratory diagnostic modalities along with the role of pcr in the diagnosis of clinicoradiological suspected cases of pott 's disease . 62 clinicoradiological suspected cases of pott 's disease with neurological complications [ table 1 ] were prospectively enrolled in this study , from 2008 to 2011 in the department of neurosurgery , at a tertiary care hospital , india . the specimens of these patients such as pus and tissue were obtained either during surgery or under ct guidance . clinicoradiological evidence of suspected pott 's disease we included clinicoradiological suspected cases of pott 's spine who underwent either open biopsy or ct guided aspiration at our institute . we excluded those subjects who did not give consent for biopsy or ct guided aspiration or biopsy showed neoplastic pathology . the specimens were examined by following methods : histopathology - the tissues stained with hematoxylin and eosin and zn stain were analyzed under the microscope for epithelioid cell granulomas with or without the presence of langerhans giant cell and afb.7periodic acid - schiff ( pas ) stain - fungal examination was performed by pas stain according to standard laboratory procedure.7zn microscopy - smears were stained using the zn method and examination for afb were done under light microscopy.8bactec 12b culture - bactec vials were incubated and interpreted as per becton dickinson ( bd , sparks , md , usa ) manual instructions.9p - nitro--acetylamino--hydroxy propiophenone ( nap ) test - the nap was done for identification and differentiation of mycobacterium tuberculosis complex ( mtbc ) and nontubercular mycobacterium from culture isolates.9molecular diagnosis - pcr for tb was done using a mtbc specific sequence is6110 ( 123 base pairs [ bp ] ) primer . histopathology - the tissues stained with hematoxylin and eosin and zn stain were analyzed under the microscope for epithelioid cell granulomas with or without the presence of langerhans giant cell and afb.7 periodic acid - schiff ( pas ) stain - fungal examination was performed by pas stain according to standard laboratory procedure.7 zn microscopy - smears were stained using the zn method and examination for afb were done under light microscopy.8 bactec 12b culture - bactec vials were incubated and interpreted as per becton dickinson ( bd , sparks , md , usa ) manual instructions.9 p - nitro--acetylamino--hydroxy propiophenone ( nap ) test - the nap was done for identification and differentiation of mycobacterium tuberculosis complex ( mtbc ) and nontubercular mycobacterium from culture isolates.9 molecular diagnosis - pcr for tb was done using a mtbc specific sequence is6110 ( 123 base pairs [ bp ] ) primer . dna extractions from tissues were done with hipura genomic dna extraction kit according to the manufacturer protocol3 . mb505 bacterial and yeast genomic dna miniprep purification spin kit , hi - media laboratories private limited mumbai , india . the reaction mixture contained 10 l pyrostart fast pcr master mix 2x ( dntp , taq polymerase with mgcl2 , fermentas , india ) , 1 l ( 10 pmole ) of each primer , 3 l water ( nuclease free ) and 5 l of extracted dna . the oligonucleotide primers11 were used forward and reverse sequence : cct gcg agc gta ggc gtc gg and ctc gtc cag cgc cgc ttc gg respectively ( sbs gene tech co. ltd . ) . these primers amplified a target fragment of 123 bp from the insertion like sequence element is6110 of mtbc . it was performed in a programmable thermal cycler ( mj research , ptc-100 , gmi , inc . each cycle comprised denaturation at 94c for 2 min , annealing at 68c for 2 min and primer extension at 72c for 1 min . after the 40 cycles , the additional extension for 10 min at 72c was carried out . the amplified products were separated on 2% agarose gel , visualized on an ultraviolet - transilluminator ( bangalore genei , bangalore , india ) . the presence of 123 bp fragment indicated as a positive test for mtbc [ figure 1 ] . the positive controls included the dna of h37rv strain and negative control included pcr grade water . polymerase chain reaction is6110 gene for detection of mycobacterium tuberculosis complex at 2% agarose gel . lane 1 ( l1 ) and l9 ladder 100 base pairs , l2 positive and l3 negative control , l4 , 5 , 6 , 8 showed positive and l7 showed negative specimens a definite treatment either by surgery ( 46 cases ) or by only anti tubercular treatment ( att ) ( 9 cases ) was offered . we followed the following major indications for surgical intervention in our setup : ( 1 ) advanced cases of neurological involvement such as marked sensory or sphincter disturbances , flaccid paralysis or severe flexor spasms . ( 2 ) prevertebral cervical abscess with difficulty in respiration and deglutition , seemingly difficult resolution on chemotherapy during conservative treatment . ( 3 ) worsening of already present neurological complications during the treatment . ( 5 ) no improvement in neurological complications / morbidity even after 4 - 6 weeks of att . a four drug regimen chemotherapy consisting of isoniazid , rifampicin , pyrazinamide and ethambutol ( hrze ) were given to all patients in their appropriate doses according to the weight of patients as per departmental protocol , following or along with surgery . four - drug regimen for at least 4 - 6 weeks to gauge the response was given ( 8 - 12 mg / kg / day rifampicin , 4 - 6 mg / kg / day isoniazid and 15 - 20 mg / kg / day ethambutol in a single daily dose ; and pyrazinamide 15 - 20 mg / kg / day in two divided doses ) , if not contraindicated the drug therapy was continued for 18 months12 . in cases of no improvements or hepatotoxicity , the patients were switched to modified att with the addition of ofloxacin and streptomycin and modification in doses of isoniazid and rifampicin or discontinuation of them . the therapeutic response was observed by clinical improvement in the form of healing sign that is an improvement in motor power / spasticity , sensory loss of limbs , autonomic dysfunctions and constitutional symptoms [ table 2 ] . the liver function test was done as per protocol to rule out the subclinical hepatotoxcity . the radiological assessment was done by using followup mri , the first at the end of 6 months of the commencement of the att and was compared with pretreatment images by the same senior radiologist of our institute . criteria for clinical improvement : ( first ( pain ) is mandatory and any other two improved symptoms / sign ) this study was approved by 41 institutional ethics committee ( a-04 pgi / imp / ec/41/28/2/2008 ) . the final diagnosis was established by using the collective results of zn microscopy , bactec culture , histopathological findings and response to att . the efficiency of test tools was calculated as ( total positive cases / total number of cases ) 100 . the positive concordance between traditional tools and pcr were assessed using the kappa coefficient ( > 0.75 , excellent agreement ; < 0.4 , poor agreement ; 0.4 and 0.75 , good to a fair agreement)13 and significance difference was analyzed by the chi square ( 2 ) test with the help of the spss 15.1 version . the cases were labeled as definite tb if at least one performed test ( zn microscopy / bactec culture / or histopathology ) was found positive . the specimens were examined by following methods : histopathology - the tissues stained with hematoxylin and eosin and zn stain were analyzed under the microscope for epithelioid cell granulomas with or without the presence of langerhans giant cell and afb.7periodic acid - schiff ( pas ) stain - fungal examination was performed by pas stain according to standard laboratory procedure.7zn microscopy - smears were stained using the zn method and examination for afb were done under light microscopy.8bactec 12b culture - bactec vials were incubated and interpreted as per becton dickinson ( bd , sparks , md , usa ) manual instructions.9p - nitro--acetylamino--hydroxy propiophenone ( nap ) test - the nap was done for identification and differentiation of mycobacterium tuberculosis complex ( mtbc ) and nontubercular mycobacterium from culture isolates.9molecular diagnosis - pcr for tb was done using a mtbc specific sequence is6110 ( 123 base pairs [ bp ] ) primer . histopathology - the tissues stained with hematoxylin and eosin and zn stain were analyzed under the microscope for epithelioid cell granulomas with or without the presence of langerhans giant cell and afb.7 periodic acid - schiff ( pas ) stain - fungal examination was performed by pas stain according to standard laboratory procedure.7 zn microscopy - smears were stained using the zn method and examination for afb were done under light microscopy.8 bactec 12b culture - bactec vials were incubated and interpreted as per becton dickinson ( bd , sparks , md , usa ) manual instructions.9 p - nitro--acetylamino--hydroxy propiophenone ( nap ) test - the nap was done for identification and differentiation of mycobacterium tuberculosis complex ( mtbc ) and nontubercular mycobacterium from culture isolates.9 molecular diagnosis - pcr for tb was done using a mtbc specific sequence is6110 ( 123 base pairs [ bp ] ) primer . dna extractions from tissues were done with hipura genomic dna extraction kit according to the manufacturer protocol3 . mb505 bacterial and yeast genomic dna miniprep purification spin kit , hi - media laboratories private limited mumbai , india . the reaction mixture contained 10 l pyrostart fast pcr master mix 2x ( dntp , taq polymerase with mgcl2 , fermentas , india ) , 1 l ( 10 pmole ) of each primer , 3 l water ( nuclease free ) and 5 l of extracted dna . the oligonucleotide primers11 were used forward and reverse sequence : cct gcg agc gta ggc gtc gg and ctc gtc cag cgc cgc ttc gg respectively ( sbs gene tech co. ltd . ) . these primers amplified a target fragment of 123 bp from the insertion like sequence element is6110 of mtbc . it was performed in a programmable thermal cycler ( mj research , ptc-100 , gmi , inc . each cycle comprised denaturation at 94c for 2 min , annealing at 68c for 2 min and primer extension at 72c for 1 min . after the 40 cycles , the additional extension for 10 min at 72c was carried out . the amplified products were separated on 2% agarose gel , visualized on an ultraviolet - transilluminator ( bangalore genei , bangalore , india ) . the presence of 123 bp fragment indicated as a positive test for mtbc [ figure 1 ] . the positive controls included the dna of h37rv strain and negative control included pcr grade water . polymerase chain reaction is6110 gene for detection of mycobacterium tuberculosis complex at 2% agarose gel . lane 1 ( l1 ) and l9 ladder 100 base pairs , l2 positive and l3 negative control , l4 , 5 , 6 , 8 showed positive and l7 showed negative specimens a definite treatment either by surgery ( 46 cases ) or by only anti tubercular treatment ( att ) ( 9 cases ) was offered . we followed the following major indications for surgical intervention in our setup : ( 1 ) advanced cases of neurological involvement such as marked sensory or sphincter disturbances , flaccid paralysis or severe flexor spasms . ( 2 ) prevertebral cervical abscess with difficulty in respiration and deglutition , seemingly difficult resolution on chemotherapy during conservative treatment . ( 3 ) worsening of already present neurological complications during the treatment . ( 5 ) no improvement in neurological complications / morbidity even after 4 - 6 weeks of att . a four drug regimen chemotherapy consisting of isoniazid , rifampicin , pyrazinamide and ethambutol ( hrze ) were given to all patients in their appropriate doses according to the weight of patients as per departmental protocol , following or along with surgery . four - drug regimen for at least 4 - 6 weeks to gauge the response was given ( 8 - 12 mg / kg / day rifampicin , 4 - 6 mg / kg / day isoniazid and 15 - 20 mg / kg / day ethambutol in a single daily dose ; and pyrazinamide 15 - 20 mg / kg / day in two divided doses ) , if not contraindicated the drug therapy was continued for 18 months12 . in cases of no improvements or hepatotoxicity , the patients were switched to modified att with the addition of ofloxacin and streptomycin and modification in doses of isoniazid and rifampicin or discontinuation of them . the therapeutic response was observed by clinical improvement in the form of healing sign that is an improvement in motor power / spasticity , sensory loss of limbs , autonomic dysfunctions and constitutional symptoms [ table 2 ] . the liver function test was done as per protocol to rule out the subclinical hepatotoxcity . the radiological assessment was done by using followup mri , the first at the end of 6 months of the commencement of the att and was compared with pretreatment images by the same senior radiologist of our institute . criteria for clinical improvement : ( first ( pain ) is mandatory and any other two improved symptoms / sign ) this study was approved by 41 institutional ethics committee ( a-04 pgi / imp / ec/41/28/2/2008 ) . the final diagnosis was established by using the collective results of zn microscopy , bactec culture , histopathological findings and response to att . the efficiency of test tools was calculated as ( total positive cases / total number of cases ) 100 . the positive concordance between traditional tools and pcr were assessed using the kappa coefficient ( > 0.75 , excellent agreement ; < 0.4 , poor agreement ; 0.4 and 0.75 , good to a fair agreement)13 and significance difference was analyzed by the chi square ( 2 ) test with the help of the spss 15.1 version . the cases were labeled as definite tb if at least one performed test ( zn microscopy / bactec culture / or histopathology ) was found positive . the study group consisted of 62 specimens from suspected pott 's spine individuals . on the basis of histopathological examination , 7 cases ( 5 specimens from open biopsy and 2 from fine needle aspiration [ fna ] ) were excluded from the study as they were diagnosed to be neoplastic . out of rest 55 cases , 46 specimens were obtained through open biopsy during surgery and 9 were ct guided fna . further , in all ( 46 ) open biopsy specimens , 7 were positive for epithelioid cell granulomas with afb , 27 revealed granulomatous lesions suggestive of tb with or without the presence of the langerhans giant cell . however , the histological diagnosis could not be reached in 12 specimens because of either suspicious granulomatous lesions or on account of inconclusive histology . five of nine ct guided fna specimens were diagnosed as tubercular and 4 specimens showed no granulomatous / no malignant lesions . the received specimens included 31 ( 56.4% ) pus and 15 ( 27.3% ) tissues through open biopsy along with 6 ( 11% ) pus and 3 ( 0.54% ) tissues were obtained through ct guided fna . the overall collected specimens were consisted 37 pus and 18 tissue specimens ( either open biopsy or by ct fna ) were analyzed . the zn microscopy was positive in total 20 ( 36.4% ) of 55 cases ( 18/37 cases in pus and 2/18 cases in tissue specimens ) . the bactec culture was positive 27 ( 49% ) of 55 cases ( 22/37 in pus and 5/18 in tissue specimens ) . the desired amplification of pcr is6110 was positive in 37 ( 67.5% ) of 55 cases ( 30/37 cases in pus and 7/18 in tissue specimens ) [ table 3 ] . the kappa coefficient of pcr with zn microscopy ( = 0.37 , [ p = 0.001 ] ) showed poor agreement and with the bactec culture ( = 0.57 , [ p < 0.001 ] ) it showed a good agreement [ figure 2 ] . result of laboratory findings bar diagram showing kappa coefficient concordance between pcr and traditional tools . ( znm = zn microscopy , bc = bactec culture and hp = histopathology ) on histopathological results ; 39 ( 71% ) cases were positive and 16 ( 29% ) were negative for tb . out of 39 positive cases , 31 ( 56.4% ) were positive on pcr also . thus , the false negative and positive of pcr was found to be 8 ( 14.4% ) and 6 ( 11% ) respectively . therefore , the kappa coefficient of pcr with histopathology was ( = 0.4 [ p = 0 . 004 ] ) slightly good [ figure 2 ] . in 55 cases , it was possible to reach a final diagnosis through the combined result of all performed tests ( without consideration of pcr results ) and with att response . hence , among all 55 cases , 45 ( 82% ) cases were tb positive test and 10 ( 18% ) were negative by all tests carried out ( zn microscopy , bactec culture and histopathology ) . although pcr showed 67.3% ( 37/55 ) sensitivity at the final diagnosis , while pcr showed 37/45 positive and no any false positive out of 10 negative cases by all traditional tests . therefore , the kappa coefficient of pcr with the combined results of all traditional tests ( = 0.63 [ p < 0.001 ] ) ultimately reached to a fair positive agreement [ figure 2 ] . the present study , the combination of ( a ) zn microscopy with bactec , the total 31 positive cases due to only 20 cases positive on zn microscopy and an additional 11 cases were positive on bactec culture . ( b ) zn microscopy with histopathology , the total positivity showed 43 cases due to 23 cases more positive on histopathology against zn microscopy . ( c ) bactec culture with histopathology , the total 45 cases to be positive due to 39 cases positive on histopathology and an additional 6 cases were positive on bactec culture , where the histopathology was negative . ( d ) the total 45 cases were positive in combination of zn microscopy + bactec culture + histopathology . ( e ) zn microscopy with pcr , the total positivity was 38 cases , in which 18 more cases were positive on pcr method . ( f ) bactec culture with pcr , the total 38 positive cases due to only 27 cases positive on bactec culture and additional 11 cases were positive on pcr . ( g ) histopathology with pcr , the total 45 cases positive on this possible battery due to 39 cases positive on histopathology and an additional 6 cases were positive on pcr . ( h ) the battery of zn microscopy + bactec culture + histopathology + pcr showed ultimately 45 cases were positive because pcr could not give any more positive against the mentioned battery d and g [ table 4 ] . the received specimens included 31 ( 56.4% ) pus and 15 ( 27.3% ) tissues through open biopsy along with 6 ( 11% ) pus and 3 ( 0.54% ) tissues were obtained through ct guided fna . the overall collected specimens were consisted 37 pus and 18 tissue specimens ( either open biopsy or by ct fna ) were analyzed . the zn microscopy was positive in total 20 ( 36.4% ) of 55 cases ( 18/37 cases in pus and 2/18 cases in tissue specimens ) . the bactec culture was positive 27 ( 49% ) of 55 cases ( 22/37 in pus and 5/18 in tissue specimens ) . the desired amplification of pcr is6110 was positive in 37 ( 67.5% ) of 55 cases ( 30/37 cases in pus and 7/18 in tissue specimens ) [ table 3 ] . the kappa coefficient of pcr with zn microscopy ( = 0.37 , [ p = 0.001 ] ) showed poor agreement and with the bactec culture ( = 0.57 , [ p < 0.001 ] ) it showed a good agreement [ figure 2 ] . result of laboratory findings bar diagram showing kappa coefficient concordance between pcr and traditional tools . on histopathological results ; 39 ( 71% ) cases were positive and 16 ( 29% ) were negative for tb . out of 39 positive cases , 31 ( 56.4% ) were positive on pcr also . thus , the false negative and positive of pcr was found to be 8 ( 14.4% ) and 6 ( 11% ) respectively . therefore , the kappa coefficient of pcr with histopathology was ( = 0.4 [ p = 0 . 004 ] ) slightly good [ figure 2 ] . in 55 cases , it was possible to reach a final diagnosis through the combined result of all performed tests ( without consideration of pcr results ) and with att response . hence , among all 55 cases , 45 ( 82% ) cases were tb positive test and 10 ( 18% ) were negative by all tests carried out ( zn microscopy , bactec culture and histopathology ) . although pcr showed 67.3% ( 37/55 ) sensitivity at the final diagnosis , while pcr showed 37/45 positive and no any false positive out of 10 negative cases by all traditional tests . therefore , the kappa coefficient of pcr with the combined results of all traditional tests ( = 0.63 [ p < 0.001 ] ) ultimately reached to a fair positive agreement [ figure 2 ] . the present study , the combination of ( a ) zn microscopy with bactec , the total 31 positive cases due to only 20 cases positive on zn microscopy and an additional 11 cases were positive on bactec culture . ( b ) zn microscopy with histopathology , the total positivity showed 43 cases due to 23 cases more positive on histopathology against zn microscopy . ( c ) bactec culture with histopathology , the total 45 cases to be positive due to 39 cases positive on histopathology and an additional 6 cases were positive on bactec culture , where the histopathology was negative . ( d ) the total 45 cases were positive in combination of zn microscopy + bactec culture + histopathology . ( e ) zn microscopy with pcr , the total positivity was 38 cases , in which 18 more cases were positive on pcr method . ( f ) bactec culture with pcr , the total 38 positive cases due to only 27 cases positive on bactec culture and additional 11 cases were positive on pcr . ( g ) histopathology with pcr , the total 45 cases positive on this possible battery due to 39 cases positive on histopathology and an additional 6 cases were positive on pcr . ( h ) the battery of zn microscopy + bactec culture + histopathology + pcr showed ultimately 45 cases were positive because pcr could not give any more positive against the mentioned battery d and g [ table 4 ] . pott 's disease , accounts for 2% of all tb infections.1 the diagnosis of pott 's disease is principally based on classical clinical manifestations of spinal infection supplemented by modern imaging like ct and mri.1415 the ct and mri in particular can detect the subtle changes in the spine , from initial changes in the intensity pattern of the vertebrae to the extreme changes like deformity of the spine including soft tissue shadow , granulation tissue and pus etc.16 however , the various pathologies including neoplastic and other inflammatory conditions simulate with pott 's diseases on imaging . the current study excluded 7 of 62 cases ; they were diagnosed to be neoplastic by the histopathological examination . it signifies that even strong clinicoradiological diagnosis based on classical or nonclassical radiological and clinical findings can be wrong on histopathological evaluation . the remaining 55 cases , 20 ( 36.3% ) were tested positive with zn microscopy and 27 ( 49% ) with bactec culture . an earlier report by jain et al.17 found 14.8% positivity on zn microscopy and 11.11% positivity on culture method in cases of spinal tb . the zn microscopy and bactec culture reports revealed higher positivity in relation to the earlier published series . our higher detection rate was because of the fact that we used the liquid culture medium bactec 12b medium . it is the rapid , sensitive and efficient method for the isolation of mycobacterium in a clinical laboratory as compared to solid medium lowenstein jensen.19 moreover , it is established today that all the traditional tests need to have a minimum concentration of bacilli for yielding positive results as mentioned in the introduction . hence , if the specimens , which are naturally having more concentration of bacilli , like sputum and pus it may become easy to detect m. tuberculosis.20 similarly , in our study the pus specimens had higher recognition of tubercular bacilli on zn microscopy and bactec culture methods [ table 3 ] . the study quoted the detection rate of eptb including pott 's spine varying from 53% to 81%.182122 the histopathological diagnosis could be reached in 71% ( 39 out of 55 ) of the cases which were either confirmed by the presence of afb in 12.7% or strongly suggestive of tubercular pathology . in our series , we found 16 cases were negative on histology . among these negative cases , 10 out of 16 cases had been under anti tubercular therapy for more than 6 months and another reason of histology negative might be a possible sampling error during the collection of the representative tissues for processing . the positivity of fna specimens seems to higher than surgical specimens , however , we require a long sample size for definite conclusion . molecular diagnostic tool , such as pcr has been shown to have higher efficiency in the diagnosis of pulmonary tb as well as eptb.4 it has been studied on clinical specimens of sputum , cerebrospinal fluid , edta - blood , pleural fluid , fluid from fistulae and pus from wounds.2324 in an attempt to increase diagnostic precision , we performed pcr is6110 , which was detected in 37/55 ( 67.3% ) cases . various studies documented the increased positivity by pcr targeting is6110 in specimens of eptb i.e. , sekar et al.25 found 63% positivity , negi et al.26 73% and tiwari et al.27 62% positivity among clinical specimens of eptb . the overall positive agreement shows a fair agreement ( = 0.63 ) between pcr and definite tb . the false negative results were observed in 14.5% cases out of 45 cases diagnosed by all performed traditional tests . the reasons of false negative results of pcr may be on account of insufficient specimens , irregular distribution of mycobacterium bacilli in specimens , the presence of extensive necrosis and presence of inhibitors.28 the limitation of our study are a low positive correlation ( = 0.4 ) of pcr with histopathological findings . this can be explained up to some extent by the specimen variation as that is different specimens ( such as pus , tissue ) from the same subjects . surgeons prefer to send tissue specimens for histology and pus for zn microscopy , bactec culture and pcr . in this study histology was done on tissue specimens ( all 55 cases ) , while zn microscopy , bactec culture and pcr were done in 37 pus and 18 tissue specimens . in our diagnostic battery , all performed traditional tests were diagnosed 45/55 cases and pcr was alone detected 37/55 cases . thus , positive results of pcr with the clinicoradiological assumption it would be possible to make a decision in suspected pott 's disease to start early treatment to prevent irreversible complications . in the present study , it is observed that no single diagnostic modality is having a good positivity rate . hence , a combination of diagnostic battery is required for rapid diagnosis and better results . the combination of bactec culture and histopathology has detected a maximum of 45 cases . while the combination of pcr and histopathology has also detected same number of cases table 4 . as culture takes long incubation time for results , the combination of pcr and histopathology can be applied for rapid detection of pott 's disease . to conclude , for diagnosis of pott 's disease , the combined results of zn microscopy , bactec culture and histopathology are most appropriate . however , for rapid diagnosis the combination of pcr amplification and histopathology offer a better prospect .

background : the diagnosis of pott 's disease is mostly based on clinicoradiological observations substantiated by the bacterial culture , staining and histopathology . since , no single technique is enough to conclude pott 's disease in diagnosis , the present study was undertaken to correlate the clinicoradiological , microbiological , histopathological and molecular method to evaluate the effectiveness in diagnosis of pott 's disease.materials and methods:62 clinicoradiologically suspected cases of pott 's disease were included in this study . the specimens for diagnostic work up were collected either during surgery or by computed tomography guided fine needle aspiration . all these specimens were tested for tuberculosis ( tb ) through ziehl - neelsen ( zn ) microscopy , bactec culture , histopathology and polymerase chain reaction ( pcr ) . the final diagnosis was established by the results of performed tests and clinicoradiological improvement of cases at the end of 6 months on anti tubercular treatment.results:out of 62 cases , 7 were excluded from this study as these were turned out to be neoplastic lesions on histopathology . amongst remaining 55 cases , the tb was diagnosed in 39 ( 71% ) on histopathology , 37 ( 67.5% ) on pcr , 27 ( 49% ) on bactec culture and 20 ( 36.3% ) on zn microscopy . ultimately 45 cases were tested as positive and 10 were detected as negative for tb in combination of zn microscopy , bactec culture and histopathology . pcr was positive in 37 of 45 cases and 10/55 cases remained negative . on clinical analysis of these 10 cases , it was noted that these were cases of relapse / poor compliance . the combination of pcr and histopathology was also shown positive for tb in 45 cases . hence , the pcr showed a fair positive agreement ( c = 0.63 ) against the combined results of all performed traditional methods.conclusions:the combination of pcr and histopathology is a rapid and efficient tool for diagnosis of pott 's disease .

it originated in germany in the 1920s and was defined by rules of a specific technique evolved in the usa in the years 19301950 , employing elements of african and native american folklore . modern dance employs all the techniques of ballet ; however , a modern dancer is not obliged to uncritically follow the classical rules of execution of these techniques . cohan writes that the most important elements of a modern dancer s work are : ground work , work with the center of gravity , and motion in space . he mentions that the most important thing in a well performed movement is self - awareness , and that modern dance consists of centering , gravitation , balance , posture , gestures , rhythm , motion in space , and breathing . although ballet is a form of art , it has much in common with professional sports . modern style choreographers often propose very dangerous dancing movements from the point of view of biomechanics of the locomotor system . movement designers are often the result of absence of basic knowledge about the capabilities and limitations of the human locomotor apparatus . modern dancers in many shows perform falls , using their upper limbs as shock absorbers , they roll around the stage , women lift men , and men not infrequently lift persons heavier than themselves , they wear heavy costumes , they walk on stilts , hang from ropes , etc . in addition , the performances often take place outdoors or in halls that are not prepared for such events . research shows that some of them , particularly during the landing phase ( during the eccentric phase of muscle work related to shock absorption ) , generate high values of the vertical component of ground reaction force ( grf ) , which may reach 7.4 bw [ 13 ] . peak forces of impact phases occur after a few dozen milliseconds . according to luke , solomon , liederbach , and nicholas [ 48 ] , the largest percentage of injuries affecting professional dancers are chronic injuries , such as soft - tissue inflammation , injuries resulting from overload , and muscle strains and tears . nevertheless , ankle joint , foot , spine , hip joint , and knee joint are the most frequently listed regions of the body injured by dancers . research by gorwa shows that modern dancers are primarily exposed to injuries of the spine , achilles tendon , knee joint , and hip joint . studies performed by gorwa also show that up to 93% of modern dancers ( female and male ) suffer from chronic injuries directly related to their profession . around 30% of persons who reported these conditions associated them with specific movements . female modern dancers complained of spine conditions such as neck pain and lumbar or sacral spine region pain ; they associated them with sudden twists and the very expressive movements so common in modern dancing style . male modern dancers listed pain and injuries of the spine , feet , and knees as chronic conditions and indicated these problems were caused by lifting dancing partners , as well as by bizarre choreography . since correct technique is a factor that significantly decreases the risk of injury , and the degree of motor habit control ( e.g. , the proper execution of a given sports technique ) determines the values of forces recorded during the landing phase , we observed kinematic values and recorded the technique of execution of a modern dance movement referred to as the stag jump . the purpose of this study was to conduct a thorough analysis of kinematic structure of the landing phase in a selected expressive dance movement called the stag jump performed as a case study of 1 male and 1 female professional modern dancer . researchers who compared the technique and morphological differences between modern and ballet dancers were bronner and ojofetimi , solomon and micheli , and solomon et al . . the methodology and measurements presented in this work are part of a project whose purpose will be to devise guidelines for new training methods to prevent injuries in dancers . the conducted kinematic analysis consisted in establishing such characteristic parameters as trajectories of joint centers and movement sequences of lower limbs , pelvis movement , angle changes in hip , knee , and ankle joints in the sagittal plane , as well as changes of foot position in relation to the ground . synchronization of kinematic measurements with the measurement of vertical components of ground reaction forces in the landing phase during this stage of the study allowed us to identify impact loads on the locomotor apparatus of the studied dancers and to forecast methods for minimizing such loads . this work is a case study of 2 professional modern dancers ( a female and a male ) who work full time in dance theatre , 6 days per week on average , and do not perform any other job . it should be stressed that although artists in ballet dance groups in poland have identical education ( national ballet schools : primary and secondary ) around 7% of members of modern dance groups begin their dance education after age 18 . the tests used for this study were carried out in the biomechanical laboratory of the chair of biomechanics at the university school of physical education in poznan , poland . kinematic values were computed with the use of apas motion analysis system and grfs were measured with the use of kistler force plate sampling at 1000 hz . the motion of test participants was recorded by 4 basler digital cameras with recording frequency of 200 hz . the images recorded by the cameras were transferred to a computer , where , with the use of apas software , the films were processed and the locations of markers positioned on dancers bodies were determined . at the same time , grfs acting during the landing phase were recorded . due to the extensiveness of performed dancing movements , the cameras were arranged to allow us to precisely determine the kinematics of the pelvis and the right lower limb during the landing phase . the number and location of markers ( figure 1 ) made it possible to determine the positions of right lower limb joint centers and , in consequence , the relative angular motion of particular lower limb segments and the pelvis . this work is a case study of 2 professional modern dancers ( a female and a male ) who work full time in dance theatre , 6 days per week on average , and do not perform any other job . it should be stressed that although artists in ballet dance groups in poland have identical education ( national ballet schools : primary and secondary ) around 7% of members of modern dance groups begin their dance education after age 18 . the tests used for this study were carried out in the biomechanical laboratory of the chair of biomechanics at the university school of physical education in poznan , poland . kinematic values were computed with the use of apas motion analysis system and grfs were measured with the use of kistler force plate sampling at 1000 hz . the motion of test participants was recorded by 4 basler digital cameras with recording frequency of 200 hz . the images recorded by the cameras were transferred to a computer , where , with the use of apas software , the films were processed and the locations of markers positioned on dancers bodies were determined . at the same time , grfs acting during the landing phase were recorded . due to the extensiveness of performed dancing movements , the cameras were arranged to allow us to precisely determine the kinematics of the pelvis and the right lower limb during the landing phase . the number and location of markers ( figure 1 ) made it possible to determine the positions of right lower limb joint centers and , in consequence , the relative angular motion of particular lower limb segments and the pelvis . the measurements allowed the authors to determine values of lower limb joint angles and pelvis position within the coordinate system ( figure 2 ) . the following angle vs. time graphs flexion / extension , abduction / adduction , and rotation ; for knee joint flexion / extension and rotation ; for talocrural joint dorsal / plantar flexion and pronation / supination . the usage of force plates allowed us to establish ground reaction forces ( grf ) acting during the landing phase that followed the performed movement . based on the results of the measurements , it was possible to assess movements performed by the dancers by analyzing the line graphs of grf and line graphs of angles in lower limb joints . this paper concentrates on the analysis of the stag jump movement performed by 2 modern dancers a woman and a man . the following diagrams ( figures 310 ) present the results of measurements performed on modern dancers . the pelvis rotation in relation to the long axis of the body was 30 for the female dancer and 25 for the male dancer . pelvis tilt in the sagittal plane is significant during the entire landing phase and fluctuates between 32 and 50 for the female dancer and 2131 for the male dancer . both pelvis rotation in relation to the vertical axis and pelvis tilt in the sagittal plane result from specific positioning of the non - supporting limb ( figure 6 ) . the line graph of joint angles in the sagittal plane ( figures 7 and 8) shows that they are similar in shape but differ in the range of motion ( rom ) . the rom in the hip joint is 50 for the female and 36 for the male . in the knee joint ( figure 8) , rom of ankle joint ( figure 9 ) in the sagittal plane was 60 for the female dancer and 59 for the male dancer . this study allowed us to describe the way a dancer positions ( coordinates ) the body during landing that follows a dance movement and the forces that act on the lower limb . the values of the vertical component of ground reaction reached the levels that equaled almost 4 times the body weight of man and a little over 3.2 times the weight of woman . considering that the values of reaction in the lower limb joints exceed the values of ground reaction , one can observe the loads that a dancer s musculoskeletal system is exposed to . such high loads are a frequent cause of injuries to dancers , as well as musculoskeletal problems that dancers already suffer from during their careers . ground reactions measured during the landings of classical dancers in other studies were even higher than in modern dancers ( 2.65 bw for females and 3.7 bw for males ) . it is directly related to the requirements imposed on the technique of dance movements , which are much more rigorous in classical dance . interestingly , the landing phase durations in this movement are very short . the impulsive character of the peak vertical component of ground reaction acting on feet during the landing phase that follows a ballet jump poses the greatest risk of joint injury , beginning with the lowest joints metatarsal and ankle joints . the short time allowed for the performance of a movement and the need to immediately transition to the following one leave dancers extremely little time to coordinate the entire body and prepare for receiving the requirement to perform each dance movement strictly according to the rules considerably limits the ability to properly absorb the impact during landing . this is why it is so important to define the best way ( from the biomechanical point of view and taking into account the artistic design ) to perform individual dance movements by dancers . it is obvious that the maximum value of the vertical component of grf depends on the way dancers will use their feet as a form of biological shock absorbers . the following observations can be formulated on the basis of the analysis of joint angles and grf peaks . at the moment the foot contacts the ground , it remains in plantar flexion in a manner that only the toes are in contact with the ground . when the vertical component of ground reaction reaches its maximum , the foot is resting flat on the ground ( parallel to the ground ) . therefore , the center of mass is transferred from toes and metatarsus to the vicinity of the ankle joint , which decreases the moment of vertical ground reaction forces ( and thus the forces generated by muscles working around the ankle joint ) . in addition , the flat positioning of the foot lowers the center of the ankle joint , shortening the moment arms of the components acting in the horizontal plane . when the vertical component of grf reaches its peak , thighs and shins assume almost vertical positions . this leads to the conclusion that when the greatest loads act on the limb , these will be mostly compressive loads , for which the long bones of the lower limb are well prepared ( they possess the best strength in the longitudinal direction ) . the analysis of foot angle in relation to the ground shows that after landing on his toes , the male dancer bends his foot dorsally and for a short period of time ( about 0.2 s ) puts weight on the entire foot and then stands on the toes . the female dancer lands in a different manner after touching the ground with the toes , she puts weight on the entire foot and keeps the foot in this position throughout most of the support phase . one of the most significant factors that lead to injuries is high dynamic loads ( forces ) of an impact nature that occur particularly during the landing phases of numerous expressive jumps . peak forces are generated over a very short time period , which makes coordinating such movement structures very difficult . it is important to realize that these forces are transmitted via the relatively small surface of the dancer s feet that are only weakly protected by characteristic footwear the forefoot region and toes . properly mastering movement technique is tremendously important in minimizing injuries . the knowledge of dynamic loads acting on the dancer s body and kinematics of movement may represent a significant contribution in the description of the proper technique of dance movements . the following values that best describe the landing phase of the stag jump were established : loads transmitted to the dancer s foot ; line graphs of joint angles in the lower limb ; changes in position of the foot in relation to the ground ; values of joint angles , as well as the angle between the foot and the ground when the highest value of the vertical component of ground reaction occurs . this allowed us to analyze the influence of movement technique on the values of external loads ( grf ) . analysis of the conducted research reveals significant differences in the technique of the same dance movement . , as well as broner and ojofeitimi , suggests that these differences in technique probably do are not due to the dancer s sex , professional experience , or educational path of the dancer . the female dancer went through all levels of ballet education in poland ( primary and secondary national ballet school ) , and the male dancer began his education as an adult at the danceweb workshop in vienna . however , such a conclusion needs to be confirmed by further research with a larger sample . observed correctly that certain dance movements can be mastered only until puberty , when the body can still be molded . during the next phases of research , the results of these measurements will be used to determine loads transmitted by musculoskeletal system when performing dancing movements . for this purpose , a mathematical model of lower limb movement will be prepared , allowing us to determine the forces generated by muscles and the forces transmitted by joint surfaces . the values thus obtained will complement the results of the research . on the basis of these results , works are being conducted in cooperation with ballet teams , choreographers , and dance teachers to devise training methods using the methodology and the potential of a biomechanical laboratory . these training methods would be aimed preventing injury and physical overload , as well as increasing dancers knowledge of biomechanics and kinesiology of the locomotor apparatus in dancing .

backgroundthis paper presents a case study of kinematic analysis of the modern dance movement known as the stag jump . detailed analysis of the kinematic structure of this movement as performed by the dancers , accompanied by measurements of impact forces during landing , will allow the authors to determine , in subsequent model - based research phases , the forces acting in knee joints of the lower landing limb.material/methodstwo professional modern dancers participated in the study : a male and a female . the study consisted in recording the values of ground reaction and body motion , and then determining and analyzing kinematic parameters of performed movements.resultsthe results of measurement of joint angles in the landing lower limb , pelvis , and foot position in relation to the ground , as well as the level of vertical components of ground reaction , provided insight into the loading response phase of the stag jump . the measurements and obtained results show differences between the man and woman in ground reactions and kinematic quantities.conclusionsthe results obtained during the research may be used in the development and teaching of dancing movements . training sessions , carried out in the biomechanical laboratory , with active participation of dancing teachers , could form a basis for a prevention model of injuries and physical overloads occurring within this occupational group.primary differences in the stag jump performance technique probably result from the different educational path the man and the woman went through .

lung cancer risk prediction models provide an estimate of individual 's risk of developing lung cancer such that at - risk subjects can be targeted for preventive and treatment interventions ( 1 ) . risk models hold promise for improving patient care by aiding the clinicians decision making process regarding choice of interventions and/or treatments . risk models can also guide selection of individuals at the population level , for screening : this ensures limited resources are focussed on those individuals who are most likely to benefit . existing lung cancer absolute risk prediction models are mostly based on traditional epidemiological and/or clinical risk factors ( 27 ) , limiting their predictive and discriminative abilities . for an improved precision , incorporation of genetic and molecular markers of disease in risk models has been advocated ( 8) and aided by recent proliferation of genetic / genomic research which has led to the identification of susceptibility genes and biological markers in many diseases ( 912 ) . common gene variants involved in lung cancer have been recently identified through a number of large , collaborative , genome - wide association studies . susceptibility genes identified to date include those on chromosomes 5p15.33 , 6p21 , and 15q24 - 25.1 ( 1315 ) . apart from these , other genetic loci have also been identified in candidate gene association studies targeting specific molecular pathways ; such as genes encoding proteins in cell cycle control , oxidant response , apoptosis , dna repair , cell adhesion and airways inflammatory response ( 16,17 ) . while genomics research has been very fruitful in identifying these common , low - risk allelic variants , there is a growing scepticism regarding their usefulness in risk prediction . it has been shown that risk profiles generated by common low - moderate susceptibility loci , in a simple additive model , provides limited discrimination ( 18,19 ) . the limited contribution of single nucleotide polymorphisms ( snps ) to risk profiling has been partly blamed on restriction to a limited number of significant alleles , methodological limitations regarding assessment of model performance and statistical approaches for incorporating the variants ( 19 ) . whilst the usual approach has been to utilise only the significant variants for risk profiling , an improved disease prediction may be attained by accounting for a large ensemble of markers ( 20 ) . for the relatively few markers arising from candidate - gene studies , incorporation of the interactive effect of these genes , through epistasis modelling , may provide better predictions beyond that afforded by the limited effect of multiple loci using additive effects ( 21 ) . models including epistatic interactions take into account the complex biological relationships among the loci and extend the traditional method that focuses only on additive score using a weighted or unweighted number of risk alleles , which assume independence between the markers ( 22 ) . in the past three decades , improvements in risk prediction models brought about by the inclusion of markers and genetic factors were quantified using changes in the area under the receiving - operating characteristic curve ( auc ) ( 23 ) . recently , an increasing popular measure of evaluating improvements in risk predictions , the net reclassification improvement was introduced ( 24 ) . this measure involves cross - tabulating categories of predicted risk for 2 models , usually one with the new marker under study and the other without it , to see how persons are classified differently when these models are used ( 25 ) . in this study , we investigated the presence of epistasis among a panel of snps previously validated individually in lung cancer ( 26 ) and used both area under the receiver operating characteristic ( auc ) analysis and net reclassification improvement ( nri ) to assess the contribution of adding an interactive epistatic effect to an extensively validated clinical - based risk model for lung cancer . details of recruitment procedure , study design and validation have been previously reported ( 3,27 ) . briefly , incident cases of histologically or cytologically confirmed lung cancer , ages between 20 and 80 years , were included . lung cancer included any of topographical subcategories of code c34 of the international classification of disease for oncology 9th revision . two population controls per case , matched on year of birth ( 2 years ) and gender , were selected from registers of general practitioners in liverpool area . the study protocol was approved by the liverpool research ethics committee , and all research participants provided written informed consent in accordance with the declaration of helsinki . in this study , we utilised complete genotype data on individuals included in the independent validation of snps identified in a candidate - gene genetic association study ( 26 ) . the data comprises of 2385 subjects ( cases=718 , controls=1667 ) selected from individuals recruited into the llp between 2000 and 2008 . of this number , 1362 ( cases=418 and controls=914 ) were included in llp case - control data used to develop the llp risk model ( 3 ) . data on epidemiological , clinical and lifestyle factors were collected using a standardised questionnaire supplemented with hospital case note reviews conducted by trained llp research nurses . information documented includes : patients smoking status ( smoking duration ) , previous history of pulmonary diseases ( pneumonia , copd and bronchitis ) , previous history of malignant diseases excluding skin melanoma , occupational exposure to asbestos , family history of lung cancer with age at onset , and case diagnosis details ( date of diagnosis , histological subtype and staging ) . genetic data consist of 20 snps independently validated from 157 snps screened in a candidate - gene discovery study ; details of selection and genotyping have been described elsewhere ( 26 ) . briefly , 157 candidate snps were screened in a discovery cohort of 439 subjects ( 200 controls and 239 lung cancer cases ) , which identified 30 snps associated with either the healthy smokers ( protective ) or lung cancer ( susceptibility ) phenotype . after genotyping this 30 snp panel in a validation cohort of 491 subjects ( 248 controls and 207 lung cancers ) and , using the same protective and susceptibility genotypes from the discovery cohort , a 20 snp panel were selected based on replication of snp associations in the validation cohort that includes variants in the metabolism of smoking - derived carcinogens ( nat2 and cyp2e1 ) , inflammatory cytokines [ interleukins 1(il1b ) , 8(il8 ) , and 18(il18 ) , tissue necrosis factor 1 receptor ( tnfr1 ) , toll - like receptor 9 ( tlr9 ) ] , smoking addiction [ dopamine d2 receptor ( drd2 ) and dopamine transporter 1(dat1 ) ] , nicotine dependency [ 5-nachr ( chrna3 ) ] , antioxidant response to smoking [ 1 anti - chymotrypsin ( serpina3 ) and extracellular super - oxide dismutase ( sod3 ) ] , cell cycle control , dna repair and apoptosis ( xpd , tp73 , bcl-2 , fasl , cerb1 , and rev1 ) and integrins ( itga11 , itgb3 ) implicated in apoptosis . genomic dna was extracted from whole blood samples by standard salt - based methods and purified genomic dna was aliquoted ( 10 ng/l concentration ) into 96-well plates . genotyping was performed on a sequenom system ( sequenom autoflex mass spectrometer and samsung 24 pin nanodispenser ) ( 26 ) . characteristics of the subjects in the cases and controls were compared using t - test for continuous variables and test or fisher 's exact test for discrete variables as appropriate . genotype and allele frequencies were checked for each snp for hardy - weinberg equilibrium ( hwe ) . the multifactor dimensionality reduction ( mdr ) and random forest ( rf ) were used to investigate gene - gene interactions by identifying snp combinations that provide the best discrimination of the status of the subjects . mdr is a non - parametric , model - free method that utilises a constructive induction technique to collapse high - dimensional genetic data into a single dimension ( 28,29 ) . it pools multi - locus genotypes into high and low risk groups using an exhaustive search to identify optimal combination of polymorphisms , which can then be evaluated for its ability to classify or predict disease status . in our implementation of mdr the relief - f algorithm as implemented in the mdr was used as a first approach to select among the 20 snps that are most likely to interact . an exhaustive search of all possible 15 loci were then explored using 10-fold cross validation as described by hahn et al ( 28 ) . cross - validation allows estimation of the prediction error by leaving out a portion of the data as an independent test set . with 10-fold cross - validation , the data are divided into 10 equal parts , the model was developed on 9/10 of the data ( i.e. the training data ) and then evaluated on the remaining 1/10 of the data ( i.e. the independent testing data ) . this is repeated for each possible 9/10 and 1/10 of the data and the resulting ten prediction errors are averaged ( 29 ) . mdr , then , seeks to find the single - locus or multi - locus predictor(s ) for explaining the outcome ( based on a balanced accuracy measure - the arithmetic mean of sensitivity and specificity ) , based on the available genomic information ( 30 ) . the prediction accuracy and cross - validation consistency defined as the number of cross - validation replicates ( partitions ) in which that same n - locus predictor(s ) was chosen as the best predictor of lung cancer status i.e. the number of replicates in which it minimised the classification error were used to select the best snps in each 1 to 5-locus combination ( 31 ) . the overall best snp classifier of lung cancer status was selected as the one with the maximum prediction accuracy and cross - validation consistency and evaluated statistically using 1000-fold permutation test . for comparison , we used the freely available willows software package for generating rf ( 32 ) . rf ranks variables by a variable importance index , a measure which reflects the importance of a variable on the basis of the classification accuracy , while considering the interaction among variables ( 33 ) . a classification tree was built by the recursive partitioning method ; each tree is constructed using a different cohort of bootstrap samples from the original cohort . approximately one - third of the samples are left out of the bootstrap ( oob ) samples and hence not used in the construction of the tree . the number of trees was set to 10,000 and the default values of the other parameters as provided by the program were used . several classification trees were created with replacement from the original data imput into the program . to determine the importance of an snp , first the values of the snp in the oob samples are randomly permuted ; then both the original oob samples and the permuted oob samples are classified by the corresponding tree . the difference in the correct classification rates between the original and permuted oob samples determines the importance of the snp , and the variable importance is obtained by averaging the differences over all trees in the random forest ( 32,34 ) . risk prediction was performed using the same risk factors included in the llp risk model ( 3 ) . multivariable logistic regression was employed to generate estimates of predicted 5-year absolute risk of lung cancer in i ) a model with epidemiological data and ii ) an extended model with both genetic and epidemiological data . the baseline risk ( , the constant term in the regression model ) for the prediction of 5-year absolute risk using the extended model with both genetic and epidemiological data was recalculated . the method for calculating the baseline from age- and gender - specific lung cancer incidence rates from the liverpool area has been described ( 3 ) . the only difference is that the probability model now includes information on rs1799732 ( drd2 ) , rs5744256 ( il-18 ) and rs2306022 ( itga11 ) . the area under the receiver - operating characteristics ( auc ) was used to i ) assess the discriminatory ability of the models , and ii ) compare the models with and without snps . the increase in auc furthermore , the net reclassification improvement ( nri ) was used to assess the added discrimination offered by the addition of snps to the risk model ( 24 ) . improvement in model calibration was assessed using akaike information criteria ( aic ) and bayesian information criteria ( bic ) . unless otherwise stated , all analyses were performed using r version 3.1.1 and stata version 13.1 ( statacorp lp , college station , tx , usa ) . details of recruitment procedure , study design and validation have been previously reported ( 3,27 ) . briefly , incident cases of histologically or cytologically confirmed lung cancer , ages between 20 and 80 years , were included . lung cancer included any of topographical subcategories of code c34 of the international classification of disease for oncology 9th revision . two population controls per case , matched on year of birth ( 2 years ) and gender , were selected from registers of general practitioners in liverpool area . the study protocol was approved by the liverpool research ethics committee , and all research participants provided written informed consent in accordance with the declaration of helsinki . in this study , we utilised complete genotype data on individuals included in the independent validation of snps identified in a candidate - gene genetic association study ( 26 ) . the data comprises of 2385 subjects ( cases=718 , controls=1667 ) selected from individuals recruited into the llp between 2000 and 2008 . of this number , 1362 ( cases=418 and controls=914 ) were included in llp case - control data used to develop the llp risk model ( 3 ) . data on epidemiological , clinical and lifestyle factors were collected using a standardised questionnaire supplemented with hospital case note reviews conducted by trained llp research nurses . information documented includes : patients smoking status ( smoking duration ) , previous history of pulmonary diseases ( pneumonia , copd and bronchitis ) , previous history of malignant diseases excluding skin melanoma , occupational exposure to asbestos , family history of lung cancer with age at onset , and case diagnosis details ( date of diagnosis , histological subtype and staging ) . genetic data consist of 20 snps independently validated from 157 snps screened in a candidate - gene discovery study ; details of selection and genotyping have been described elsewhere ( 26 ) . briefly , 157 candidate snps were screened in a discovery cohort of 439 subjects ( 200 controls and 239 lung cancer cases ) , which identified 30 snps associated with either the healthy smokers ( protective ) or lung cancer ( susceptibility ) phenotype . after genotyping this 30 snp panel in a validation cohort of 491 subjects ( 248 controls and 207 lung cancers ) and , using the same protective and susceptibility genotypes from the discovery cohort , a 20 snp panel were selected based on replication of snp associations in the validation cohort that includes variants in the metabolism of smoking - derived carcinogens ( nat2 and cyp2e1 ) , inflammatory cytokines [ interleukins 1(il1b ) , 8(il8 ) , and 18(il18 ) , tissue necrosis factor 1 receptor ( tnfr1 ) , toll - like receptor 9 ( tlr9 ) ] , smoking addiction [ dopamine d2 receptor ( drd2 ) and dopamine transporter 1(dat1 ) ] , nicotine dependency [ 5-nachr ( chrna3 ) ] , antioxidant response to smoking [ 1 anti - chymotrypsin ( serpina3 ) and extracellular super - oxide dismutase ( sod3 ) ] , cell cycle control , dna repair and apoptosis ( xpd , tp73 , bcl-2 , fasl , cerb1 , and rev1 ) and integrins ( itga11 , itgb3 ) implicated in apoptosis . genomic dna was extracted from whole blood samples by standard salt - based methods and purified genomic dna was aliquoted ( 10 ng/l concentration ) into 96-well plates . genotyping was performed on a sequenom system ( sequenom autoflex mass spectrometer and samsung 24 pin nanodispenser ) ( 26 ) . characteristics of the subjects in the cases and controls were compared using t - test for continuous variables and test or fisher 's exact test for discrete variables as appropriate . genotype and allele frequencies were checked for each snp for hardy - weinberg equilibrium ( hwe ) . the multifactor dimensionality reduction ( mdr ) and random forest ( rf ) were used to investigate gene - gene interactions by identifying snp combinations that provide the best discrimination of the status of the subjects . mdr is a non - parametric , model - free method that utilises a constructive induction technique to collapse high - dimensional genetic data into a single dimension ( 28,29 ) . it pools multi - locus genotypes into high and low risk groups using an exhaustive search to identify optimal combination of polymorphisms , which can then be evaluated for its ability to classify or predict disease status . in our implementation of mdr the relief - f algorithm as implemented in the mdr was used as a first approach to select among the 20 snps that are most likely to interact . an exhaustive search of all possible 15 loci were then explored using 10-fold cross validation as described by hahn et al ( 28 ) . cross - validation allows estimation of the prediction error by leaving out a portion of the data as an independent test set . with 10-fold cross - validation , the data are divided into 10 equal parts , the model was developed on 9/10 of the data ( i.e. the training data ) and then evaluated on the remaining 1/10 of the data ( i.e. the independent testing data ) . this is repeated for each possible 9/10 and 1/10 of the data and the resulting ten prediction errors are averaged ( 29 ) . mdr , then , seeks to find the single - locus or multi - locus predictor(s ) for explaining the outcome ( based on a balanced accuracy measure - the arithmetic mean of sensitivity and specificity ) , based on the available genomic information ( 30 ) . the prediction accuracy and cross - validation consistency defined as the number of cross - validation replicates ( partitions ) in which that same n - locus predictor(s ) was chosen as the best predictor of lung cancer status i.e. the number of replicates in which it minimised the classification error were used to select the best snps in each 1 to 5-locus combination ( 31 ) . the overall best snp classifier of lung cancer status was selected as the one with the maximum prediction accuracy and cross - validation consistency and evaluated statistically using 1000-fold permutation test . for comparison , we used the freely available willows software package for generating rf ( 32 ) . rf ranks variables by a variable importance index , a measure which reflects the importance of a variable on the basis of the classification accuracy , while considering the interaction among variables ( 33 ) . a classification tree was built by the recursive partitioning method ; each tree is constructed using a different cohort of bootstrap samples from the original cohort . approximately one - third of the samples are left out of the bootstrap ( oob ) samples and hence not used in the construction of the tree . the number of trees was set to 10,000 and the default values of the other parameters as provided by the program were used . several classification trees were created with replacement from the original data imput into the program . to determine the importance of an snp , first the values of the snp in the oob samples are randomly permuted ; then both the original oob samples and the permuted oob samples are classified by the corresponding tree . the difference in the correct classification rates between the original and permuted oob samples determines the importance of the snp , and the variable importance is obtained by averaging the differences over all trees in the random forest ( 32,34 ) . risk prediction was performed using the same risk factors included in the llp risk model ( 3 ) . multivariable logistic regression was employed to generate estimates of predicted 5-year absolute risk of lung cancer in i ) a model with epidemiological data and ii ) an extended model with both genetic and epidemiological data . the baseline risk ( , the constant term in the regression model ) for the prediction of 5-year absolute risk using the extended model with both genetic and epidemiological data was recalculated . the method for calculating the baseline from age- and gender - specific lung cancer incidence rates from the liverpool area has been described ( 3 ) . the only difference is that the probability model now includes information on rs1799732 ( drd2 ) , rs5744256 ( il-18 ) and rs2306022 ( itga11 ) . the area under the receiver - operating characteristics ( auc ) was used to i ) assess the discriminatory ability of the models , and ii ) compare the models with and without snps . the increase in auc furthermore , the net reclassification improvement ( nri ) was used to assess the added discrimination offered by the addition of snps to the risk model ( 24 ) . improvement in model calibration was assessed using akaike information criteria ( aic ) and bayesian information criteria ( bic ) . unless otherwise stated , all analyses were performed using r version 3.1.1 and stata version 13.1 ( statacorp lp , college station , tx , usa ) . seven hundred and eighteen cases and 1667 population controls were successfully genotyped for 20 snps , which had been independently validated from 157 snps screened in a candidate - gene discovery study ( 26 ) . men constituted the majority of the study population cases ( 57.7% ) and ( 58.1% ) controls . the proportion of ever smokers was significantly higher in cases ( 93.2% ) compared with controls ( 65.2% ) . significant differences were observed in other risk factors including smoking duration , prior diagnosis of pneumonia , occupational exposure to asbestos , and prior diagnosis of tumour ( p<0.001 ) . heterozygosity for rs1799732 ( drd2 ) , rs5744256 ( il-18 ) and rs2306022 ( itga11 ) conferred an increased risk for lung cancer in reference to the wild - type genotype [ or 1.74 ( 95% ci 1.312.32 ) ; 1.42 ( 95% ci 1.161.72 ) and 2.53 ( 95% ci 2.063.12 ) , respectively ] . the homozygote genotype for rs16969968 ( chrna3/5 ) , rs13181 ( ercc2 ) , rs5744256 ( il-18 ) and rs2306022 ( itga11 ) increased the risk of developing lung cancer with reference to the wild - type [ or 1.46 ( 95% ci 1.111.93 ) ; 1.46 ( 95% ci 1.131.89 ) ; 4.07 ( 95% ci 3.115.31 ) 4.09 ( 95% ci 2.217.56 ) , respectively ] . table iii summarises the result obtained from the mdr analysis investigating epistatic effects among the snps . the best candidate classifiers of lung cancer status based on five snp loci selected using the cross - validation consistency , training and testing accuracy were as follows : single locus : rs2306022 ( itga11 ) ; 2 loci : rs5744256 ( il-18 ) , rs2306022 ( itga11 ) ; 3 loci : rs1799732 ( drd2 ) , rs5744256(il-18 ) , rs2306022 ( itga11 ) ; 4 loci : rs1696998 ( chrna3/5 ) , rs1799732 ( drd2 ) , rs5744256 ( il-18 ) , rs2306022 ( itga11 ) ; 5 loci : rs1799732 ( drd2 ) , rs763110 ( fasl ) , rs5744256 ( il-18 ) , rs4073 ( il-8 ) , rs2306022 ( itga11 ) . the 3 loci consisting of snps rs1799732 ( drd2 ) , rs5744256 ( il-18 ) and rs2306022 ( itga11 ) appears to be the overall best classifier of lung cancer status . these loci had training and testing accuracy of 0.6592 and 0.6572 respectively , and the cross validation consistency of 10/10 ( model selected as the best of 3 in 10 cv ) p<0.0001 ( permutation test ) . rf ranks variables by a variable importance index , which is an indication of the importance of a variable on the basis of classification accuracy while considering interaction among variables . the three snps [ rs1799732 ( drd2 ) , rs5744256 ( il-18 ) and rs2306022 ( itga11 ) ] selected as the overall best classifier of lung cancer status in mdr were also ranked top 3 by rf using variable importance index . table v summarises reclassifications for cases and controls using epidemiological model and models with snps [ rs1799732 ( drd2 ) , rs5744256 ( il-18 ) and rs2306022 ( itga11 ) ] . subjects were categorised into three different thresholds ; low - risk ( < 0.91 ) , intermediate risk ( 0.91 to 5.12 ) , and high - risk ( > 5.12 ) groups . the threshold values were defined from the predicted 5-year absolute risks for the original llp control samples ( n=1,272 ) , assuming the risk distribution in this group is similar to that of the general liverpool population . the upper threshold ( 5.12 ) corresponds to the value for the top 20% of predicted absolute risks in the population ; individuals whose 5-year predicted absolute risk is equal to or above this value are designated as high risk group . the lower threshold value of 0.91 corresponds to the bottom 40% of absolute risks in the control population and represents the low risk group . this definition of high risk and low risk groups was used in an earlier study ( 13 ) . overall , 42.7% of cases ( 311/727 ) and 35.7% of controls ( 592/1657 ) had their predicted risks re - classified into other risk groups when snps were incorporated into risk prediction model . this reclassification showed improvement ( upward shift ) in approximately 25% of cases and became worse ( downward shift ) for 18% resulting in a net gain of ~6% . the net gain was higher for controls ( 10% ) with overall improvement in risk ( downward shift ) for 23% and worse performance ( upward shift ) for 13% . table vi depicts the odds ratios ( or ) and 95% confidence intervals ( 95% ci ) of the multivariate logistic regression models for the epidemiological model and the extended model with snps . the ors and 95% ci for both models were comparable which suggests the absence of any serious confounding effects of snps on the relationship between each of the other clinical and epidemiological risk factors and lung cancer risk . model fit was assessed using akaike information criterion ( aic ) and bayes information criteria ( bic ) . there was an improvement in model fit as indicated by the reduction of the aic from 2098.42 from the epidemiological model to 1930.14 for the extended model with snps . likewise , a similar reduction was observed in bic from 2167.75 from the epidemiological model to 2016.80 for the extended model with snps . the apparent auc of the epidemiological model without snps was 0.75 ( 95% ci 0.730.77 ) . when epistatic data were incorporated in the extended model , the auc increased to 0.81 ( 95% ci 0.790.83 ) which corresponds to 8% increase in auc for the model with snps ( delong 's test p=2.2e- ) . after correction for optimism , the auc was 0.73 for the epidemiological model and 0.79 for the extended model . this study demonstrates the use of comprehensive analytical techniques for investigating the contribution of adding an interactive effect of a panel of genetic markers ( snps ) to the prediction of individual absolute risk of developing lung cancer , using a risk model similar to the llp model ( 3 ) . using genotype data from 2385 individuals included in the independent validation of snps identified in a candidate - gene genetic association study from the llp case - control study , we found the 3 loci genotype interaction rs2306022 ( itga11 ) , rs5744256 ( il-18 ) and rs1799732 ( drd2 ) provided the best classifier of disease status using both mdr and rf . adding these snps to a clinically - based lung cancer risk model lead to an increase in auc ( 0.75 to 0.81 ) ; and we utilised two different approaches ; discrimination and reclassification to evaluate the contribution of adding an interactive epistatic effect to a risk model for lung cancer . auc is the most popular metric used for measuring the discriminatory power of a model to correctly classify subjects with or without a disease . our result showed 8% increase in auc ( delong 's test p=2.2e- ) for risk prediction in the extended model with snps ( auc=0.81 ) compared with the epidemiological model without snps ( auc=0.75 ) , which is higher than that reported by li et al ( 9 ) . li et al , in a chinese case - control study , genotyped five snps identified in genome wide association study of 5068 subjects . the genetic risk scores based on these snps were estimated by two approaches : a simple risk alleles count ( cgrs ) and a weighted method ( wgrs ) . their auc in combination with the bootstrap resampling method was used to assess the predictive performance of the genetic risk score for lung cancer . smoking history contributed significantly to lung cancer ( p<0.001 ) risk [ auc=0.619 ( 0.6030.634 ) ] , and incorporated with wgrs gave an auc value of 0.639 ( 0.6210.652 ) after adjustment for over - fitting ( 9 ) . for clinical risk prediction , it is expedient that a new risk model correctly classify individuals into higher or lower risk categories ( 37 ) . pencina et al introduced a new metric , the nri that assesses the improvement in model performance by quantifying the degree of correct classification ( 24 ) . by applying the nri , we demonstrated that the addition of snps lead to a 17.5% improvement in the risk classification of the subjects . this study is the first to replicate the association between the itga11 locus and lung cancer described by young and colleagues ( 26 ) . itga11 ( integrin 11 ) belongs to the family of transmembrane receptors that mediate physical interactions between cells and extracellular matrix protein collagens ( 38 ) . itga11 is localised to stromal fibroblast and commonly overexpressed in non - small cell lung cancer ( nsclc ) ( 38 ) . earlier studies have reported that the interactions of tumour cells with the stroma play a crucial role in tumour growth , invasion , metastases , angiogenesis , and chemoresistance ( 3841 ) . it has been shown that carcinoma - associated fibroblasts in nsclc express higher levels of itga11 . one of the factors which are affected by higher levels of itga11 during tumour growth is igf2 ( 38,42 ) . higher levels of igf2 , in turn , can stimulate growth of tumour epithelial cells leading to tumour progression and metastasis ( 38 ) . il18 ( interleukin-18 ) is a multifunctional cytokine ( an extracellular signalling molecule ) that augments ifn- production and affects tumour immune response , leukocyte recruitment , cancer proliferation , and angiogenesis ( 43,44 ) . an earlier study reported the presence of il-18 in induced sputum of lung cancer patients ( 45 ) . farjadfar et al also reported an association between il-18 and lung cancer in a case - control study including 73 lung cancer patients ( 53 squamous carcinoma and 20 small cell lung carcinoma ) , and 97 healthy regional aged - matched individuals ( 46 ) . they suggested that their finding may be attributed to the disruption of the potential of camp responsive element - binding protein site and subsequent reduction in il-18 production as observed in other cancer types ( 46 ) . reduced production of il-18 can result in decreased ifn- synthesis , imbalanced th1/th2 differentiation , insufficient activation of natural killer cells and cd8 lymphocytes ( 46,47 ) impairment of cancer cell apoptosis and efficient angiogenesis ( 47,48 ) . ddr2 is a receptor tyrosine kinase that binds collagen as its endogenous ligand ( 49 ) . it has been previously shown to promote cell migration , proliferation , and survival when activated by ligand binding and phosphorylation ( 49,50 ) . harmmerman et al reported that ddr2 mutations are present in 4% of small cell lung carcinomas ; gain - of - function mutations in this gene are important oncogenic events and are amenable to therapy with dasatinib ( 49 ) . since epistasis is known to contribute to unexplained genetic variation of common diseases , some genetic variants may have a weak and insignificant independent effect , but strong epistatic effect ( biological interaction ) with other variants . the integration of genetic variants in risk prediction models beyond the traditional epidemiological covariates have been applauded as the way forward in lung cancer risk prediction modelling ( 8) . genetic factors function primarily through complex mechanisms that involve interactions between multiple genes and environmental factors ( 21,22 ) . however , the effect of interaction will be disregarded if the genetic effect is examined in isolation , without taking cognisance of potential interactions with other unknown factors ( 31 ) . the inherent nonlinearity implies that epistasis can occur among polymorphisms even in the absence of independent effect of the components , presenting computational intensive difficulties and statistical challenges because an infinite number of combinations that needs to be evaluated ( 21,22 ) . the use of nonparametric and genetic model - free machine learning algorithms such as mdr ( 28,29 ) and rf ( 32,33 ) have been proposed to overcome the caveat of the traditional parametric statistics and have proven to be useful in this study . here we see that the addition of the three snps increases the auc , indicating that the interaction of these loci may be important . there was an improvement in model fit , as indicated by the reduction of the aic and bic . furthermore , the snps used in this study were internally validated using a two stage design as described by young et al ( 26 ) and the use of hwe to minimise genotyping error are methodological advantages utilised to minimise false positive results . to the best of our knowledge , this is the first study to evaluate the addition of these specific interactions of snps to a lung cancer risk model . however , the result of this study must be considered in the light of a number of limitations . first , our prediction model used covariates in the llp risk model but did not include other risk factors for lung cancer such as chronic obstructive pulmonary diseases . however , the objective of this study is to evaluate the contribution of adding an interactive effect of a panel of genetic snps to the llp risk model and the model has been validated in three independent external datasets with good discrimination and calibration ( 27 ) . second , our study demonstrates how a modest increase in auc can lead to a substantial improvement in reclassification as quantified by the nri . this finding supports a suggestion by pencina et al that a small increase in auc might still be suggestive of a meaningful improvement ( 24 ) . third , the llp comprise predominantly caucasians and therefore , the lack of ethnic diversity implies that this model may be less applicable in non - white population . fourth , our approach to reclassification did not distinguish between persons with competing events and those without an event because both are classified as not having the event of interest . fifth , the lack of validation of the epistatic model in an independent population is a limitation , however , the application of bootstrap correction for optimism addresses in part the lack of independent validation . sixth , many of the 20 snps from table ii failed to replicate in the current study , particularly given the larger sample size ( 718 cases , 1667 controls ) in the current study when compared with previous study ( 248 cases , 207 controls ) . a plausible explanation for this observation may be due to the fact that the non - significant snps play lesser or no role in epistatic interaction . finally , our threshold values for risk classification was based on the predicted 5-year absolute risk for original llp control samples but the appropriateness of these threshold values in other populations is uncertain . using different values could have affected the results of our reclassification analyses and subsequent clinical implications . in conclusion , our result shows in principle how an snp epistatic factor can be incorporated into an epidemiological risk prediction model . in this study , inclusion of snps rs1799732 ( drd2 ) , rs5744256 ( il-18 ) , rs2306022 ( itga11 ) resulted in a modest improvement in lung cancer risk prediction .

incorporation of genetic variants such as single nucleotide polymorphisms ( snps ) into risk prediction models may account for a substantial fraction of attributable disease risk . genetic data , from 2385 subjects recruited into the liverpool lung project ( llp ) between 2000 and 2008 , consisting of 20 snps independently validated in a candidate - gene discovery study was used . multifactor dimensionality reduction ( mdr ) and random forest ( rf ) were used to explore evidence of epistasis among 20 replicated snps . multivariable logistic regression was used to identify similar risk predictors for lung cancer in the llp risk model for the epidemiological model and extended model with snps . both models were internally validated using the bootstrap method and model performance was assessed using area under the curve ( auc ) and net reclassification improvement ( nri ) . using mdr and rf , the overall best classifier of lung cancer status were snps rs1799732 ( drd2 ) , rs5744256 ( il-18 ) , rs2306022 ( itga11 ) with training accuracy of 0.6592 and a testing accuracy of 0.6572 and a cross - validation consistency of 10/10 with permutation testing p<0.0001 . the apparent auc of the epidemiological model was 0.75 ( 95% ci 0.730.77 ) . when epistatic data were incorporated in the extended model , the auc increased to 0.81 ( 95% ci 0.790.83 ) which corresponds to 8% increase in auc ( delong 's test p=2.2e-16 ) ; 17.5% by nri . after correction for optimism , the auc was 0.73 for the epidemiological model and 0.79 for the extended model . our results showed modest improvement in lung cancer risk prediction when the snp epistasis factor was added .

skin wounds are caused by the loss of connection in the skin and recovery requires cellular and biochemical reactions . chronic wounds arise from the lack of physiological processes and the disease due to complications ( e.g. infection and amputation ) has devastating consequences on societies . according to the principles of iranian traditional medicine ( itm ) this is a systematic review study that involved gathering data from three traditional medical textbooks , namely canon of medicine , tib - e - akbari and exir - e - azam with the keyword chronic wound or few tables were developed for nutrition measures as well as edible and topical medicinal herbs . electronic databases such as pubmed , scopus , web of science , and cochrane library were searched for relevant articles and those related to effective medicinal herbs in the treatment of wounds were obtained . depending on appearance and secretions , wounds are divided into two categories , namely simple and compound wounds . the prognostic factors are based on the age , weight , accompanied disease , as well as the quality and quantity of the wound secretions . patient s diet is very important and oral medications have a major role in the whole body detoxification . topical medications are used together with the above - mentioned treatments ; noting that without detoxification , these medications are not effective entirely . body detoxification is the first medical step , after which topical medications could lead to a better wound healing result .

background : skin is the body s first defense against stressful factors . skin wounds are caused by the loss of connection in the skin and recovery requires cellular and biochemical reactions . chronic wounds arise from the lack of physiological processes and the disease due to complications ( e.g. infection and amputation ) has devastating consequences on societies . according to the principles of iranian traditional medicine ( itm ) , these disorders can be treated in a step - by - step procedure.methods:this is a systematic review study that involved gathering data from three traditional medical textbooks , namely canon of medicine , tib - e - akbari and exir - e - azam with the keyword chronic wound or ghorha . in the next step , few tables were developed for nutrition measures as well as edible and topical medicinal herbs . electronic databases such as pubmed , scopus , web of science , and cochrane library were searched for relevant articles and those related to effective medicinal herbs in the treatment of wounds were obtained.results:depending on appearance and secretions , wounds are divided into two categories , namely simple and compound wounds . the prognostic factors are based on the age , weight , accompanied disease , as well as the quality and quantity of the wound secretions . wound treatments include medical nutrition therapy , edible medications , and manipulation therapies . patient s diet is very important and oral medications have a major role in the whole body detoxification . topical medications are used together with the above - mentioned treatments ; noting that without detoxification , these medications are not effective entirely.conclusion:wound healing requires physiological processes within the body . from the perspective of itm , body detoxification is the first medical step , after which topical medications could lead to a better wound healing result .

in generalized ligamentous laxity , the range of motion across various joints in an individual is increased compared to the mean range of motion in the general population ( 1 ) . although this could occur as a result of genetic disorders affecting the connective tissue , in most individuals there is no genetic aberrancy and it exists in isolation ( 2 ) . the prevalence of generalized ligamentous laxity varies among different races from 5% to 57% of the general population ( 3 ) . it is well known that generalized ligamentous laxity is implicated in various musculoskeletal injuries , especially in the lower extremities , such as cruciate ligament injuries , patellar instability , and ankle injuries ( 4 , 5 ) . although generalized ligamentous laxity is a predisposing factor for shoulder instability ( 6 ) , to the best of the authors knowledge no study has investigated the role of generalized ligamentous laxity in acute and chronic shoulder injuries in athletes . this study aimed to determine whether generalized ligamentous laxity can be a predisposing factor for acute and chronic shoulder injuries in athletes . this cross - sectional study was conducted from april to october 2015 . to obtain a representative sample of athletes with shoulder injuries , we sought all athletes who had sustained shoulder injuries in hamadan province in iran over the previous three years according to the documents of the local branch of the iranian federation of sports medicine in hamadan province . all the relevant data were recorded , including demographic information , type of sport , duration of sporting activity , and the beighton score for generalized ligamentous laxity ( table 1 ) . our inclusion criteria were being 17 - 37 years old and having a history of sports activity for at least six months ( if there was a shoulder injury , then the requirement was at least six months of sports activity before the injury ) . the exclusion criteria were having any deformity or disorder that interfered with the beighton score assessment and a lack of documents proving at least six months of regular sports activity . several athletes were excluded , mainly because of a lack of documentation proving sports participation . using a sampling formula for cross - sectional studies where = 0.05 , d = 0.065 , and p ( ligament laxity prevalence according to other studies ) = 0.15 , all the volunteers signed a consent form to confirm their participation in the study , and the study was approved by the ethics committee of hamadan university of medical sciences ( no . : ir.umsha.rec.1395.101 ) we considered generalized ligamentous laxity as point 4 or more of the beighton score and divided all the participants into two groups : the participants in group a had ligamentous laxity ( with ligamentous laxity ) and those in group b did not ( without ligamentous laxity ) . any acute or chronic shoulder injuries and functional issues among the participants were also assessed according to the standard farsi ( contemporary persian ) translation of the quickdash measure . a total of 118 volunteer athletes participated in our study , which included 32 athletes with shoulder injuries and 86 athletes without shoulder injuries . they were divided into two groups according to the beighton score : group a ( with ligamentous laxity ) consisted of 43 participants and group b ( without ligamentous laxity ) consisted of 75 participants . we then compared the following factors between the two groups : acute shoulder injury , chronic shoulder injury , shoulder instability , chronic shoulder pain , and dash scores ( table 2 ) . a statistical analysis was carried out using the statistical package for social sciences ( spss ) version 20 software ( spss inc . , chicago , il ) . the chi - squared test was used to analyze the effect of the generalized ligamentous laxity on the nominal scale values , namely chronic shoulder pain , and chronic and acute shoulder injuries . spearman s test was used to analyze the generalized ligamentous laxity effect on the last value , successful return to sports activities , for which the dash score was ordinal . first , we evaluated the relationship between ligamentous laxity and chronic shoulder pain ( table 3 ) . sixteen participants ( 37.2% ) in group a and 13 participants ( 17.3% ) in group b suffered from chronic shoulder pain ( p = 0.016 ) . we then evaluated the number of chronic shoulder injuries in both groups ( table 4 ) . thirteen participants in group a ( 30.2% ) and 11 participants in group b ( 14.6% ) had chronic shoulder injuries , which was statistically significant ( p = 0.032 ) . with respect to acute shoulder injuries , 11 participants in group a ( 25.5% ) and 19 participants in group b ( 25.3% ) had chronic shoulder injuries ( table 5 ) . we then assessed upper limb function using the quickdash measure ( table 6 ) and found that there was a significant difference between the functional status of the two groups ( p = 0.030 ) . finally , we compared the history of acute and chronic instability between the two groups based on dislocation documentation or a history of shoulder pain with at least two positive clinical tests for instability ( table 7 ) . eleven participants in group a ( 25.5% ) and five participants in group b ( 6.6% ) had instability , which was significant ( p = 0.004 ) . the shoulder joint gets injured frequently in sports like volleyball , handball , swimming , basketball , and overhead sports , and an abnormal increase or decrease in range of motion is an important factor in sports injuries involving the shoulder ( 7 ) . generalized ligamentous laxity refers to an increased range of joint motion compared to that of the general population . its prevalence is 5% - 15% but varies among different race groups , with an incidence as high as 57.3% among africans ( 1 , 6 ) . although there is no standard for defining ligamentous laxity , there are several clinical scoring systems for measuring joint laxity in individuals and populations . the beighton score is considered an excellent method for screening generalized ligamentous laxity ( 8 , 9 ) . bin abd razak et al . evaluated generalized ligamentous laxity in the musculoskeletal injuries of 100 young patients in a primary care center for comparison with a healthy control group ( 1 ) . they found that the individuals who had musculoskeletal injuries were 3.35 times more likely to have generalized ligamentous laxity compared to healthy people . several studies have demonstrated the relationship between generalized ligamentous laxity and sports injuries in the lower extremities , including cruciate ligament injuries ( 4 ) and patellar dislocation ( 5 ) . found a significantly increased risk of knee joint injury among hypermobile participants who played contact sports ( 10 ) . notwithstanding , little evidence exists regarding the relationship between generalized ligamentous laxity and shoulder injuries . studied generalized ligamentous laxity and increased external rotation of the shoulder as a predisposing factor for primary shoulder dislocation in young , healthy individuals . they found that men with shoulder dislocation were 6.8 times more likely to have generalized ligamentous laxity ( 6 ) . to the best of our knowledge , no study has investigated a range of shoulder injuries in athletes with or without ligamentous laxity . hence , we evaluated the relationship between generalized ligamentous laxity and various shoulder injuries in athletes who had participated in sports activities for at least six months . the athletes with generalized ligamentous laxity had more chronic shoulder pain , chronic shoulder injuries , shoulder instability ( acute and chronic ) , and less functionality according to their dash scores compared to the athletes without generalized ligamentous laxity . interestingly , there was no difference in the number of acute shoulder injuries between the two groups . this was because we considered fractures around the shoulder to be acute injuries in the study . if we had omitted fractures in both groups , the difference in acute injuries would have been significant . the strength of our study was the evaluation of all the volunteer professional athletes in our province who had participated in at least six months of regular sports activity , including professional athletes with shoulder injuries . since the treatment of injured athletes is the responsibility of the federation of sports medicine in iran , this federation had a complete list of all the injured athletes in the province . the limitation of our study was that we only evaluated athletes in one province of iran . although hamadan province is ethnically diverse and includes most iranian ethnic groups ( azeri , kurd , lur , and fars ) , and parallel research results will thus be quite compatible with the current research findings , it is suggested that the same study be conducted in different provinces of iran with a greater number of athletes . a sports injury can be influenced by the athlete s level of professionalism . although we excluded athletes who had participated in professional sports activities for less than six months , it is not clear if their knowledge and ability to prevent sports injuries would be the same as those with more experience . this study highlights the importance of having screening programs in athletic clinics , sports clubs , and sports offices at high schools and universities to identify individuals with generalized ligamentous laxity , and emphasizes the need for physicians and surgeons to practice prophylactic measures . there may be a role for shoulder - specific proprioceptive and strength training protocols for shoulder injuries in individuals with generalized ligamentous laxity who participate in high - risk sports , especially contact sports .

backgroundgeneralized ligamentous laxity is defined as an increased range of joint motion compared to that of the general population . it is a predisposing factor for sports injuries , especially in the lower extremities . nevertheless , there is little evidence about the relationship between generalized ligamentous laxity and sports injuries in the upper extremities.objectivesto evaluate the relationship of generalized ligamentous laxity with acute and chronic shoulder injuries in athletes.patients and methodsour study comprised 118 volunteer athletes with a history of at least six months of sports activities and a shoulder injury in the three years prior to participation in our study . the athletes were divided into two groups : those with or without generalized ligamentous laxity . acute and chronic shoulder injuries , shoulder pain , shoulder instability , and functional status assessed via the quickdash measure were determined and compared between the two groups . a p value of less than 0.05 was considered significant.resultsgroup a ( with ligamentous laxity ) consisted of 43 participants ( 36.4% ) and group b ( without ligamentous laxity ) consisted of 75 participants ( 63.6% ) . the athletes in group a had more shoulder pain ( p = 0.016 ) , chronic shoulder injuries ( p = 0.032 ) , and shoulder instability ( p = 0.004 ) , and less functionality ( p = 0.030 ) than those in group b. if fracture were not considered an acute injury in both groups , the athletes with generalized ligamentous laxity would have had more acute shoulder injuries.conclusionsgeneralized ligamentous laxity is an important predisposing factor for acute and chronic shoulder injuries in athletes . prescreening programs for beginners and rehabilitation shoulder programs for sports athletes at high risk are strongly recommended .

to describe the surgical technique and initial experience with a single - port retroperitoneal renal biopsy ( sprrb ) . a single 1.5 cm incision was performed under the 12th rib at mid - axillary line , and an 11 mm trocar was inserted . after lower pole exposure , a laparoscopic biopsy forceps was introduced through the nephroscope working channel to collect a renal tissue sample . the mean operative time was 32 minutes , and mean estimated blood loss was less than 10 ml . the hospital stay of all patients was two days because they were discharged in the second postoperative day , after remaining at strict bed rest for 24 hours after the procedure . sprrb is a simple , safe and reliable alternative to open and videolaparoscopic approaches to surgical renal biopsy . image - guided percutaneous renal biopsy is the most widely used method to sample renal parenchyma for the evaluation of malignancy or diffuse renal disease . the risks of this procedure are minimal and the overall success rate of all renal biopsies varies from 70 to 100% ( 1 ) . its major indications rest on diagnosis and follow - up of several systemic and nephrological conditions that lead to glomerular damage and renal function impairment , providing useful data for treatment and prognosis . it may also be used for evaluation of solid renal masses and cystic renal lesions ( 1 ) . however , this method has absolute and relative contraindications that may hamper or preclude it , such as the presence of a solitary kidney , uncontrolled arterial hypertension , coagulation disorders , renal artery aneurysm , previous percutaneous needle biopsy failure and obesity . bleeding and inadequate amounts of renal tissue for diagnosis are not infrequent , and constitute potential disadvantages of the procedure . in addition , children may be unable to cooperate , requiring general anesthesia . in these settings , open and laparoscopic approaches are well - established alternatives and should be considered , although with a higher level of invasiveness and complexity . in search for an alternative that could minimize surgical aggressiveness of these procedures and hence spread its use , we outlined a renal biopsy technique through a single retroperitoneal laparoscopic access using standard urological instruments . the aim of this paper is to describe the technique now standardized in our institution and our initial experience with the single port retroperitoneal renal biopsy ( sprrb ) . after receiving general anaesthesia , orogastric and bladder catheterization , the patient is usually positioned in the left flank position , as the kidney is more easily accessible at the right side due to its lower position . a 1.5 cm incision is carried out just below the tip of the 12th rib , at the mid - axillary line , and is followed by blunt access to the retroperitoneum space . an initial digital dissection is done aiming to identify the lower renal pole , while also displacing the peritoneum anteriorly . during this step , care must be taken in order to avoid peritoneal tearing , as the pneumoperitoneum resulting from gas insufflation would hamper the maintenance of adequate retroperitoneal working space . next , a rubber balloon is positioned between the kidney and the posterior abdominal wall , and is filled with 300 - 400 cc of saline , creating a virtual cavity . the saline is drained after a few minutes , to achieve hemostasis , and the balloon is then removed . an 11 mm trocar is inserted and carbon dioxide is used to maintain pneumoretroperitoneum at 12 to 15mmhg . retroperitoneal inspection and identification of the psoas muscle and the lower pole of the kidney are now performed with a standard 26 french nephroscope , as shown in figure-1 . it is frequently possible to expose the renal surface bluntly , by using gentle movements of the tip of the scope to drag the perirenal fat away from the intended site of biopsy . alternatively , standard laparoscopic surgical aspirator , scissors or hooks can be inserted through the working channel of the nephroscope , and then be used to dissect , cut and coagulate nearby structures , allowing a clear renal surface to be assessed . once the biopsy site is cleared from fat , one or two samples are taken with the aid of a toothed biopsy forceps , also through the nephroscope ( figure-2 ) . bleeding is expected to be negligible , as the injury caused by the forceps is shallow ( figure-3 ) , but the parenchyma can be coagulated with the same instruments , and a cellulose hemostatic bolster can be applied , if needed . finally , the pneumoretroperitoneum is evacuated and , if no bleeding is observed , the trocar is removed and the access port is closed . figure 2sampling renal parenchyma with a toothed biopsy forceps , through the nephroscope working channel . figure 3aspect of kidney surface after a tissue sample was taken , with only minimal bleeding . at our institution , laparoscopic retroperitoneal renal biopsy is currently often performed for pediatric patients with nephrological conditions ( 2 ) . as the surgical team s experience progressed and the procedure was standardized , however , we felt that it should be even less invasive , especially for this very young population . additionally , in order to spread and popularize its execution , we devised how to use instruments that are already present in a regular urological operating room , such as the nephroscope and laparoscopic scissors and forceps , in a different fashion . a similar approach has been described previously , in pediatric surgery , for appendectomies and varicocelectomies , but with only one case of renal biopsy ( 3 ) . between january and april/2013 , five children underwent sprrb in our hospital , referred from the nephrology clinic for renal biopsy . informed consent was previously obtained from parents , respecting our institution s ethics committee recommendations and approval . the procedure was successfully performed with the technique above described , by a supervised resident in - training . the overall mean operative time was 32 minutes , and mean estimated blood loss was less than 10 ml . no open conversion was needed . the hospital stay of all patients was two days because they were kept in absolute bed rest for 24 hours after the procedure , before being discharged home . pain and analgesics use were low , and there were no significant detected complications . regarding the obtained samples , the average number of glomeruli present in the specimens was 31 , and the histopathological findings showed focal proliferative lupus glomerulonephritis in two cases , diffuse mesangial proliferative glomerulonephritis in another two , and nephritis related to henoch - schnlein purpura in one child . these results are comparable to those previously shown by us , with laparoscopic renal biopsy in children , regarding operative time , blood loss , hospital stay and success in obtaining adequate samples ( 2 ) . table 1clinical features of patients submitted to single - port retroperitoneal renal biopsy.patientgenderage ( years)bmi ( kg / m)ot ( min.)bl ( ml)gndiagnosiscomplications1m0723.625423diffuse mesangial proliferative glomerulonephritisnone2f0924.5371338nephritis related to henoch - schnlein purpuranone3f1121.827530focal proliferative lupus glomerulonephritisnone4m1024.0401743diffuse mesangial proliferative glomerulonephritissmall skin ecchymosis5f1232.031621focal proliferative lupus glomerulonephritisnoneaverage-9.825.1832931 - - bmi = body mass index ( kg / m ) ; ot = operative time ( minutes ) ; bl = blood loss ( milliliters ) ; gn = number of glomeruli per biopsy . bmi = body mass index ( kg / m ) ; ot = operative time ( minutes ) ; bl = blood loss ( milliliters ) ; gn = number of glomeruli per biopsy . although it is likely that the same approach could be used in adult patients as well . our experience with this very initial group was composed entirely of children , and sprrb has been shown to be a very simple , safe and reliable alternative to other laparoscopic approaches . the use of a nephroscope , instead of a regular laparoscope , obviates the need to place an additional trocar for using an auxiliary instrument to dissect the perirenal fat , as is the standard practice ( 4 , 5 ) . its working channel finely substitutes that , sparing one incision , the cost of another trocar and also surgical time to place it . because a second trocar traditionally would be only 5 mm wide , it may seem that the benefit here is not strongly relevant in terms of postoperative pain or cosmetic results , but it is our understanding that no technical difficulty was added whatsoever , by using only one access . moreover , especially children could benefit the most even of a small effect , and coincidently they constitute the majority of patients requiring a surgical renal biopsy in our hospital . mini - perc nephroscopes are not available at our institution at this time , but its use could be a step forward , in this regard , and further decrease the required size of the access port incision . additionally , the ease for urologists in using regular urological equipment , and the possibility that the surgeon simultaneously controls both the camera and laparoscopic scissor / biopsy forceps , are other advantages of this alternative method . in our hospital , retroperitoneal laparoscopy is the procedure of choice for renal biopsy in children and the sprrb is an even less invasive option for these patients , performed through a single incision and with very satisfactory results and only minor pain .

objective to describe the surgical technique and initial experience with a single - port retroperitoneal renal biopsy ( sprrb).materials and methods between january and april 2013 , five children underwent sprrb in our hospital . a single 1.5 cm incision was performed under the 12th rib at mid - axillary line , and an 11 mm trocar was inserted . a nephroscope was used to identify the kidney and dissect the perirenal fat . after lower pole exposure , a laparoscopic biopsy forceps was introduced through the nephroscope working channel to collect a renal tissue sample.results sprrb was successfully performed in five children . the mean operative time was 32 minutes , and mean estimated blood loss was less than 10 ml . the hospital stay of all patients was two days because they were discharged in the second postoperative day , after remaining at strict bed rest for 24 hours after the procedure . the average number of glomeruli present in the specimen was 31.conclusion sprrb is a simple , safe and reliable alternative to open and videolaparoscopic approaches to surgical renal biopsy .

the interaction between follicle - stimulating hormone ( fsh ) and the fsh receptor ( fshr ) is essential for normal oogenesis and spermatogenesis [ 115 ] . in the male , fsh is fundamental for sertoli cell function and the induction and maintenance of qualitatively and quantitatively normal spermatogenesis by a specific receptor ( fshr ) that is a member of the g protein - coupled receptor family [ 8 , 11 ] . the fshr gene spans a region of 54 kb on chromosome 2p21 and consists of 10 exons and 9 introns [ 3 , 8 , 11 ] . the extracellular domain is encoded by exons 1 to 9 ; whereas exon 10 encodes the c - terminal part of the extracellular domain , the complete transmembrane , and the intracellular domain [ 5 , 8 , 11 ] . the activity of this gene is driven by a core promoter spanning 225 bp , which represents a tata - less promoter with no evident regulatory elements beside an e - box [ 8 , 9 ] and a more recently identified initiator element ( inr ) . mutation screening of the fshr gene revealed various single nucleotide polymorphisms ( snps ) both in the core promoter and in the coding region [ 1 , 8 , 9 , 11 , 12 ] . ( 29g > a ) results in a g / a exchange in a potential ggaa binding domain for an e-26 transcription factor , which is altered [ 3 , 11 ] . the other most common snp in the coding region occurs at nucleotide 2039 in exon 10 , in which a / g transitions cause amino acid exchange from asparagine to serine at codon 680 ( n680s ) [ 7 , 8 , 11 , 12 , 14 ] . investigations on the distribution of these snps produced varying results . in the normal and infertile men and women , some studies revealed that there is no difference in the distribution of snp and they have no effect on serum fsh levels [ 2 , 7 , 11 , 13 , 17 , 18 ] ; whereas other investigations found significant differences between patients and controls [ 6 , 8 , 15 , 19 , 20 ] , suggesting that ethnic differences could be involved . this is the first study to determine the polymorphism of the fshr core promoter at position 29 , alone and in combination with the snp at codon 680 in exon 10 , and to evaluate the possible role of these two fshr snps on serum levels of fsh in southeast turkey . the study population consists of 240 proven fathers ( sperm count > 20 10/ml and serum fsh levels < 7 iu / l ) , and 270 infertile men ( 150 nonobstructive azoospermic and 120 severe oligozoospermic in which sperm count < 10 10/ml ) referred to human genetic department of dicle university hospital . there were not seen karyotype abnormalities and y chromosome long arm microdeletions in the study population . since infertile men are a quite heterogeneous population and spermatogenesis can vary qualitatively and quantitatively in individual subjects , to increase the stringency of the study we selected only men with nonobstructive azoospermia and oligozoospermia compared them to proven fathers with normal spermatogenesis . this study was approved by the hospital ethics committee ( b.30.2.dic.0.01.00.00/80 ) , and written informed consent was obtained from all participants . genomic dna was extracted from peripheral blood leucocytes by standard procedures [ 21 , 22 ] before being analyzed by multiplex pcr . the snps at positions 29 of promoter and at nucleotide 2039 ( codon 680 ) of exon 10 were analyzed using the polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) technique with the primers which were designed based on the published sequence of the human fshr gene . for position 29 ( rs1394205 ) we used forward primer : 5-tgg tga aca gca agg aga ctt-3 , reverse primer : 5-ttg gca gag aaa aac cct gt-3 , whereas for nucleotide 2039 genotyping ( codon 680 ) ( rs6166 ) , forward primer : 5-ccc aaa ttt ata gga cag-3 , reverse primer : 5-gag gga caa gta tgt aag tg-3 . the pcr products were then digested with the restriction enzymes ( mboii for 29 and bsri for snp ser680asn ) , according to the manufacturer 's protocol . the pcr fragment following 2.5% agarose gel electrophoresis shows three different patterns for 29 . the uncleaved fragment , homozygous for a , has a size of 404 bp ; whereas the cleaved fragment , homozygous for g , gives rise to 289 and 115 bp fragments . the asn 680 allele gives an undigested fragment of 520 bp ; whereas the ser 680 allele gives two fragments of 413 and 107 bp . for heterozygous ( asn / ser ) , agarose gel electrophoresis allows visualization of three bands 520 bp , 413 bp , and 107 bp . serum concentrations of fsh were measured by electrochemiluminescence immunoassays ( eclias ) , using roche elecsys 1010 ( roche diagnostics , mannheim , germany ) according to the manufacturer 's instructions . statistical analysis was performed by applying a commercially available software package ( spss 15.0 for windows , spss , chicago , illinois , usa ) . and one - way analysis of variance ( anova ) tests were used for the analysis of the data . statistical significance was set at p .05 . the separate analysis for snp at nucleotide position 29 did not show any difference in genotypic frequencies between proven fathers and infertile patients ( = 1.182 , p > snp at nucleotide position 29 was not associated with different fsh concentrations in each group ( p > .05 , anova ) ( table 1 ) . when the snp at amino acid positions 680 was separately analyzed , a statistically significant difference was found in the genotype frequency between three groups ( = 22.87 , p < .001 ) . further testing by two - by - two statistics revealed significant difference for genotype asn - ser between proven fathers and nonobstructive azoospermic groups ( = 5.26 , p < .05 ) , for genotype ser - ser between proven fathers and nonobstructive azoospermic groups ( = 6.67 , p < .05 ) , for genotype asn - ser between proven fathers and severe oligozoospermic groups ( = 5.18 , p < .05 ) , for genotype asn - ser between nonobstructive azoospermic and severe oligozoospermic ( = 15.36 , p < .001 ) , and for genotype ser - ser between nonobstructive azoospermic and severe oligozoospermic groups ( = 11.86 , p < .05 ) . to assess whether the polymorphism at 680 influences fsh levels , we compared fsh concentrations among genotypes . the fsh concentrations were not different between the fshr genotypes for each group of patients and proven fathers ( p > .05 , anova ) . when we analyzed the haplotypes determined by the two snps at position 29 and codon 680 , our results show that four possible haplotypes result from all the two snps of the fshr gene : a - asn , g - asn , a - ser , and g - ser . these haplotypes account is combined into the 10 major combinations shown in table 2 , in which nine groups are presented since the two possible allelic combinations of group 5 ( double heterozygous ) can not be distinguished and are considered together . further testing by chi - square revealed the significant difference for g - asn / g - ser and g - ser / g - ser genotype in men with proven fathers and infertile ( nonobstructive azoospermic and severe oligozoospermic ) . we then calculated the overall frequency of the four fshr haplotypes in proven fathers and infertile men . as shown in table 3 , no statistically significant difference between the groups was found ( p > .05 by test ) . to assess whether the haplotypes influences fsh levels , we compared fsh concentrations among genotypes . there were no significant differences in the fsh levels among the fshr genotypes in both the two groups of infertile and proven fathers ( p > .05 , anova ) ( table 2 ) . test for deviation from hardy - weinberg proportions has been performed , and the deviation from the hardy - weinberg equilibrium ( excess of homozygosity ) takes place almost for all investigated groups ( except for snp at nucleotide position 29 for severe oligozoospermic group and snp 680 fshr for nonobstructive azoospermic group ) . the impact of 29 snp , alone or in combination with exon 10 snps , is less clear but does not seem to influence the clinical parameters or plasma fsh concentrations both in women and men [ 8 , 10 , 11 ] . our data showed that the genotype distribution of snp 29 is similar both in proven fathers and infertile men and does not influence serum fsh levels when considered alone . this result was in agreement with those reported in the previous similar studies [ 8 , 10 , 11 ] , but there are differences in the proportions of genotype distribution between our study and others . the possible ethnic differences might be responsible for this difference . in women with normal ovarian function the polymorphism at codon 680 of the fshr is an important determinant of ovarian sensitivity to fsh [ 6 , 8 , 19 , 25 ] . identified a loss - of - function mutation of fsh receptor in ovarian dysgenesis due to ala189val . the snp at position 680 was then confirmed and has been well studied by perez mayorga et al . . the distribution was 29% for the asn / asn , 45% for the asn / ser , and 26% for the ser / ser fshr variant . in japan , sudo et al . reported 522 ovulating women who visited the university hospital . the proportions of genotype asn - asn , asn - ser , and ser - ser were 41.0 , 46.9 , and 12.1% , respectively . in contrast to observations in women , snps in exon 10 of the fshr have no effect on serum levels of fsh and other clinical parameters in men with either normal or impaired spermatogenesis [ 4 , 7 , 9 ] . simoni evaluated asn - asn , asn - ser , and ser - ser ( 37.2 , 45.4 , and 17.4% , resp . ) in populations with proven fertility and ( 32.0 , 48.0 , and 20.0% , resp . ) infertility , and no significant differences were observed . in another study , shimoda et al . reported that the proportions of asn - asn , asn - ser , and ser - ser were 38.2 , 47.3 , and 13.1% , respectively , in subject with proven fertility and 49 , 42 , and 8% in infertile patients , and there were no significant differences between the two . in our study , the genotype distribution of snp at codon 680 is different between proven fathers and infertile men but does not influence serum fsh levels when considered alone . the differences in the genotype frequency might represent genetic factors contributing to phenotypic expression of severe spermatogenetic impairment . when considered in combinations with the snp in 29 and exon 10 ( codon 307 and codon 680 ) , there are a few reports on affected groups with diverse ethnic backgrounds , and the results are not in agreement . a study in german men investigated fshr snp genotypes ( 29 , codon 307 and codon 680 ) alone or in combinations . the authors concluded that while no fshr haplotype was associated with different serum fsh levels , the a - ala - ser and the g - thr - asn alleles might represent genetic factors contributing to severe spermatogenetic impairment . a recent meta - analyses of fshr snp and male infertility revealed that there is no any association with fsh serum levels or sperm output . a study in italian men also investigated the same three snp genotypes and their combinations . the authors concluded that the genotypes had no influence on fsh concentrations in normal or infertile males and did not associate with spermatogenetic impairment . however , very recently a study in japanese men investigated codon 307 and codon 680 genotypes and concluded that heterozygous combination of thr / ala ( codon 307 ) and ser / asn ( codon 680 ) was significantly increased in infertile patients compared with the controls but not ser / asn alone . in our study , the combination of the snp at position 29 and codon 680 gives rise to four haplotypes as these alleles show a statistically similar distribution ( except the two allelic variants g - asn / g - ser g - ser / g - ser ) in infertile men compared to proven fathers and suggests that these alleles might not represent a risk factor for male infertility . in the test for deviation from hardy - weinberg proportions , the deviation from the hardy - weinberg equilibrium ( excess of homozygosity ) takes place almost for all investigated groups ( except for snp at nucleotide position 29 for severe oligozoospermic group and snp 680 fshr for nonobstructive azoospermic group ) . this deviation is very important with regard to high parental consanguinity in populations like turkey . in conclusion , our data demonstrate that fshr gene polymorphisms seem not to have a direct influence on spermatogenesis , but are differently distributed , identifying an additional genetic factor possibly contributing to the multigenic origin of male infertility . the discrepancies are most likely due either to study subjects by chance or to study different genetic backgrounds in different populations . in populations like turkey , high parental consanguinity could bring out genetic factors or provide permissive background for complex disorders . additional studies on well - defined populations of infertile men will probably clarify these aspects .

the influence of fsh receptor ( fshr ) variants on male infertility is not completely understood . the present investigation is the first screening study for snp at nucleotide position 29 in the core promoter region and codon 680 in exon 10 of the fshr and the effect of the serum levels of fsh on male infertility in southeast turkey . the snps in codon 680 and at position 29 of the fshr gene were analyzed by pcr - rflp technique in 240 men with proven fathers , and 270 infertile men ( 150 nonobstructive azoospermic and 120 severe oligozoospermic ) . the separate analysis for snp at nucleotide position 29 did not show any difference in genotypic frequencies and serum fsh levels . the genotype distribution of snp at position 680 was different but does not influence serum fsh levels . together the two snps form four discrete haplotypes ( a - thr - asn , g - thr - asn , a - ala - ser , and g - ala - ser ) occurring in 10 combinations . a statistically significant difference in the allelic distribution of g - asn / g - ser and g - ser / g - ser genotype between proven fathers and infertile men but there were not any statistically significant difference in the overall frequency of the four fshr haplotypes . we conclude that the fshr haplotype does not associate with different serum fsh levels but it is differently distributed in proven fathers and infertile men .

six a17 bac and one fosmid library were used to create mt3.5 ( table s1 ) . most were processed by sanger paired - end sequencing of 3 - 6 kb shotgun libraries . sequences were downloaded in february / march 2009 with scaffolding performed by aligning all bac and fosmid ends against contigs and then anchored and ordered primarily by optical mapping . separately , 25 gb of illumina sequence was generated using short ( 375 nt ) inserts plus 2.1 gb from a 5 kb mate - pair library , then assembled using clcbio ( www.clcbio.com ) and soap ( http://soap.genomics.org.cn/ ) . five tissues were used for rna - seq analysis with ~10 million illumina 36 bp reads per library ( table s12 ) . three tissues were used for small rna analysis with ~3 million reads per illumina library ( figures s17-s18 , table s16 , datafile s9 ) . six a17 bac and one fosmid library were used to create mt3.5 ( table s1 ) . most were processed by sanger paired - end sequencing of 3 - 6 kb shotgun libraries . sequences were downloaded in february / march 2009 with scaffolding performed by aligning all bac and fosmid ends against contigs and then anchored and ordered primarily by optical mapping . separately , 25 gb of illumina sequence was generated using short ( 375 nt ) inserts plus 2.1 gb from a 5 kb mate - pair library , then assembled using clcbio ( www.clcbio.com ) and soap ( http://soap.genomics.org.cn/ ) . five tissues were used for rna - seq analysis with ~10 million illumina 36 bp reads per library ( table s12 ) . three tissues were used for small rna analysis with ~3 million reads per illumina library ( figures s17-s18 , table s16 , datafile s9 ) .

legumes ( fabaceae or leguminosae ) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria , a process that takes place in a specialized structure known as the nodule . legumes belong to one of the two main groups of eurosids , the fabidae , which includes most species capable of endosymbiotic nitrogen fixation 1 . legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago ( mya ) . papilionoids are the largest clade , dating nearly to the origin of legumes and containing most cultivated species 2 . medicago truncatula ( mt ) is a long - established model for the study of legume biology . here we describe the draft sequence of the mt euchromatin based on a recently completed bac - assembly supplemented with illumina - shotgun sequence , together capturing ~94% of all mt genes . a whole - genome duplication ( wgd ) approximately 58 mya played a major role in shaping the mt genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation . subsequent to the wgd , the mt genome experienced higher levels of rearrangement than two other sequenced legumes , glycine max ( gm ) and lotus japonicus ( lj ) . mt is a close relative of alfalfa ( m. sativa ) , a widely cultivated crop with limited genomics tools and complex autotetraploid genetics . as such , the mt genome sequence provides significant opportunities to expand alfalfa s genomic toolbox .

hydroxyapatite ( hap ) with multiple substitutions at all sites and containing ~4% to 6% carbonate is the primary component of bones ( 70% wt ) and teeth ( 96% wt ) [ 1 , 2 ] . several authors have reported on the structure and properties of human dental carbonate hap [ 35 ] . they study the enamel part of the tooth with a focus on the crystallographic structure and the carbonate substitution because carbonate affects important properties of the physiological hap such as reactivity and solubility . studies on the graded nature and texture of dental enamel by comparison of the microstructures of slices of human adult and baby canine enamel have been reported [ 7 , 8 ] . human deciduous and permanent enamel samples were studied by fourier transform infrared ( ftir ) spectroscopy to determine quantitatively the b - type ( carbonate for phosphate ) and a - type ( carbonate for hydroxyl ) carbonate contents in human enamel . the mineral content , crystallite size , and mechanical properties of aging ( transparent ) human dentin were compared with the ones in normal human dentin in recent studies [ 10 , 11 ] . we study the average crystal structure properties in bulk human dental apatite as a function of age in the range 587 years without separating the enamel from the dentin . we have undertaken a research project that requires a large and diverse origin of specimens in order to correlate the average crystal structure properties of aging dental apatite with the parameters that create the structural changes . understanding the fundamental science of the dental mineral phase as a function of age could be helpful in efforts of remineralization of human dental apatite . here we report some preliminary results on systematic trends of average crystal structure parameters and carbonate content in bulk human dental apatite as a function of age by using powder x - ray diffraction ( xrd ) , thermogravimetric analysis ( tga ) , and ftir spectroscopy . two local dental offices provided teeth samples for this research with the informed consent of their patients . the teeth - samples were selected in the age range 1787 years old . one healthy 5 year old deciduous tooth was offered by a family member of the first author . the cleanest pieces of each tooth ( free of fillings , etc . ) were selected under magnification , powdered with an agate mortar and pestle , and passed through a 125 sieve . the way of preparation of the samples implies that the average crystal structure properties of the dental apatite are studied . each specimen was labeled with a capital t followed by a number that represents the age of the donor of the tooth . powder x - ray diffraction measurements were performed using a siemens d5000 powder diffractometer operating at 45 kv and 40 ma with cu - k radiation and a diffracted beam monochromator . data were collected in the 2 range of 890 with a step size of 0.02 and a counting time of 20 seconds at each step . the data bank from the international center for diffraction data ( icdd ) was used in a search / match program for phase identification . the rietveld refinement method in the gsas program was used for crystal structure analysis of the diffraction patterns only for the hap phase . the crystallographic model used space group p63/m with isotropic atomic displacement parameters . first , the scale factor , background , peak profile ( pseudovoight function ) and lattice parameters were simultaneously refined ; then , the atomic positions and isotropic displacement parameters were refined for all the atoms except for the oxygen and hydrogen of the hydroxyl site in the diffraction patterns from the older age teeth , because combination of low occupancy at the channel site , poor crystallization and peak overlapping would create instability of the refinement . for the same reason , the fractions of the ca1 , ca2 , and p atoms were refined in diffraction patterns up to 45-year - old teeth . the loss of weight as a function of temperature from samples of 50100 mg was recorded using a perkinelmer thermogravimetric analyzer tga-6 . the heating rate was 10c / min in the temperature range 25c to 950c . ftir spectroscopy was used to study the carbonate presence in the hap structure of the specimens . the ftir spectra were recorded on a bruker optic ifs66v / s interferometer equipped with an attenuated total reflectance ( atr ) unit . the atr unit permits the spectra collection without any special sample preparation and it is used for characterization and quantitative estimations in several materials . at a minimum two samples from each age were measured . the range of frequencies was 500 to 4000 cm and the spectra were recorded in ambient conditions with a resolution of 2 cm . in order to obtain a good signal - to - noise ratio , more than a hundred scans figure 1 shows the x - ray diffraction pattern collected from the sample t17 in comparison with the hap phase pdf number 9 - 432 . while this is the main identified phase in the diffraction patterns of all samples , minor unidentified phase(s ) exist even in the young - age teeth , although within the detection limits of the method in the later . the identified secondary phases vary with the tooth - age qualitatively and quantitatively , as deduced from the bragg peaks and their relative intensities . poor crystallinity , broadening , and overlapping of the diffraction peaks would make the phase identification ambiguous especially in old age teeth - samples . possible secondary phases include the biologically relevant calcium compounds : ca2(p4o12).4h2o ( no . 50 - 582 ) , ca8h2 ( po4)65h2o ( no . 71 - 2392 ) and ca2p2o7 ( 73 - 440 ) [ 6 , 15 ] . the x - ray diffraction patterns of figure 2 reveal a systematic decrease of the crystallinity of human dental apatite from 587 years old . it is quite noticeable that patterns collected from teeth up to 45 years old show highly crystallized materials ( with the exception of t43 ) , whereas the patterns from older - age teeth display an increasing broadening of the bragg peaks that indicates an increasing loss of crystallinity of the human dental apatite as a function of age . as it has been demonstrated , acids produced by plaque bacteria , acidic food , or drink cause a partial dissolution of dental apatite . then it is reasonable to expect that aging of dental apatite favors such dissolution which is followed by decrease of the crystallite size . this decrease as a function of age is demonstrated by the broadening of the diffraction peaks of figure 2 . the average crystallite size was calculated from the fwhm of the ( 002 ) , and ( 310 ) bragg peaks using the scherrer formula = k / cos . these two peaks were chosen because they do not overlap with others . it was found that the average crystallite - size in the specimens varies from ~12 nm ( older age teeth ) up to ~38 nm ( younger age teeth ) . these numbers are in agreement with values found for crystallites in dentin and enamel . the average crystallographic properties of the specimens were found from rietveld refinement of the powder diffraction patterns . the weighted r factors of the refined patterns of all the samples were 0.15 rwp 0.18 except for the t87 with rwp = 0.28 . the reduced were 1.2 1.6 and the rbragg were 0.06 rbragg 0.13 . the low counting rate , presence of secondary phases , and nanoscale crystallite size explain such high residuals combined with low goodness of fit . the occupancies of the ca and p sites refine to values less than one as expected from the chemical composition of dental apatite [ 1 , 2 ] . a systematic decrease of the a - lattice constant with the tooth - age is demonstrated in figure 4 . decrease of the a - lattice parameter in carbonate apatites is associated with an increase in carbonate content [ 1 , 2 , 17 ] . higher number of planar carbonate ions substituting for the tetrahedral phosphate ions in the apatite structure is followed by an increased crystal structure disorder and reduction of the crystallinity as it is demonstrated by the broadening of the diffraction peaks in figure 2 . this is biologically important because the increase in carbonate content as a function of age also means an increase of the solubility of the dental apatite and consequently the formation of calcium phosphate phases that alter the composition of the dental mineral . on the other hand , as figure 5 shows , no significant changes of the c - lattice constant with the tooth - age were found with the exception of one sample . accordingly , no significant substitution variations occur at the channel ( hydroxyl ) site as a function of the tooth - age . the refined interatomic distances between the atoms of the phosphate tetrahedron as calculated from the rietveld refinements of the x - ray diffraction patterns are plotted in figure 6 versus age . notice that while in young - age teeth the tetrahedral distances p - o2 ( triangles ) and p - o3 ( circles ) are distributed around the ideal value of 1.54 ( marked with the dashed line ) , they show disturbance in older - age teeth . moreover , the p - o1 distances ( squares ) are noticeably disturbed in all samples compared to the p - o2 and p - o3 bond lengths . distortion of the phosphate tetrahedron is correlated with the well - known lattice disorder caused by the carbonate for phosphate ( b - type ) substitution in natural and synthetic apatites , referred to as the carbonate substitution problem [ 18 , 19 ] . notice that this distortion is different from the one observed in carbonate natural fluorapatites and synthetic haps that was studied earlier in the sense that in those both the p - o1 and p - o2 interatomic distances of the atoms on the mirror plane of the phosphate tetrahedron were distorted by 3 - 4% because of the carbonate for phosphate substitution . further investigation is required to draw conclusions on this subject regarding the dental carbonate hap . the wt% of the carbonate loss from several samples versus the tooth - age is plotted in figure 7 , as evaluated from differential thermogravimetric analysis in the temperature range above 600c up to 950c . weight losses of absorbed , adsorbed water , or possible organic compounds that take place at temperatures less than 600c were evaluated . an increase of carbonate content with the age is demonstrated in this figure confirming the correlation between the decrease of the a - lattice constant as a function of age of figure 4 with an increased carbonate content . figure 8 shows the 1750800 cm region of the ftir spectra as collected from samples in the age range 586 years old . these spectra are characteristic of bio - apatites ; the phosphate bands are identified by peaks at ~962 cm ( 1 po4 stretching ir mode ) , and the 3 po4 region which appears as a very strong broad asymmetric b and at ~1015 cmand consists of at least three submodes . strong peaks assigned to the b - type carbonate substitution ( carbonate for phosphate ion ) are observed at 872 cm(2 co3 mode ) and at 1405 , 1450 cm(3 co3 ) . the weak bands in the 3 co3 region are attributed either to co3 replacing po4 ions without an adjacent oh ion ( at 1480 cm ) , or to the a - type carbonate substitution ( weak shoulders at 880 cm , ~1495 cm , and ~1530 cm ) . organic phase related bands , mainly due to dentin , have been observed in the raman spectra of tooth samples [ 22 , 23 ] . in particular , the raman bands peaked at 1250 , 1450 and 1670 cm were related with the amide iii , amide ii , and amide i bands , respectively . the amide bands have been observed in the ir spectra of tooth samples above 1500 cm . the broad feature above 1600 cm that consists of two subbands at 1610 and 1650 cm can be attributed to overlapping bands of carbonate containing phases other than hap ( carbonate probably at the channel sites ) , with amide iii bands . the 1650 cm band dominates over the 1600 cm in the spectra from teeth older than 45 years old . usually the higher - frequency subband is stronger at dentin untreated samples , while it loses intensity at enamel samples or upon treatment . according to the aforementioned assignment the subbands behavior can be related either with the different content of dentin and enamel in the samples with age or with the secondary phases observed in the x - ray diffraction patterns . in favor of the secondary phases ' explanation , some other weak bands at the 2 co3 region also imply the presence of carbonate in slightly different environments than a- and b - type as mentioned earlier for the weak bands in the 3 co3 region . in a previous work we have used the atr technique for a quantitative estimation of the relative carbonate content in specimens of synthetic and natural carbonate apatites from the ratios of the intensities of the 2 co3 modes to the 1 po4 . figure 9 presents the ratio of the ir intensities of the 2[b ] co3 mode to the 1 po4 ( b - type carbonate substitution marked with squares ) as a function of age . in the same figure the intensity ratios of the 2[a ] co3 to the 1 po4 mode ( a - type carbonate substitution ) as a function of age are marked with circles . figure 9 clearly demonstrates a trend of increasing carbonate content with the tooth age , which is in a good agreement with the results of the xrd experiments shown in figure 4 and the actual measurements of the carbonate loss of figure 7 . the maximum b- to a - type relative carbonate content is approximately 5 , a value that is close to other estimates in biological apatites [ 9 , 15 ] . other authors have found similar results by using the relative intensities of the 1415 cm ( 3[b ] co3 ) to the 603 cm ( 4 po4 ) band and the 1545 cm ( 2[a ] co3 ) to the 4 po4 band , respectively . we prefer to use the ratios as in figure 9 because there is no coexistence of more than one a- and b - type bands in the 2 co3 region as in the 3 , hence we avoid a possible fitting procedure uncertainty . consistent , systematic variations of average crystal structure properties of human dental apatite as a function of age were found in this study from xrd , tga , and ftir spectroscopy experiments . the decrease of the a - lattice constant versus age in dental apatite that is associated with increased carbonate content is related to increasing solubility which in turn results in a decrease of crystallinity and disturbance of the local lattice order of the biomineral . the approximately age - independent c - lattice parameter implies that the phosphate tetrahedron remains the main site of the carbonate substitution in the apatite lattice ( b - type substitution ) in the studied age - range . ftir spectra also show an increase of the b and a - type carbonate contents as a function of the age of the dental mineral phase with the b - type substitution up to 5 times greater than the a - type . these trends of the average crystal structure properties of human dental apatite as a function of age could be useful in understanding the details of structural modifications in aging teeth . however , further research is required using specimens from a large , diverse pool in order to acquire statistical information considering that the tooth bioactivity is greatly affected by diet , diseases , or other local factors that consequently affect the evolution of the mineral phase in aging human teeth .

studies of the average crystal structure properties of human dental apatite as a function of age in the range of 587 years are reported . the crystallinity of the dental hydroxyapatite decreases with the age . the a - lattice constant that is associated with the carbonate content in carbonate apatite decreases with age in a systematic way , whereas the c - lattice constant does not change significantly . thermogravimetric measurements demonstrate an increase of the carbonate content with the age . ftir spectroscopy reveals both b and a - type carbonate substitutions with the b - type greater than the a - type substitution by a factor up to ~5 . an increase of the carbonate content as a function of age can be deduced from the ratio of the 2co3 to the 1po4 ir modes .

radiologically , they are similar to ependymomas , and histopathologically , they are similar to neuroendocrine tumors . most of them are non - secreting tumors , although they are of sympathetic origin . back pain is the most common symptom , and they are not associated with tachycardia , flushing , or hypertension . the treatment of choice is total surgical resection , and no additional treatment is needed with total resection . most of them are benign , but , recurrence can occur in cases involving a failure of the total resection . we describe a rare case of a paraganglioma that was adherent to the cauda equina that presented with low back pain and radiating pain in both the lower extremities . a 76-year - old female visited our department with complaints of low back pain and radiating pain in both the lower extremities for more than two months . magnetic resonance ( mr ) imaging revealed a lobulated sausage - like intradural mass ( fig . 1 ) . she underwent a total laminectomy and near - total tumor removal . during the operation , after opening the dura , a reddish , friable , and well - marginated mass was observed ( fig . we removed the mass near - totally except for the lesion that was adherent to the cauda equina . extradural paragangliomas may compress the spinal cord , resulting in paraparesis5 ) . from a pathologic point of view , paragangliomas are similar to parasympathetic tumors . however , functional paragangliomas are rare6 ) . histopathologically they are similar to parasympathetic tumors that occur in the carotid body or glomus region . althogh they are also similar to catecholamine - secreting tumors , symptoms of flushing , tachycardia , or hypertension are rare . however , a case in which hypertension was controlled after thoracic paraganglioma removal has been reported12 ) . preoperative diagnosis is difficult with radiological tools . mr images of paragangliomas in the cauda equina region are non - specific and are relatively isointense on t1-weighted images and hyperintense on t2-weighted images . immunohistochemically , neuron - specific enolase , synaptophysin , and chromogranin staining are positive2 ) . in the cauda equina region , it is very difficult to distinguish paragangliomas from ependymomas . cauda equina paragangliomas are classified as grade i on the world health organization ( who ) classification . recurrence after surgery on a paraganglioma in the cauda equina region , especially if it is encapsulated , is rarely encountered2 ) . the surgical goal is complete total resection with preservation of the surrounding nerve roots1 ) . thus , we did not treat the patient any additional therapy , and conducted a close follow - up . after incomplete surgical resection , prolonged postoperative observations are mandatory because of the slow evolution of paragangliomas4 ) . distant metastases very rarely occur5 ) , and metastatic paragangliomas of the spine have been reported7,11 ) . total resection was difficult in this case due to adhesion to the cauda equina , and close follow - up may therefore be needed .

paragangliomas are rare among intradural spinal tumors . most of them are benign , but aggressive behavior and local recurrence can occur . cases of paraganglioma are , difficult to diagnose radiologically ; hence , diagnosis is confirmed histopathologically . radiologically , paragangliomas are similar to ependymomas , and , histopathologically , they are similar to neuroendocrine tumors . we evaluated the case of a 76-year - old woman with a spinal paraganglioma that was associated with back pain and radiating pain in both the lower extremities . she underwent an operation , and her symptoms were relieved . here , we describe a rare case of paraganglioma that was adherent to the cauda equina .

ameloblastomas are rare benign odontogenic tumours , most commonly found in a patient s lower jaw . although rarely metastatic , the growth of these lesions can result in defects in the jaw easily destroying surrounding bony tissues . for this reason , the treatment is often excision of the lesion , with wide excision margins , resulting in the absence of a part , or all , of the mandible . the surgical approach for correction of this mandibular defect is often reconstruction of the mandible in view of inserting dental implants at a later date . despite surgical advancements , , bone grafting with non - vascularized bone remains a good option where facilities for soft tissue flaps are not readily accessible . creating an effective wound closure and reducing the incidence of infection is an important stage in the success of the operation , and has been combated here by tissue expanding the submucosa , using osmed pellets , prior to surgery . this is a relatively new technique to the mandibular region and has the potential to revolutionize the success of microsurgeries in this area . in this report , i describe a woman who underwent a repair of her mandibular defect by a combination of innovative techniques , in order to reduce the risk of complications and get her back to an aesthetically pleasing level . a 35-year - old lady presented to the craniofacial surgeons looking for a permanent fixture to the bony defect in her lower - right mandible . in 1991 she had her first operation to remove the ameloblastoma and then had it further excised 6 years later due to a reoccurrence . this resulted in an absence of teeth from the canines back and removal of bone down to the inferior alveolar nerve . the patient has since been clear of any further reoccurrences and wears lower dentures for aesthetic reasons . she has no significant past medical history nor family history , nor does she have any known drug allergies . she does not smoke or drink alcohol . on clinical examination , the patient experiences mild parasthesia in the distribution of her right inferior dental nerve , particularly the lateral aspect of the lower lip . on examination , there is clear absence of teeth on the lower - right side from the canines back ( to include the premolar , molar and wisdom teeth ) and presence of healthy oral mucosa with laxity to move the submucosa medially . there is a loss of the vertical height of the posterior body of the mandible of about 50% and the vestigial remnants of the lingual plate are just palpable and visible on plain radiograph and ct . scans show that the superior margin of the resection is well corticated under the inferior dental nerve ( fig . , the patient wishes to get dental implants . the surgical approach taken , prior to the implants , ( b ) ct scan of head imaging of the patient 's mandibular defect pre - surgically . ( a ) radiolocal image of patient 's jaw . ( b ) ct scan of head the patient was initially given the option between a bone graft or vascularized tissue ( free fibular flap ) and informed of the complications a bone graft carries a risk of rejection and vascularized tissue needs an increased amount of post - operative care to ensure that the tissue remains vascularized . the patient was not keen on a free flap and given the fact that the bony deformity was < 5 cm , non - vascularized iliac bone graft was deemed to be sufficient to carry out this operation . the first step was achieving significant tissue expansion ( > 2 cm ) using osmed pellets , guided into the submucosa and kept over the alveolar ridge for several months ( fig . ( c ) osmed pellets before insertion into the submucosa image of patient 's oral cavity during insertion of osmed pellets . ( a ) incisions made in the submucosa . ( c ) osmed pellets before insertion into the submucosa two months later , the patient underwent the main bulk of her operation . the osmed pellets were removed from the subperiosteum ( fig . 3a and b ) and the iliac bone graft was taken , made up of cortical and cancellous bone and placed to one side ( fig . an epidural catheter was placed inside the soft tissue of the graft site and left overnight . it had a continuous fusion of a local anaesthetic to improve post - operative pain and encourage early mobilization . ( b ) open site where iliac bone graft is harvested from ( c ) cortical and cancellous iliac bone submucosa after adequate tissue expansion . ( b ) open site where iliac bone graft is harvested from ( c ) cortical and cancellous iliac bone previously , using ct scans , a titanium construct was made to guide the outline of the bone graft and complete the missing height and width of the mandible . once fitted to the patients jaw , it was packed with the bone graft and screwed down into position ( fig . a slight groove was made on the mandible to allow space for the inferior dental nerve to lay , so that the construct did not compact the nerve and cause functional problems . figure 5:titanium construct packed with iliac bone graft and screwed into position on the lower mandible titanium construct packed with iliac bone graft and screwed into position on the lower mandible to complete the surgery , the periosteum was sutured over the construct and then the buccal muscosa was sutured over again to form a double - layered closure ( fig . 6a c).the purpose of this was to prevent infection and rejection , by ensuring that there was an increased distance between the foreign object ( titanium construct ) and the outside environment . the dental implants are scheduled to be inserted several months after this operation . figure 6:image of patient 's surgical site . ( c ) image of post - surgical site image of patient 's surgical site . bony defects in the maxillofacial area are a huge problem in clinical orthodontics , caused by a wide range of injuries and diseases presenting as either cosmetic or functional defects and in some instances both . it is the importance of accounting for the anatomical , functional and aesthetic aspects that make the reconstruction so challenging . often patients suffering from ameloblastomas undergo resection as treatment and are left with bony defects that require reconstruction . in this case , the intention was to realize an aesthetically pleasing appearance for the patient and it has been shown that bone grafting with subsequent dental implants is a successful method . as in this case , it is recommended that non - vascularized autogenous bone grafting can be used when a two - layer watertight closure is attainable . sources of bone could have been harvested from either local or distant sites but in this case , an extra - oral site ( iliac bone ) was used as a moderate amount of bone was needed . the surgery lasted 5 h and incorporated several novel techniques to improve the outcome of the operation . the use of osmed pellets was innovative , as pellets of this size have only recently been experimented on goats maxilla . as well as allowing for space to put the construct , the pellets allowed for a tension - free closure . otherwise , the result would have been a high - tension closure , leading to rapid breakdown of the graft providing an entrance for infection , a common complication occurring when titanium plates are exposed . advantages of the osmed tissue expanders are that the material is safe with a low complication rate and low risk of infection due to small incision site and minimal trauma . the titanium construct is another relatively novel approach and was invaluable in the success of this surgery , as it guided reconstruction of the mandible , allowing for dental implants to be put in at a later date . at present , an alloplastic material such as titanium is often used for mandibular reconstruction as it is resistant to corrosion , adaptable and biocompatible . although the insertion of dental implants is the end goal for this surgery , the importance of this case report is to highlight the novel techniques involved in making this surgery a success . the use of small osmed pellets in the submucosa of the mouth to cause sufficient tissue expansion and create a tension - free closure , combined with the titanium construct packed with grafted iliac bone to rebuild the jaw was an approach recommended by the doctors for the repair of bony defects of < 5 cm . i suggest prudent management of future cases post - operatively to ensure a continued success of the graft .

this case report describes a 35-year - old caucasian radiographer who presented with a significant mandibular bony defect following multiple excisions of an ameloblastoma . as a result , there was an absence of teeth on the lower - right mandible and a clear defect in the mandible . the treatment objectives were to rebuild the mandibular defect , with a long - term view of inserting dental implants . in a novel approach outlined in this presentation , tissue expansion of the submucosa , a titanium construct and an iliac bone graft were used to rebuild the patient s jaw . this surgical technique is recommended for the reconstruction of bony defects .

asthma in adults is a heterogeneous disease usually characterized by chronic airway inflammation with hyperresponsiveness of airways to various stimuli . the prevalence estimates of asthma , in general , and the prevalence of severe asthma , in particular , are well known and sufficiently illustrative . according to the global asthma report 2014,1 asthma affects 334 million people with projections of 400 million in 2025 . in addition , asthma still accounts for one of every 250 deaths worldwide , and almost all of these deaths are avoidable.2 in spain , prevalence rates of 5% and 10% have been estimated in adults and children , respectively , with more than three million people with asthma.35 in addition , there is a large geographical variability is asthma prevalence , with a trend toward stabilization following an increase in the prevalence of the disease observed in the past years.3,6 this complex disease affects patients of all ages . although its diagnosis is usually easily established and most patients respond to therapy , approximately between 3% and 10% of adult asthma patients have disease that is difficult to control despite taking maximal doses of inhaled medications.7 patients with therapy - resistant or difficult - to - control asthma require a rigorous and systematic approach to their diagnosis and treatment . despite rigorous , optimized follow - up treatment , 75% of severe asthma patients did not achieve adequate symptom control and presented with impaired quality of life.8 although much progress has been made in the understanding of difficult - to - control asthma , with consensus documents and clinical practice guidelines focused on severe uncontrolled asthma with proposals for a stepwise diagnostic procedure and phenotype - targeted treatment,9,10 improvements in clinical practice are still limited . in addition , involvement of pulmonology services in the specialized approach for difficult - to - control asthma is insufficient . in a survey carried out in spain , 47 ( 68.1% ) of a total of 69 pulmonology services met criteria for an important level of health care activity in asthma , but only 29 ( 42% ) had a monographic consultation for difficult - to - control asthma and 37 ( 53.6% ) had implemented an education program . as for post - graduate education , only 31 ( 44.9% ) provided their resident physicians with specific asthma training.11 in 2015 , the asthma area of the spanish society of pneumology and thoracic surgery ( separ ) addressed the task of establishing the necessary requirements for the provision of official accreditation standards of the different levels of care for asthma units already existing in hospitals of the spanish national healthcare system . accreditation levels included basic units , specialized units , or specialized units of high complexity , with or without the distinctive of excellence , according to the fulfillment of a series of criteria established by the society . the development and implementation of this nationwide strategic plan had several objectives , including improving the care and quality of life of asthma patients , particularly patients with severe difficult - to - control asthma in order to achieve a decrease and prevention of acute exacerbations , with a subsequent reduction in the number of visits to the emergency department , need of in - patient care , and inadequate use of asthma medications . all these actions have been complemented by the development and implementation of education and self - management strategies ( where nursing plays a key and indispensable role ) and follow - up of patients in monographic consultations for asthma and in the framework of multidisciplinary involvement of health care professionals . in a recent scientific meeting of health care professionals of the different asthma units , which underwent specialized accreditation , it was agreed that a perspective article should be drafted to achieve greater dissemination of the details regarding the characteristics , service portfolio , and resources available in these units . the present document written and approved by all attendees of this event and members of units provides information and describes the initial global experience of asthma units accredited in spain . it also aims to contribute to raising awareness among pulmonologists and other professionals about the problem of severe refractory asthma and the response offered by asthma units to improve patient care and to minimize the complications and burden of this complex pathology . in this respect , we believe that the spanish experience may be useful and potentially applicable to other settings , in particular with similar health care systems . however , workup details and assessment of outcomes were outside the scope of this report . significant progress and advances in the understanding of asthma and in the care of asthma patients with an increase in the number of medications and development of new protocolized therapeutic strategies have been associated with a substantial reduction in asthma - specific mortality and hospitalization rates for asthma . however , despite all these improvements and the development of clinical practice guidelines with specific recommendations for the control of asthma , it is well known that in clinical practice , adequate asthma control is achieved in only one - third of patients.1214 uncontrolled severe asthma accounts for ~15% of all asthma patients but is associated with a higher morbidity and mortality as well as an important impact on health care costs and the consumption of resources.15 the asmacost study16 based on data of a prospective cohort of 627 patients throughout spain with asthma diagnosed according to the guidelines of the global initiative for asthma ( gina)17 and the adapted spanish criteria ( gua espaola de manejo del asma [ gema])9 and followed up for 12 months revealed that the total societal cost for asthma was 1,726 per patient annually ( 1,533 to the national health service ) , with higher costs for patients older than 65 years and for those with a more severe disease ( 2,635 for severe asthma ) . based on these findings , the total annual cost of asthma in spain was estimated to be 1,480 million euros . on the other hand , 70% of total costs were attributed to the poor control of the disease , which further reinforces the need to achieve clinical stability of the patients as it has been repeatedly emphasized in clinical practice guidelines.9,17 from another perspective , poor asthma control is a determining factor of the high indirect costs related to absenteeism and reduced productivity . in a naturalistic and observational study ( tenor study ) of 4,756 asthma patients recruited in 283 study sites from diverse geographical areas in the usa and followed up for 24 months , the mean annual costs for uncontrolled patients with difficult - to - treat asthma were more than double of those for controlled patients throughout the study.18 different studies carried out in other countries have obtained similar results.1921 moreover , chronic comorbidities contribute to the burden and costs of persistent asthma,22 and poor adherence to asthma medication regimens , including patients with difficult - to - control asthma , is a key problem contributing to poor disease control.23 adherence to asthma treatment in patients with uncontrolled difficult asthma is highly relevant , since treatments currently considered for step - up therapy ( biologics , bronchial thermoplasty)9,17 are very expensive and , in many cases , are prescribed without adequate adherence to drug regimens in the lower steps.24 a further interesting aspect concerns to deficiencies in the development and implementation of effective education programs for asthma patients , promotion of health , and social support . it has been demonstrated that education in self - management of asthma with symptom or peak flow monitoring , combined with regular medical visits and written action plans , is effective in improving health outcomes in adults with asthma.25,26 in a 1-year cluster randomized controlled multicenter study with the participation of 230 adults with mild - to - moderate persistent uncontrolled asthma , an asthma educational program based on a repeated short intervention , given in four face - to - face sessions at 3-month intervals was effective in improving asthma symptom control , future risk , and quality of life.27 in this study , the education program included administration of a written personalized action plan and training on inhaler technique.27 other experiences reported similar results . in a controlled clinical trial in which a comprehensive asthma intervention program was evaluated in a population of medicaid - insured asthmatic children , a significant improvement in health outcomes ( emergency department visits , hospitalizations ) and asthma health care costs was observed in the intervention group in the year after enrollment.28 in a randomized patient selection study with crossover , a vigorous medical regimen and intensive educational program were able to decrease hospital use among a group of adult asthmatics who had previously required repeated readmissions for acute asthma exacerbations.29 in a large teaching hospital ( glasgow royal infirmary ) where asthma management was audited prospectively for 1 year , treatment of asthma patients in wards with a specialist interest in respiratory medicine was associated with a reduction in the rate of readmissions compared to patients admitted to general wards without this special interest ( 2% vs. 20%).30 given the complexity and multiple factors involved in the control of asthma , there is a need for establishing asthma units involved in the care of asthma patients , especially those patients with severe difficult - to - control disease . however , up to the present time , a few studies have demonstrated that assessment and management of patients in specific units for severe asthma are associated with substantial benefits in terms of health ( asthma control , quality of life ) and reduction in economic burden . in a study of 346 patients with severe asthma referred to specialist centers across the uk and followed up for a median of 286 days , significant reductions in health - care use ( primary care or emergency department visits ) , hospital admissions , and steroid dose were observed , which were accompanied by significant improvements in quality of life and asthma control.31 in a crossover study carried out in spain , treatment of patients in asthma clinics was cost - effective and beneficial in asthma management in comparison with standard outpatient services.32 the national asthma program in finland , probably one of the most outstanding health care networks for asthma patients , has shown that integration of different health care levels involved in the management of asthma ( pneumologists , primary care physicians , and pharmacists ) improves control of the disease and reduces the morbidity of asthma.33 the separ has promoted the task of accrediting the levels of the different asthma units already existing in our country , with the following objectives : 1 ) to improve the level of care of asthma patients , ensuring a framework of quality of care ; 2 ) to establish resources and facilitating their management ; 3 ) to promote the development of training plans in asthma and to advance in the concept of accreditation of knowledge ; 4 ) to favor collaboration with professionals from other clinical disciplines in a cooperative environment ; and 5 ) to promote asthma research . within the separ training framework , a manual on severe asthma and difficult - to - control asthma has been published , which includes a chapter on the provision and organization of a severe asthma unit.34 briefly , three levels of specialized asthma care have accredited based on available resources : specialized unit for highly complex asthma , specialized asthma unit , and basic asthma unit . regardless of the level of accreditation obtained , the distinction of excellence could be granted when more requirements were met at each level . in order to obtain accreditation at each of the levels , there were indispensable requirements ( irs ) that had to be met ; also , there were two other criteria , which included evaluable criteria ( ec ) and recommended criteria ( rc ) . the certification of each level was achieved if at least 80% of the ec corresponding to each category were fulfilled . the quality of each level was excellent if the result of the formula ( ec + rc ) 100/(total number of ec + rc items at the level ) was 80% . the integral care of the patient with severe asthma in a specialized multidisciplinary and high - quality setting presents many advantages , particularly related to the diagnosis of asthma , including identification of patients according to phenotypes , treatment of comorbidities , protocolized followup , optimization of the therapeutic arsenal , indication of specific treatments , and emphasis on health education for both patients and health care professionals . for instance , to have available a specialized nurse well trained in asthma education and use of inhaler devices will result in a better understanding of the disease , adherence to treatment , and control of the disease . in addition , there is a high prevalence of psycho - comorbidity in asthma , and acting at this level can improve the quality of life of the patients , control of anxiety , low consumption of resources , etc . in this respect , likewise , the possibility of implementing complementary programs contributes to better control of the disease . this approach is associated with cost savings as a result of a better and rational use of the resources . in addition , accredited asthma units could help patients with asthma through identifying misdiagnosed cases of asthma and providing more appropriate treatment / referral . misdiagnosis of non - asthmatic conditions as uncontrolled asthma has been reported to be as high as 12%30%.35,36 the implementation of databases with relevant and updated clinical information is an essential tool for the independent analysis of the data of each unit or aggregated data from different units . this is effectively a proposal for the control and improvement of care provided to our patients , the results of which are pending to be collected and evaluated . we believe that diagnosis and treatment by highly specialized and experienced personnel in asthma will result in the achievement of a better control of disease . a recent study carried out in an asthma clinic of a university - affiliated hospital in lugo in 2012 showed that all cost variables except drugs and diagnostic tests were significantly reduced in comparison with standard outpatient services , giving an annual saving per patient of 338.32 the assessment and management of patients with severe difficult - to - control asthma in accredited asthma units aims to improve the quality of care and control of the disease . in addition , specialized asthma units can improve the cost - effectiveness of pharmaceutical expenditure , especially regarding the new and costly therapies . health care professionals involved in the management of asthma should continue pursuing for unifying the quality of care of patients with asthma in a multidisciplinary collaborative setting .

this paper , developed by consensus of staff physicians of accredited asthma units for the management of severe asthma , presents information on the process and requirements for already - existing asthma units to achieve official accreditation by the spanish society of pneumology and thoracic surgery ( separ ) . three levels of specialized asthma care have been established based on available resources , which include specialized units for highly complex asthma , specialized asthma units , and basic asthma units . regardless of the level of accreditation obtained , the distinction of excellence could be granted when more requirements in the areas of provision of care , technical and human resources , training in asthma , and teaching and research activities were met at each level . the spanish experience in the process of accreditation of specialized asthma units , particularly for the care of patients with difficult - to - control asthma , may be applicable to other health care settings .

several factors have been associated with this trend range from changes in the physical , social and economic way of life and environments of the people , to genetic and physiologic factors . education is one of the media that influences greatly the attitude , social , economic and physiologic behaviors of the people . many studies have established relationship between education and socioeconomic status ( ses)[46 ] . while education can be seen to influence both ses and obesity , a number of works has been done on the relationship of obesity and ses , but the same can not be said for obesity and education . part of the growing concern in the increasing rate of obesity and overweight is the varying rates at which they are distributed across social groups , by levels of education , ses and ethnic background . lipowics commented on the increase in the association between obesity and higher social class among men , women and children in developing countries , but noted a strong inverse relationship between obesity and social class only among women in developed countries . while sobal and stunkard noted inconsistent and general non - linear direction in the relationship of obesity and ses in men and children , they noted consistent relationship with women , where obesity was more prevalent in women of low ses , than those of upper ses . in view of the present increase in the prevalence of obesity in developing countries and accelerated increase in blood pressure ( bp ) amongst black population adopting western lifestyle . this work has been designed to determine the impact of education on obesity and bp in the ibo ethnic group of nigeria . this was based on a random sampling of 567 subjects aged 20 to 80 years of the ibo ethnic group of nigeria . this is of the three major ethnic groups ( ibos , hausa and yoruba ) in nigeria having a population density of over 800- 1500 per square kilometer . after focused grouped discussions on the objective of the study , and with the consent of the participants , questionnaires were distributed to them and bp and anthropometric measurements were taken . pregnant women and hypertensive patients and individuals with structural deformities were excluded from the measurement . the first group called the primary education level consisted of candidates who had no form of formal education at all and also candidates who did not go beyond the first 6years of formal primary education . the second group called the secondary education level included candidates that did not go beyond the secondary or college education . the third group , tertiary education level , included candidates that have gone beyond the secondary education to tertiary education . participants were allowed to rest at least for 10minutes in a seated position with arms supported at the level of the heart . the bp was measured three times with the cuff completely evacuated and recovery allowed between readings . the average of the readings was used as the systolic ( sbp ) and diastolic blood pressures ( dbp ) . height ( ht ) was measured to the nearest 0.5 cm using a vertical scale of portable stadiometer with the participant in erect position without shoes and head held in the frank fort plane . weight was measured to the nearest 0.1 kg with a bathroom scale with the participant lightly clothed . bmi was calculated as weight divided by square of height ( kg / m2 ) . hip circumference ( hc ) was measured at the largest posterior extension of the buttocks . arm circumference ( ac ) and forearm circumference ( fac ) were taken at the midpoints respectively . skin fold thickness was measured on the subject 's body at 3 sites ( triceps , subscapular and calf ) with a large caliper ( cambridge , md ) . the triceps skin fold ( tsf ) was measured in midline of the posterior aspect of the arm over the triceps muscles , midway between the lateral projections of acromion process of the scapula and the inferior margin of the ulna olecranon process . the subscapular skin folds ( ssf ) calf skin fold thickness was measured in the midline over the level of maximum protrusion . in each case a double thickness of skin and underlying tissue were raised and measured . based on the who definition for cardiovascular disease risk , the following were accepted as cut - off points for obesity , bmi > 29.9 kg / m , wc>102 cm for men and 88 cm for women , whr > 1.0 in men and 0.85 in women . whtr > 0.05 was used as a cut - off point . following who standard for definition of elevated blood pressure ; the following were accepted as elevated bp ; sbp > 140mmhg and/ or dbp > 90mmhg . the population characteristics , anthropometric indicators , sbp and dbp for both rural and urban populations are shown as means and standard deviations . pearson correlation between the blood pressure indicators with adiposity indicators were carried out and also differences between the various adiposity indications between the two populations were also noted using the t - test . participants were allowed to rest at least for 10minutes in a seated position with arms supported at the level of the heart . the bp was measured three times with the cuff completely evacuated and recovery allowed between readings . the average of the readings was used as the systolic ( sbp ) and diastolic blood pressures ( dbp ) . height ( ht ) was measured to the nearest 0.5 cm using a vertical scale of portable stadiometer with the participant in erect position without shoes and head held in the frank fort plane . weight was measured to the nearest 0.1 kg with a bathroom scale with the participant lightly clothed . bmi was calculated as weight divided by square of height ( kg / m2 ) . hip circumference ( hc ) was measured at the largest posterior extension of the buttocks . arm circumference ( ac ) and forearm circumference ( fac ) were taken at the midpoints respectively . skin fold thickness was measured on the subject 's body at 3 sites ( triceps , subscapular and calf ) with a large caliper ( cambridge , md ) . the triceps skin fold ( tsf ) was measured in midline of the posterior aspect of the arm over the triceps muscles , midway between the lateral projections of acromion process of the scapula and the inferior margin of the ulna olecranon process . the subscapular skin folds ( ssf ) calf skin fold thickness was measured in the midline over the level of maximum protrusion . in each case a double thickness of skin and underlying tissue were raised and measured . based on the who definition for cardiovascular disease risk , the following were accepted as cut - off points for obesity , bmi > 29.9 kg / m , wc>102 cm for men and 88 cm for women , whr > 1.0 in men and 0.85 in women . whtr > 0.05 was used as a cut - off point . following who standard for definition of elevated blood pressure ; the following were accepted as elevated bp ; sbp > 140mmhg and/ or dbp > 90mmhg . the population characteristics , anthropometric indicators , sbp and dbp for both rural and urban populations are shown as means and standard deviations . pearson correlation between the blood pressure indicators with adiposity indicators were carried out and also differences between the various adiposity indications between the two populations were also noted using the t - test . participants were allowed to rest at least for 10minutes in a seated position with arms supported at the level of the heart . the bp was measured three times with the cuff completely evacuated and recovery allowed between readings . the average of the readings was used as the systolic ( sbp ) and diastolic blood pressures ( dbp ) . height ( ht ) was measured to the nearest 0.5 cm using a vertical scale of portable stadiometer with the participant in erect position without shoes and head held in the frank fort plane . weight was measured to the nearest 0.1 kg with a bathroom scale with the participant lightly clothed . bmi was calculated as weight divided by square of height ( kg / m2 ) . waist circumference ( wc ) was measured in centimeters at the narrowest waist . hip circumference ( hc ) arm circumference ( ac ) and forearm circumference ( fac ) were taken at the midpoints respectively . skin fold thickness was measured on the subject 's body at 3 sites ( triceps , subscapular and calf ) with a large caliper ( cambridge , md ) . the triceps skin fold ( tsf ) was measured in midline of the posterior aspect of the arm over the triceps muscles , midway between the lateral projections of acromion process of the scapula and the inferior margin of the ulna olecranon process . the subscapular skin folds ( ssf ) calf skin fold thickness was measured in the midline over the level of maximum protrusion . in each case a double thickness of skin and underlying tissue were raised and measured . based on the who definition for cardiovascular disease risk , the following were accepted as cut - off points for obesity , bmi > 29.9 kg / m , wc>102 cm for men and 88 cm for women , whr > 1.0 in men and 0.85 in women . whtr > 0.05 was used as a cut - off point . following who standard for definition of elevated blood pressure ; the following were accepted as elevated bp ; sbp > 140mmhg and/ or dbp > 90mmhg . the population characteristics , anthropometric indicators , sbp and dbp for both rural and urban populations are shown as means and standard deviations . pearson correlation between the blood pressure indicators with adiposity indicators were carried out and also differences between the various adiposity indications between the two populations were also noted using the t - test . a total number of 567 individuals from the ages of 18 to 80 years were used for this study . 325 ( 57.3% ) of the individuals were males while 254 ( 42.7% ) were females . the mean age of the study group was 33.6 + 14 years , and 33.7 + 14 and 33.5 + 13 years , for males and females respectively . the mean ages of the males and females of the various groups and also the mean values of all the anthropometric measurements , blood pressure values , and obesity indicators have been summarized in table 1 . summary of the means and standard deviations of the various anthropometric and blood pressure parameters by sex across the educational levels the largest mean value of subcutaneous fat for both males and females was noted in the primary education group , followed by the secondary education group . the bmi assessment has the smallest mean value for both males and females in the tertiary education group . in the measurements of the central obesity indicators , the largest mean wc was noted within the males and females of the primary education group while the least was noted in the tertiary education group . both mean values of whr and whtr were noted to be smallest in the males and females of the tertiary education group . the mean blood pressure values were also largest for the primary education group and least for the tertiary education group . for all the indicators of subcutaneous fat ( ssf , tsf , csf , and sts ) , general obesity ( bmi ) , and central obesity ( wc , whr , whtr ) largest mean deposition of both subcutaneous , general and abdominal fats were noted amongst women of the primary education group . the males showed larger mean bp values than the females in all the groups . in the summary of the obesity and cardiovascular disease risk occurrence , shown in table 2 , the primary education group was noted to be at most risk as against the tertiary education group . prevalence of obesity and cardiovascular disease risk by various indicators by sex across educational levels the prevalence of obesity and cardiovascular disease risk was found to be highest amongst females of the primary education group . all the obesity and blood pressure indicators used in this study ( wc , bmi , sbp , dbp , whr , whtr , sts ) showed inverse correlations with educational levels , which are significant for wc , sbp , dbp , and whtr . the highest and most consistent relationship was noted with blood pressure ( sbp , dbp ) . it is an important risk factor for major chronic diseases , such as diabetes , heart disease , stroke and certain cancers and its link with education and socio - economic status in the various communities and ethnicities has been the query of most researches . education has been shown to be a major determinant of health status , particularly in poor countries . varying results have been reported on the effect of education on obesity and blood pressure from the different researches on various ethnicities and countries . higher mean values of the measured anthropometric dimensions and ratios were observed in the individuals that had little or no education than in those that had better education . this same group also was more vulnerable to obesity and all the related cardiovascular disease risks . both general , subcutaneous and abdominal obesities were worse for all the females of the various education group and worst for the females of the least educated group . this work has noted a strong education gradient in obesity for the studied population with the education gradient in obesity being stronger in women than in men . similar findings have being reported for other countries and ethnicities . increasing education at any point along that spectrum cutler and lleras - muney found that those with more years of schooling are less likely to smoke , drink a lot , to be overweight or obese or to use illegal drugs . grossman and kaestner in their work effects of education on health concluded that years of formal schooling is the most important correlate of good health . various explanations have been proffered on the reasons for this strong relationship between education and obesity . educated individuals have been reported to make better use of health related information than those who are less educated . the lack of education about energy contents of foods has also been suggested to be a contributor to the effects of social class on obesity . cutler and lleras - muney found that those with more years of schooling are less likely to smoke , drink a lot , to be overweight or obese or to use illegal drugs . yoon suggested that it is possible that more highly educated people have the knowledge to develop healthy lifestyles and have more awareness of the health risks associated with being obese . the positive effect of education on obesity can summarily be attributed to greater access to health - related information and improved ability to handle such information by the educated , clearer perception of the risks associated with lifestyle choices and improved self - control and consistency of preferences over time . the establishment of a strong relationship between obesity and blood pressure within any community , group or country is important in the development of the most suitable education - based policies to counteract the present increase in obesity and related chronic diseases both in developed and developing communities and countries . this relationship between education and health has been traced to the health information which is part of education . so health education programs aimed at promoting healthy lifestyles might in principle generate similar effects to those associated with school education by providing relevant health information . to this effect government and other relevant policy making bodies are called upon to intensify actions toward organizing and funding of public health education campaigns . education , whether through formal schooling or health promotion campaigns , has been identified as one of the factors that can be used to combat the present increase in the prevalence of elevated blood pressure , obesity and obesity related diseases .

background : increase in the prevalence of hypertension , obesity and obesity related diseases has become significant cause of disability and premature death in both developing and newly developed countries , with over bearing demand on national health budgets.aim:to evaluate the impact of various levels of education on obesity and blood pressure.materials and method:325 male and 254 female nigerians of ages 20 - 80 years of the ibo ethnicity through random sampling , were selected for this study . the participants were broken into three major groups based on their educational levels ; primary , secondary and tertiary levels . systolic and diastolic blood pressure ( sbp & dbp ) levels , body mass index ( bmi ) , waist hip ratio ( whr ) , waist height ratio ( whtr ) , waist circumference ( wc),various skin fold thicknesses , and other anthropometric parameters were measured.result:for all the indicators of subcutaneous fat , general obesity , and central obesity , largest mean deposition was noted to be highest in the lowest education group and least in the highest education group . mean blood pressure parameters were also highest in the least education group . while fat deposition was noted to be highest in all the females of all the groups , the males showed larger mean bp values . education was noted to have a significant inverse relationship with most of the fat indicators and blood pressure parameters and cardiovascular disease risk highest in the least education groups.conclusion:education showed a significant impact on obesity and blood pressure and could be one of the major tools to reduce the high prevalence of obesity , hypertension and other obesity associated diseases .

a 45year old woman was admitted to our hospital and surgery was planned for right staghorn calculus ( figure 1 ) . ultrasound showed multiple calculi in the right kidney ; 38 mm in the pelvis , 21 mm in the lower pole and 16 mm in the upper pole with moderate hydronephrosis . intravenous urography showed right renal staghorn calculus with delayed excretion suggestive of impaired right renal function . ray showing the burden of calculi in the right kidney . under general anesthesia , initial ureteric the procedure was carried out with the patient in prone position using a c arm and bull 's eye technique . lower pole puncture was done and the stones in the lower calyx , middle calyx and pelvis were cleared . an upper pole puncture was done under c arm guidance and bile was observed gushing out of the needle when the stiletto was removed . a 20 fr foley catheter was kept as a percutaneous nephrostomy tube and the patient was put on antibiotics according to urine culture and sensitivity along with metronidazole . in the immediate post operative period , the patient was closely monitored with parameters such as pulse rate , blood pressure , abdominal girth monitoring , water balance and nasogastric tube aspirate . the patient had two episodes of vomiting in the immediate post operative period and mild distension of the abdomen . an ultrasound was performed twenty four hours after surgery and revealed minimal collection in the gall bladder fossa ( < 10 ml ) with pelvic collection ( < 50 ml ) . a 72 hour scan was done which showed resolution of the gall bladder fossa collection . the patient had no signs of peritonitis and responded well to conservative line of management . she was discharged home on post operative day five and was followed up after two weeks with renal function test and liver function tests which were within normal limits . according to clavien dindo grading system for surgical complications this case fits into grade 2 . percutaneous nephrolithotomy is the treatment of choice for large ( > 2 cm ) renal staghorn calculi [ 1 , 2 ] . it constitutes a small number of all complicated visceral injuries during pcnl . till date , only six cases have been documented in literature . other organ injuries such as spleen , liver and colon have been documented , with gall bladder injuries being the least common . a well distended gall bladder is in close proximity to the right kidney and medial right sided percutaneous renal access may increase the risk of gallbladder injury . it is significant to bear in mind that gall bladder injury is not a common complication but is still commonly encountered in thin individuals . most of the cases in the literature have undergone immediate cholecystectomy . in our case , pcnl was not abandoned and the patient did not undergo cholecystectomy ; our case was instead managed conservatively . in the post operative period , close monitoring is required , both clinical and by means of ultrasound . if there is deterioration in the clinical scenario , then immediate cholecystectomy has to be done . in our case , the leak subsided and spontaneous closure of perforation occurred , most probably due to the use of a two piece diamond tip needle , which is supposed to be less traumatic as compared to other conventional needles .

percutaneous nephrolithotomy ( pcnl ) has been an excellent option for the management of kidney stones . there have been many complications in regards to solid organ injury during pcnl . here we discuss an interesting case of 45-year - old woman , who underwent pcnl for right renal staghorn calculus , and had an accidental puncture of the gall bladder . post operatively , the patient was conservatively managed and recovered well . a small number of cases has been reported until now in literature .

we conducted a prospective cohort study in 951 young people aged 1220 years with type 1 diabetes of minimum 2 years duration at a tertiary hospital in australia between 1990 and 2007 as previously described ( 14 ) . all were free of retinopathy at baseline and had a minimum of two diabetes complication assessments . the number of people with baseline photographs suitable for grading was 736 ( 77% ) . no baseline urine samples were available for 70 ( 10% ) who were excluded from the study . a1c in those without urine samples at baseline was higher ( 8.9 vs. 8.5% ; p = 0.04 ) but no other baseline differences were observed . 511 had normal aer at baseline ( aer 7.5 g / min ) and 155 had elevated aer . baseline characteristics are described for all 666 participants ( table 1 ) . participants were prospectively followed using standardized interviews , examinations , and laboratory investigations at each visit as previously described ( 15 ) . the study was approved by the children s hospital at westmead ethics committee , and written informed consent was obtained from all participants . baseline characteristics by renal function status ( aer > 7.5 g / min ) through the study period incident renal dysfunction was defined as an aer > 7.5 g / min from three overnight , timed urine collections . this cutoff is above the 95th percentile of the normal adolescent population and predictive of microalbuminuria ( 3,4 ) . urinary albumin was measured using a polyclonal radioimmunoassay ( pharmacia ria , uppsala , sweden ) from 1997 to march 2000 , changed to nephelemetry ( immage analyzer ; beckman coulter , sydney , australia ) until 2003 , and then changed to immulite analyzer ( siemans , los angeles , ca ) from 2004 onwards . a cross - validation analysis found no significant differences or systematic bias between methods in the estimation of urinary albumin concentration . the slope of the fitted regression between the values obtained from each of the assays did not differ significantly from unity , nor did an intercept term differ significantly from zero . mydriatic seven - field stereoscopic fundal photography was performed using a topcon fundus camera ( trc50-vt ; tokyo - optical , tokyo , japan ) using film and , subsequently , digital photography ( 16 ) . camera settings , including angle of retinal photography , remained unchanged . all film images prior to september 2004 were individually scanned in a high - resolution scanner ( 3071 2048 pixels ) in raw format to avoid pixel and color compression ( canoscan fs2710 ; canon , tokyo , japan ) . each image was selected and deemed appropriate for retinal vascular geometry grading by a single grader ( m.b.s . ) . retinopathy was assessed by a single ophthalmologist masked to participants clinical characteristics , according to the modified airlie house classification ( 12 ) . incident retinopathy was defined as at least one microaneurysm / hemorrhage in either eye ( early treatment of diabetic retinopathy study [ etdrs ] level 21 , minimal nonproliferative diabetic retinopathy or greater ) . right - eye , digitized retinal photographs of each patient were analyzed by a single grader ( m.b.s . ) , masked to participants characteristics , using a semiautomated , computer - assisted image program ( singapore i vessel assessment or siva ) as previously described ( 14,15 ) . the software then combined the individual measurements into summary indices : ldr was calculated as the length from the midpoint of the first branch to the midpoint of the second branch , divided by the diameter of the parent vessel at the first branch . st was calculated as the ratio between the actual path length of the vessel segment ( measured by tracking ) and the straight - line length of the same segment . retinal vessel caliber measurements , represented by the central retinal arteriolar equivalent ( crae ) and the central retinal venular equivalent ( crve ) , were calculated as previously described ( 17 ) . branching angle in degrees ( ) represented the angle between two daughter vessels ( 18 ) . for quality control of retinal vascular measures , we performed intragrader reliability analysis on 120 randomly selected images to which the grader was masked . the intragrader correlation coefficients ( iccs ) for each parameter were as follows : ldrv 0.80 , arteriolar ldr 0.84 , stv 0.81 , arteriolar st 0.96 , crae 0.79 , crve 0.89 , arteriolar branching angle 0.65 , and venular branching angle 0.4 . the population was classified into three groups : 1 ) normal renal function throughout the study ( n = 337 ) ; 2 ) incident renal dysfunction ( n = 174 ) , and 3 ) renal dysfunction at baseline ( n = 155 ) . the latter group was excluded from multivariate analyses exploring predictors of incident nephropathy , leaving 511 participants . descriptive statistics are presented as mean and sd for normally distributed data or median and iqr for skewed distributions . anova was used to determine differences between renal function groups with tukey test for post hoc analysis for normally distributed variables . the kruskal - wallis test was used to examine between group differences for skewed data , and differences in categorical variables were compared using the test ( table 1 ) . the effect of diabetes duration on retinal vascular measures was explored by comparing groups with shorter ( 5 years ) and longer ( > 5 years ) diabetes duration using independent sample student t tests ( table 2 ) . cox proportional hazard regression analysis was used to study threshold effects for ldr and st using quartiles as predictors of time to development of early renal dysfunction and retinopathy . predictive gee univariate and multivariate models longitudinal analysis was performed using generalized estimating equations ( gees ) with presence of renal dysfunction at the respective visit as the outcome variable . predictive models were fitted to explore the association between retinal vascular geometry and development of renal dysfunction ( aer > 7.5 g / min ) for those with normal renal function at baseline ( n = 511 ) . the use of gee allowed all visits to be included in the analysis and accounted for correlations between repeated observations for a given patient . age , sex , diabetes duration , a1c , systolic blood pressure ( sbp ) sds , diastolic blood pressure ( dbp ) sds , bmi sds , total cholesterol , and insulin therapy intensity ( as a binary variable multiple daily injections ( mdis ) greater than or equal to four insulin injections per day versus less than four ) were included in the models as explanatory variables . baseline arteriolar and venular st and ldr were log transformed for analysis as continuous variables and categorized into quartiles to examine for threshold effects . results of significant variables in gee models are reported as odds ratio ( or ) and 95% cis , and results of cox regression are reported as hazard ratio ( hr ) and 95% ci . statistical procedures were performed using pasw version 18.0 ( 2009 spss inc . , chicago , il ) . incident renal dysfunction was defined as an aer > 7.5 g / min from three overnight , timed urine collections . this cutoff is above the 95th percentile of the normal adolescent population and predictive of microalbuminuria ( 3,4 ) . urinary albumin was measured using a polyclonal radioimmunoassay ( pharmacia ria , uppsala , sweden ) from 1997 to march 2000 , changed to nephelemetry ( immage analyzer ; beckman coulter , sydney , australia ) until 2003 , and then changed to immulite analyzer ( siemans , los angeles , ca ) from 2004 onwards . a cross - validation analysis found no significant differences or systematic bias between methods in the estimation of urinary albumin concentration . the slope of the fitted regression between the values obtained from each of the assays did not differ significantly from unity , nor did an intercept term differ significantly from zero . mydriatic seven - field stereoscopic fundal photography was performed using a topcon fundus camera ( trc50-vt ; tokyo - optical , tokyo , japan ) using film and , subsequently , digital photography ( 16 ) . all film images prior to september 2004 were individually scanned in a high - resolution scanner ( 3071 2048 pixels ) in raw format to avoid pixel and color compression ( canoscan fs2710 ; canon , tokyo , japan ) . each image was selected and deemed appropriate for retinal vascular geometry grading by a single grader ( m.b.s . ) . retinopathy was assessed by a single ophthalmologist masked to participants clinical characteristics , according to the modified airlie house classification ( 12 ) . incident retinopathy was defined as at least one microaneurysm / hemorrhage in either eye ( early treatment of diabetic retinopathy study [ etdrs ] level 21 , minimal nonproliferative diabetic retinopathy or greater ) . right - eye , digitized retinal photographs of each patient were analyzed by a single grader ( m.b.s . ) , masked to participants characteristics , using a semiautomated , computer - assisted image program ( singapore i vessel assessment or siva ) as previously described ( 14,15 ) . the software then combined the individual measurements into summary indices : ldr was calculated as the length from the midpoint of the first branch to the midpoint of the second branch , divided by the diameter of the parent vessel at the first branch . st was calculated as the ratio between the actual path length of the vessel segment ( measured by tracking ) and the straight - line length of the same segment . retinal vessel caliber measurements , represented by the central retinal arteriolar equivalent ( crae ) and the central retinal venular equivalent ( crve ) , were calculated as previously described ( 17 ) . branching angle in degrees ( ) represented the angle between two daughter vessels ( 18 ) . for quality control of retinal vascular measures , we performed intragrader reliability analysis on 120 randomly selected images to which the grader was masked . the intragrader correlation coefficients ( iccs ) for each parameter were as follows : ldrv 0.80 , arteriolar ldr 0.84 , stv 0.81 , arteriolar st 0.96 , crae 0.79 , crve 0.89 , arteriolar branching angle 0.65 , and venular branching angle 0.4 . the population was classified into three groups : 1 ) normal renal function throughout the study ( n = 337 ) ; 2 ) incident renal dysfunction ( n = 174 ) , and 3 ) renal dysfunction at baseline ( n = 155 ) . the latter group was excluded from multivariate analyses exploring predictors of incident nephropathy , leaving 511 participants . descriptive statistics are presented as mean and sd for normally distributed data or median and iqr for skewed distributions . anova was used to determine differences between renal function groups with tukey test for post hoc analysis for normally distributed variables . the kruskal - wallis test was used to examine between group differences for skewed data , and differences in categorical variables were compared using the test ( table 1 ) . the effect of diabetes duration on retinal vascular measures was explored by comparing groups with shorter ( 5 years ) and longer ( > 5 years ) diabetes duration using independent sample student t tests ( table 2 ) . cox proportional hazard regression analysis was used to study threshold effects for ldr and st using quartiles as predictors of time to development of early renal dysfunction and retinopathy . predictive gee univariate and multivariate models longitudinal analysis was performed using generalized estimating equations ( gees ) with presence of renal dysfunction at the respective visit as the outcome variable . predictive models were fitted to explore the association between retinal vascular geometry and development of renal dysfunction ( aer > 7.5 g / min ) for those with normal renal function at baseline ( n = 511 ) . the use of gee allowed all visits to be included in the analysis and accounted for correlations between repeated observations for a given patient . age , sex , diabetes duration , a1c , systolic blood pressure ( sbp ) sds , diastolic blood pressure ( dbp ) sds , bmi sds , total cholesterol , and insulin therapy intensity ( as a binary variable multiple daily injections ( mdis ) greater than or equal to four insulin injections per day versus less than four ) were included in the models as explanatory variables . baseline arteriolar and venular st and ldr were log transformed for analysis as continuous variables and categorized into quartiles to examine for threshold effects . results of significant variables in gee models are reported as odds ratio ( or ) and 95% cis , and results of cox regression are reported as hazard ratio ( hr ) and 95% ci . statistical procedures were performed using pasw version 18.0 ( 2009 spss inc . , chicago , il ) . at baseline , median age was 13.5 ( iqr 12.814.9 ) years and diabetes duration 4.8 ( 3.37.5 ) years . those with renal dysfunction at baseline were older and had higher aer , a1c , sbp , and dbp than the other groups ( table 1 ) . overall baseline crae was smaller in those who had renal dysfunction at baseline , whereas crve was not significantly different . in the incident renal dysfunction group , arteriolar branching angle was significantly greater among those with longer diabetes duration , whereas venular branching angles did not differ ( table 1 ) . of the 511 participants with normal aer at baseline , median follow - up was 3.7 ( iqr 2.35.7 ) years , with 3.0 ( 2.04.0 ) visits per participant and 1,898 clinical assessments . incident renal dysfunction was evident in 34% ; those who developed incident renal dysfunction had higher mean baseline aer ( p < 0.001 ) and were generally younger ( p = 0.02 ) than those who had normal aer throughout follow - up . no significant differences were observed with respect to sex distribution , diabetes duration , bp sds , bmi sds , insulin therapy intensity , or glycemic control ( a1c ) between these groups ( table 1 ) . a greater proportion of those who developed incident renal dysfunction also developed incident retinopathy ( p < 0.001 ) . there was no significant correlation between ldrv and stv ( pearson correlation 0.04 , p = 0.4 ) . in cox proportional hazard regression analysis ( n = 511 ) , ldrv in the highest quartile ( hr 2.0 [ 95% ci 1.32.9 ] ) , stv in the lowest quartile ( 1.5 [ 1.12.1 ] ) , and higher dbp sds and a1c were associated with a higher cumulative risk of early renal dysfunction ( fig . 1 ) . there were no significant differences in risk between quartiles 13 for ldrv and quartiles 24 for stv . a : cumulative hazard curves for risk of early renal dysfunction by ldrv quartiles : quartile 4 vs. quartiles 13 ( hr 2.0 [ 95% ci 1.32.9 ] ) adjusted for sex , a1c , and dbp sds . b : cumulative hazard curves for risk of early renal dysfunction by stv : quartile 1 vs. quartiles 24 ( 1.5 [ 1.12.1 ] ) adjusted for sex , a1c , and dbp sds . in univariate gee analysis ( n = 511 ) , log - ldrv , as a continuous variable , predicted incident renal dysfunction ( or 1.9 ) , whereas log - stv as a continuous variable , was not statistically significant ( or 1.00 [ 95% ci 0.981.01 ] ; p = 0.4 ) . when analyzing for a threshold effect , those in the highest ldrv quartile ( or 1.5 ) and lowest stv quartile ( or 1.5 ) were at greater risk of incident renal dysfunction . other significant risk factors for incident renal dysfunction included longer diabetes duration , higher a1c , and male sex ( table 2 ) . in multivariate gee analysis , log - ldrv predicted incident renal dysfunction ( or 2.4 ) after adjusting for age , sex , diabetes duration , a1c , dbp sds , bmi , and total cholesterol . when analyzing for a threshold effect , those in the highest ldrv quartile ( or 1.7 ) and lowest stv quartile ( or 1.6 ) were at greater risk of incident renal dysfunction ( table 2 ) . in this longitudinal study of young people with type 1 diabetes , normal aer , and no retinopathy at baseline , greater retinal venular ldr and lower venular tortuosity predicted incident renal dysfunction independent of established risk factors including diabetes duration , glycemic control , bp , and total cholesterol . we also confirmed our recent findings that lower arteriolar ldr and greater arteriolar tortuosity were associated with incident retinopathy ( data not shown ) ( 14 ) . we recently proposed that , in the diabetic milieu , neuroretinal hypoxia leads to a compensatory increase in afferent blood flow through increased vessel density ( earlier arteriolar branching resulting in lower arteriolar ldr ) and a corresponding increase in arteriolar tortuosity ( 14 ) . although this described the afferent changes in blood flow , it is important to understand the adaptations at the efferent ( venular ) end and their significance . whereas arteriolar changes may best represent localized organ tissue changes ( 14,15 ) determinants of resistance to blood flow within a vascular network include the individual blood vessel size ( caliber and length ) , the organization of the vascular network ( series and parallel ) , and the flow characteristics ( laminar versus turbulent ) . although the microcirculation provides most of the systemic vascular resistance , it has low reynolds numbers and therefore turbulence at this level is unlikely to occur . in this setting , hydrostatic and oncotic pressures , viscosity , and wall shear stress would be most influential . hyperglycemia causes significant hemodynamic and rheological changes ( 19 ) , including increased retinal blood flow ( 20 ) and blood viscosity ( 19 ) . furthermore , changes to particulate components of blood include greater platelet aggregation ( 19 ) and increased leukocyte numbers with higher integrin - mediated adhesion properties promoting leukostasis ( 11 ) . we propose that decreased venular network complexity together with early vascular tone dysregulation and non - newtonian influences contribute to increased capillary hydrostatic pressure , greater wall shear stress , and vessel damage . thus , individuals with a simplified venular network may be at greater risk of microvascular complications such as renal dysfunction . baseline characteristics ( table 1 ) demonstrated overall narrower arteriolar calibers in those with renal dysfunction . however , longer diabetes duration was associated with wider vessel calibers ( both arteriolar and venular ) . this may describe the natural history of microvascular changes in diabetes : 1 ) early impairment of microvascular autoregulation with inappropriate vasoconstriction , 2 ) subsequent compensatory dilation , 3 ) loss of smooth muscle cells and pericytes ( or podocytes in the kidney ) , and 4 ) loss of wall structural integrity and , finally , irreversible dilation . early hyperglycemia results in a downregulation of large - conductance , calcium - activated potassium channels necessary for vascular smooth muscle cell relaxation , leading to early vasoconstriction and neuroretinal hypoxia ( 21 ) . diabetic rat models demonstrated a similar dysregulation in the renal microvasculature early in diabetes with severe insulin deficiency . it has been speculated that tissue hypoxia beyond a certain threshold may override the vasoconstrictive effect of diabetes ( 12 ) . finally , large population - based studies associated wider retinal venules with proteinuria in adults with type 1 diabetes of longer duration ( 13 ) . changes in vessel caliber are most influential in the acute regulation of blood flow . according to the poiseuille equation , vessel resistance is inversely proportional to the vessel radius to the fourth power ( r ) . thus , even small changes in the diameter of arterioles and venules can lead to significant changes in blood flow and capillary pressures . capillary hypertension has been associated with both retinopathy ( 12 ) and nephropathy ( 22 ) . classical renal micropuncture studies demonstrated that glomerular hypertension ensued in diabetes models even in the setting of normal bp ( 23 ) . diabetes led to an impairment of glomerular circulatory autoregulation , with greater vasodilatation of the afferent arteriole than the efferent arteriole , which resulted in greater intraglomerular capillary pressure ( 23 ) . ultimately , this exposes the glomeruli to increased intracapillary pressure , increased shear stress , and mechanical stretch . mechanical stretch has been observed to favor an accumulation of extracellular matrix ( 24 ) and a decrease in podocyte number through effects on cell proliferation , apoptosis , and cell adhesion to the basement membrane ( 22 ) . in this setting , even minor increments in systemic bp would exacerbate glomerular hypertension in a self - perpetuating cycle of ongoing hypertensive injury with impaired microvascular autoregulation . the efficacy of ace inhibitors in retarding the progression of dn may in part be due to their vasodilatory effect on the efferent glomerular arteriole , thus decreasing glomerular capillary pressure ( 25 ) . although we observed higher bp in those with baseline renal dysfunction ( table 1 ) , bp measured at each visit did not predict renal dysfunction in gee longitudinal analysis ( table 2 ) . a longer follow - up period may be required to examine this relationship with adequate statistical power . in our study , a1c was an independent predictor of early renal dysfunction in keeping with previous reports ( 26 ) . loss of capillary autoregulation , higher glomerular capillary pressure , and decrease in podocytes are related to the severity and chronicity of hyperglycemia ( 22 ) . this study of normotensive young people with no intercurrent medications provides an insight into the preclinical renal dysfunction associated with diabetes and may assist in identifying those at greatest risk where early intervention may yield greatest benefit . the lack of a statistically significant association between bp and incident renal dysfunction in our multivariate models contrasts with previous findings regarding retinopathy ( 27 ) . it is noteworthy that a significantly greater proportion of those who developed incident renal dysfunction also developed incident retinopathy . thus , changes in bp may only become apparent later in the course of renal disease where capillary bed autoregulation may no longer be able to compensate for metabolic demands . this also suggests that retinal hypoxic demands may have a greater influence in bp autoregulation than changes in renal microvasculature . this is consistent with our previous observations between bp and retinopathy in young people with type 1 diabetes ( 27 ) . our findings suggest that a simplified venular network ( i.e. , greater ldrv and lower stv ) results in a greater risk of early renal dysfunction . as previously described , lower ldr can result from shorter axial length due to earlier branching points and/or wider vessel calibers ( 14 ) . our current software does not report vessel length ; however , our results suggest venular axial length was increased in view of the wider venules observed with longer diabetes duration . these early venular differences may represent greater tissue metabolic demands , mechanical stress , and an increased propensity for early nephropathy . our measures of tortuosity and ldr had good reproducibility ; however , branching angles are particularly vulnerable to parallax error as reflected in the relatively low intraobserver correlation . not unexpectedly , therefore , there were no significant associations in gee models between branching angles and renal dysfunction . the strengths of this study include the longitudinal design , a large patient cohort with multiple visits per individual , and standardized , quantitative evaluation of retinal vascular measures by a single grader masked to participants clinical status . our young cohort without comorbidities avoids confounders present in older groups with established complications and medical therapy . the use of gees allowed inclusion of every available patient visit in the analysis and accounts for uneven follow - up and missing data . the reproducibility of ldr and st measures ( iccs ranging from 0.80 to 0.96 ) was comparable with retinal vascular caliber measurements in the atherosclerosis risk on community study ( iccs ranging from 0.79 to 0.83 ) ( 28 ) . this cohort from a tertiary referral center may be biased toward closer monitoring , tighter metabolic control , and , thus , underrepresentation of early renal dysfunction ; conversely , this underscores the robust nature of the measures studied . limitations of our measurements include the use of the central retinal field only and the lack of reporting absolute values for vessel length . therefore , we can not generalize our findings to the whole microvascular bed involving peripheral retinal areas that need further study both structurally and functionally . the spherical nature of the retinal surface can not be accurately assessed in two - dimensional photographs and has an inherent degree of parallax error when evaluating branching angles . nevertheless , our results demonstrate the potential utility of novel quantitative measurements of retinal vascular geometry from this central field , which is practical and reproducible , in people with diabetes . in summary , retinal vascular geometry parameters , specifically retinal venular ldr and tortuosity , are independent predictors of incident renal dysfunction in young people with type 1 diabetes . these noninvasive retinal measures may further our understanding of early mechanistic pathways for microvascular complications . our study highlights the role of venular ldr and tortuosity as tools for risk stratification of individuals at high risk for microvascular complications , and in monitoring of disease progression and therapeutic benefits .

objectiveto examine the relationship between retinal vascular geometry parameters and development of incident renal dysfunction in young people with type 1 diabetes.research design and methodsthis was a prospective cohort study of 511 adolescents with type 1 diabetes of at least 2 years duration , with normal albumin excretion rate ( aer ) and no retinopathy at baseline while attending an australian tertiary - care hospital . aer was quantified using three overnight , timed urine specimen collections and early renal dysfunction was defined as aer > 7.5 g / min . retinal vascular geometry ( including length - to - diameter ratio [ ldr ] and simple tortuosity [ st ] ) was quantified from baseline retinal photographs . generalized estimating equations were used to examine the relationship between incident renal dysfunction and baseline venular ldr and st , adjusting for age , diabetes duration , glycated hemoglobin ( a1c ) , blood pressure ( bp ) , bmi , and cholesterol.resultsdiabetes duration at baseline was 4.8 ( iqr 3.37.5 ) years . after a median 3.7 ( 2.35.7 ) years follow - up , 34% of participants developed incident renal dysfunction . in multivariate analysis , higher retinal venular ldr ( odds ratio 1.7 , 95% ci 1.22.4 ; quartile 4 vs. 13 ) and lower venular st ( 1.6 , 1.12.2 ; quartile 1 vs. 24 ) predicted incident renal dysfunction.conclusionsretinal venular geometry independently predicted incident renal dysfunction in young people with type 1 diabetes . these noninvasive retinal measures may help to elucidate early mechanistic pathways for microvascular complications . retinal venular geometry may be a useful tool to identify individuals at high risk of renal disease early in the course of diabetes .

it was previously suggested that atherothrombotic infarction ( ati ) and lacunar infarction ( li ) , as two different subtypes of ischemic stroke , might also differ in the set of the relevant risk factors , with ati being more associated to the atherogenic risk factors in contrast to li . . however , decreased insulin sensitivity ( is ) , that is , insulin resistance , was observed both in ati and li , which was frequently accompanied with compensatory hyperinsulinemia in t2d patients as well as in nondiabetics [ 2 , 3 ] . simultaneously , it has been shown that impaired balance between products of oxidative stress and the level of antioxidant enzyme activities might be the important mechanism underlying the occurrence of ischemic stroke . moreover , it was elucidated that the brain has only moderate content of glutathione - dependent enzymes , for example , glutathione peroxidase ( gsh - px ) , glutathione reductase ( gr ) , and superoxide dismutase ( sod ) , together with the fact that the intact antioxidant defense could provide first line of protection from initiation and exacerbation of ischemic cerebral injury . in addition , changes in enzymatic antioxidative defense mechanisms in patients with stroke are still controversial . previous results implied that the majority of antioxidant enzyme activity was significantly reduced in acute ischemic stroke , possibly as a consequence of increased oxidative stress while the recent finding suggested increased levels of glutathione dependent enzymes as an adaptive mechanisms during acute cerebral ischemia . finally , due to novel facts , oxidative stress can be an important component for astrocytic cell death following metabolic stress . until now , experimental studies provided evidence of an association between ischemic stroke and increased oxidative stress [ 8 , 9 ] , but data in humans are still heterogeneous and limited . therefore , our study was aimed to determine is levels and three different types of antioxidant enzyme activities gsh - px , gr , and sod , in t2d with ati and li . in this study we included a total of 93 patients with t2d , ascribed to the following groups : t2d patients with ati ( group a , n = 30 ) , and t2d with li ( group b , n = 30 ) , and t2d without ischemic stroke ( group c , n = 33 ) . simultaneously , we involved a total of 93 nondiabetics , matched with the t2d patients regarding gender and age , and also comprising the following groups : nondiabetics with ati ( group d , n = 30 ) , nondiabetics with li ( group e , n = 30 ) , and nondiabetics without stroke ( group f , n = 33 ) . t2d was diagnosed in accordance with the criteria of the world health organization . diagnosis of ati and li was done by a neurologist due to clinical features and brain imaging methods such as cranial computerized scan and/or magnetic resonance imaging in two consecutive examinations , during the first 7 days from the appearance of ischemic stroke . the patients with ati or li were included in the study provided that they had not shown signs of cardioembolic cerebral infarction , or coronary heart disease based on a history of myocardial infarction with definite elevation of serum cardiac enzymes or coronary angiography . t2d patients were treated with insulin therapy , and/or ingestion of antioxidant supplements and drugs , which might affect free radical and antioxidant activity potential ; likewise patients who had other endocrine disease or autoimmune diseases , renal or hepatic failure , current infections , neoplasms , polycythemia , or rheumatic diseases were also excluded as well as the patients with history of trauma or operation within the last 3 months . no patient had uncontrolled hypertension , severe alcohol consumption , acute infection , or an inflammatory disease during the last 4 weeks . all the patients , with or without ati and li , showed the similar level of their physical activity . in addition , they were required not to smoke at least 12 hr before the tests were performed . the patients were fully informed about the study and gave the inform consent to participate . the study was conducted at the clinic for endocrinology , diabetes and metabolic diseases and at the clinic for neurology , clinical centre of serbia , faculty of medicine , university of belgrade , and was approved by the institutional ethics committee . the interview , physical examination , metabolic test , and evaluation of antioxidant enzyme activities were performed in all the patients included in the study , for each patient within the same day . hypertension was diagnosed according to world health organisation criteria ( systolic / diastolic blood pressure 140/90 mm hg ) or by the use of antihypertensive agents . the metabolic tests were implemented at least after 6 months from the occurrence of the ischemic stroke . the evaluation of insulin sensitivity was done by frequently sampled intravenous glucose tolerance test ( fsigt ) with minimal model analysis . briefly , before testing , each patient was required to be at a 12 hr fasting state . during the fsigt , 0.3 g / kg body weight of glucose was injected and the blood samples for plasma glucose ( pg ) and plasma insulin ( pi ) determination were taken immediately before and 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 12 , 14 , 16 , 20 , 23 , 24 , 25 , 27 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , 120 , 160 , and 180 minutes after intravenous glucose stimulation . insulin was injected as a continuous infusion 4 mu / kg / min between minutes 20 and 25 in order to avoid the effect of the potentially blunted insulin response . the insulin sensitivity index ( si ) was calculated from the results of pg and pi levels by computerized minimal model analysis , using the minmod program ( kindly provided by dr . determination of the antioxidant enzymes sod , gsh - px , gr was conducted using the commercial assays ( produced by randox laboratories ltd . , uk ) , based on spectrophotometer determination methods as described previously . pg was determined by glucose oxidase method using a beckman glucose analyser ( beckman instruments , fullerton , ca ) . pi was tested by radioimmunoassay ( inep , zemun , rs , double antibody kits ) . total cholesterol , hdl cholesterol , and triglyceride concentrations were determined with the chromatography method using commercial kits ( produced by boehringer mannheim ) . the continuous variables within each subtype of ischemic stroke were analyzed with analysis of variance ( anova ) with a post hoc bonferroni test . all analyses were performed with the spss statistical package ( version 16.0 for windows ) . the clinical characteristics and biochemistry parameters of t2d patients and nondiabetics with ati or li as different subtypes of ischemic stroke are shown in table 1 . the age , gender , duration of diabetes , and duration from the onset of ischemic stroke were similar in t2d patients and nondiabetics with different subtypes of ischemic stroke , together with hba1c levels , implying satisfactory metabolic control before metabolic investigation was done . however , ldl - c level was significantly higher in t2d patients with ati compared to the t2d patients with li and t2d patients without stroke and also in nondiabetics with ati compared to nondiabetics with li and healthy controls . there is no difference in prevalence of hypertension in patients with t2d and ati or li and t2d without ischemic stroke , while it was significantly higher in nondiabetics with ati or li than in healthy controls . we found that si levels were significantly lower in t2d patients with ati ( group a ) and li ( group b ) compared to t2d patients without ischemic stroke ( group c ) ( 1.14 0.58 and 1.00 0.26 versus 3.14 0.62 min / mu / l 10 , resp . also , the results showed significantly lower si levels in nondiabetics with ati ( group d ) and li ( group e ) compared to healthy controls ( group f ) ( 3.38 0.77 and 3.03 0.72 versus 6.03 1.69 min / mu / l 10 , resp . simultaneously , pi levels were higher in t2d patients , ati ( group a ) , and li ( group b ) than in t2d patients without ischemic stroke ( group c ) ( 20.94 4.31 and 20.07 0.88 versus 16.06 0.91 mu / l , respectively , p < 0.001 ) and in nondiabetics with ati ( group d ) or li ( group e ) in comparison to healthy controls ( group f ) ( 15.57 1.86 and 15.59 1.26 versus 7.54 2.03 mu / l , respectively , p < 0.001 ) ( figure 2 ) . when we evaluated antioxidant enzyme activities in t2d patients with ati ( group a ) and li ( group b ) and without ischemic stroke ( group c ) and in nondiabetics with ati ( group d ) and li ( group e ) and healthy controls ( group d ) , we detected the levels of the gsh - px and gr activity being significantly lower in group a and b versus c and in group d and e versus f ( gshpx : a : 21.96 3.56 versus b : 22.51 1.23 versus c : 25.12 1.67 u / ghb , p < 0.001 ; d : 24.75 3.02 versus e : 25.57 1.92 versus f : 28.56 3.91 u / ghb , p < 0.001 ; gr : a : 44.37 3.58 versus b : 43.50 2.39 versus c : 48.58 3.67 u / ghb , p < 0.001 ; d : 24.75 3.02 versus e : 25.57 1.92 versus f : 28.56 3.91 u / ghb , resp . , p < 0.001 ) ( figure 3 ) , while the sod levels did not differ between the investigated groups ( a : 769.57 72.36 versus b : 768.97 34.50 versus c : 789.18 60.28 , d : 795.23 48.28 versus e : 797.80 69.21 versus f : 813.88 45.80 mu / mghb , resp . this analysis identified that decreased insulin sensitivity si and the decreases of gr were related to both ati and li in t2d patients . simultaneously , this model identified decreased insulin sensitivity si and the level of gr and gsh - px in nondiabetics with ati , but predominantly decreased insulin sensitivity si in nondiabetics with li ( table 2 ) . in this study , we directly measured the is level , together with three different types of antioxidant enzyme activities , gsh - px , sod , and gr , in t2d patients and nondiabetic individuals with two different subtypes of ischemic stroke in type 2 diabetics . our results have shown decreased is level in t2d patients with two different subtypes of ischemic stroke , ati and li , compared to t2d without stroke , while we could not demonstrate the difference between the subtypes , and the same pattern of is changes was found in the nondiabetics . to our knowledge , there are scarce data regarding the changes in is in t2d with ischemic stroke subtypes . previous study also suggested that insulin resistance measured using different methodology , the short insulin tolerance test , is independently associated with markers of atherosclerosis detected on carotid arteries in t2d patients . simultaneously , an association has been documented between insulin resistance and other markers at the large vessels , such as the pulsatility index on cerebral arteries . on the other hand , it has been shown that insulin resistance , evaluated by the homeostasis model assessment ( homa ) index , was higher in diabetic in contrast to nondiabetic patients with li being a small vessel disease . additionally , it has been suggested that diabetes , hypertension , and metabolic syndrome which share insulin resistance as a common mechanism , contribute to li occurrence [ 1820 ] . however , detailed mechanisms of the possible link between the presence of ischemic stroke subtypes and insulin resistance remain to be clarified . in addition , when we measured the level of insulinemia in these patients , the significantly higher level of insulinemia was detected in both groups of t2d patients , with ati and li . the increases in insulin levels might be primarily consequence of the simultaneous presence of insulin resistance in the relevant groups . however , the increases in insulin might contribute to the appearance of the ischemic stroke subtypes . insulin , as a growth factor , might interfere with the beneficial effects of nitric oxide ( no ) on the vasculature . moreover , insulin infusion during euglycemic insulin clamp was able to suppress endothelium - dependent vasodilation in large arteries , which is reported to be based on increased availability of endothelin-1 , leading to downregulation of nad(p)h oxidase and superoxide anion production , and endothelial dysfunction is proposed to play an important role in the pathogenesis of cerebral small vessel disease [ 23 , 24 ] . also , our results demonstrated lower values of antioxidant enzymes in t2d patients with ati or li than in type 2 diabetics without ischemic stroke . the patients with ati and li did not differ regarding the level of antioxidant enzymes . these findings could be explained by the fact that in diabetes there is already reduced capacity of antioxidant protection , which is further significantly disturbed in the acute phase of ischemic stroke [ 4 , 5 ] . in our data , in patients with ati and li , we found the decreases in glutathione - dependent enzymes activity in contrast to other types of antioxidant enzyme activity , for example , sod . these results imply a prolonged and severe depression of gluthatione dependent antioxidative defense mechanisms irrespective of stroke subtypes . since it has been documented that free radicals are extremely difficult to measure directly , antioxidant enzymes have been proposed to represent indirect markers of oxidative stress . numerous , but mostly experimental , studies provided evidence of an association between ischemic stroke and decreased antioxidant enzyme activity , although this possible association in humans has been less investigated [ 9 , 25 ] . moreover , the analyses of treatment with agents in stroke showing an ability to prevent further depression of antioxidant protection and scavenging reactive free radicals were reported to fail to restore gsh - px and gr activities [ 26 , 27 ] . generally , a recently study that aimed to assess total antioxidant capacity and oxidative stress in diabetic and nondiabetic acute stroke patients with 2 different stroke subtypes , large and small vessel disease strokes , concluded that oxidative stress and counterbalancing antioxidant capacity are more pronounced in diabetic acute stroke patients than in nondiabetics . the study provided different data in comparison to the previous investigations showing decreased gsh - px activity in both diabetic and nondiabetic patients with coronary heart disease when compared to controls [ 17 , 29 , 30 ] . however , the results from our study have shown the diminished activity of both gsh - px and gr in t2d patients with ati and li , which is consistent with previous reports of decreased gsh - px levels patients with stroke [ 6 , 8 , 31 , 32 ] . the levels of sod are found to exhibit great variations in the previous study in patients with the stroke [ 6 , 8 , 26 , 29 , 3137 ] . our results could not detect the differences in the sod levels in different groups of patients , and thus they are in line with data showing no changes in respect to sod level in both diabetics and nondiabetics irrespective of different subtypes of ischemic stroke [ 33 , 34 , 37 ] . the tentative explanation for the inconsistent findings regarding sod might reflect the predominant role of intracellular versus extracellular fraction of sod in free radical scavenging . in our study , the detected antioxidant enzyme activities were not affected by other important factors potentially influencing the enzyme levels , for example , by hyperglycemia , aging , duration of diabetes , and presence of macrovascular complications , due to the fact that in all groups of t2d patients had similar levels of metabolic and satisfactory metabolic control and that patients were matched in respect of age , duration of diabetes , and the prevalence of macrovascular complications . the multiple regression analysis applied to our data has demonstrated that decreased is together with the decreases in gr are related to both ati and li in t2d patients . in nondiabetics , the decreases in si levels and the diminished gr the analysis reveals the potential difference in the mechanisms underlying the onset of the two subtypes of the stroke in t2d patients compared to nondiabetics . the results imply that higher levels of insulin resistance combined with lower levels of gr , detected in t2d patients compared to nondiabetics , were underlying the onset of li together ati in t2d in contrast to the findings in nondiabetics . in this context , our results are consistent with the findings that li represents the most frequent ischemic stroke subtype in t2d . taken together , our data imply that insulin resistance exerts its pathogenic influence on the level of gluthatione dependent antioxidant enzymes , especially in t2d . in conclusion , our results suggest that the presence of different subtypes of ischemic stroke is associated with insulin resistance and diminished antioxidant enzyme activity in both subtypes of ischemic stroke in t2d . the results also imply that atherogenic influence of decreased is in the different subtypes of stroke in t2d might be exerted through a significantly reduced glutathione dependent antioxidant enzyme activity , while the mechanisms relating the effects of insulin resistance and decreased antioxidant enzyme activity remain to be clarified .

we analyzed ( a ) insulin sensitivity ( is ) and ( b ) glutathione peroxidase ( gsh - px ) , glutathione reductase ( gr ) , and superoxide dismutase ( sod ) antioxidant enzyme activity in type 2 diabetic ( t2d ) patients with atherothrombotic infarction ( ati ) ( group a ) , lacunar infarction ( li ) ( b ) , or without stroke ( c ) and in nondiabetics with ati ( d ) , li ( e ) , or without stroke ( f ) . ati and li were confirmed by brain imaging is levels were determined by minimal model ( si index ) , and the enzyme activity by spectrophotometry . in t2d patients , si was lower in a and b versus c ( 1.14 0.58 , 1.00 0.26 versus 3.14 0.62 min1/mu / l 104 , p < 0.001 ) and in nondiabetics in d and e versus f ( 3.38 0.77 , 3.03 0.72 versus 6.03 1.69 min1/mu / l 104 , p < 0.001 ) . also , gsh - px and gr activities were lower in a and b versus c ( gsh - px : 21.96 3.56 , 22.51 1.23 versus 25.12 1.67 ; gr : 44.37 3.58 , 43.50 2.39 versus 48.58 3.67 u / ghb ; p < 0.001 ) and in d and e versus f ( gsh - px : 24.75 3.02 , 25.57 1.92 versus 28.56 3.91 ; gr : 48.27 6.81 , 49.17 6.24 versus 53.67 3.96 u / ghb ; p < 0.001 ) . decreases in si and gr were significantly related to both ati and li in t2d . our results showed that decreased is and impaired antioxidant enzymes activity influence ischemic stroke subtypes in t2d . the influence of insulin resistance might be exerted on the level of glutathione - dependent antioxidant enzymes .

for the invasive treatment of symptomatic aortic stenosis several attractive options became available beyond conventional cardiosurgical implantation of stented bioprostheses or mechanical valves . besides stentless prostheses , in particular transcatheter procedures are of growing importance . nevertheless the number of implanted stented bioprostheses in germany expanded or at least remained stable over the last years . thus , 9.704 bioprostheses were implanted in germany in 2010 , according to 84% of all implanted prostheses in aortic position . implantation of bioprostheses was associated with a 30-day - mortality of 3,3 % , which is higher than for mechanical valves ( 1,5% ) due to the fact that patients for bioprostheses are significantly older or suffer from more complex co - morbidities . the purpose of this review is to summarize the features of the currently available bioprostheses in the light of competing procedures . there has been a continuous development to improve the design of stented bioprostheses with respect to hemodynamic profile , biological durability and ease of implantation . various designs are available , allowing a valve selection according to individual patient factors or anatomical criteria including techniques for intra - annular and supra - annular positioning [ 3 , 4 ] . it would have the same biological and hemodynamic properties as a normal valve and would not undergo degeneration . stented bioprostheses have proven to be effective even in small aortic roots regarding hemodynamic improvement , left ventricular mass reduction and improvement of the patient s quality of life . in vitro examinations showed pericardial valves to be slightly superior to porcine valves with regard to gradient and orifice area . associated with the scalloped designs of stented bioprostheses a tension - free implantation with avoidance of paravalvular leaks can be expected . implantation techniques evolved over the last decades which resulted in short cross - clamp times in normal findings . surgeons learned that meticulous care in decalcifying the aortic annulus and sizing helps to prevent paravalvular leaks . additionally , accurate sizing prevents a patients - prosthesis - mismatch which could result in poor postoperative clinical performance . avoiding the use of running suture for stented valve implantation the evolvement of low profile valves reduces the risk of coronary occlusion in patients with a small distance between annulus and coronary ostia . the currently available and established bioprostheses show rates of degeneration at 20 years around 15% in patients aged 65 or higher . the freedom from repeat aortic valve replacement reaches over 85% after 20 years in patients older than 60 years and 65% in all age groups . along with these convincing results of durability and freedom of re - implantation stented bioprostheses became also attractive for younger patients . if a repeated valve replacement is necessary due to functional deterioration the operative mortality is acceptable with 4 to 6% and is mainly due to active endocarditis and comorbidities . lately transcatheter aortic valve implantation ( tavi ) with valve - in - valve implantation into the degenerated xenograft showed to be an additional option in high risk patients with the need for redo valve replacement . stentless porcine valves were initially believed to have superior hemodynamic properties resulting in more effective reduction of left ventricular mass and better clinical performance according to nyha class . these findings could nt be yet approved by long - term follow up investigations [ 15 , 16 ] . bearing this in mind calls the more demanding implantation technique of stentless prostheses in question . stented bioprostheses are still used more commonly than stentless ones because of their relative ease of implantation , their extensively documented long - term results , and the low risk associated with reoperation . another biological approach is the use of an autograft like the ross procedure , where the patient s pulmonic valve is transferred to the aortic position . these pulmonic autografts have excellent hemodynamic properties as well as low rates of thrombosis , degeneration , and endocarditis . the ross procedure is suitable for children and young adults because it is compatible with further growth of the aortic root . nevertheless since the pulmonary valve has to been replaced by a bioprosthesis it is a demanding two valve procedure with prolonged cross clamping times . aside from this , special means are necessary for pulmonary autograft stabilization to prevent its degeneration . there are only a few current randomized trials comparing the long - term results of biological and mechanical valves . a large - scale review revealed no difference in survival rates at 10 years and a slightly higher survival rate at 15 years for patients with mechanical prostheses . the reoperation rate for mechanical valves in the aortic position is less than 5% at 10 years and less than 10% at 15 years , while the corresponding figures for bioprostheses are 10% and 30% , respectively . hemorrhagic complications are significantly more common in patients with mechanical valves because of anticoagulation . chronic atrial fibrillation is no longer a contraindication to bioprostheses since permanent oral anticoagulation can be avoided if concomitant ablative surgery results in permanent conversion to sinus rhythm . end stage renal failure was also defined a contraindication to bioprostheses since a rapid degeneration was feared due to altered metabolism in these patients . it has been found that life expectancy is already curtailed to such an extent that bioprosthesis degeneration often does not occur in the remaining lifetime . the supposed advantage of the longer durability of a mechanical valve is also offset by the potential complications of oral anticoagulation , especially because anticoagulation is more difficult to manage in dialysis patients than in others . nevertheless aortic valve replacement using a tissue valve remains controversial for patients younger than 60 years since there are studies reporting reduced mid - term results . some younger patients are averse to oral anticoagulation and therefore prefer a biological valvular prosthesis . thus , younger patients opting for a bioprosthesis can enjoy a normal quality of life without anticoagulation for many years but may need to undergo a second valve replacement procedure with an acceptable degree of risk . implantation of conventional prostheses can be performed through a limited direct access like a hemi - sternotomy or a lateral access . reported large series show that aortic valve operations can be safely performed in experienced centers . substantial progress in valve technology led to the development of self - expanding valves . since a few years a number of bioprostheses are available for suture - less implantation which is usually combined with a limited surgical access . . future will show the benefits of this procedure characterized by short cross clamping times and whether it can coexist with tavi procedures . after its clinical inauguration by cribier in 2002 the transcatheter aortic valve implantation gained widespread use in high risk patients with aortic stenosis . in germany a massive increase of its application can be observed ; in 2010 nearly every fourth aortic valve replacement was performed as a tavi procedure . the partner trial was a randomized trial comparing tavi with standard - of - care therapies in high risk patients . a 2-year follow - up of patients in the partner trial supports lately tavi as an alternative to surgery in high - risk patients . the two treatments were similar with respect to mortality , reduction in symptoms , and improved valve hemodynamics , but paravalvular regurgitation was more frequent after tavi and was associated with increased late mortality . there are a growing number of publications reporting promising short term results in high risk patients . nevertheless there is still a lack of evidence that tavi is superior to open valvular replacement as the gold standard regarding long - term results . current guidelines recommend the use of tavi restricted to patients with contraindications for open surgery or inacceptable perioperative risks . current studies reveal that bioprostheses of the most recent generation last longer than earlier types . because life expectancies in general have risen , more and more elderly patients are presenting for valve replacement and for these patients a bioprosthesis is usually chosen . in addition the cost effectiveness of stented bioprostheses appears unbeatable and the surgical ease of implantation allows for cross - clamp times between 30 and 60 minutes . overall , even in face of more innovative biological alternatives the implantation of stented bioprostheses is still a very interesting option and represents actually the most frequent valve implantation technique for aortic stenosis . especially in the light of growing use of interventional valve replacement there is the urgent need for complete nationwide registry with adequate long term follow up , quality of life information and relevant subgroup analysis to define new standards in the treatment of patients with aortic stenosis .

introductionbiological stented prostheses are currently the main type of prosthetic valve used for aortic valve replacement . the ratio of bioprotheses to mechanical prostheses has switched in the last 15 years ; the percentage of biological prostheses implanted has risen from 30 % to 85 % . moreover the total number of implanted stented bioprostheses remained stable over the last years despite competing procedures like stentless prostheses or transcatheter aortic valve implantation.methodsa literature search of all published aortic valve replacement studies was performed from january 2000 through may 2012.resultsthe recommendations guiding the type of heart valve replacement have been revised in recent years . of particular interest are the new generation of biological prostheses with extended durability , a decrease in mortality of reoperation and an increase in life expectancy . comorbidities such as chronic renal insufficiency or chronic atrial fibrillation are no longer contraindications to bioprostheses.conclusionoverall , even in face of more innovative biological alternatives the implantation of stented bioprostheses is still a very interesting option and represents actually the most frequent valve implantation technique for aortic stenosis .

primary mitochondrial disorders due to impaired oxidative phosphorylation ( oxphos ) are a well - established cause of severe disability and precocious death in both children and adults ( koopman et al . , 2012 ) . no effective therapies are currently available for these conditions , but encouraging results have recently been obtained by stimulating mitochondrial biogenesis acting on either the ppar system ( wenz et al . , 2008 ) or the amp - kinase ( ampk)/pgc1 axis ( viscomi et al . , 2011 ) . importantly , these approaches can in principle be extended to several mitochondrial diseases with different genetic causes , as they do not point to the correction of a specific defect but are based on a more general strategy aimed at increasing the overall residual activity of the respiratory chain . additional targets able to activate the mitochondriogenic program and boost mitochondrial function are sirtuins 17 ( houtkooper et al . , 2012 ) , the mammalian orthologs of the yeast silent information regulator ( sir ) 2 gene ( imai et al . , 2000 ; sirtuins have different subcellular localization , sirt1 and sirt6 being mainly found in the nucleus , sirt2 in the cytosol , sirt3sirt5 in mitochondria , and sirt7 in the nucleolus . sirtuins are important regulators of several proteins , including key metabolic players , acting as either deacetylases or adp - ribosylases ( houtkooper et al . , 2012 ) . the most - investigated member of the family is sirtuin1 ( sirt1 ) , a nad - dependent type iii nuclear deacetylase that utilizes nad as a cosubstrate to remove acetyl groups from lysine residues of a target protein . known targets of sirt1 are the tumor suppressor p53 , the myocyte - specific enhancer factor 2 ( mef2 ) , the forkhead box o ( foxo ) , and pgc1 , all of which regulate transcriptional programs related to increased mitochondrial function ( andreux et al . , 2013 ) . sirt1 activity is directly regulated by nad availability , by substrate - dependent activation , raising the hypothesis that nad acts as a metabolic sensor . for instance , both nad levels and sirt1 activity increase in mammalian tissues in response to energy / nutrient stresses such as exercise ( cant and auwerx , 2009 , 2010 ) and fasting ( cant and auwerx , 2010 ; chen et al . recent studies have shown that sirt1 activation can prevent diet - induced obesity in mice . this effect was achieved by increasing the content of nad in cells and tissues essentially through ( 1 ) dietary supplementation of suitable nad precursors , such as nicotinamide riboside , nr ( cant et al . , 2012 ) , or ( 2 ) inhibition of nad - consuming enzymes , such as the poly(adp - ribose ) polymerase 1 , parp1 ( bai et al . , 2011 ) . here , we have tested the therapeutic efficacy of these strategies on a genetic mitochondrial disease model , the sco2 knockout / knockin ( sco2 ) mouse ( yang et al . , 2010 ) . sco2 encodes a metallochaperone involved in the formation of the copper redox centers into nascent complex iv ( cytochrome c oxidase , cox ) ( leary et al . , 2009 ) . mutations in this gene lead to infantile fatal encephalocardiomyopathy ( papadopoulou et al . , 1999 ) . most of the patients carry at least one allele encoding the common mutation p.e140k , corresponding to the p.e129k mutation in the knockin murine sco2 allele . the second knockout allele in this animal model is functionally null ( yang et al . , 2010 ) . while homozygous knockout individuals are embryonic lethal , the sco2 mice show a predominantly myopathic phenotype characterized by exercise intolerance and associated with ubiquitous cox deficiency . to test the effects of persistent increase of the nad pool on the sco2 mouse , we first crossed it with a constitutive parp1 mouse ( de murcia et al . , 1997 ) , which shows increased levels of nad in skeletal muscle ( bai et al . , 2011 ) . the sco2-parp1 double mutants showed reduced fasting blood glucose levels , body weight and epididymal white adipose tissue ( wat ) compared to sco2 littermates , whereas no differences were observed between wt and parp1 littermates ( see figure s1 available online ) . the endurance motor performance of sco2-parp1 double mutants , sco2 , parp1 , and wt littermates ( four animals per group ) was monitored weekly by a standard treadmill test for 4 weeks starting at 2 months of age . while sco2 mice showed markedly reduced motor performance compared to wt littermates throughout the observation time , the double mutant individuals performed as well as the wt and parp1 littermates ( figure 1a ) . biochemically , significant reduction of complex iv activity and , to a lesser extent , complex iii as well ( yang et al . , 2010 ) , was measured in skeletal muscle of sco2 mice . in the muscle homogenate of sco2-parp1 double mutants , these activities were comparable to that of parp1 and wt littermates , whereas complex i and ii activities were even higher ( figure 1b ; table s1 ) . accordingly , the intensity of the histochemical staining specific to cox was increased in skeletal muscle of the double mutants , compared to the sco2 mutants ( figure 1c ) . the activities of complex iii and iv ( figure 1d ; table s2 ) were also increased in the brains of double mutants relative to sco2 animals , but remained significantly lower than those measured in parp1 and wt littermates ; the histochemical reaction to cox was concordant with the biochemical data ( figure 1e ) . to test the effects of pharmacological intervention , we first administered nr to sco2 mice and wt littermates ( n = 4/group ) , as a food admix ( 400 mg / kg ) ( cant et al . , 2012 ) for four weeks . metabolic parameters in the treated groups , including reduced blood glucose , plasma fatty acids , and epididymal wat , mirrored those of parp1 mice ( figure s2 ) , confirming that nr , or its derivative nmn , was pharmacologically active in vivo . nr - treated sco2 mice significantly improved their motor performance compared to the vehicle - treated sco2 group , rapidly achieving the levels of motor endurance displayed by treated and untreated wt mice ( figure 2a ) , which showed no difference to each other . these results suggest that nr treatment increases mitochondrial function in the sco2 mice through nad activation of sirt1 ( cant et al . , 2012 ) . accordingly , the nad / nadh ratio was significantly increased ( figure 2b ) , and the ratio between acetylated and total foxo1 , a direct target of sirt1 , was clearly reduced ( figure 2c ) in skeletal muscle of nr - treated versus vehicle - treated sco2 and wt animals . we did not observe differences in mtdna content ( data not shown ) and citrate synthase ( cs ) activity ( figure 2f ) , but mrna levels of several genes related to either fatty acids oxidation ( fao ) , including acox and cd36 , or oxidative phosphorylation ( coxi , coxii , coxiv , coxva ) were significantly increased in nr - treated versus vehicle - treated sco2 but not in wt animals ( figure 2d ) . tfam , a key factor of mtdna transcription , was also increased , and ucp3 and pdk4 , which were downregulated in sco2 mice , returned to control levels upon nr treatment . as expected , we found no change in transcripts specific to pgc1 , which is activated posttranscriptionally by sirt1 , and to two pgc1 partners , nrf1 and nrf2 . accordingly , western blot immunovisualization demonstrated increased levels of several nuclear- and mitochondrial - dna - encoded oxphos - related proteins in nr - treated versus vehicle - treated skeletal muscle samples ( figure 2e ) . in the same specimens , mitochondrial respiratory chain activities were significantly increased ( figure 2f ; table s3 ) in nr - treated versus vehicle - treated sco2 mice , but not in wt animals . the histochemical staining for cox reflected the biochemical results ( figure 2 g ) . in both c. elegans and mammalian cells , nr - dependent sirt1 activation can induce the mitochondrial unfolded protein response ( mtupr ) , a stress - response protective mechanism which can improve mitochondrial function ( durieux et al . we found that the mtupr - specific transcripts clpp , hsp60 , and sod2 were significantly increased in muscle samples of nr - treated sco2 mice , whereas sod3 , which is unrelated to mtupr , was unchanged ( figure 2h ) . no effect of the nr treatment was observed on the mitochondrial respiratory chain activities in the brain of our animals ( table s4 ) . next , we administered a pan - parp inhibitor ( mrlb-45696 , ic50 for parp < 1 nm ; pirinen et al . , 2014 , in this issue of cell metabolism ) at 50 mg / kg as a food admix for 4 weeks . in mrlb-45696-treated sco2 mice , we observed metabolic effects similar to those of nr treatment ( figure s3 ) . weekly treadmill tests showed progressive increase , up to normal , of the motor endurance in mrlb-45696-treated sco2 , whereas no change was seen in wt mice ( figure 3a ) . the nad / nadh ratio was significantly increased in treated versus untreated wt , but not in the sco2 , animals ( figure 3b ) , whereas the acetylated / total foxo1 ratio was reduced in both treated groups ( figure 3c ) , indicating activation of sirt1 by mrlb-45696 . similar to nr , mrlb-45696 increased the mrna expression levels of oxphos- and fao - related genes in both sco2 and wt mice , whereas mtdna content ( data not shown ) and cs activity ( figure 3f ) remained unchanged . in treated sco2 but not in wt animals , western blot analysis showed increased content of mitochondrial respiratory chain subunits ( figures 3d and 3e ) , which was paralleled by significantly increased mitochondrial respiratory chain activities ( figure 3f ; table s3 ) . histochemistry for cox showed increased staining in treated versus vehicle - treated sco2 mice ( figure 3 g ) . again , we found that expression of the mtupr genes hsp60 , clpp , and sod2 was significantly increased , unlike the mtupr - unrelated gene sod3 ( figures 3h and 3i ) . in contrast to the nr treatment , the mrlb-45696 treatment determined significant increase of cox transcripts ( figure 4a ) , and respiratory chain activities in the brain ( figure 4b ; table s4 ) . the intensity of cox staining was increased as well ( figure 4c ) . similar results were obtained in both skeletal muscle and brain by using pj34 , a commercially available pan - parp inhibitor ( figures s3 and s4 ; table s4 ) . the nad pool is set by the balance between de novo and salvage biosynthetic pathways and utilization by nad - consuming enzymes . nad is synthesized de novo from tryptophan , but the main source of nad is from salvage pathways ( houtkooper et al . , 2010 ) . these require the uptake of other nad precursors from the diet , including nr . upon its entry in the cell , nr is phosphorylated by nr kinases into nicotinamide mononucleoside ( nmn ) , which is then converted to nad by nmn adenylyltransferase ( bieganowski and brenner , 2004 ) . nad biosynthesis and cellular levels are also controlled by a circadian clock related to the feeding / fasting cycle . for example , in the mitochondrial compartment nad levels regulate sirtuin 3 , a deacetylase targeting respiratory chain subunits ( peek et al . , 2013 ) , while in the nucleus nad is a substrate of both parp1 and sirt . parp1 , the highest consumer of nad in mammalian tissues , is activated upon binding to damaged or abnormal dna ( durkacz et al . , 1980 ) and catalyzes the formation of poly(adp - ribose ) polymers ( par ) using nad as a substrate , onto different acceptor proteins , including parp1 itself ( adamietz , 1987 ) . ablation of the parp1 gene , supplementation of nr or administration of parp inhibitors ( parpi ) can expand the nad pool and activate sirtuins , particularly sirt1 , a master regulator of mitochondrial homeostasis . these effects can protect mice from high - fat ( hf)-induced metabolic disease ( cant et al . , we have shown here that these treatments can correct the biochemical and clinical phenotype of the sco2 mouse , a model of genetically determined mitochondrial disease . increased transcription of genes related to both oxphos and mtupr was associated with activation of oxidative metabolism , increase of mitochondrial respiratory chain activities , and normalization of the endurance motor deficit , displayed by naive sco2 animals . notably , these effects were hardly seen in wt littermates , suggesting that mitochondrial dysfunction sensitizes muscle and possibly other tissues to activators of mitochondriogenic programs . increased mitochondrial function can be achieved in wt animals only by much longer - term treatments ( > 6 months ) ( pirinen et al . , 2014 ) . in contrast with previous results , we found no change in mtdna copy number and cs activity in nr- or parpi - treated versus untreated animals , possibly because of the shorter timeframe of our experimental protocol ( 4 weeks ) compared to that of other studies ( 12 weeks ) . this observation suggests time - dependent activation of different mitochondriogenic programs , with induction of oxphos- and fao - related genes occurring much earlier than stimulation of mitochondrial proliferation and increase in mtdna content . likewise , prolonged nr supplementation up to 6 months induced mitochondrial biogenesis in the brain and improvement of cognitive dysfunction of an alzheimer disease mouse model ( gong et al . , 2013 ) . while we observed hardly any effect of nr in our 4 week trial , two pan - parp inhibitors did correct the respiratory chain defect in the brains of our sco2 mice . this observation is particularly relevant , as the brain is an exquisite target of mitochondrial dysfunction , and progressive encephalopathy is the most frequent clinical presentation of mitochondrial disease in infancy and childhood . our work supports the idea that the increase of nad levels in critical tissues is an effective therapeutic option for mitochondrial disease . nr is a natural vitamin with no known adverse effects , which could be administered as a dietary supplement , particularly in case of isolated mitochondrial myopathy . our results are concordant with very recent works reporting beneficial effects of nad precursors in mouse models characterized by reduced nad / nadh ratio , such as aging ( gomes et al . , 2013 ) or complex i deficiency ( karamanlidis et al . , 2013 ) initially shown to boost oxidative metabolism in diet - induced models of obesity , parp1 ablation or inhibition has recently been reported to remarkably rescue pharmacological models of liver cirrhosis , partly by correcting the associated mitochondrial impairment ( mukhopadhyay et al . , 2013 ) . several parp inhibitors are currently under clinical trial as anticancer molecules , and seem to be associated with relatively mild side effects ( bundred et al . , 2013 ; tutt et al . , however , more work is needed to evaluate their use in chronic conditions such as primary mitochondrial disorders in view of their potential genotoxic effects . mouse tissues were homogenized in 15 volumes of 10 mm potassium phosphate buffer ( ph 7.5 ) . mitochondrial - enriched fractions were collected after centrifugation at 800 g for 10 min in the presence of protease inhibitors , and frozen and thawed three times in liquid nitrogen . aliquots , 70 g each , were run through a 12% sds - page and electroblotted onto a nitrocellulose membrane , which was then matched with different antibodies . carlo besta neurological institute , in accordance with guidelines of the italian ministry of health . mice were maintained in a temperature- and humidity - controlled animal - care facility , with a 12 hr light / dark cycle and free access to water and food . a standard treadmill apparatus ( columbus instruments , columbus , oh ) was used to measure motor exercise endurance , as described in viscomi et al . series of 8 m thick sections were stained for cox and sdh , as described ( sciacco and bonilla , 1996 ) . muscle quadriceps samples stored in liquid nitrogen were homogenized in 10 mm phosphate buffer ( ph 7.4 ) , and the spectrophotometric activity of ci , cii , ciii , and civ , as well as cs , was measured as described ( bugiani et al . , 2004 ) . note that in all panels the activity of cii has been multiplied by 10 for visualization clarity . nad was extracted using acidic and alkaline extraction methods , respectively ( yang and sauve , 2006 ) . tissue nad was analyzed with mass spectrometry as previously described ( yang and sauve , 2006 ) . mtdna content and transcripts analysis was carried out by sybr green real - time pcr , as described ( viscomi et al . , 2011 ) .

summarymitochondrial disorders are highly heterogeneous conditions characterized by defects of the mitochondrial respiratory chain . pharmacological activation of mitochondrial biogenesis has been proposed as an effective means to correct the biochemical defects and ameliorate the clinical phenotype in these severely disabling , often fatal , disorders . pathways related to mitochondrial biogenesis are targets of sirtuin1 , a nad+-dependent protein deacetylase . as nad+ boosts the activity of sirtuin1 and other sirtuins , intracellular levels of nad+ play a key role in the homeostatic control of mitochondrial function by the metabolic status of the cell . we show here that supplementation with nicotinamide riboside , a natural nad+ precursor , or reduction of nad+ consumption by inhibiting the poly(adp - ribose ) polymerases , leads to marked improvement of the respiratory chain defect and exercise intolerance of the sco2 knockout / knockin mouse , a mitochondrial disease model characterized by impaired cytochrome c oxidase biogenesis . this strategy is potentially translatable into therapy of mitochondrial disorders in humans .

catheter - based radiofrequency ablation ( rfa ) delivered during endoscopic retrograde cholangiopancreatography ( ercp ) has recently emerged as a possible therapeutic option within the bile duct 1 2 3 4 . intrabiliary extension of neoplasm remains an important challenge in the endoscopic eradication of complex ampullary lesions 5 6 , and rfa may represent a viable treatment adjunct for this problem . recently , the use of rfa at the ampulla and within the distal bile duct has been described 7 8 . herein we present 4 cases assessing the technical feasibility , safety , and treatment outcomes of rfa employed at the time of ercp to treat ampullary lesions with intraductal extension . the study was conducted at the medical university of south carolina ( musc ) from july 1 , 2014 through october 1 , 2015 . after institutional review board approval , we retrospectively identified eligible adult subjects through the musc endoscopy report database ( endoworks , olympus america , center valley , pa ) by searching for reports that contained the keywords radiofrequency ablation ( rfa ) and endoscopic retrograde cholangiopancreatography ( ercp ) . we excluded patients who underwent rfa of a stricture not associated with an ampullary lesion . all procedures were performed by an experienced pancreaticobiliary endoscopist under general anesthesia using a side - viewing duodenoscope . ampullary resection was performed either en bloc or in piecemeal fashion by delivering electrosurgical current through a snare with or without prior submucosal lift . intraductal extension of the lesion was assessed cholangiographically ( fig . 1 ) and/or visually ( fig . 2 ) . in some cases , a biliary sphincterotomy extension and papillary balloon dilation was performed to expose the inside of the terminal bile duct for assessment and therapy . ablative therapy was delivered using a standard argon plasma coagulation ( apc ) probe ( erbe usa inc . , mariette , ga ) at a flow rate of 1.0 l / min to 1.2 l / min and 30 to 40 maximum watts ( w ) and/or the habib endohpb rfa bipolar cautery probe ( emcision united kingdom , london , united kingdom ) at 10 w for 60 to 90 seconds , extrapolating from manufacturer s recommendations of 7 to 10 w 120 seconds 9 . given the proximity to the pancreatic orifice and the benign nature of the target lesions , a shorter duration of treatment was chosen . in general , apc was reserved for treating exposed target tissue in the duodenum or very distal duct , whereas rfa was reserved for treating hidden or difficult to access tissue within the duct . all patients undergoing rfa received a temporary pancreatic stent ( 5 fr , 2 5 cm ) and rectal indomethacin to reduce the risk of post - ercp pancreatitis ( pep ) , as well as a plastic endobiliary prostheses to prevent biliary obstruction and cholangitis . cholangiogram showing a filling defect in the distal bile duct ( arrow ) representing bulky intraductal extension of an ampullary adenoma . endoscopic view of the papilla after ampullectomy demonstrating intraductal extension of the adenoma ( arrow ) . technical success was defined as the ability to successfully position the rfa probe across the biliary orifice and deliver thermal energy to the region of the papilla and terminal bile duct , resulting in coagulation of the visualized target areas . clinical success was defined as endoscopic absence of polypoid or adenomatous - appearing tissue at the treatment site and histologic absence of neoplasm based on extensive follow - up biopsies from the papilla , pancreaticobiliary septum , biliary orifice , and distal bile duct . when the distal bile duct was not fully exposed by prior sphincterotomy , a pediatric biopsy forceps was introduced into the distal duct to acquire tissue . patient demographics , procedure indications , and treatment outcomes are listed in table 1 . four eligible patients were identified , all of whom were men with a mean age of 63 years ( range 54 84 ) . three patients ( 75 % ) had a history of familial adenomatous polyposis ( fap ) . three patients were treated for ampullary adenoma and 1 for ampullary adenoma with a focus of adenocarcinoma ( he declined surgical evaluation ) . fap , familial adenomatous polyposis ; lgd , low - grade dysplasia ; hgd , high - grade dysplasia ; imc , intramucosal cancer ; apc , argon plasma coagulation ; rfa , radiofrequency ablation ; pep , post - endoscopic retrograde cholangiopancreatography pancreatitis ; eus , endoscopic ultrasound video 1 presents a synopsis of 2 representative cases . all rfa procedures were technically successful , resulting in a perceptible tissue effect ( fig . 3 ) . rfa was performed immediately following endoscopic resection in 1 case and during a subsequent session in the remaining cases . the mean number of rfa sessions per patient was 1.5 ( range 1 3 ) . all patients were discharged uneventfully after the procedure without any immediate adverse events ( aes ) . one patient developed obstructive jaundice due to a fibro - inflammatory bile duct stricture at the level of prior rfa that manifested 3 days after biliary stent removal ( approximately 6 weeks after the rfa ) and has required ongoing endobiliary stent therapy in excess of 3 months . 3 patients had visual and histologic evidence of complete eradication ; the patient with a focus of adenocarcinoma who declined surgery developed overt invasive ampullary cancer . video 1 this footage consists of 2 video clips demonstrating catheter - based rfa of intraductal ampullary adenoma although endoscopic ampullectomy is the preferred treatment for noninvasive ampullary lesions with a success rate reported as high as 92 % 10 , biliary extension of neoplasm represents a significant obstacle to endoscopic eradication . exposure and eversion of the adenoma through a biliary sphincterotomy to allow resection or ablation has been described in amenable cases 5 11 12 . however , broad adenomatous involvement of the distal bile is associated with limited treatment success ( < 50 % ) and has been considered an indication for surgical resection 5 . based on its ease of use and the ability to precisely position the probe within the distal duct , radiofrequency ablation may represent the first viable treatment adjunct for this challenging scenario . to date , only single case reports of rfa for benign ampullary lesions have been described ; we aimed to expand our understanding of this technology by presenting our experience in 5 patients . catheter - based rfa was technically successful in all cases , and based on short - term follow up in a small sample , may be safe and clinically effective . however , because rfa induces thermal injury and subsequent necrosis of the bile duct wall and beyond , several safety concerns exist . first , while rfa has been associated with a favorable safety profile when applied to malignant biliary strictures 1 2 3 4 , it remains unclear whether rfa in the intra - pancreatic portion of the bile duct without the protective buffer of a surrounding tumor especially in the vicinity of the pancreatic orifice will be associated with an increased risk of pancreatitis . until additional data on the risk of post - ercp pancreatitis in this context if the pancreatic and biliary orifices are in close proximity , especially if adenoma appears to involve the pancreaticobiliary septum , it may be best to perform the rfa adjacent to a guidewire which has already been placed in the pancreatic duct ( subsequently guaranteeing pancreatic access for stent placement ) rather than adjacent to a plastic pancreatic stent which may be damaged or even fractured during rfa . another safety concern is the development of clinically important post - rfa biliary strictures that occurred in 1 of our patients , akin to what has been observed in the esophagus after rfa of barrett s epithelium 13 . this concern is particularly relevant in the context of benign ampullary disease in which patients do not typically undergo long - term stent placement , as is the case when rfa is performed for palliation of malignant strictures . along these lines , until additional data are available , we have attempted to minimize rfa across the cystic duct takeoff to avoid thermal injury - related obstruction of the cystic duct , which has intentionally been induced by electrohydraulic lithotripsy to treat refractory bile leak 14 . in our series , rfa appears to have provided effective adjunctive therapy in all 4 cases of benign pathology but was ineffective in the setting of early adenocarcinoma , underscoring the concept that surgical resection remains first - line therapy for ampullary cancer ( our patient declined surgery and chemoradiation ) . despite the apparent effectiveness for benign lesions , it is important to consider that intrabiliary extension is often nodular in nature , leading to heterogeneous contact between the rfa probe and the target tissue ; this may lead to incomplete therapy and/or an increased risk of buried neoplasm as is the concern when rfa is used to treat nodular barrett s esophagus . moreover , it can be technically challenging to ensure circumferential contact of the probe and the target tissue within a dilated bile duct , even when luminal air is suctioned to induce collapse of the duct around the probe . in these cases , a balloon - based rfa device that flattens nodular tissue and maximizes treatment contact may be of value . an additional consideration is that the proximal extent of neoplasm is often difficult to assess cholangiographically and the role of cholangioscopy to guide probe placement should be further explored . prospective studies are necessary to evaluate these issues and determine the long - term effectiveness of this modality . in summary , catheter - based rfa after endoscopic resection of ampullary lesions that extend up the bile duct is technically feasible . concerns regarding injury to the pancreas and bile duct as well as incomplete treatment of nodular target tissue exist and will be addressed by additional clinical experience and research .

background and study aims : catheter - based radiofrequency ablation ( rfa ) delivered during endoscopic retrograde cholangiopancreatography ( ercp ) may represent a viable treatment option for intraductal extension of ampullary neoplasms , however , clinical experience with this modality is limited . after ampullary resection , 4 patients with intraductal extension underwent adjunctive rfa of the distal bile duct . all patients received a temporary pancreatic stent to reduce the risk of pancreatitis , as well as a plastic biliary stent to prevent biliary obstruction . three patients were treated for adenoma and 1 for adenoma with a focus of adenocarcinoma . during a short follow - up period , 3 patients experienced complete eradication of the target lesion , whereas the patient with a focus of adenocarcinoma had progression to overt invasive cancer . there were no immediate adverse events . one patient developed a post - rfa bile duct stricture , which has required additional endoscopic therapy . catheter - based rfa of ampullary lesions that extend up the bile duct is technically feasible . additional research is necessary to understand the risks and long - term benefits of this technique .

techniques and equipment to accomplish endotracheal ( et ) intubation were the precursor to modern day invasive mechanical ventilation . in recent years , however , the popularity of the et tube has waned . clinically , the et tube is seen as an impediment to spontaneous breathing , a transit route for bacteria to the lower airway , and with the advent of noninvasive ventilation a device to be avoided when possible . of particular interest has been the effect of the et tube on work of breathing and methods to eliminate this work . commonly , pressure support ventilation ( psv ) has been suggested as the technique of choice for eliminating imposed work due to the et tube . more recently , the technique of automatic tube compensation ( atc ) has become available to specifically address this issue . in this issue of critical care , maeda and colleagues compare the technique of atc , as provided by the drager evita 4 ( dragerwerks , lubeck , germany ) and the puritan bennett 840 ( carlsbad , ca , usa ) , versus psv in reducing imposed work of breathing in a lung model . before i comment on the merits of the study , clearly , before the advent of pressure support ventilation in the early 1980s , patients were successfully weaned using t - piece trials and intermittent mandatory ventilation , with no apparent untoward effects . in 1986 , shapiro and coworkers presented data from three normal volunteers breathing through et tubes at a constant tidal volume of 500 ml . that report is widely quoted but is limited by the use of unintubated normal individuals and the requirement for a constant tidal volume during increasing respiratory rates . additionally , close review of the data demonstrates that with a size 8.0 et tube the work of breathing in joules per minute does not become excessive until minute ventilation exceeds 15 l / min . brochard and colleagues compared work of breathing during assisted ventilation , four levels of pressure support , continuous positive airway pressure , and after extubation in an effort to determine the role of pressure support in overcoming the imposed work presented by the et tube . that trial evaluated both patients with normal lungs and those with chronic obstructive pulmonary disease ( copd ) . the authors concluded that the pressure support level that eliminated imposed work was between 3 and 14 cmh2o . interestingly , this was determined retrospectively by matching the work of breathing after extubation to the level of pressure support that resulted in equivalent work of breathing during mechanical ventilation . recent work suggests that the work of breathing postextubation may actually increase compared with work of breathing through the et tube , raising questions about the conclusion of the study by brochard and colleagues . weissman evaluated flow - volume loops in 18 postoperative patients intubated with size 7.0 and 8.0 et tubes and found that only ' minimal limitation to airflow ' occurred at volumes and frequencies associated with tidal breathing . that study is one of the very few that contemplate the idea that et tube resistance may not be important when the appropriate size is chosen and the patients pulmonary function has improved to the point that weaning may be considered . of course , there are numerous other studies that suggest that et tube resistance may represent a significant impediment to spontaneous breathing . however , it is important to note that although there remains concern about et tube resistance , and the literature is replete with lung model studies of increased work of breathing , there is not a single clinical trial that suggests that spontaneous breathing through the et tube results in untoward outcomes . the report by maeda and coworkers in this issue of critical care evaluates the two most popular methods of overcoming et tube resistance , namely psv and atc . the authors concluded that tube compensation could not overcome the pressure time product associated with triggering and that pressure support is as effective as atc at 100% . there are , however , tremendous disadvantages to a lung model study in comparing these techniques . calculating the pressure time product and work includes the work required to trigger the ventilator , which can only be overcome by direct measurement of tracheal pressure and triggering from the tracheal signal . this method has been proposed by many , and advanced by the group from gainesville . additionally , although the lung model allows consistency , it can not react to differences in gas delivery . in several human studies comparing psv with atc , the slow flow and longer inspiratory time associated with psv has been associated with dysynchrony . when used in a patient with copd , the patient 's high airway resistance and increased compliance allow even small amounts of psv to cause hyperinflation and auto - peep ( positive end - expiratory pressure ) . alterations in pulmonary mechanics , along with the preference of the copd patient for short inspiratory times and long expiratory times , result in neuromechanical dysynchrony during psv . the main proposed advantages of atc over psv are reduced work of breathing as patient demand varies , preservation of a normal , variable breathing pattern , and improved synchrony . in a recent trial of spontaneous breathing before extubation , haberthur and coworkers failed to show any advantage of atc over psv or t - tube trials . one issue not addressed in the study by maeda and coworkers is the role of expiratory compensation . one distinct difference in the operation of the drager evita 4 and puritan bennett 840 is that evita 4 provides both inspiratory and expiratory compensation for et tube resistance . in these cases , the airway pressure may be allowed to drop below peep to facilitate expiratory flow . new modes and new techniques are developed in the hope of resolving clinical problems and often concentrate on a short - term physiologic end - point . conventional wisdom suggests that the et tube is an impediment to efficient spontaneous breathing , yet clinical evidence during spontaneous breathing trials appears to argue to the contrary . the real question may be whether overcoming et tube resistance is necessary , not whether atc is as good as or better than psv . the future of atc , like many new techniques , may not be in overcoming et tube resistance , but as a method of support during spontaneous breathing that improves patient - ventilator synchrony as compared with psv . additional clinical studies are required to complement the excellent laboratory work by the group from osaka reported in this issue . atc = automatic tube compensation ; copd = chronic obstructive pulmonary disease ; et = endotracheal ; peep = positive end - expiratory pressure ; psv = pressure support ventilation .

concerns about the work of breathing imposed by the endotracheal tube have led clinicians to routinely use pressure support to overcome this resistive component . more recently , ventilator manufacturers have introduced systems to automatically overcome endotracheal tube resistance , regardless of tube diameter or patient demand for flow . despite the theoretical advantages , neither method appears to provide superior performance . stepping back , the real question may be , is overcoming endotracheal tube resistance really important ?

in this review , pubmed and sciencedirect databases were used to identify relevant articles published until the 3rd of april 2013 ( date of search ) . the search was conducted using the electronic library of king saud bin abdul aziz university for health sciences , riyadh , saudi arabia . pubmed search identified 3 groups of articles as follows : disease of interest ( keyword : autism ) , geographical location ( keywords : saudi , uae , oman , kuwait , qatar , and bahrain ) , and epidemiological terms ( keywords : epidemiology , prognosis , diagnosis , pattern , odds , risk , incidence , prevalence , impact , trends , and biomarker ) . finally , the boolean operator and combined the results for each group of articles , a total of 22 articles met the inclusion criteria . epidemiology of autism and each of the gcc countries individually , which yielded a total of 79 results . the titles of the final list consisting of 101 results were reviewed , 85 articles were excluded because they were not related to epidemiology of autism , and the abstracts of the remaining 16 articles were obtained . after reviewing the 16 abstracts , 6 more articles were eliminated : reviews ( n=3 ) , 2 were qualitative studies and abstract / full text of the sixth was irretrievable . a search was conducted for the full text of the remaining 10 articles , of which 8 were located and 2 were not . the first was an article published as a brief communication,16 and the second is an article published during the writing up of this review.17 literature search flowchart . studies were identified from 3 gcc countries only ; oman , saudi arabia , and uae . the 10 included articles , 3 were prevalence studies , and the rest discussed potential risk factors or biomarkers for autism in patients from gcc countries . prevalence studies were conducted in uae,18 saudi arabia,19 oman,20 and bahrain.17 the prevalence of asd was 1.4 per 10,000 in oman , and 29 per 10,000 for pdd in uae , and 4.3 per 10,000 in bahrain.17,18,20 the saudi study documented patients characteristics and reasons for referral for group of saudi autistic patients.19 male gender and history of developmental delay were significantly associated with autism prevalence in all 3 studies . consanguinity was present in 28.6% of saudi patients , and behavioral problems such as hyperactivity or aggression were evident in 45% of patients ( table 1).19 summary of epidemiological studies on autism / autism spectrum disorder in the gulf cooperation countries . the included studies were conducted in saudi arabia , oman , or bahrain , and all employed the case - control study design . sample size varied between these studies from 52 to 204 patients ; and the risk factors investigated were : suboptimal breastfeeding,21 lead exposure,22 serum osteopontin,23 maternal and paternal age , cesarian section , and prenatal complications . delayed initiation of breastfeeding and no colostrum intake was associated with increased risk of asd . exclusive and prolonged breastfeeding ( up to 24 months or more ) markedly reduced the risk of developing asd.21 higher blood levels of lead22 and osteopontin23 were found in autism patients . occurrence of autistic disorder was correlated with maternal ( or 1.83 ) and paternal ( or 2.08 ) age above 30 years at time of birth . the disease was also more common among children of mothers who delivered with a cesarian section or had antenatal complications16,17 ( table 1 ) . lipid peroxidation,24 glutathione peroxidase,25 superoxide dismutase , sodium - potassium adenosine triphosphatase,21 lactate , saturated fatty acids,26 and homocysteine26 levels were significantly higher among autistic patients . inversely , levels of vitamin e,24 glutathione,24 folate , vitamin b12,27 and some polyunsaturated acids were much lower in autistic patients . serum osteopontin levels were higher among autistic patients,25 and levels were positively related to the severity of autism ( table 1 ) . prevalence studies were conducted in uae,18 saudi arabia,19 oman,20 and bahrain.17 the prevalence of asd was 1.4 per 10,000 in oman , and 29 per 10,000 for pdd in uae , and 4.3 per 10,000 in bahrain.17,18,20 the saudi study documented patients characteristics and reasons for referral for group of saudi autistic patients.19 male gender and history of developmental delay were significantly associated with autism prevalence in all 3 studies . consanguinity was present in 28.6% of saudi patients , and behavioral problems such as hyperactivity or aggression were evident in 45% of patients ( table 1).19 summary of epidemiological studies on autism / autism spectrum disorder in the gulf cooperation countries . the included studies were conducted in saudi arabia , oman , or bahrain , and all employed the case - control study design . sample size varied between these studies from 52 to 204 patients ; and the risk factors investigated were : suboptimal breastfeeding,21 lead exposure,22 serum osteopontin,23 maternal and paternal age , cesarian section , and prenatal complications . delayed initiation of breastfeeding and no colostrum intake was associated with increased risk of asd . exclusive and prolonged breastfeeding ( up to 24 months or more ) markedly reduced the risk of developing asd.21 higher blood levels of lead22 and osteopontin23 were found in autism patients . occurrence of autistic disorder was correlated with maternal ( or 1.83 ) and paternal ( or 2.08 ) age above 30 years at time of birth . the disease was also more common among children of mothers who delivered with a cesarian section or had antenatal complications16,17 ( table 1 ) . lipid peroxidation,24 glutathione peroxidase,25 superoxide dismutase , sodium - potassium adenosine triphosphatase,21 lactate , saturated fatty acids,26 and homocysteine26 levels were significantly higher among autistic patients . inversely , levels of vitamin e,24 glutathione,24 folate , vitamin b12,27 and some polyunsaturated acids were much lower in autistic patients . serum osteopontin levels were higher among autistic patients,25 and levels were positively related to the severity of autism ( table 1 ) . the prevalence of asd and pdd was highly variable between included studies.18,20 the difference between both rates could be attributed , in part , to setting , method of assessment , and age of participants . the omani study20 included a larger sample with wide age range to determine prevalence of formally diagnosed asd from medical records . conversely , the emirati study18 screened a sample of preschool children for undiagnosed pdd , and the case definition for pdd is more inclusive than asd . for example , studies in europe recorded a prevalence that ranged from 1.9 up to 72.6 per 10,000 , and the range has been wider ( 2.8 - 94 per 10,000 ) in china.1 prevalence is also affected by accessibility to a specialized autism care center and source of case identification ( mainly families ) . a low prevalence rate is expected in areas with no facilities or centers for case reporting . over time , healthcare professionals and families have learned more about the disorder , and the increased awareness resulted in more comprehensive diagnostic criteria . this emphasizes the need to establish a reliable autism - screening tool that can be applied in community - based studies to produce more precise prevalence in gcc countries . collaborative efforts should target increasing awareness of the community toward asd to encourage early consultation and diagnosis . also , availability and accessibility to diagnosis and treatment centers should be assessed in light of the burden of this disorder . the male to female ratio in gcc countries was consistent with studies from elsewhere showing male predilection,1 and there is no evidence to date that explains this finding.12 one possible reason is that female children are more able to mask their behavioral difficulties than males.19 moreover , culture in developing countries may be a contributing factor , as some families may pay more attention to the development of male children compared with females . as the burden of reporting cases falls on parents ; there could be a lack of detection or lack of willingness to report certain behavior exhibited by a female child . this cultural perception could explain the older age for females at autism diagnosis in saudi arabia.19 either way ; more research is needed to explain other reasons behind this significant gender difference . still , with consanguinity rates in saudi arabia that range from 34 - 80% , depending on rural , or urban setting , this risk factor deserves further investigation.19,28 lead toxicity has been associated with autism,22 which is in agreement with other research.29 however , the nature of environmental exposure that might have contributed to lead toxicity was not clarified in the study . a strong dose - response protective effect of breastfeeding21 is in agreement with other studies,30,31 but potential for recall bias remains high as data collection depends on parents memory and reporting . findings for various biomarkers were comparable with studies carried out elsewhere.29,32 - 35 still , more research is needed to clarify applicability of these biomarkers for early screening or monitoring of autistic patients . first , is the sample size , as numbers of cases and controls used were small and can not be considered representative . also , the recruitment of cases and controls was from specialized clinics / tertiary care institutions , which might be problematic when extrapolating results to community autistic children in gcc countries . second , is that cases were recruited from pediatric wards , and none come from psychiatric clinics . the diagnosis of autism must include more comprehensive behavioral / psychological considerations to form a full picture about the disorder . the current study s limitations were the small number of studies identified and absence of studies from 3 gcc countries , which precluded comparing prevalence of autism across countries . generally , there is an evident lack of research into risk factors affecting the etiology of autism in gcc countries . consanguinity , multiparity , and closely - spaced pregnancies are common in the gcc region , and provide an exceptional opportunity to learn more about genetic determinants of the disease . also , dietary habits should be looked into as they could help in i ) investigating the risk and prognostic factors of the disease and , ii ) assisting families by identifying high - risk foods that could affect their children . more focus should be given to children in psychiatric wards to identify autistic symptoms among them . in conclusion , population - based studies should focus on quantifying the burden of asd in gcc countries . knowing the burden and extent of disease could help design screening tools that are applicable , culturally acceptable , and cost - effective to identify individuals who can benefit the most from early diagnosis and intervention . furthermore , raising asd awareness among parents , preschool / elementary school teachers are invaluable in helping autistic children cope with different challenges . generally , there is an evident lack of research into risk factors affecting the etiology of autism in gcc countries . consanguinity , multiparity , and closely - spaced pregnancies are common in the gcc region , and provide an exceptional opportunity to learn more about genetic determinants of the disease . also , dietary habits should be looked into as they could help in i ) investigating the risk and prognostic factors of the disease and , ii ) assisting families by identifying high - risk foods that could affect their children . more focus should be given to children in psychiatric wards to identify autistic symptoms among them . in conclusion , population - based studies should focus on quantifying the burden of asd in gcc countries . knowing the burden and extent of disease could help design screening tools that are applicable , culturally acceptable , and cost - effective to identify individuals who can benefit the most from early diagnosis and intervention . furthermore , raising asd awareness among parents , preschool / elementary school teachers are invaluable in helping autistic children cope with different challenges .

objective : to assess the current state of knowledge on the epidemiology of autism in arab gulf countries , and identify gaps for future research.methods:pubmed and sciencedirect databases were used to identify relevant articles published until the 3rd of april 2013 ( date of search ) . the search was conducted using the electronic library of king saud bin abdulaziz university for health sciences , riyadh , saudi arabia . studies were eligible for inclusion if they concerned the epidemiology of autism , conducted in any arab gulf country , and published in english.results:twelve articles met the inclusion criteria . studies showed a prevalence ranging from 1.4 to 29 per 10,000 persons . identified risk factors were metabolic , autoimmune , and environmental in nature . the following determinants were found as possible contributing factors for autism : suboptimal breast - feeding , advanced maternal and paternal age , cesarean section , and prenatal complications.conclusion:only a few studies explored the epidemiology of autism in arab gulf countries and none have investigated the burden of the disease on the child , family , or society . more research is needed to better identify the burden and risk factors of autism in gulf countries .

cancer is the second leading cause of death worldwide , accounting for about 600,000 deaths in the united states in 2012 . despite significant improvements in treatment , early detection remains the most important prognostic factor predicting of better outcome [ 24 ] . current cancer screening methods , including mammography for breast cancer , colonoscopy for colon cancer , computed tomography for lung cancer , prostate - specific antigen for prostate cancer , and papanicolaou stains for cervical cancer , have demonstrated some limitations in terms of sensitivity , specificity , complexity , cost , and compliance . serum tumor - associated antigens ( taas ) have been extensively studied for early cancer detection because of the simplicity and reliability of the tests used for their determination , such as western blot and enzyme - linked immunosorbent assay ( elisa ) . unfortunately , they are transiently secreted and rapidly eliminated from blood circulation [ 6 , 7 ] and usually reach a detectable concentration only in advanced stage of the disease . along with taas , autoantibodies are frequently detected in sera from patients affected by different types of neoplasms . this finding has been interpreted as an attempt of the immune system to block invasion and spreading of cancer cells in the organism . circulating autoantibodies have biological and biochemical characteristics that render them particularly suitable to screen subjects at cancer risk . in fact , they may develop early in the process of tumorigenesis , when premalignant or malignant cells begin to express altered molecules as a result of cell transformation [ 10 , 11 ] . in addition , they can easily be detected in the serum because of the usual high concentration and long - time stability . for these reasons , great efforts have been made in recent years to identify circulating autoantibodies directed against cancer - related proteins in order to build up tests for the early detection of neoplastic disease [ 1215 ] . in this study , we investigated the production of autoantibodies against lectin galactoside - binding soluble 3 binding protein ( lgals3bp ) in patients affected by different types of cancer . lgals3bp , also known as 90k or mac-2 bp , has been largely regarded as a taa , since it is present at elevated concentrations in the blood of cancer patients and is overexpressed in the vast majority of cancer tissues . both high serum and tumor levels of lgals3bp have been associated with a poor outcome in patients with different types of neoplasms [ 1821 ] . in fact , it can bind important molecules associated with the membrane of tumor cells , such as galectin-3 , galectin-1 , and 1-integrins [ 16 , 22 , 23 ] . additionally , lgals3bp can interact with extracellular matrix proteins such as collagen , fibronectin , and laminin [ 23 , 24 ] . here we show that patients with different types of cancer , but not healthy subjects , develop autoantibodies against lgals3bp . this finding discloses the capability of lgals3bp to trigger a humoral immune response in cancer patients and provides the basis for further investigation on a possible use of anti - lgals3bp antibodies as biomarkers for early diagnosis of cancer . the study population consisted of 71 patients with different types of cancers , 15 gastrointestinal cancers , 13 non - small - cell lung cancers ( nsclc ) , 12 breast cancers , 10 neuroendocrine tumors ( net ) , 10 urogenital tract cancers , 7 melanomas , and 4 others ( 2 gliomas , 1 tongue cancer , and 1 osteosarcoma ) . serum was collected from total blood and stored at 20c after adding 0.01% sodium azide . human recombinant lgals3bp was immunoaffinity - purified from serum - free supernatant of human embryonic kidney ebna-293 cells ( invitrogen , carlsbad , ca , usa ) transfected with lgals3bp cdna . ninety - six well microtiter plates ( nalge nunc , denmark ) were coated with recombinant purified lgals3bp protein ( 5 g / ml in pbs ) at 4c overnight . the plate was saturated with 1% bsa and 0.05% tween-20 in pbs ( saturation buffer ) at 37c for 2 h and then incubated with 100 l of serum from healthy donors or patients at 37c for 1 h. the serum was diluted 1 : 100 in saturation buffer . after 3 washes with 0.05% tween-20 in pbs ( washing buffer ) , a second incubation was performed with 100 l of biotin - conjugated anti - human igg ( sigma , st louis , mo , usa ) , diluted 1 : 2000 in saturation buffer , at 37c for 1 h. after washing , a third incubation was performed with peroxidase - conjugated extravidin ( sigma , st louis , mo , usa ) diluted 1 : 4000 in saturation buffer , at 37c for 45 min . after washing , 100 l of tmb substrate was added to each well and the plate was shaken at room temperature for 15 min . eventually , the reaction was stopped by adding 100 l of 1 m h2so4/well and color revealed by reading absorbance at 450 nm in an automatic elisa reader . to measure serum concentration of lgals3bp a commercially available elisa kit ( diesse , siena , italy ) purified human recombinant lgals3bp ( 10 g / well ) was separated by 8% sds - page under reducing conditions and transferred to nitrocellulose using standard procedures . membrane was saturated in blocking buffer phosphate buffered saline with 0.05% tween-20 ( pbs - t ) , 5% low - fat milk , 1% bsa and at 4c overnight . after washing in pbs - t , membrane was incubated with serum from controls or patients affected by cancer , diluted in an equal volume with pbs - t , at room temperature for 1 h. after washing in pbs - t , membrane was incubated with biotinylated anti - human igg ( sigma , st louis , mo , usa ) diluted 1 : 1000 in blocking buffer at room temperature for 1 h and then with extravidin peroxidase ( sigma , st louis , mo , usa ) 1 : 500 in blocking buffer for 30 min . to identify the presence of antibodies bound to lgals3bp , the colorimetric substrate dab ( 3,3-diaminobenzidine ) each sample was assayed in triplicate and the mean value was used for statistical analyses . the normal upper cut - off value of anti - lgals3bp antibody in sera was set at the value of the mean + 2sd of the absorbance in 54 healthy donors . differences in the proportion over the cut - off limit were evaluated by fisher 's exact test . patients affected by different types of cancers showed significant increased levels of lgals3bp autoantibodies ( p < 0.001 ) compared to healthy subjects ( figure 1(a ) ) . the specific binding of antibodies to lgals3bp was confirmed in western blot , where the presence of anti - lgals3bp igg was detected as two bands at about 97 kda and 66 kda , the exact size of the protein in its full length and cleaved form , respectively ( figure 1(b ) ) . among patients , nsclc , gastrointestinal cancer , urogenital tract cancer , and net reached the highest levels of autoantibodies , while there was no significant increase in breast cancer and melanoma ( table 2 ) . setting the normal upper cut - off limit of elisa at od 0.99 ( the mean + 2sd of the absorbance in sera from healthy individuals ) , the assay showed a sensitivity of 33% ( 26/71 patients were positive ) and a specificity of 98% ( only 1 out of 54 controls was positive ) . all cancer groups , but melanoma , showed autoantibody levels significantly above the cut - off limit ( table 2 ) . as expected , the protein was significantly higher in patients with cancer compared to normal subjects ( 13.19 versus 6.36 g / ml , p < 0.001 ) ( table 3 ) , but values did not correlate with the levels of autoantibodies ( data not shown ) . using elisa technique , we show that lgals3bp is able to elicit host immune response with igg autoantibodies production in patients affected by different types of cancer . anti - lgals3bp igg concentrations were higher in patients with nsclc , gastrointestinal cancer , urogenital tract cancer , and net than in those with breast cancer and melanoma , but the number of patients in each subgroup was insufficient to yield a statistically reliable comparison . it is generally accepted that tumor proteins perceived as nonself by the immune system and able to trigger an immune response are often overexpressed , mutated , misfolded , or endowed with posttranslational changes , such as alterations of glycosylation and phosporylation [ 28 , 29 ] . consistently , lgals3bp may evoke autoantibody production because it is overexpressed in cancer cells , and also because it may carry posttranslational alterations in its glycidic moiety . qualitative and quantitative changes in o- and n - glycosylation of proteins are frequent events in malignancies [ 30 , 31 ] and differences in the glycosylation pattern of lgals3bp have been reported in some cancer cell lines . finalistically , antitumor directed antibodies are generated in order to halt tumor initiation and progression . as this process initiates early in cancerogenesis , in a preclinical phase of the disease , autoantibodies production has been considered a useful biomarker for early cancer diagnosis [ 3235 ] . in this study , the serum levels of anti - lgals3bp igg detected in cancer patients were not correlated with those of lgals3bp , indicating that even small amount of the protein , as expected in the initial phase of cancer growth , may generate high concentrations of autoantibodies . this evidence suggests a possible role for anti - lgals3bp igg in the early detection of cancer . although it is not possible to exclude that the presence of autoantibodies might affect the correct quantification of lgals3bp by elisa , the identification of anti - lgals3bp igg in western blot indicates that the epitopes recognized by these autoantibodies are different from those recognized by the antibody used in elisa . in fact , western blot performed under reducing conditions can detect only autoantibodies directed to epitopes expressed on the primary structure of lgals3bp , while the monoclonal antibody contained in the commercially available elisa kit , known as sp2 , recognizes a conformational epitope shaped in the native form of the protein and , for this reason , is not suitable for western blotting . in cancer patients , autoantibodies are frequently directed against cellular proteins that play key roles in tumor progression , including molecules involved in cell cycle , signal transduction , proliferation , and apoptosis [ 3638 ] . as a consequence , the identification of the molecular target of autoantibodies might be of relevance in designing new antitumor agents . we can , therefore , speculate that lgals3bp could be a candidate for targeted therapies against cancer . in the past few years , the growing interest in autoantibodies as a possible tool for the early diagnosis of cancer and the identification of new targets for molecular therapy has made the development of high - throughput techniques such as serex ( serological analysis of tumor antigens by recombinant cdna expression cloning ) , phage display , protein microarray , serpa ( serological proteome analysis ) , and mapping ( multiple affinity protein profiling ) able to detect simultaneously multiple autoantibodies and their cognate taas . with these methods , several new targets have been identified , but collectively single antigens have shown low sensitivity and specificity to be used in clinical screening . to increase sensitivity , autoantibody diagnostic tests combining two or more taas [ 4143 ] or evaluating well - known biomarkers in combination with autoantibodies have been developed . for example , a large screening study of high - risk individuals for lung cancer has validated a test measuring autoantibody levels against a panel of six taas ( p53 , ny - eso-1 , cage , gbu4 - 5 , annexin 1 , and sox2 ) . in another study , the combination of p53 autoantibodies and ca125 levels increased sensitivity for ovarian cancer from 73.8% ( ca125 ) to 85.7% ( ca 125 plus p53 autoantibodies ) . the determination of anti - lgals3bp igg presented in this study showed a very high specificity ( 98% ) , but a low sensitivity ( 33% ) , comparable to that reported for autoantibodies against single taa , ranging between 10% and 30% . therefore , our anti - lgals3bp elisa lacks sufficient sensitivity to be used in early cancer diagnosis . nevertheless , the determination of autoantibodies against lgals3bp might be useful to increase the sensitivity of tests combining multiple autoantibodies . preliminary results indicate that using a set of different autoantibodies combined with autoantibodies for lgals3bp will increase the sensitivity for breast cancer patients to 50% and maintain the high specificity ( 98% ) . these preliminary results should be tested for other types of cancers as well . in summary , our study demonstrated the presence of autoantibodies against lgals3bp in the serum of patients with different types of cancers . these autoantibodies may be used in developing screening tests for early - stage cancer detection .

purpose . circulating autoantibodies have been extensively investigated as possible markers for early diagnosis of cancer . the present study was carried out to investigate whether anti - lgals3bp igg autoantibodies could be classified as a biomarker for malignant tumors . methods . an in - house developed enzyme - linked immunosorbent assay was used to detect autoantibodies to lgals3bp in sera from 71 patients with various types of cancers and 54 healthy subjects matched by age and gender . results . patients with cancer have significant higher circulating levels of anti - lgals3bp antibodies as compared to control subjects ( p < 0.001 ) . the test has a sensitivity of 33% and a specificity of 98% . conclusions . anti - lgals3bp igg autoantibodies are a promising biomarker for malignant tumors and could play a role in the development of a multimarker assay for the early detection of cancer .

cell metabolism governing the growth and functioning of each cell and a whole organism refers to chemical transformations and enzyme - catalyzed energy producing and energy utilizing reactions of carbohydrates , proteins , and lipids . amongst the most metabolically active organs are liver , brain , gut , kidneys , and heart [ 13 ] . although the rate of metabolic reactions is lower in skeletal muscles , they account for around 20% of the total energy expenditure due to a 5060% contribution to a total body mass . several micrornas were reported to control processes related to metabolism such as insulin secretion ( mir-9 , mir-375 ) , adipocyte differentiation ( mir-143 ) , fatty acid metabolism ( mir-122 ) , and myogenesis ( mir-1 , mir-133a , mir-133b , and mir-206 ) ( reviewed in ) . of potential meaning is also mir-378a , located in the gene encoding master metabolic regulator , peroxisome proliferator - activated receptor gamma , coactivator 1 beta ( pgc-1 ) . mir-378a was found to affect lipid and xenobiotic metabolism , lipid storage , mitochondrial function , and shift towards a glycolytic pathway ( warburg effect ) [ 5 , 6 ] . because nutrients supply for metabolic processes is a matter of circulation , metabolically active tissues require high vascular density . recently , mir-378a was reported to regulate tumor angiogenesis mainly via inhibition of tumor suppressors sufu and fus-1 [ 8 , 9 ] . thus , a growing body of evidence suggests a role of mir-378a as a mediator controlling reciprocally dependent processes such as metabolism , muscle differentiation / regeneration , and angiogenesis . micrornas ( mirnas ; mirs ) are small noncoding rna molecules with an average length of 21 - 22 nucleotides which can regulate gene expression posttranscriptionally by targeting mostly the 3untranslated region ( 3utr ) of mrnas . however , mirna target sites were also found on the 5utr regions of human mrna . since their discovery in c. elegans in 1993 , mirnas currently can be recognized as potent players in wide spectrum of biological processes like development , differentiation , cellular defense mechanisms , and others . conservative estimates state that over 30% of mrna expression is regulated by mirnas [ 12 , 13 ] . however , others suggest that even up to 60% of the mrna expression is targeted by mirnas . mirnas are often located in the introns of coding genes or noncoding sequences but can also be located in exons . intronic mirnas can be expressed together with their host gene mrna being derived from a common rna transcript [ 15 , 16 ] . other mirnas can also have their own promoters , which enable independent expression , or can be organized in clusters sharing a common transcriptional regulation [ 17 , 18 ] . mirnas transcription is rna polymerase ii - dependent . in the case of mirnas that are encoded in their own genes , the primary mirna transcript ( pri - mirna ) is several kilobases long , while mirnas encoded in intronic regions of other genes ( mirtrons ) have shorter transcripts . the mirna stem loop is excised from pri - mirna by endoribonuclease drosha / dgcr8 ( microprocessor complex ) and a hairpin called pre - mirna is exported from the nucleus by exportin-5 in a ran - gtp dependent manner . an endoribonuclease dicer removes the hairpin loop sequence from pre - mirna , creating a double stranded mirna duplex . depending on the relative stability of the mirna duplex , one or , more rarely , both strands can be incorporated in a multiprotein rna - induced silencing complex ( risc ) . when there is perfect pairing between the mirna sequence and its target site , mrna is cleaved by a protein part of the risc called argonaute ( ago ) . if the pairing is partial , deadenylation of the mrna via recruitment of the ccr4-not complex by the gw182 proteins inside the risc takes place and the poly - a tail is lost , leaving the mrna vulnerable to rnase activity , ubiquitination , and mrna degradation . alternatively , mirna - induced risc can also cause repression of translation by mechanisms such as , for example , the promotion of ribosome drop - off from the mrna transcript or destabilization of the mrna binding cap protein ( figure 1 ) ( reviewed in [ 20 , 21 ] ) . the pre - mir gives rise to a leading strand ( mir-378a-3p , previous ids for murine sequence : mmu - mir-422b , mmu - mir-378 , and mmu - mir-378 - 3p ; for human : hsa - mir-422b and hsa - mir-378 ) and a passenger strand ( mir-378a-5p , previous ids for murine sequence : mmu - mir-378 , mmu - mir-378 , and mmu - mir-378 - 5p ; for human : hsa - mir-378 and hsa - mir-378 ) . mirna-378a-3p mature strand was first identified in 2004 in humans ( originally named mir-422b ) . recently , other mirs with similar sequences but other localizations in the genome have been discovered and named : mmu - mir-378b , c , d in mouse and hsa - mir-378-b , c , d1,d2,e , f , g , h , i , j in human [ 2327 ] ( table 1 ) . in humans , mir-378a is by far the most expressed of the mir-378 sequences , with 7030 reads per million , in 78 experiments during deep sequencing , compared with 1013220 reads per million , in 4272 experiments for the other forms , respectively . in mice , mir-378a and mir-378b have similar expression levels , at 11700 and 11000 reads per million ( mirbase , version 21 , september 2015 ) . the sequence of mir-378a mature strands is highly conserved between species , with the mir-378a-5p strand being identical in both human and mice and the mir-378a-3p strand only differing in one nucleotide ( table 2 ) [ 6 , 27 ] . pgc-1 may regulate several facets of energy metabolism such as mitochondrial biogenesis , thermogenesis , and glucose and fatty acid metabolism . both strands of mir-378a are coexpressed with pgc-1 as shown , for example , in the liver and during adipocyte differentiation [ 6 , 29 ] . the coexpression of mir-378a with its host gene implies they may share the same transcriptional activators , and mir-378a might be involved in similar processes as pgc-1. accordingly , high levels of ( porcine ) mir-378 - 1 ( table 2 ) expression are found in developing muscle , postnatal muscle , and myocardium and in brown adipose tissue [ 29 , 30 ] . to date , only a limited number of mir-378a targets , which can be predicted based on in silico analysis , have been experimentally validated . the latter , however , imply a role of mir-378a in mitochondrial energy homeostasis , glycolysis , and skeletal muscle development and in tumor angiogenesis and other processes ( table 3 ) . a major source of energy production comprises oxidation of glucose in glycolysis followed by oxidation of pyruvate in well - oxygenated cells ( or followed by lactic acid fermentation in cancer , the warburg effect ) and from -oxidation of lipids , which yields even more atp per gram then carbohydrates metabolism . a location of mir-378a in the gene encoding pgc-1 implies an involvement of mir-378a in metabolic pathways . unlike its homologue , pgc-1 , the expression of pgc-1 is not elevated in response to cold exposure but occurs in response to hypoxia , exercise , caloric restriction , or aging ( reviewed in ) . pgc-1 is preferentially expressed in tissues with relatively high mitochondrial content , such as heart , skeletal muscle , and brown adipose tissue . in 2002 , pgc-1 was first cloned and shown to be upregulated in the liver during fasting . pgc-1 strongly activates hepatic nuclear factor 4 ( hnf4 ) and ppar , both of these nuclear receptors being important for the adaptation of hepatocytes to the effects of fasting . these findings could hint to a possible role of pgc-1 in the regulation of gluconeogenesis and fatty acid oxidation in the liver . pgc-1 is also involved in the regulation of energy expenditure or in the pathway of estrogen receptor - related receptors ( errs ) [ 3337 ] . since mirnas originating in the introns of host genes may modulate the protein encoded by their parental genes and may be involved in the same mechanisms [ 3840 ] , mir-378a is proposed to be involved in the metabolic pathways affected by pgc-1 . it was reported that mice lacking the first intron of the ppargc1b gene ( and thus mir-378a ) have a significantly higher oxygen capacity and mitochondrial function . they identified a mediator complex subunit 13 ( med13 ) , involved in nuclear receptor signaling , and carnitine acetyltransferase ( crat ) , a mitochondrial enzyme involved in fatty acid metabolism , as targets of mir-378a-5p and mir-378a-3p , respectively . regulated also cytochrome p450 2e1 ( cyp2e1 ) being involved in the metabolism of , for example , drugs and toxins . in addition , it has been discovered that transcription factor nuclear respiratory factor-1 ( nrf-1 ) , a critical regulator of the expression of some important metabolic genes in mitochondria regulating cellular growth , is inhibited by mir-378a-3p . thus , mir-378a can be considered as a regulator of mitochondrial function in cells overexpressing mir-378a . moreover , mir-378a-5p inhibits the mrnas of err and ga - binding protein- in breast cancer , which both interact with pgc-1 and together control oxidative metabolism . this leads to a reduction of tricarboxylic acid gene expression and oxygen consumption and an increase in lactate production , which shifts cells from an oxidative towards a glycolytic pathway . in this way , mir-378a-5p is believed to be a switch regulating the warburg effect in breast cancer . moreover , in situ hybridization experiments in this study showed that mir-378a-5p expression correlates with progression of breast cancer . the proposed regulating role of mir-378a-5p on the warburg effect is in parallel with the effects of pgc-1 , which mediates gluconeogenesis and fatty acid metabolism after periods of fasting or intense exercise . coactivation by pgc-1 of err and ppar makes muscle fibers in pgc-1 transgenic mice more rich in mitochondria and highly oxidative . accordingly increased glycolytic rates and increased cell proliferation can be related to lactate production by lactate dehydrogenase ( ldh ) . ldha was found to be a direct target of mir-378a in the study of mallat et al . . hsa - mir-378a-3p represses cell growth and increases cell death by targeting ldha . of note , hsa - mir-378a-3p and hsa - mir-378a-5p had opposite effects on ldha expression . in addition , mir-378a is also considered as an important factor in adipogenesis and lipid storage . there is a complex family of factors regulating those processes such as insulin , insulin - like growth factors ( igfs ) , glucagon , and thyroid hormones t3 and t4 ( reviewed in [ 4649 ] ) . as mentioned before , it was demonstrated that mir-378a - knockout mice do not get fat after 8 weeks of high fat diet . such animals show an enhanced mitochondrial fatty acid metabolism and have elevated oxidative capacity of tissues targeted by insulin ( e.g. , liver , muscles , and adipose tissues ) . in accordance with that , it was shown that mature strands of bta - mir-378 - 1 ( table 1 ) are expressed at higher level in cows with high ( versus low ) amount of back fat . similarly , an inhibition of both mmu - mir-378a-3p and its host gene , pgc-1 , by the flavonoid fisetin lowered the accumulation of fat in the liver . interestingly , mmu - mir-378a-5p was downregulated in mice that were fed a high fat diet for five months . overexpression of mir-378a-3p/-5p during adipogenesis increased the transcriptional activity of ccaat / enhancer - binding proteins ( cebp ) alpha and beta , which can stimulate the expression of leptin , a hormone produced mainly by adipocytes which controls the homeostasis of body weight ( reviewed in [ 52 , 53 ] ) . on the other hand , tnf- , il-6 , and leptin are reported to increase the expression of mir-378a-3p in mature human adipocytes in vitro . these cytokines are mainly secreted in the adipose tissue and are suggested to be involved in development of insulin resistance [ 55 , 56 ] . in addition , mir-378a-3p was shown to target insulin growth factor 1 receptor ( igf1r ) and reduce the akt signaling cascade in cardiomyocytes during cardiac development . moreover , in tissues where igf1 levels were high ( e.g. , fibroblasts and fetal hearts ) , mir-378 - 3p levels were very low , showing an inverse relation and suggesting a negative feedback loop between mir-378a-3p , igf1r , and igf1 . the latter functions as a master regulator of adipogenesis and is involved in the formation of peroxisomes and the catabolism of very long chain fatty acids [ 58 , 59 ] . accordingly , the amount of adipose tissue does not increase in mice lacking ppar when they are fed a high fat diet . it was also reported that in cultured adipocytes mmu - mir-378a and pgc-1 expression is ppar , or rosiglitazone ( a ppar ligand ) , dependent , finding two peroxisome proliferator response elements in the mir-378a loci . on the other hand , overexpression of mir-378a elevated the expression of ppar isoform 2 , suggesting positive feedback loop and confirming the involvement of mir-378a in the storage of fat . there are several activators known to induce expression of ppar such as the members of the e2f transcription factor family and prostaglandin j-2 ( pgj-2 ) [ 6365 ] . the latter may act through rar - related orphan receptor alpha ( rora ) , which is frequently found in myocardium . in addition to ppar , rora regulates also myod , a major transcription factor involved in skeletal muscle differentiation [ 67 , 68 ] . interestingly , rora is a possible ( but not yet validated ) target for mir-378a-3p . a proteomics - based study revealed several other proteins that are potentially targeted by rat mir-378a-3p or mir-378a-5p . mir-378a-3p was shown to regulate mannose-1-phosphate guanylyltransferase ( gdp ) , dimethylarginine dimethylaminohydrolase 1 ( ddah1 ) , and lactate dehydrogenase a ( ldha ) ; all those proteins are participating in metabolic processes . on the other hand , tropomyosin beta chain , which is involved in the regulation of atpase activity , high levels of murine and rat mir-378a-3p , mir-378a-5p , and porcine mir-378 - 1 are reported in both developing and adult skeletal muscles [ 7 , 30 , 44 ] . myod and myog play a role in the processes of myogenesis and muscle regeneration , in which dormant satellite cells are activated upon muscle damage and start proliferating and differentiating into muscle fibers ( reviewed in [ 70 , 71 ] ) . it has been shown that mir-378a-3p targets the myogenic repressor myor during myoblast differentiation , which directly inhibits myod . on the other hand , myod is upregulated in response to mir-378a-3p overexpression and , conversely , the level of mir-378a-3p may be enhanced by myod . thus , there is evidence for a feedback loop in which mir-378a-3p regulates muscle differentiation via inhibiting myor , leading to an increase of myod , which in turn enhances mir-378a-3p . that mir-378a may also control the development of skeletal muscle mass after training . in this study , mir-378a ( strand not specified ) was significantly downregulated in men who obtained low training - induced muscle mass gain compared to men who obtained high training - induced muscle mass gain . mir-378a-3p is expressed mostly by cardiomyocytes , but not by nonmuscle cells , whereas the level of mir-378a-5p was reported to be very low in the heart . fang et al . showed that mir-378 - 3p is significantly downregulated both in vitro in cardiomyocytes cell cultures exposed to hypoxia and in vivo during myocardial injury in rats . overexpression of mir-378a-3p enhanced cell viability and inhibited apoptosis via caspase-3 inhibition . in contrast to this finding , another study found that mir-378a-3p downregulation enhanced the survival of cardiac stem cells via focal adhesion kinase activation and releasing connective tissue growth factor ( ctgf ) , the latter being a target of mir-378a-3p . overexpression of mir-378a-3p in the study of knezevic et al . increased apoptosis of cardiomyocytes via the direct targeting of igf1r leading to a decrease of akt signaling . this is in opposition to the previously mentioned study of fang et al . which showed apoptosis the converse findings of the studies could be explained by different models used by knezevic et al . and because of those discrepancies , the role of mir-378a in apoptosis of cardiomyocytes requires further investigation . the finding that mir-378a-3p affects both igf1r and the akt pathway was confirmed in a study which found that overexpression of mir-378a-3p in rhabdomyosarcoma suppressed igf1r expression and affected phosphorylation of the akt protein . mir-378a-5p was shown to target heat shock protein 70.3 ( hsp70.3 ) in mouse hearts in normoxic conditions , but in hypoxic conditions a transcript variant of hsp70.3 without mir-378a-5p target site in its 3-utr is not repressed and can exert its cytoprotective properties . mir-378a-3p prevented cardiac hypertrophy by targeting either ras signaling or the mitogen - activated protein kinase ( mapk ) pathway [ 77 , 78 ] . more studies on the effect of mir-378a expression in muscle disorders would also be desirable . in both golden retriever muscular dystrophy dogs and duchenne muscular dystrophy patients , all in all , these findings suggest mir-378a-3p can be considered as an important player in cardiac development , remodeling , and hypertrophy . angiogenesis comprises development of new blood vessels from existing ones , regulated by cytokines and growth factors such as , for example , vascular endothelial growth factor ( vegf ) , platelet - derived growth factor ( pdgf ) , and angiopoietin-1 ( ang-1 ) . their expression can be posttranscriptionally controlled by micrornas such as mir-126 , mir-296 , mir-210 , mir-21 , and the mir-17~92 cluster ( reviewed in ) . skeletal muscles and heart muscle are tissues which , due to their oxygen and energy consumption , need to be sufficiently vascularized . one of the major regulators of angiogenesis is the hypoxia - inducible factor-1 ( hif-1 ) , which controls over 100 genes involved mainly in the glycolytic pathway and blood vessel formation , including vegf - a or interleukin-8 [ 8385 ] . vegf is generally induced by hypoxia , while il-8 in at least some cancers and endothelial cells can be diminished by hif-1 via inhibition of c - myc and sp-1 transcription factors [ 86 , 87 ] . c - myc , known as a regulator of cell cycle progression , apoptosis , and cellular transformation , is also a potent activator of pgc-1 and , in turn , mir-378a-3p , upregulating their expression . in addition , mir-378a has been shown to affect vegf - a in two ways . human hsa - mir-378a-5p ( by the study of hua et al . named as mir-378 ) can directly affect vegf - a by competing with hsa - mir-125a for the same seed - region in the vegf - a 3utr causing upregulation of vegf - a . mir-378a-5p can also indirectly regulate vegf - a affecting sonic hedgehog ( shh ) signaling via sufu inhibition , which is an inhibitory component of this signaling pathway . the shh pathway in turn can upregulate vegf - a and also other regulators of blood vessels formation , ang-1 and ang-2 expression [ 9092 ] . increased expression of vegf - a , as well as pdgf and tgf1 , was also seen in mesenchymal stromal cells ( mscs ) transfected with rno - mir-378a-5p . in skeletal muscles , vegf - induced angiogenesis appears not to be regulated by the well - known hif pathway but by pgc-1 , which coactivates estrogen - related receptor alpha ( err- ) on binding sites in the promoter and the first intron of the vegf gene , inducing its expression . this angiogenic pathway shows new roles for pgc-1 and err- , which are important regulators of mitochondrial activity in response to stimuli like exercise . if there might be a role for pgc-1 in this pathway , it is yet to be examined . it is noteworthy , however , that mir-378a-5p is known to affect the estrogen receptors by inhibiting err , another estrogen - related receptor . a role for mir-378a in cell cycle regulation and stimulation of cell growth is also proposed . in human mammary epithelial and breast cancer cell lines , mir-378a-3p can target the antiproliferative protein tob2 , which is a suppressor of cyclin d1 , which in turn is required for cell cycle g1-phase to s - phase progression . whether mir-378 affects endothelial cell proliferation by regulation of cell cycle remains to be established . the role of mir-378a in the formation of blood vessels nourishing tumor and enabling tumor growth was revealed . mir-378a was found to be differentially regulated in different types of cancers being downregulated in gastric cancer [ 96 , 97 ] , oral , and colon carcinoma , while being upregulated in renal and lung cancer [ 9 , 101 ] . since it is also changed in serum or plasma of patients with prostate cancer , renal cancer [ 100 , 103 ] , and gastric cancer and frequently found to be overexpressed in cryopreserved bone marrow mononuclear cells from acute myeloid leukemia patients , mir-378a might be considered as a biomarker . the role of mir-378a in tumorigenesis , tumor growth , and tumor vascularization was revealed for the first time by lee and coworkers in glioblastoma . they showed that mir-378a-5p enhances cell survival , reduces caspase-3 activity , and promotes tumor growth and angiogenesis , through repression of two tumor suppressors , sufu and fus-1 . strikingly , nude mice injected with mir-378a-5p transfected cancer cells formed tumors of bigger volume and with larger blood vessels compared to gfp - transfected cells . on the other hand , high expression of mir-378a-5p in nsclc correlated with brain metastases due to higher cell migration , invasion , and tumor angiogenesis . another study confirmed the downregulation of fus-1 by mir-378a-5p and showed that in the hepg2 liver cancer cells mir-378a-5p overexpression enhanced proliferation , migration , and , when injected in mice , invasion . also in rhabdomyosarcoma , enhanced expression of mir-378a , vegf , and mmp9 correlated with increased vascularization and metastasis . taken together , these studies suggest that mir-378a may serve as a prognostic marker in cancer due to its effects on angiogenesis . our recent data confirmed the proangiogenic effect of mir-378a ( both strands ) in non - small cell lung carcinoma ( nsclc ) and pointed at its correlation with heme - degrading enzyme , heme oxygenase-1 ( ho-1 ) . an involvement of ho-1 in angiogenesis and vegf - a as well as il-8 signaling was shown by us previously ; however , its action in tumors seems to be complex . in nci - h292 cell line overexpressing ho-1 , mir-378a ( both strands ) also overexpression of the mir-378a precursor sequence diminished ho-1 expression . conditioned medium from nci - h292 cells overexpressing mir-378a enhanced angiogenic potential of hmec-1 endothelial cell line . tumors formed by such cells in subcutaneous xenografts showed enhanced growth , vascularization , oxygenation , and distal metastasis in vivo . these interactions between mir-378a and ho-1 were confirmed in our studies on the role of the nrf-2 transcription factor / ho-1 axis in nsclc cell lines [ 110 , 111 ] . on the other hand , enhanced expression of mmu - mir-378a-5p in 4t1 murine breast cancer cells decreased the proliferation , migration , and invasiveness of these cancer cells in vitro and in vivo by targeting fibronectin , resulting in inhibition of tumor growth . recent study showed that mir-378a may act as a biomarker for response to antiangiogenic treatment in ovarian cancer . low expression of mir-378a was associated with longer progressive - free survival in patients with recurrent ovarian cancer treated with the antiangiogenic drug bevacizumab . overexpression of the mir-378a precursor in ovarian cancer cells altered expression of genes associated with angiogenesis ( alcam , ehd1 , elk3 , and tln1 ) , apoptosis ( rpn2 , hipk3 ) , and cell cycle regulation ( swap-70 , lsm14a , and rdx ) . high mir-378a ( strand not specified ) expression in renal carcinoma correlated with higher levels of endothelial surface marker cd34 in these tumors . notably , a recent study suggested clinical relevance for mir-378a in metastatic colorectal cancer , in which enhanced mir-378a expression significantly improved the sensitivity to cetuximab treatment in these patients . mir-378a-5p transfection of mscs has been shown to enhance their survival and angiogenic potential under hypoxic conditions in vitro . in coculture with human umbilical vein endothelial cells ( huvecs ) , mir-378a-5p - transfected mscs formed a larger number of vascular branches on matrigel . in the mscs transfected with mir-378a-5p , the expression of bcl-2-associated x protein ( bax ) , which is an important proapoptotic regulator , was decreased , leading to a better survival . it still has to be determined if the proangiogenic effect of mir-378a in vivo is confined to tumor angiogenesis , or if this effect is also present in physiological angiogenesis and regenerative neovascularization . recently , it was reported that anti - mir-378a-5p enhances wound healing process by upregulating integrin beta-3 and vimentin . it was reported that pgc-1 induces angiogenesis in skeletal muscle , enhancing the expression of vegf both in vitro and in vivo after ( transgenic ) overexpression . accordingly , it was also found that vegfa is upregulated in c2c12 myoblast cell line with pgc-1 overexpression . however , after a pcr - based gene array of 84 known angiogenic factors and further rt - pcr of individual genes , they concluded that pgc-1 triggered an antiangiogenic program . after inducing hind limb ischemia in pgc-1 overexpressing mice , an impaired reperfusion was noticed when compared to wild type littermates . the role of inflammation in angiogenesis is studied the most in the context of cancer ( e.g. , reviewed in [ 119 , 120 ] ) but is certainly not limited to this pathology . both lymphoid ( reviewed in [ 121 , 122 ] ) and myeloid ( reviewed in ) derived inflammatory cells affect angiogenesis in a stimulating or inhibitory manner . the role of mir-378a in inflammatory cells was reported and its anti - inflammatory effect could be suggested . nk cells exert potent cytotoxic effects when activated by type i ifn from the host once infected . mir-378a was found to be downregulated in activated nk cells and further proved to target granzyme b. thus , ifn- activation decreases mir-378a expression and in turn augments nk cell cytotoxicity . accordingly , suppression of mir-378 targeting granzyme b in nk cells resulted in inhibition of dengue virus replication in vivo . macrophages are known to play either inhibitory or stimulatory roles in angiogenesis ( reviewed by ) . mirnas have been proposed to regulate activation and polarization of macrophages ( reviewed by [ 127 , 128 ] ) . in a study of rckerl mir-378a-3p was identified as a part of the il-4-driven activation program of anti - inflammatory macrophages ( m2 ) . mir-378a-3p was highly upregulated after stimulation with il-4 of peritoneal exudate cells of mice injected with the parasite brugia malayi compared to controls and infected il-4-knockout mice . the study identified several targets for mir-378a-3p within the pi3 k / akt signaling pathway , which are important for proliferation but only partially responsible for m2 phenotype . another study found mir-378a ( strand not specified ) expression upregulated after stimulation with cytokines like , for example , tnf- and il-6 . in line with its potential role in macrophages , mmu - mir-378a ( strand not specified ) has been found to be upregulated during osteoclastogenesis in vitro . furthermore , serum levels of mir-378a-3p have been shown to correlate with bone metastasis burden in mice injected with mouse mammary tumor cell lines 4t1 and 4t1.2 . a growing body of evidence suggests a role for mir-378a as a mediator controlling reciprocally dependent processes in metabolism , muscle differentiation / regeneration , and angiogenesis . as mir-378a was found to be differentially regulated in different types of cancers and its level is changed in serum of prostate , renal , and gastric cancer patients , it can be considered as a biomarker for those diseases . the correlation between mir-378a expression and disease progression in lung cancer , liver cancer , and rhabdomyosarcoma suggests a further role of this microrna as a prognostic marker . if more research will be done to the mechanisms of action , possibilities for therapeutic use of mir-378a could be sought in the field of metabolic disorders , obesity , or tumors . more studies on the effect of mir-378a expression in muscle disorders would also be desirable . the proangiogenic effect of mir-378a was observed in tumors ; however , no studies have been performed on the angiogenic effects of mir-378a in physiological settings or diseases where angiogenesis plays important roles , such as diabetes and cardiovascular diseases . more study has to be done to assess the mechanisms of mir-378a function in blood vessel formation . of note , in contrast with proangiogenic role of mir-378a , inhibition of mir-378a-5p enhanced wound healing process . this might suggest a role for mir-378a-5p in diseases such as diabetes or in decubitus ulcers , in which wound healing is impaired . of note is the confusion that has arises because of a disarray in nomenclature with studies describing the same molecule , mir-378a , as mir-422b , mir-378 , or mir-378 . in addition , it is not always clear which of the two mature strands of mir-378a is studied . this could lead to misunderstandings and errors in interpreting the data published so far .

microrna-378a ( mir-378a , previously known as mir-378 ) is one of the small noncoding rna molecules able to regulate gene expression at posttranscriptional level . its two mature strands , mir-378a-3p and mir-378a-5p , originate from the first intron of the peroxisome proliferator - activated receptor gamma , coactivator 1 beta ( ppargc1b ) gene encoding pgc-1. embedding in the sequence of this transcriptional regulator of oxidative energy metabolism implies involvement of mir-378a in metabolic pathways , mitochondrial energy homeostasis , and related biological processes such as muscle development , differentiation , and regeneration . on the other hand , modulating the expression of proangiogenic factors such as vascular endothelial growth factor , angiopoietin-1 , or interleukin-8 , influencing inflammatory reaction , and affecting tumor suppressors , such as sufu and fus-1 , mir-378a is considered as a part of an angiogenic network in tumors . in the latter , mir-378a can evoke broader actions by enhancing cell survival , reducing apoptosis , and promoting cell migration and invasion . this review describes the current knowledge on mir-378a linking oxidative / lipid metabolism , muscle biology , and blood vessel formation .

cancer is the leading cause of deaths worldwide with breast cancer being second in the list of cancer related deaths.1 among all of the available treatment options , chemotherapy is the treatment of choice for advanced stage breast cancer that is refractory to hormonal therapy.2,3 although traditional breast cancer chemotherapy regimens include combinations of cyclophosphamide , methotrexate , and 5-flurouracil,4 anthracycline- and taxane - based chemotherapy is being commonly used nowadays for the treatment of breast malignancy due to its better performance in patient s overall survival rate.57 though breast cancer is considered to be highly chemosensitive with response rates as high as 80%,8 the majority of initially chemoresponsive tumours develop resistance to once effective chemotherapeutic agents.9 therefore , a switch to other chemotherapy regimens is ineffective because of the tumour s cross - resistance to multiple structurally and functionally unrelated chemotherapy drugs.10 abc transporter represents the key component of energy - dependent efflux transport system contributing to the development of multidrug - resistant phenotype in cancer.11 abcb1 transporter or p - glycoprotein , the first cytotoxic drug efflux pump to be identified,12 functions by actively extruding drugs including anthracyclines , taxanes , and vinca alkaloids out of cancer cells , thus reducing intracellular drug concentration below the threshold level required for cell killing.10,11 the poor chemotherapy response in breast cancer is generally correlated to the extent of abcb1 gene expression.1316 a second type of abc transporter discovered in a multidrug resistant breast cancer cell line is abcg2,17 though there are no conclusive data currently available to correlate its expression with chemotherapy efficacy in breast cancer.18 however , judging from the ability of abcg2 transporter in extruding doxorubicin , a very commonly used anthracycline drug,19 it might be reasonable to correlate abcg2 expression with development of clinical multidrug resistance in cancer . although low molecular weight pharmacologically active compounds have been developed to circumvent multidrugresistant phenotypes by either directly or indirectly modulating the abc transporter efflux activity,20 the first generation modulators failed in clinical trials because of their inherent toxicities21 while the second generation molecules were associated with undesirable pharmacokinetic interactions.22,23 thus , no drug has been clinically approved for the reversal of cancer multidrug resistance.24 a new promising approach to enhance chemosensitivity without undesirable side effects , other than the structure - based drug design , is to prevent the biosynthesis of abc transporters by selectively inhibiting the expression of abc transporters through gene silencing technologies.25 in the last decade , ribozymes2628 and antisense oligonucleotides2931 were introduced to modulate cancer multidrug resistance through inhibition of abc transporter gene expression . very recently , small interfering rnas ( sirnas ) , a double - stranded rna of between 2128 nucleotides that selectively degrade mrna and thus block production of a particular protein,32 have been applied in vitro for reversal of abc transporter - mediated drug efflux by targeting both abcb1 and abcg2 transporters . a pioneering study using exogenous sirna demonstrated suppression of abcb1 expression in conjunction with reversal of doxorubicin and paclitaxel resistance in human breast cancer cells.33 reversal of multidrug - resistant abcg2 phenotype was also investigated with a sirna - mediated knockdown study in several human cancer cells.34 currently , we have established ph - sensitive carbonate apatite as a potential tool to efficiently deliver sirna across the cell membrane and silence cyclin b1 gene transcript , thus inducing apoptosis of cervical cancer cells in synergy with anti - cancer drugs.35,36 here , we reveal that carbonate apatite - mediated delivery of the sirna targeting either abcg2 or abcb1 gene transcript in mcf-7 , a human breast cancer cell line constitutively expressing abcg2 and abcb1 transporters , led to an increased chemosensitivity to doxorubicin , paclitaxel , and cisplatin , depending on the doses of the individual drug . moreover , co - delivery of two specific sirnas targeting abcb1 and abcg2 transcripts caused a dramatic enhancement of chemosensitivity activity in mcf-7 cells , indicating the reversal of abc transporter - mediated multidrug resistance . dulbecco s modified eagle medium ( dmem ) was purchased from biowhittaker ( walkersville , md ) . dmem powder , fetal bovine serum ( fbs ) and trypsin - ethylenediamine tetraacetate ( trypsin - edta ) were obtained from gibco brl ( carlsbad , ca ) . calcium chloride dihydrate ( cacl22h2o ) , sodium bicarbonate , diammineplatinum(ll ) dichloride , dimethyl sulphoxide ( dmso ) , doxorubicin hydrochloride , paclitaxel , and thiazolyl blue tetrazolium bromide ( mtt ) were from sigma - aldrich ( st louis , mo ) . the sirna sequence targeting abcg2 ( genbank accession no nm_004827 ) and abcb1 ( genbank accession no nm_000927 ) purchased from qiagen ( valencia , ca ) correspond to coding regions of these genes . one target sequence ( 5-ctggtctaatttattaatcta-3 ) was selected for abcg2 ( abcg2_v1 ) and one ( 5-gacagaaagcttagtaccaaa-3 ) for abcb1 ( abcb1_v4 ) . the 21-nucleotide sirnas having a 3-dtdt extension were chosen based on recommendations made by others.37,38 sirna was supplied in lyophilised form and upon delivery , the sirna ( 1 nmol ) was reconstituted with rnase - free water provided by manufacturer to obtain a stock solution of 20 m . human breast cancer cell line mcf-7 was grown in 25 cm culture flask in dmem , supplemented with 10% heat - inactivated fbs in a humidified atmosphere containing 5% co2 at 37c . once the monolayer cells had reached 80%90% confluency , the medium was removed and the cells were washed with phosphate - buffered saline ( pbs ) . trypsin - edta was added to detach the cells from the flask and the suspended cells were collected in a fresh medium and transferred to new 25 cm flasks . the cells were then sub - cultured at densities of 1.05.0 10 cells/10 ml every 3 to 4 days . exponentially growing mcf-7 cells ( log phase ) were trypsinised and fresh medium was added to wash remaining cells from the bottom of the culture flask . the cells were counted using hemocytometer and appropriate dilutions were made using culture medium to produce a cell suspension with a concentration of 5.0 10 cells / ml . one ml of the prepared cell suspension was subsequently added into each of the wells in 24-well plate and allowed to attach overnight at 37c and 5% co2 before sirna transfection . on the day of sirna transfection , 100 ml of dmem was prepared using 1.35 g of dmem powder and 0.37 g of sodium bicarbonate . ph of the prepared dmem solution was adjusted to 7.4 using 0.1 m hydrochloric acid . dmem was then filtered using 0.2 a m syringe filter in a laminar flow hood , followed by transferring 1 ml of the filtered medium into 1.5 ml microcentrifuge tubes . 3 l of 1 m calcium chloride was then added into the microcentrifuge tubes , followed by addition of 1 or 10 nm sirna35,36 and incubation at 37c for 30 minutes . after the incubation , ten nm to 100 m of a drug ( doxorubicin , paclitaxel , or cisplatin ) prepared from serial dilution using 1 mm stock solution was then added into the respective microcentrifuge tubes . culture medium from the wells seeded one day before was aspirated and replaced with 1 ml of the prepared medium containing sirna - loaded carbonate apatite nanoparticles and free drugs . plates were then incubated at 37c and 5% co2 for 2 consecutive days . following two days of sirna transfection , the fraction of viable mcf-7 cells was determined using mtt assay as previously described.39 briefly , 50 l of mtt ( 5 mg / ml in pbs ) was added aseptically into each of the wells in sirna transfected plates , followed by incubation at 37c and 5% co2 for 4 hours . after the incubation period , medium containing mtt was aspirated and the purple formazan crystals at the bottom of each well were dissolved by mixing with 300 l of dmso solution . absorbance of the resulting formazan solution was then determined spectrophotometrically at wavelength 595 nm using a microplate reader ( dynex technologies inc . each experiment was performed in triplicate and the data were plotted as mean standard deviation ( sd ) of three independent experiments . dulbecco s modified eagle medium ( dmem ) was purchased from biowhittaker ( walkersville , md ) . dmem powder , fetal bovine serum ( fbs ) and trypsin - ethylenediamine tetraacetate ( trypsin - edta ) were obtained from gibco brl ( carlsbad , ca ) . calcium chloride dihydrate ( cacl22h2o ) , sodium bicarbonate , diammineplatinum(ll ) dichloride , dimethyl sulphoxide ( dmso ) , doxorubicin hydrochloride , paclitaxel , and thiazolyl blue tetrazolium bromide ( mtt ) were from sigma - aldrich ( st louis , mo ) . the sirna sequence targeting abcg2 ( genbank accession no nm_004827 ) and abcb1 ( genbank accession no nm_000927 ) purchased from qiagen ( valencia , ca ) correspond to coding regions of these genes . one target sequence ( 5-ctggtctaatttattaatcta-3 ) was selected for abcg2 ( abcg2_v1 ) and one ( 5-gacagaaagcttagtaccaaa-3 ) for abcb1 ( abcb1_v4 ) . the 21-nucleotide sirnas having a 3-dtdt extension were chosen based on recommendations made by others.37,38 sirna was supplied in lyophilised form and upon delivery , the sirna ( 1 nmol ) was reconstituted with rnase - free water provided by manufacturer to obtain a stock solution of 20 m . human breast cancer cell line mcf-7 was grown in 25 cm culture flask in dmem , supplemented with 10% heat - inactivated fbs in a humidified atmosphere containing 5% co2 at 37c . once the monolayer cells had reached 80%90% confluency , the medium was removed and the cells were washed with phosphate - buffered saline ( pbs ) . trypsin - edta was added to detach the cells from the flask and the suspended cells were collected in a fresh medium and transferred to new 25 cm flasks . the cells were then sub - cultured at densities of 1.05.0 10 cells/10 ml every 3 to 4 days . exponentially growing mcf-7 cells ( log phase ) were trypsinised and fresh medium was added to wash remaining cells from the bottom of the culture flask . the cells were counted using hemocytometer and appropriate dilutions were made using culture medium to produce a cell suspension with a concentration of 5.0 10 cells / ml . one ml of the prepared cell suspension was subsequently added into each of the wells in 24-well plate and allowed to attach overnight at 37c and 5% co2 before sirna transfection . on the day of sirna transfection , 100 ml of dmem was prepared using 1.35 g of dmem powder and 0.37 g of sodium bicarbonate . ph of the prepared dmem solution was adjusted to 7.4 using 0.1 m hydrochloric acid . dmem was then filtered using 0.2 a m syringe filter in a laminar flow hood , followed by transferring 1 ml of the filtered medium into 1.5 ml microcentrifuge tubes . 3 l of 1 m calcium chloride was then added into the microcentrifuge tubes , followed by addition of 1 or 10 nm sirna35,36 and incubation at 37c for 30 minutes . after the incubation , ten nm to 100 m of a drug ( doxorubicin , paclitaxel , or cisplatin ) prepared from serial dilution using 1 mm stock solution was then added into the respective microcentrifuge tubes . culture medium from the wells seeded one day before was aspirated and replaced with 1 ml of the prepared medium containing sirna - loaded carbonate apatite nanoparticles and free drugs . following two days of sirna transfection , the fraction of viable mcf-7 cells was determined using mtt assay as previously described.39 briefly , 50 l of mtt ( 5 mg / ml in pbs ) was added aseptically into each of the wells in sirna transfected plates , followed by incubation at 37c and 5% co2 for 4 hours . after the incubation period , medium containing mtt was aspirated and the purple formazan crystals at the bottom of each well were dissolved by mixing with 300 l of dmso solution . absorbance of the resulting formazan solution was then determined spectrophotometrically at wavelength 595 nm using a microplate reader ( dynex technologies inc . each experiment was performed in triplicate and the data were plotted as mean standard deviation ( sd ) of three independent experiments . since abc transporter gene expression is essential in causing multidrug - resistant phenotype in breast cancer cells , knockdown of this gene might enhance sensitivity of the cancer cells towards chemotherapeutic agents , and possibly reverse cancer multidrug resistance . therefore , mcf-7 , the widely used human breast model cell line intrinsically expressing abcg2 ( alternatively called brcp or breast cancer resistance protein),17 was transfected with the target sirna / carbonate apatite complexes in presence or absence of traditionally used chemotherapeutic agents ( doxorubicin , paclitaxel , or cisplatin ) for a consecutive period of 48 hours prior to cell viability assessment by mtt assay . as shown in figure 1a , while the viability of the cancer cells was reduced to 36% due to the potent cytoxicity of 100 nm doxorubicin , the concerted effect of sirna delivery and drug exposure resulted in 26% and 24% reduction in cell viability for 1 nm and 10 nm of sirna initally used for complex formation , respectively . thus , the combination of sirna and doxorubicin killed a much higher number of cells than the drug and the sirna alone , suggesting that silencing of the abcg2 gene might have inhibited to a significant extent the efflux of doxorubicin across the membrane , and raised intracellular concentration of the drug for effective killing of the cancer cells ( table 1 ) . abcg2 gene knockdown similarly accelerated chemosensitivity of paclitaxel ( 100 nm ) particularly at the higher dose of sirna ( 10 nm ) , while demonstrating an insignificant response with cisplatin ( figure 1b , table 1 ) . the apparent enhancement of chemosensitivity to cisplatin , as shown in table 1 , was virtually due to the silencing effect of sirna ( figure 1c ) alone . indeed , the decrease in viability of the cells treated with sirna / nanoparticle complexes in comparison with those untreated or treated with nanoparticles alone indicates the role of abc expression in delaying apoptosis of cancer cells.4042 based on the notion that anti - cancer drugs could dose - dependently influence the expression profile or susbtrate specificity of abc transporters , a range of relatively lower concentrations of the drugs was investigated in the knockdown study of abc transporters using 1 nm of target sirnas . as shown in figure 2 and table 2 , with drug doses ranging from 10 pm to 1 nm for doxorubicin as well as paclitaxel and 100 pm to 10 nm for cisplatin , silencing of abcg2 gene expression resulted in significant enhancement of chemosensitivity towards doxorubicin and paclitaxel in all of the drug concentrations used , whereas a weak response to 100 pm of cisplatin was observed probably due to the poor cytotoxic effect of cisplatin at the particular concentration . the finding thus suggests that the chemosensitivity of mcf-7 breast cancer cells due to the knockdown of abcg2 gene is virtually similar among the three drugs in respect to the maximum enhancement and dependent on the dose of the invidual drug . the enhancement of chemosensitivity to the popular anti - cancer drugs by virtue of silencing abcg2 gene expression triggered us to carry out a similar study in the same breast cancer cells using the sirna targeting the gene of abcb1 , the most extensively studied abc transporter involved in development of multi - drug resistance in various cancer cells including mcf-7.13 as shown in figure 3 , in synergy with 100 nm of paclitaxel , anti - abcb1 sirna at both 1 and 10 nm led to killing of much more cancer cells than the target sirna ( 1 and 10 nm ) or the drug ( 100 nm ) alone , resulting in 14% and 33% enhancement of chemosensitivity respectively . on the contrary , the apparent enhancement of c hemosensitivity ( to the same extent ) towards both 100 nm of doxorubicin and cisplatin following knock - down of abcb1 transporter ( table 3 ) was solely due to the effect of gene silencing killing more cells than the individual drugs or the target sirna alone ( figure 3 ) . however , when the doses of the individual drugs were reduced to 10 pm to 1 nm for doxorubicin and paclitaxel , sirna - mediated cleavage of abcb1 gene transcript surprisingly increased chemosensitivity towards each of the two drugs ( table 4 ) with demonstration of significantly higher level of cytotoxicity than sirna or drugs alone ( figure 4 ) , suggesting the broader substrate - specificity of abcb1 transporter at lower concentrations of the individual cancer drugs . on ther other hand , the apparent increase in chemosensitivity for 100 pm to 10 nm of cisplatin was statistically insignificant , which is consistent with the fact that cisplatin is not a substrate for abcb1 . regardless of the type of sirna ( abcg2 or abcb1 ) used in this study , the enhancement of chemosensitivity after single sirna treatment was only modest . this observation was similar to that made by other groups , where no complete reversal of cancer multidrug resistance was noted even at sirna concentration as high as 200 nm.33,43 such incomplete reversal of cancer multidrug resistance may be attributed to the inherent nature of the target protein under study . due to its long half - life ( from 16 hours44 to 72 hours45 ) and high cellular abundance , abc transporter protein remains a challenging target for sirna silencing.46 delivery of single sirna targeting either abcg2 or abcb1 transporter with carbonate apatite nanoparticles demonstrated a modest 15% to 30% enhancement in c hemosensitivity in mcf-7 cells ( tables 14 ) . to explore whether the combined delivery of two specific sirnas simultaneously targeting the two different transporters could further improve the chemo - sensitivity towards the anti - cancer drugs , mcf-7 cells were transfected with the sirnas ( at 1 nm each ) targeting the gene transcripts of abcg2 and abcb1 in presence of 1 nm of the individual drugs . as shown in figure 5 , nanoparticle - facilitated intracellular delivery of single sirna targeting a particular transporter killed fewer cells than co - delivery of the two target sirnas , suggesting that both of the transporters , which are intrinsically expressed in mcf-7 cells,13,17 are important for survival of the cells.4042 on the other hand , simultaneous delivery of the two target sirnas into mcf-7 cells in presence of cisplatin , paclitaxel , or doxorubicin , was found to dramatically enhance cytotoxicity with enhancement of chemosensitivity by 50% , 49% and 43% , respectively ( table 5 ) , in comparison to the individual drugs and the sirnas co - delivered without a drug . this suggests that both abcg2 and abcb1 are fully functional at a time in extruding any of those popular anti - cancer drugs from the cytoplasm of mcf-7 cells and inhibition or knockdown of a single transporter could only partly block the cellular efflux process , allowing drug efflux to continue through the other channel(s ) . since abc transporter gene expression is essential in causing multidrug - resistant phenotype in breast cancer cells , knockdown of this gene might enhance sensitivity of the cancer cells towards chemotherapeutic agents , and possibly reverse cancer multidrug resistance . therefore , mcf-7 , the widely used human breast model cell line intrinsically expressing abcg2 ( alternatively called brcp or breast cancer resistance protein),17 was transfected with the target sirna / carbonate apatite complexes in presence or absence of traditionally used chemotherapeutic agents ( doxorubicin , paclitaxel , or cisplatin ) for a consecutive period of 48 hours prior to cell viability assessment by mtt assay . as shown in figure 1a , while the viability of the cancer cells was reduced to 36% due to the potent cytoxicity of 100 nm doxorubicin , the concerted effect of sirna delivery and drug exposure resulted in 26% and 24% reduction in cell viability for 1 nm and 10 nm of sirna initally used for complex formation , respectively . thus , the combination of sirna and doxorubicin killed a much higher number of cells than the drug and the sirna alone , suggesting that silencing of the abcg2 gene might have inhibited to a significant extent the efflux of doxorubicin across the membrane , and raised intracellular concentration of the drug for effective killing of the cancer cells ( table 1 ) . abcg2 gene knockdown similarly accelerated chemosensitivity of paclitaxel ( 100 nm ) particularly at the higher dose of sirna ( 10 nm ) , while demonstrating an insignificant response with cisplatin ( figure 1b , table 1 ) . the apparent enhancement of chemosensitivity to cisplatin , as shown in table 1 , was virtually due to the silencing effect of sirna ( figure 1c ) alone . indeed , the decrease in viability of the cells treated with sirna / nanoparticle complexes in comparison with those untreated or treated with nanoparticles alone indicates the role of abc expression in delaying apoptosis of cancer cells.4042 based on the notion that anti - cancer drugs could dose - dependently influence the expression profile or susbtrate specificity of abc transporters , a range of relatively lower concentrations of the drugs was investigated in the knockdown study of abc transporters using 1 nm of target sirnas . as shown in figure 2 and table 2 , with drug doses ranging from 10 pm to 1 nm for doxorubicin as well as paclitaxel and 100 pm to 10 nm for cisplatin , silencing of abcg2 gene expression resulted in significant enhancement of chemosensitivity towards doxorubicin and paclitaxel in all of the drug concentrations used , whereas a weak response to 100 pm of cisplatin was observed probably due to the poor cytotoxic effect of cisplatin at the particular concentration . the finding thus suggests that the chemosensitivity of mcf-7 breast cancer cells due to the knockdown of abcg2 gene is virtually similar among the three drugs in respect to the maximum enhancement and dependent on the dose of the invidual drug . the enhancement of chemosensitivity to the popular anti - cancer drugs by virtue of silencing abcg2 gene expression triggered us to carry out a similar study in the same breast cancer cells using the sirna targeting the gene of abcb1 , the most extensively studied abc transporter involved in development of multi - drug resistance in various cancer cells including mcf-7.13 as shown in figure 3 , in synergy with 100 nm of paclitaxel , anti - abcb1 sirna at both 1 and 10 nm led to killing of much more cancer cells than the target sirna ( 1 and 10 nm ) or the drug ( 100 nm ) alone , resulting in 14% and 33% enhancement of chemosensitivity respectively . on the contrary , the apparent enhancement of c hemosensitivity ( to the same extent ) towards both 100 nm of doxorubicin and cisplatin following knock - down of abcb1 transporter ( table 3 ) was solely due to the effect of gene silencing killing more cells than the individual drugs or the target sirna alone ( figure 3 ) . however , when the doses of the individual drugs were reduced to 10 pm to 1 nm for doxorubicin and paclitaxel , sirna - mediated cleavage of abcb1 gene transcript surprisingly increased chemosensitivity towards each of the two drugs ( table 4 ) with demonstration of significantly higher level of cytotoxicity than sirna or drugs alone ( figure 4 ) , suggesting the broader substrate - specificity of abcb1 transporter at lower concentrations of the individual cancer drugs . on ther other hand , the apparent increase in chemosensitivity for 100 pm to 10 nm of cisplatin was statistically insignificant , which is consistent with the fact that cisplatin is not a substrate for abcb1 . regardless of the type of sirna ( abcg2 or abcb1 ) used in this study , the enhancement of chemosensitivity after single sirna treatment was only modest . this observation was similar to that made by other groups , where no complete reversal of cancer multidrug resistance was noted even at sirna concentration as high as 200 nm.33,43 such incomplete reversal of cancer multidrug resistance may be attributed to the inherent nature of the target protein under study . due to its long half - life ( from 16 hours44 to 72 hours45 ) and high cellular abundance , abc transporter protein remains a challenging target for sirna silencing.46 delivery of single sirna targeting either abcg2 or abcb1 transporter with carbonate apatite nanoparticles demonstrated a modest 15% to 30% enhancement in c hemosensitivity in mcf-7 cells ( tables 14 ) . to explore whether the combined delivery of two specific sirnas simultaneously targeting the two different transporters could further improve the chemo - sensitivity towards the anti - cancer drugs , mcf-7 cells were transfected with the sirnas ( at 1 nm each ) targeting the gene transcripts of abcg2 and abcb1 in presence of 1 nm of the individual drugs . as shown in figure 5 , nanoparticle - facilitated intracellular delivery of single sirna targeting a particular transporter killed fewer cells than co - delivery of the two target sirnas , suggesting that both of the transporters , which are intrinsically expressed in mcf-7 cells,13,17 are important for survival of the cells.4042 on the other hand , simultaneous delivery of the two target sirnas into mcf-7 cells in presence of cisplatin , paclitaxel , or doxorubicin , was found to dramatically enhance cytotoxicity with enhancement of chemosensitivity by 50% , 49% and 43% , respectively ( table 5 ) , in comparison to the individual drugs and the sirnas co - delivered without a drug . this suggests that both abcg2 and abcb1 are fully functional at a time in extruding any of those popular anti - cancer drugs from the cytoplasm of mcf-7 cells and inhibition or knockdown of a single transporter could only partly block the cellular efflux process , allowing drug efflux to continue through the other channel(s ) . due to the heterogenous nature of tumours where several drug resistance mechanisms may coexist and simultaneously contribute to the development of cancer multidrug resistant - phenotype , 11 the use of sirna combinations might represent a potential mechanism for circumventing this problem . additionally , the concern about saturating the cellular rna interference machinery by exogenous addition of two sirnas may have been potentially overcome by combining sirnas at the lowest possible concentration . thus , the synergism achieved with combination of abcg2 and abcb1 targeting sirnas would provide valuable insight for complete reversal of clinical cancer multidrug resistance otherwise deemed impossible to be achieved by currently available therapeutics .

backgroundmultidrug resistance , a major impediment to successful cancer chemotherapy , is the result of overexpression of atp - binding cassette ( abc ) transporters extruding internalized drugs . silencing of abc transporter gene expression with small interfering rna ( sirna ) could be an attractive approach to overcome multidrug resistance of cancer , although delivery of sirna remains a major hurdle to fully exploit the potential of sirna - based therapeutics . recently , we have developed ph - sensitive carbonate apatite nanoparticles to efficiently carry and transport sirna across the cell membrane , enabling knockdown of the cyclin b1 gene and consequential induction of apoptosis in synergy with anti - cancer drugs.methods and resultswe report that carbonate apatite - mediated delivery of the sirnas targeting abcg2 and abcb1 gene transcripts in human breast cancer cells which constitutively express both of the transporter genes dose - dependently enhanced chemosensitivity to doxorubicin , paclitaxel and cisplatin , the traditionally used chemotherapeutic agents . moreover , codelivery of two specific sirnas targeting abcb1 and abcg2 transcripts resulted in a more robust increase of chemosensitivity in the cancer cells , indicating the reversal of abc transporter - mediated multidrug resistance.conclusionthe delivery concept of multiple sirnas against abc transporter genes is highly promising for preclinical and clinical investigation in reversing the multidrug resistance phenotype of breast cancer .

the course is divided into 5 modules , where the leading experts in the field provide online lectures within their area of expertise ( table i ) . during the first module of the course therefore , topics that are covered during the introduction week are the nomenclature for the different subpopulations of evs as well as an introduction to the diversity of organisms releasing evs and the tissues and body fluids where evs can be found . furthermore , one of the pioneers , professor emeritus philip stahl , shares the story about how he and his colleagues discovered exosomes in the early 1980s ( 4 ) . the second module focuses on the biogenesis and release of evs and how this differs between the ev subpopulations : exosomes and microvesicles . additionally , the different uptake mechanisms of evs when they are encountered by a recipient cell are covered in depth ( 10 ) . as evs have been shown to contain functional rnas , proteins and lipids , this module also covers the different types of molecules present in evs as well as a brief overview on what the potential functions of these molecules are . furthermore , the techniques that are commonly used to detect these molecules and to analyse the cargo of evs will be highlighted . in the third module , the focus is on the collection and processing of cell culture media and body fluids prior to isolation of evs . here , considerations and guidelines that are important during the collection of the ev - containing material and when isolating evs from these fluids are discussed ( 11,12 ) . this module will help the students to reflect over the many different choices , such as anticoagulants , collection time points and protein inhibitors , which are important for the outcome when working with a particular body fluid compared with conditioned media or other body fluids . this module also illustrates some examples of studies on evs from body fluids such as blood , urine , breast milk and cerebrospinal fluid and why it is of interest to analyse evs from these bodily fluids . the basic concepts and some guidelines for methods such as differential ultracentrifugation , density gradient centrifugation , size - exclusion chromatography and kit - based precipitation are covered . furthermore , this module covers how the techniques are used in the field of evs as well as their limitations and benefits . the importance of evaluating the heterogeneity , purity and characteristics of the isolated vesicles regardless of isolation method is also highlighted ( 13 ) . the fifth module covers some of the different techniques that can be used to characterise evs . here , the basic concepts for techniques such as electron microscopy , cryo - transmission electron microscopy ( cryo - tem ) , flow cytometry , atomic - force microscopy ( afm ) and nanoparticle - tracking analysis ( nta ) are covered . furthermore , this module covers how the techniques are used in the field of evs as well as their limitations and benefits . this course is recommended for anyone interested in the field of evs including biology and medical students and phd students without previous experience in the field as well as clinicians , cell and molecular biologists and researchers who want to broaden their understanding of the field and deepen their knowledge about particular techniques . the course contains 5 modules , where each module contains 47 recorded lectures ( 635 min / lecture ) . each module contains in total 12.5 h of recorded materials , and all lectures are in english . each of the 5 modules is followed by a quiz in the format of multiple choice questions . after completing the course , the student should be able to : discuss the nomenclature and subgroups of evs , describe the release and uptake mechanisms of evs , describe the rna , protein and lipid content of evs , explain the considerations that are important during the collection and isolation of evs from different body fluids , describe the basic concepts about the most common isolation and characterisation techniques and how these techniques are used in the ev field andstate the benefits and limitations of the most common isolation and characterisation techniques for evs . discuss the nomenclature and subgroups of evs , describe the release and uptake mechanisms of evs , describe the rna , protein and lipid content of evs , explain the considerations that are important during the collection and isolation of evs from different body fluids , describe the basic concepts about the most common isolation and characterisation techniques and how these techniques are used in the ev field and state the benefits and limitations of the most common isolation and characterisation techniques for evs . the initial response to the course has been overall positive with high ratings , and one student commented the course as : this course was really well organized and paced but packed full of a lot of really good information from great sources and leaders in the field . i really did n't know anything about exosomes before i started this course and now i feel like i can even teach the people in my own lab a few tricks . this course was really well organized and paced but packed full of a lot of really good information from great sources and leaders in the field . i really did n't know anything about exosomes before i started this course and now i feel like i can even teach the people in my own lab a few tricks . we are pleased to see this initial feedback to the course and isev will now initiate the work of producing more online courses on other related topics such as the biological functions of evs in health and disease . this course was funded by the international society for extracellular vesicles and supported by grants for pedagogic development from the sahlgrenska academy , university of gothenburg . ysg is the inventor of patents for using evs as therapeutics , diagnostics and vaccines and is the founder of aeon medix and rosetta exosome and own stock in the company . jl is the co - owner of patents for using exosomes as therapeutics and is currently an employee of codiak biosciences , inc . in parallel with his position at university of gothenburg .

the international society for extracellular vesicles ( isev ) has organised its first educational online course for students and beginners in the field of extracellular vesicles ( evs ) . this course , basics of extracellular vesicles , uses recorded lectures from experts in the field and will be open for an unlimited number of participants . the course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles . the first module is an introduction to the field covering the nomenclature and history of evs . module 2 focuses on the biogenesis and uptake mechanisms of evs , as well as their rna , protein and lipid cargo . module 3 covers the collection and processing of cell culture media and body fluids such as blood , breast milk , cerebrospinal fluid and urine prior to isolation of evs . modules 4 and 5 present different isolation methods and characterisation techniques utilised in the ev field . here , differential ultracentrifugation , size - exclusion chromatography , density gradient centrifugation , kit - based precipitation , electron microscopy , cryo - electron microscopy , flow cytometry , atomic - force microscopy and nanoparticle - tracking analysis are covered . this first massive open online course ( mooc ) on evs was launched on 15 august 2016 at the platform coursera and is free of charge .

paul ehrlich described mast cells for the first time more than 100 years ago , through their characteristic metachromatic staining with aniline dyes . he noticed that these cells had abundant and dense granules ; he assumed that the granules were caused by excessive feeding . these cells were abundant in areas near tumors , so ehrlich postulated that they played a role in the protection against cancer . since then , many functions were proposed for mast cells in the regulation of organism homeostasis , but experimental evidence to support these assumptions was lacking . the discovery of mice with a diminished number of mast cells , and their reconstitution with bone marrow - derived mast cells , provided a tool to test these hypotheses and to extend our knowledge about the functions of mast cells in the body . mast cells derive from hematopoietic precursors that are released from the bone marrow into the bloodstream and are recruited to connective tissues , where they complete their differentiation . mast cells can live for months in the tissues , where they proliferate in response to different signals . the tissues that usually contain abundant mast cells are those that interact with the external environment ( the skin and the gastrointestinal , urogenital and respiratory tracts ) , where mast cells are located near lymphatics and blood vessels , or close to nerve terminals or smooth muscle cells . the morphology of mast cells depends on their anatomical location , ranging from rounded - shaped to elongated forms , when they are associated with blood vessels . their size ranges from 5 to 20 m and their cytoplasm contains abundant granules , with approximately 1,000 granules per cell . mast cell granules contain several chemical mediators , including histamine , proteoglycans , cytokines like tnf- and il-4 , and neutral proteases like tryptase , chymase , carboxypeptidase and cathepsin g. granule composition is not identical in all mast cells : human mast cells that reside in the skin have all the aforementioned neutral proteases , while epithelial lung mast cells have significant amounts of tryptase , but lack chymase , carboxypeptidase and cathepsin g. accordingly , human mast cells are classified as either mct ( skin ) or mt ( lung ) . in addition to these pre - formed mediators that are stored in granules , mast cells are efficient producers of lipid - derived mediators , such as platelet - activating factor ( paf ) and the arachidonic acid - derived prostaglandins , thromboxanes and leukotrienes . mast cells are also efficient producers of many soluble protein mediators , such as cytokines , chemokines and antimicrobial peptides [ 6 , 7 ] . activation of mast cells induces the release of one group of these mediators or of all of them , depending on the triggering signal . however , not all the mediators are released at the same time : chemical mediators from the granules are secreted in seconds after stimulation , while lipid - derived mediators are produced minutes after activation and protein soluble mediators are detected hours after the initial trigger . mast cells produce abundant histamine , and since the discovery that histamine is a crucial element for the development of type i hypersensitivity , the relevance of mast cells for the initiation of allergic reactions was underscored . by far , the most studied mast cell - activating signal is the cross - linking of fcri by ige and allergen , an event that leads to mast cell degranulation and to the release of several chemical mediators that cause the immediate inflammatory response characteristic of type i hypersensitivity . as a consequence , much of our knowledge of mast cell biology is biased towards their participation in this pathological process . type i hypersensitivity , or allergic inflammation , is an acute inflammation produced in sensitized individuals when they are exposed to the corresponding allergen . sensitization occurs when dendritic cells capture an allergen in the airways , migrate to the regional lymph nodes and activate nave t cells . in the presence of il-4 ( derived from mast cells , eosinophils or innate lymphoid cells ) , these cytokines promote class - switch recombination in b cells , which then produce allergen - specific ige . ige binds to fcri on mast cells , which are then primed for activation upon re - exposure to the allergen . during this subsequent exposure , the allergen cross - links fcri - bound ige , which leads to mast cell activation and degranulation . the mediators derived from activated mast cells cause bronchoconstriction , vasodilation , increased vascular permeability , mucus production and recruitment of eosinophils and neutrophils . repetitive or persistent exposure to the allergen leads to a chronic allergic inflammation , which causes tissue injury and remodeling ; this last process includes the severe narrowing of the airway lumen observed in asthmatic patients . however , mast cells are conserved in different groups of vertebrates , and from an evolutionary point of view , it makes no sense that humans should have conserved a cell population that causes a harmful and even deadly response if it conferred no evolutionary advantage . so , in the last years , the study of mast cells has been redirected to investigate their participation in processes such as innate immune response to pathogens , regulation of inflammation , wound healing and regulation of tumoral growth . in this review , we focus on the mechanisms used by mast cells in the lung to keep the proper function of this vital organ . mast cells have a vast collection of cell membrane receptors that allow them to sense the external environment . a set of these receptors are pattern recognition receptors ( prr ) that recognize pathogen associated molecular patterns ( pamp ) . these receptors are crucial for the initiation of the innate immune response , and they are classified in five groups : toll - like receptors ( tlr ) , nod - like receptors ( nlr ) , c - type lectin receptor ( clr ) , rig - i - like receptor ( rir ) and scavenger receptors . mast cells express at least one receptor of each group , allowing them to recognize a multitude of microorganisms , including viruses , bacteria , protozoa , fungi and multicellular parasites [ 13 - 23 ] ( fig . 1 ) . the first evidence that mast cells are crucial elements of the immune response to pathogens came from a study of the infection of the rat gastrointestinal tract with multicellular parasites . there are few studies that investigate if mast cells play a role in the containment of parasitic diseases in the lungs . one of the firs studies that established a role for mast cells in fungal infections in vivo reported that , in a mouse model of infection with sporothrix schenckii , mast cell depletion decreased the severity of cutaneous lesions . whether mast cells in the lungs plays a role in fungal infections needs to be evaluated , but in vitro experiments suggest that they do have a role , because they are activated by aspergillus fumigates . identified for the first time that mast cells are essential for the early containment of bacterial infections in the lungs ; klebsiella pneumonie , a gram - negative bacteria that causes community - acquired pneumonia , was among the bacteria tested in this study . the authors observed that the lung bacterial load was higher in mice deficient in mast cells , compared with wild - type mice , and this correlated with a defective recruitment of neutrophils to the lung . these results indicated that mast cells did not have a direct antimicrobial activity , but they induced inflammation through tnf- , which allowed the recruitment of neutrophils . mast cells also participate in the immune response to mycoplasma pneumoniae , a bacteria that is among the smallest free - living organisms and lacks a classical bacterial cell wall . airway infection of mice deficient in mast cells induced faster weight loss , more severe pneumonia , higher mortality and increased bacterial burden in the lungs , compared with wild - type mice . this work showed that mast cells play an important role in innate immune response against bacteria that infect lungs . tularemia is a life - threatening disease caused by francisella tularensis , a gram - negative coccobacillus that can be spread through aerosols . intranasal infection of wild - type mice with f. tularensis induces an increased accumulation of mast cells in the lungs . accordingly , mast cell - deficient mice showed impaired bacterial control and a significant decrease in il-4 production . in fact , mast cells confer protection by inhibiting bacterial entry and replication in macrophages , thus showing a different mechanism by which mast cells confer protection against bacterial infection in the lungs . it is caused by mycobacterium tuberculosis ( mtb ) , a bacteria that has a cell wall unique among bacteria , and that infects and usually persists in the lungs . studies with humans have shown an increase of mast cells in the lymph nodes of patients with tuberculous lymphadenitis . in vitro studies demonstrated that mtb activates mast cells , inducing degranulation , production of il-6 and tnf- and even bacterial internalization through lipid rafts [ 36 , 37 ] . moreover , in vivo experiments have suggested a role of mast cells in the containment of mtb infection . in one of the first studies , mice received c48/80 , a compound that induces mast cell degranulation , before infection with mtb . these mice showed an altered production of cytokines and chemokines , which induced a decrease in lung inflammation and an increase in mtb load , compared with untreated mice . a second study used tlr-2 knockout mice , which are highly susceptible to mtb infection . when these mice received mast cells that express tlr-2 before infection with mtb , they had an improved inflammatory response , with myeloid cell recruitment and granuloma formation that correlated with increased protection against the bacilli . these studies reinforce the notion of mast cells as crucial players of the lung innate immune response against bacterial pathogens . cytomegalovirus is a beta - herpes virus that causes interstitial pneumonia in immunocompromised patients and is a serious problem in patients with an organ transplant . infection of mast cell - deficient mice with cytomegalovirus is followed by high viral replication and delayed virus clearance from the lungs , compared with wild - type mice . interestingly , this phenomenon was associated with a defective production of the chemokine ccl5 ( also known as rantes ) and a decreased recruitment of cd8 + t cells into the infected lungs , providing evidence that mast cells can recognize viral infection and induce the recruitment of essential effector cells to the site of infection in the lung . influenza is a frequent infectious disease of the human respiratory tract , which is caused by a group of viruses from the orthomyxoviridae family ; the most common of these viruses is influenza a. these viruses have attracted attention because they are associated with pandemics that have severely affected human health , like the spanish flu of 1918 . interestingly , studies with this strain have reported alterations in lung architecture , which are caused by an excessive inflammatory response promoted by an uncontrolled production of inflammatory cytokines and chemokines , also known as cytokine storm . in a mouse model of infection with a pathogenic strain of h5n1 virus , lung lesions were reduced if mice were treated with ketotifen , a well - known mast cell degranulation inhibitor . moreover , mice survival was dramatically increased if mice received a combined treatment of ketotifen and oseltamivir , an antiviral drug . these results suggest that mast cell activation correlated with pathology development during h5n1 infection , and that inhibition of mast cell activation improved host health . recently showed that the pathology associated with influenza a virus infection is significantly decreased in mast cell - deficient mice , compared with wild - type mice . this event is associated with a lower production of the inflammatory cytokine tnf- and the chemokines ccl2 , ccl3 , ccl4 , cxcl2 and cxcl10 in the lungs , suggesting a critical role of mast cells during the induction of the cytokine storm . the studies in mice described above show that mast cells play a pro - inflammatory role against bacterial and viral infections , with both beneficial and detrimental effects . on the one hand , mast cells are critical for the innate immune response because they recruit effector cells that are fundamental for the containment of the pathogens during the early stages of infection ; this event has been observed in other organs besides the lungs . on the other hand , excessive mast cell activation by pathogens can lead to damage and loss of lung function , as evinced by influenza studies . this excessive mast cell activation has also been observed in calves with paroxystic respiratory distress syndrome caused by bovine respiratory syncitial virus . recent evidence indicates that mast cells also play a predominant role in the development of pathology during dengue virus infection . st john a , et al . observed a direct correlation between the serum levels of chymase and the severity of dengue disease in humans ; interestingly , treatment of mice with drugs that block mast cell degranulation or with an antibody that blocks leukotriene receptors improved the vasculopathy associated with dengue virus infection . these studies point to mast cells as a new therapeutic target to limit the damage provoked by excessive inflammation during infectious diseases , such as influenza and dengue . in contrast with their classical pro - inflammatory function , it has been reported that mast cells can produce significant amounts of anti - inflammatory cytokines , like il-10 . mast cell - produced il-10 plays a crucial role in limiting the sterile inflammation provoked by allergic contact dermatitis or uv radiation . moreover , mast cell - produced il-10 is essential for the induction of immune suppression by uv exposure [ 49 , 50 ] . recently demonstrated that mast cells produce il-10 during in vivo bladder infection with uropathogenic escherichia coli , and this event was necessary to maintain the tolerance that allowed bacterial persistence . this study demonstrated that microorganisms can tune mast cells into a pro - inflammatory or an anti - inflammatory program in order to subvert the immune response . choi et al , recently showed that salmonella typhimurium injects a potent phosphatase ( sptp ) into the cytosol of mast cells ; sptp interferes with mast cell activation , even with a very strong signal ( fcri cross - linking ) . this strategy is also exploited by yersinia pestis , which uses a phosphatase ( yoph ) with high homology to sptp to dampen mast cell activation . pathogenic bacteria are not the only ones that effectively suppress mast cell activation , symbiotic bacteria have established a delicate balance with the immune response in order to colonize and survive in different anatomical locations of the host . breakdown of such balance usually results in the development of different pathologies . so , it is not surprising that bacteria that usually colonize the gastrointestinal tract can regulate mast cell activation . one of the first evidences of this strategy came from a study in which murine mast cells were stimulated with non - pathogenic e. coli from human gut . unexpectedly , mast cells did not degranulate , even after incubation with a high bacterial load , which contrast with the swift degranulation in response to pathogenic e. coli . furthermore , mast cells incubated with non - pathogenic e. coli lost the ability to degranulate in response to strong activators , such as a23187 and fcri cross - linking . a similar phenomenon is also observed with lactobacillus and bifidobacterium , other probiotic and symbiotic bacteria that reside in the human gut [ 56 , 57 ] . these results are of great interest in view of the association that exists between the use of probiotics and the prevention and treatment of several atopic diseases . however , how this prevention and treatment are achieved is still a matter of discussion , and several mechanisms have been proposed to explain this phenomenon . recent evidence derived from a mouse model of allergic inflammation points out that probiotics have the ability to decrease the effector phase in allergic diseases , even in the presence of specific ige antibodies . this effect is related to the inhibition of mast cell activation through the alteration of the signaling pathways triggered by fcri . the probiotics bifidobacterium breve and lactobacillus rhamnosus have been proposed as treatments for atopic diseases because of their potent effect in suppressing mucosal mast cell degranulation during chronic asthma . as described in the previous section , mast cells play an essential role in the initiation of the innate immune response to pathogens . in fact , all the cells that participate in type i hypersensitivity also participate in type 2 immune responses , which protect against infections with bacteria , viruses and multicellular parasites , and participate in the clearance of venoms , xenobiotics and irritants .type 2 innate lymphoid cells and th2 lymphocytes produce il-5 , which induces eosinophil activation and recruitment ; they also produce il-9 , which causes basophil and mast cell proliferation , and il-13 , which increases mucus production and causes fibrosis . these cell populations constantly interact with each other : for example , type 2 innate lymphoid cells produce il-13 in response to mast cell - derived prostaglandin d2 , for which they have a specific receptor , crth2 . since mast cells play a fundamental role during the early stages of type i hypersensitivity and also during the initiation of the innate immune response to pathogens , two important questions arise : 1 . is the response of mast cells to pathogens affected in an allergic environment ? 2 . can pathogens exacerbate type i hypersensitivity through mast cell activation ? in relation to the first question , there is considerable epidemiological evidence that asthma is a risk factor for viral and bacterial infections in the lungs , but what promotes this increased susceptibility to infections is poorly understood [ 63 - 67 ] . a deficient immune response could explain this increased susceptibility ; for example , jung j et al . observed that asthmatic patients have decreased titers of specific pneumococcal antibodies when compared to not - asthmatic individuals . other authors suggest a defective innate immune response as the cause of this increase in the susceptibility to infections . several mechanisms of the innate immune response are altered in allergic patients , including a defective epithelial barrier , an excessive mucus production and a decreased production of cytokines . however , this defective innate immune response apparently does not affect the potential of mast cell to combat infections , as is demonstrated in a mouse model of infection with mycoplasma pneumonie in an allergic setting . in relation to the second question , infectious diseases , especially those caused by bacteria and viruses , are considered important environmental elements that exacerbate atopic diseases . several reports indicate that infections with bacteria and viruses that affect the respiratory tract induce the production of ige antibodies specific for bacterial or viral components . infection of newborn mice with respiratory syncital virus ( rsv ) induces the production of virus - specific ige , and reinfection of these mice caused the airway symptoms classically associated with an allergic response . moreover , in a mouse model of acute infection with influenza a virus , an enhanced susceptibility of the host to develop a severe allergic response to house dust - mite was described . these studies suggest that the induction and the exacerbation of allergic responses by pathogens are associated with a modification of the environment that favors ige production ; however , another mechanism could be the exacerbation of mast cell responses . as we discussed earlier , several bacterial and viral pathogens can induce mast cell activation , inducing a pro - inflammatory environment that recruits effector immune cells . but how does a mast cell respond if it is simultaneously activated through fcri and through receptors that sense infection ? early studies suggested that this double stimuli results in an increased activation of mast cells . for example , mast cell lines activated through fcri and infected with rhinovirus release more histamine and produce more cytokines than mast cell lines activated with a single stimuli . patients with allergic rhinitis that were experimentally infected with rhinovirus showed increased levels of histamine and eosinphil recruitment in bronchoalveolar lavages , compared with allergic individuals that were not infected with rhinovirus , suggesting an altered mast cell response . several reports also associate infection with pathogenic bacteria with the development of an environment that facilitates or exacerbates the development of type i hypersensitivity . for example , the pathogenic bacteria mycoplasma pneumoniae , streptococcus pneumoniae , haemophilus influenza and chlamydia pneumoniae induce the production of ige specific for bacterial components , suggesting that infection promotes an environment favorable for the development of allergies [ 76 - 78 ] . however , little information exists about how these pathogenic bacteria could affect mast cell activation through fcri . as mentioned above , the only bacteria that have been analyzed in this respect are symbiotic bacteria from the gastrointestinal tract ( e. coli , lactobacillus spp and bifidobacterium spp ) , and these bacteria ameliorated fcri - mediated activation of mast cells , showing a therapeutic potential of these bacteria for allergic reactions in the gut . however , other symbiotic bacteria that colonize a different environment , the vagina , apparently do not mimic this effect . interestingly , new evidence has demonstrated that the human respiratory tract is home to several symbiotic bacteria , and environments that were thought to be sterile , like the bronchial tree , in fact contain about 2,000 bacterial genomes per cm . moreover , asthmatic patients showed altered bacterial communities in their airways , when compared with healthy individuals , with an increase in haemophillus spp and a decrease in prevotella spp . how these changes in microbiota airway affect the progress of type i hypersensitivity and how they alter mast cell function are fields that need to be clarified in the future . this process develops in an orderly fashion , involving hemostasis , inflammation , cell proliferation and remodeling of the affected tissue . alterations in the sequence , duration or intensity of any of these stages result in an impaired tissue repair that is usually reflected in the development of different pathologies . several cell populations are involved in this process , and cells of the immune system have been implied in different steps of the process . first , they tend to accumulate and degranulate in the wounded tissue , favoring the recruitment of effector cells , mainly neutrophils and macrophages . the neutral proteases chymase and tryptase favor the degradation of the extracellular matrix [ 83 , 84 ] ; mast cell production of arachidonic acid - derivatives induces the production of type i collagen by fibroblasts , and mast cell production of fibroblast growth factor ( fgf ) , vascular endothelial growth factor ( vegf ) and transforming - growth factor 1 ( tgf-1 ) is considered important for fibroblast proliferation and for the formation of new blood vessels . however , much of the knowledge about the role of mast cells during wound healing comes from studies in the skin , and little work has been performed in the lung , perhaps because of the lack of an appropriate model of lung injury , making this an interesting field that needs more research to clarify the role of mast cells in this environment . paul ehrlich described that mast cells were abundant in areas near tumors , and assumed that these cells played an important role in the regulation of tumor growth more than 100 years ago . since then , several reports have confirmed that mast cells usually accumulate in areas near tumors , and that this accumulation is associated with a poor prognosis in breast cancer , melanoma , hodgkin s lymphoma , oral squamous cell carcinoma , renal carcinoma , pancreatic cancer , endometrial cancer , cervical cancer , etc . in 2000 , imada et al . observed that patients with stage i non - small cell lung adenocarcinoma had an increased number of mast cells in tumor areas around blood vessels , and those patients with higher mast cells counts had a worse prognosis . it has been suggested that tumor cells produce chemotactic mediators that attract mast cells to the tumor , but few of these chemotactic mediators have been identified . one example is stem cell factor ( scf ) , which is produced by several tumor - derived cell lines , including a human alveolar adenocarcinoma cell line . scf interacts with the receptor c - kit , which is expressed on mast cells , and directs their migration to the tumor . how mast cells contribute to tumor growth is still matter of research , but three main mechanisms have been proposed : 1 ) production of tumor growth factors , 2 ) facilitation of tumor angiogenesis and 3 ) modification of the interaction between the epithelial cells and the stroma . a mast cell product that leads to the proliferation of tumor cells is histamine , which promotes the proliferation of alveolar basal carcinoma cells . adrenomedullin - stimulated mast cells produce cytokines , such as fibroblast growth factor ( fgf ) and vascular endothelial growth factor ( vegf ) , which are associated with increased angiogenesis in vivo . observed an increase in the number of mast cells that produce vegf in patients with lung adenocarcinoma . in contrast with previous reports by several authors , welsh et al . reported that , in non - small cell lung adenocarcinoma , the number of mast cells in the tumor stroma had no correlation with tumor progression , while an increased number of mast cells in the tumor cell islets correlated with an improved prognosis . these results suggested an anti - tumor role for the infiltrating mast cells , but the mechanism was not investigated , and further studies are needed to clarify this issue . mast cells can trigger different mechanisms that contribute to the homeostasis and adequate function of the lungs . disequilibrium in the function of mast cells can lead to pathological states , of which asthma is the most recognized . mast cells play an essential role in type i hypersensitivity ( allergic inflammation ) and also during the early stages of the innate immune response to pathogens . several causes , from infections to tumor growth , alter the function of mast cells ( fig . the regulation of mast cell activation by conventional drugs or microorganisms , either pathogenic or symbiotic , opens a new window of opportunities to develop new treatments that could preserve and restore lung health .

lungs are indispensable organs for the respiratory process , and maintaining their homeostasis is essential for human health and survival . however , during the lifetime of an individual , the lungs suffer countless insults that put at risk their delicate organization and function . many cells of the immune system participate to maintain this equilibrium and to keep functional lungs . among these cells , mast cells have recently attracted attention because of their ability to rapidly secrete many chemical and biological mediators that modulate different processes like inflammation , angiogenesis , cell proliferation , etc . in this review , we focus on recent advances in the understanding of the role that mast cells play in lung protection during infections , and of the relation of mast cell responses to type i hypersensitivity - associated pathologies . furthermore , we discuss the potential role of mast cells during wound healing in the lung and its association with lung cancer , and how mast cells could be exploited as therapeutic targets in some diseases

gastric cancer is the second most frequent neoplasm of the alimentary tract after the large intestine . 5,103 people in poland were affected by it in 2008 . the case - to - death ratio of around 1 indicates unfavourable prognosis as to recovery from this disease [ 1 , 2 ] . this poor result is determined by the fact that it is rarely ( only in around 8% ) detected in the form of early gastric cancer , in the mildly symptomatic or asymptomatic phase . in most patients it is diagnosed at a higher degree than the 1st degree of disease progression and its classic symptoms are weight loss , continuous and dull pain in the epigastrium , loss of appetite , nausea , vomiting and chronic bleeding [ 3 , 4 ] . the existence of gastric cancer metastasis to the ureter has been described twice in the literature to date . a female patient , age 67 , was diagnosed at the district hospital ( 22.04 - 30.04.2010 ) because of intensified symptoms of left - sided renal colic . based on the conducted usg and single - phase computed tomography tests of the abdominal cavity and the pelvis , dilation of the ureter was found because of its infiltration by a pathological focus with the dimensions of 28 mm 15 mm . another lesion was located nearby at the level of the left iliac muscle 30 mm 27 mm 20 mm , adhering to the sigmoid colon . tissue infiltration of the pelvis minor wall was found descending in the direction of the left appendages . colonoscopy was conducted and in this test the large intestine was described without pathology , while the gynaecological usg test confirmed the presence of fluid in the pelvis . during her stay the patient was treated with analgesic and diastolic medication and then referred for further treatment at the regional oncology centre . because of reported pain complaints , she was immediately admitted to the department of oncological surgery on the day of her visit to the outpatient clinic ( 04.05.2010 ) . the urologist consulting the patient indicated the possibility of kidney damage due to ureteral obstruction with recommendation for an accelerated operation . therefore , pre - operative diagnostics were not extended beyond the tests received from the district hospital . the patient underwent surgery on 10.05.2010 and intraoperatively , besides the expected neoplastic tumour of the left ovary with infiltration of the ureter , numerous neoplastic foci were also found : a sigmoid colon tumour , a caecum tumour , a tumour of the body of the stomach and two single tumours in the omentum . because of the resectability of the neoplastic foci described above , the operation plan adopted earlier was changed and the following were performed in succession with palliative intention : partial gastrectomy by the rydygier method , right - sided hemicolectomy , left ovariectomy and sigmoid resection . after restoring the continuity of the alimentary tract , the tumour occluded the lumen , which is why segmental ureterectomy was performed with end - to - end anastomosis over a pigtail catheter . the patient passed the post - operative period without complications , except a two - day fever . because of the expected alimentary tract failure , parenteral nutrition was included on the 1st day after the procedure and blood deficits were supplemented with 2 units of erythrocyte mass and 7 units of plasma . on the 9th day after the operation , the patient was discharged home in a good general condition with recommendations for further treatment . the received postoperative histopathological protocol indicated that the stomach was the origin of the neoplastic process . to lauren ) , type i ( acc . to goseki ) , of a stomach adenocarcinoma with a g2 malignancy degree , with occupation of the whole thickness of the stomach wall , with the following immunohistochemical characteristics : ck7(+++ ) , ck20(++ ) , ca125(+ ) , mucicarmine(+ ) . neoplastic infiltrations along nerves and neoplastic embolisms of blood vessels , as well as metastatic foci in the greater omentum , were observed in the specimen . because of the palliative partial gastrectomy type , only 3 lymph nodes were described in this specimen and all of them contained neoplastic cells . besides the above , histopathological confirmations were obtained of the metastatic character of the foci in the ovary , caecum and sigmoid colon . the cancer occupied large and small intestine walls without infiltration of the mucous membrane and the immunohistochemical characteristics were identical as for the primary lesion . according to the above protocol , excision within tissues with a healthy margin of these organs was achieved . the widening of the left ureter is visible ( marked by arrow ) focus of metastatic carcinoma in the ureter wall . microscopic section , he staining , magnification 40 neoplastic invasion in the ureter wall . microscopic section , he staining , magnification 100 it is interesting biologically and constitutes the basis for this paper that the ureteral tumour was described by the pathologists not as a neoplastic infiltration encroaching on the ureter , but as a metastatic focus to the ureteral wall . after the operative treatment , the patient was qualified for palliative chemotherapy and received it from 28.07.2010 . the first treatment course was according to the eox regimen ( oxaliplatin and capecitabine ) , but because of the occurrence of neutropenic fever the regimen was changed to pf ( cisplatin and 5-fluorouracil ) . she again received only one course and the treatment was changed once more because newly formed metastatic foci in the liver were located in imaging tests . next , three chemotherapy courses were administered according to the folfiri regimen ( irinotecan , leucovorin and 5-fluorouracil ) , but only until 11.11.2010 because due to progression of changes in the liver described in examinations the chemotherapy was discontinued and the patient was qualified for symptomatic treatment . according to our knowledge treatment of gastric cancer in the disseminated phase of the disease aims at extension of life and achieving a good palliative effect . the recommended methods , allowing the above to be achieved , are chemotherapy or combination radiochemotherapy . in an advanced stage , palliative operations on this organ are performed for life reasons and serve to eliminate complications such as bleeding , perforation or obstruction of the organ . it is indicated that the above complications forced surgical intervention in 1/4 of the patients previously disqualified from operative treatment because of the presence of metastatic foci . according to literature data , patients operated on by palliative resection in the disseminated phase of the disease achieve a survival time of between 9 and 15 months . the survival time is limited by the number of metastatic foci ; when their number is higher than two foci , no statistically significant differences are observed in this scope . the value of these procedures is increasing because of the reported low perioperative mortality and the observed significant improvement in the further quality of life for these patients [ 513 ] . it seems that the operative procedure conducted in the presented patient allowed a good palliative effect to be achieved along with loss of severe colic and maintenance of kidney function . the literature often presents , as characteristic of gastric cancer , blood - borne metastasis to the ovary , termed a krukenberg tumour . this is a term generally defining metastasis to the ovary , mainly gastric cancer and next colon cancer . metastases from other organs , such as the lungs , the mammary gland and the uterus , are also possible , though much rarer . krukenberg tumours are encountered in the course of 2 - 4% of disseminated neoplastic processes . this is a negative prognostic factor , with varying median survival time after its diagnosis : 12 - 13 months in the course of gastric cancer compared to 17 - 29 months in the course of large intestine cancer . this undoubtedly results from biological differences in the course of these neoplasms [ 1423 ] . in summary , the available literature sources have described this only twice to date ( in 1976 and 2000 ) . however , nephroureterectomy was conducted in these quoted cases [ 24 , 25 ] . in the presented patient the kidney was spared and healing of the ureter after end - to - end suture was achieved . the dissemination of a neoplasm with extremely rarely encountered symptomatology described above is an interesting experience which we wanted to share . the beginning of the symptomatic disease as a left - sided renal colic is , in itself , a previously unreported case .

the typical symptoms of advanced cancer of the stomach are well known in clinical practice . the presented case concerns a patient with symptoms of left - sided renal colic , caused by a malignant tumour involving the ureter , which was diagnosed with a ct scan . the multifocal process , involving the stomach , two parts of the colon , the left ovary and the side of the pelvis , was confirmed only during surgery.the resection or partial resection of the above - mentioned organs involved by the malignant process and reconstruction of the alimentary tract as well as the ureter were performed at time of this operation . the patient 's recovery was without any complications . the histopathological findings support the diagnosis of this malignant process as disseminated stomach cancer.in the available literature only two cases of stomach cancer metastasis to the ureter have been described . in both cited examples resection of the ureter with nephrectomy was performed . the review of the literature supports the value of stomach palliative resection in prolonging life and improving quality of life .

the study was designed as a multicenter , retrospective collection of cases with using a case reporting form . only patients who underwent an ia recanalization procedure due to acute anterior circulation ischemia in the internal carotid artery ( ica ) and the middle cerebral artery ( mca ) , and this was performed within the past five years , were included in this review . this form included the basic clinical information , the initial imaging results , the angiography and procedure - related information , the follow - up imaging results and the clinical outcome of each patient together with the actual imaging data . we requested that the clinical and procedure - related parts of the form be filled out by the radiologist at each center . the basic clinical information included age , gender , national institute of health stroke scale ( nihss ) score at the time of presentation and the presence of underlying diabetes , hypertension and/or heart disease . selection of the initial imaging modality , the time from symptom onset to the time of imaging , the time from symptom onset to the initiation of angiography ( the first angiographic run ) , and the time of day of the procedure ( i.e. , during or after working hours ) were analyzed . the pre- and post - procedure ct / mr images and angiographic findings were evaluated by the board - certified radiologist who had more than five years of experience with ia thrombolysis . the ct results were analyzed for the presence of early ct signs , involvement of basal ganglia and the presence of hyperdense mca signs . the mr findings were analyzed to determine the extent of the lesion on the diffusion - weighted images ( dwi ) , involvement of the basal ganglia , the presence of perfusion / diffusion mismatch and presence of microbleeds . the angiographic results were analyzed for the anatomic location of the culprit steno - occlusive lesions and the initial and post - procedure thrombolysis in myocardial infarction ( timi ) grades . we regarded a post - procedure timi grade of ii or iii as indicative of successful recanalization . the immediate post - procedure ct was reviewed for the presence of hemorrhages and parenchymal hyperdensity that was observed due to the contrast media used during the thrombolytic procedure . the follow - up ct or mr findings were analyzed for occurrence of hemorrhagic transformation , which was evaluated by the european cooperative acute stroke study ( ecass ) classification method . we calculated the rates of occurrence of any hemorrhage and any radiologically significant hemorrhage , with the latter regarded as hemorrhage with a mass effect , such as parenchymal hematoma type 2 or some cases of hemorrhagic infarction type 2 . the clinical outcome analysis was performed using the modified rankin scale ( mrs ) , which was recorded at various time points after the procedure ( at 1 - 12 months , except for the patients who died in the interim ) . if the clinical outcome data were not available , the case was dropped from the clinical outcome analysis . we calculated the percentage of patients with each mrs score , as well as the mortality rate , which was defined as the rate of procedure - related deaths within one month . univariate logistic regression methods were used to analyze the factors that had an influence on successful recanalization , significant hemorrhagic transformation and poor functional outcome results . the proportion of patients with a mrs of more than 3 was used for the latter . variables with p values less than 0.05 on the univariate analyses were chosen as the variables for the multivariate logistic regression analysis . in both analyses , the basic demographic characteristic of the 163 patients from seven domestic institutes are summarized in table 1 . the mean time interval from the symptom onset to the initial ct scanning was 139145 minutes and the mean time interval from the symptom onset to the first angiography was 280178 minutes ( table 2 ) . out of the 149 patients for whom we were able to obtain the time of day of their angiography , 92 ( 62% ) had the procedures performed during normal working hours ( 09:00 - 18:00 ) . the initial imaging modalities were ct in 46 patients ( 28% ) , mr in 63 ( 39% ) , and both ct and mr in 54 ( 33% ) . we were able to review the ct images of 69 of the 100 ( 69% ) patients who initially underwent ct . the basic initial ct and mr findings are summarized in tables 3 and 4 , respectively . the site of arterial stenosis ( timi grade 1 , n = 18 ) or occlusion ( timi grade 0 , n = 145 ) was the ica , including the carotid ' t ' occlusion , in 62 patients ( 38% ) , the mca , including m2 occlusion , in 99 ( 61% ) and the anterior cerebral artery in two patients ( 1.2% ) ( table 5 ) . before the initiation of ia thrombolysis , 73 patients ( 45% ) were administered intravenous tissue plasminogen activator ( t - pa , 0.6 or 0.9 mg / kg ) . urokinase at a median dosage of 400,000 iu ( range 40,000 - 1,000,000 iu ) was the ia fibrinolytic agent in 139 patients ( 85% ) , t - pa at a median dosage of 10 mg ( range 5 - 20 mg ) was used in 11 patients ( 7% ) , and the procedure was performed without ia fibrinolytics in 13 patients ( 8% ) . in the latter case , mechanical methods were primarily applied , except for one patient , who received an ia infusion of abciximab . various mechanical methods were applied , along with local infusion of fibrinolytics , in 82 patients ( 50% ) , including those patients who were treated with primary mechanical thrombolysis . the methods employed included mechanical disruption of the clot using a micro - guidewire ( n = 49 , 60% ) , stenting of the steno - occlusive lesion using various kinds of metallic stents ( n = 17 , 21% ) , balloon catheter angioplasty or clot disruption ( n = 12 , 14% ) and aspiration of the clot using a large - bore guiding catheter ( n = 4 , 5% ) . a combination of these methods was used in patients for whom the primary mechanical method was ineffective in recanalization . the final timi grades were zero in 21 patients ( 14% ) , one in 23 ( 15% ) , two in 31 ( 20% ) and three in 77 ( 51% ) . various patterns of parenchymal high densities were observed in 54 patients ( 47% ) , and 24 ( 21% ) of them showed obvious hematoma formation and/or high density with a mass effect . hemorrhagic transformation of the lesion was evaluated in 155 patients whose follow - up ct and/or mr images were available for the review , and 55 ( 35% ) of these patients showed variable amounts of hemorrhage . the hemorrhage pattern was hemorrhagic infarction ( hi ) type 1 in 15 patients ( 27% ) , hi type 2 in six ( 11% ) , parenchymal hematoma ( ph ) type 1 in 17 ( 31% ) and ph type 2 in 17 ( 31% ) . the follow - up period varied from one to 12 months , except for those patients who died in the interim . we noted mrs scores of zero in 17 patients ( 11% ) , one in 27 ( 17% ) , two in 19 ( 12% ) , three in 20 ( 13% ) , four in 33 ( 21% ) and five in 24 ( 15% ) ; these findings were comparable with those of other major thrombolysis trials ( fig . 1 ) . out of the 158 patients , 18 died , yielding a mortality rate of 11% . among the various factors considered , an initial nihss score of less than 15 was the only factor that positively influenced successful recanalization ( p = 0.001 ) . table 6 shows the univariate analysis of the factors that influenced significant hemorrhage after the procedure , and table 7 shows the factors that influenced the poor functional outcome . subsequent multivariate analyses failed to show any statistically significant variables both for significant hemorrhage and for a poor functional outcome . the basic demographic characteristic of the 163 patients from seven domestic institutes are summarized in table 1 . the mean time interval from the symptom onset to the initial ct scanning was 139145 minutes and the mean time interval from the symptom onset to the first angiography was 280178 minutes ( table 2 ) . out of the 149 patients for whom we were able to obtain the time of day of their angiography , 92 ( 62% ) had the procedures performed during normal working hours ( 09:00 - 18:00 ) . the initial imaging modalities were ct in 46 patients ( 28% ) , mr in 63 ( 39% ) , and both ct and mr in 54 ( 33% ) . we were able to review the ct images of 69 of the 100 ( 69% ) patients who initially underwent ct . the basic initial ct and mr findings are summarized in tables 3 and 4 , respectively . the site of arterial stenosis ( timi grade 1 , n = 18 ) or occlusion ( timi grade 0 , n = 145 ) was the ica , including the carotid ' t ' occlusion , in 62 patients ( 38% ) , the mca , including m2 occlusion , in 99 ( 61% ) and the anterior cerebral artery in two patients ( 1.2% ) ( table 5 ) . before the initiation of ia thrombolysis , 73 patients ( 45% ) were administered intravenous tissue plasminogen activator ( t - pa , 0.6 or 0.9 mg / kg ) . urokinase at a median dosage of 400,000 iu ( range 40,000 - 1,000,000 iu ) was the ia fibrinolytic agent in 139 patients ( 85% ) , t - pa at a median dosage of 10 mg ( range 5 - 20 mg ) was used in 11 patients ( 7% ) , and the procedure was performed without ia fibrinolytics in 13 patients ( 8% ) . in the latter case , mechanical methods were primarily applied , except for one patient , who received an ia infusion of abciximab . various mechanical methods were applied , along with local infusion of fibrinolytics , in 82 patients ( 50% ) , including those patients who were treated with primary mechanical thrombolysis . the methods employed included mechanical disruption of the clot using a micro - guidewire ( n = 49 , 60% ) , stenting of the steno - occlusive lesion using various kinds of metallic stents ( n = 17 , 21% ) , balloon catheter angioplasty or clot disruption ( n = 12 , 14% ) and aspiration of the clot using a large - bore guiding catheter ( n = 4 , 5% ) . a combination of these methods was used in patients for whom the primary mechanical method was ineffective in recanalization . the final timi grades were zero in 21 patients ( 14% ) , one in 23 ( 15% ) , two in 31 ( 20% ) and three in 77 ( 51% ) . various patterns of parenchymal high densities were observed in 54 patients ( 47% ) , and 24 ( 21% ) of them showed obvious hematoma formation and/or high density with a mass effect . hemorrhagic transformation of the lesion was evaluated in 155 patients whose follow - up ct and/or mr images were available for the review , and 55 ( 35% ) of these patients showed variable amounts of hemorrhage . the hemorrhage pattern was hemorrhagic infarction ( hi ) type 1 in 15 patients ( 27% ) , hi type 2 in six ( 11% ) , parenchymal hematoma ( ph ) type 1 in 17 ( 31% ) and ph type 2 in 17 ( 31% ) . the clinical follow - up data were available for 158 patients . the follow - up period varied from one to 12 months , except for those patients who died in the interim . we noted mrs scores of zero in 17 patients ( 11% ) , one in 27 ( 17% ) , two in 19 ( 12% ) , three in 20 ( 13% ) , four in 33 ( 21% ) and five in 24 ( 15% ) ; these findings were comparable with those of other major thrombolysis trials ( fig . 1 ) . out of the 158 patients , 18 died , yielding a mortality rate of 11% . among the various factors considered , an initial nihss score of less than 15 was the only factor that positively influenced successful recanalization ( p = 0.001 ) . table 6 shows the univariate analysis of the factors that influenced significant hemorrhage after the procedure , and table 7 shows the factors that influenced the poor functional outcome . subsequent multivariate analyses failed to show any statistically significant variables both for significant hemorrhage and for a poor functional outcome . the results of our analysis provide an overview on the current practice status of ia thrombolysis in korea . although more than 30 centers in korea actively perform neurointerventional procedures ( see the 2005 member list of the korean society of interventional neuroradiology ) , only seven of these centers participated in this study . it is likely that many of the other centers have been reluctant to perform ia thrombolysis , primarily due to a shortage of trained personnel . in western countries also , ia thrombolysis is performed less frequently than the iv method due to a shortage of interventional specialists ( 15 ) . we found that from the symptom onset , the mean time before the imaging was 2.3 hours , and the mean time before the treatment was 4.7 hours ; these results are comparable with those reported in the proact ii trial ( 7 ) . the mean delay between the ct and mr imaging was about 60 min , which was one of the in - hospital delay factors for ia thrombolysis ( 18 ) . about 40% of these procedures were performed before or after normal working hours , and the functional outcome results of these patients were generally poorer than those of the patients who were treated during normal working hours . one of the primary reasons for the poorer outcome in the former group of patients may be the in - hospital delay . a well - organized stroke team and an on - call system may improve the results for these patients . one notable finding of our survey was the preference for mr as the initial imaging modality . mr was the sole initial imaging modality in 39% of patients , and it was combined with the ct imaging in 33% . although mr has the potential disadvantage of time delay , it has shown an advantage for the initial evaluation of acute ischemic stroke ( 19 - 22 ) . however , a delay in the initiation of the optimal treatment due to logistic limitations of mr imaging can not be justified . many centers use multi - detector ct imaging to overcome the inevitable logistic limitation of mr imaging ( 21 , 23 ) . it was notable that about 10% of the cases in the study were in violation of the ' one - third rule ' , which is one of the most important exclusion criteria of the european thrombolysis trial ( 24 ) and the proact ii trial ( 7 ) . out of all the patients evaluated by the mr imaging , 26% showed dwi lesions covering more than half of the involved vascular territory . this was probably due to a lack of certain inclusion or exclusion criteria for mr imaging in respect to the patient selection for ia thrombolysis . although we limited our study to patients with anterior circulation infarction and so increasing the homogeneity of the study group , we found a wide variation in the occlusion site , from the proximal ica to the proximal portion of the mca . our observed recanalization rate , 71% , was slightly higher than that reported in the proact ii trial ( 7 ) , which may have been due , at least in part , to the liberal use of mechanical methods ( 14 ) . we were disappointed to find that only one factor , the lower initial nihss score , had a significant effect on the successful recanalization rate . in contrast to reports on the combined or sequential thrombolytic treatments ( 25 - 27 ) , we found that the use of iv thrombolytics prior to the ia procedure did not affect the recanalization rate . however , in many of these patients , however , the initial iv administration of thrombolytics was ineffective . about half of the patients received a combination of ia and mechanical thrombolysis , although the use of mechanical methods failed to improve the recanalization rate ( p = 0.083 ) . one of the major concerns after the ia thrombolytic treatment is hemorrhagic transformation of the affected lesion ( 28 , 29 ) . we found that the rate of any amount hemorrhagic transformation was 35% , and the rate of the significant hemorrhagic transformation was 13% . the univariate analysis showed that several factors that had influence on significant hemorrhagic transformation , including the presence of hypertension , severe initial neurological symptoms , involvement of the basal ganglia on ct / mr and the performance of procedures after normal working hours . in addition , the risk of significant hemorrhage was increased by the presence of any contrast density on the ct obtained immediately after the thrombolysis procedure . violation of the one - third rule on the initial ct tended to increase the occurrence of significant hemorrhage ( p = 0.08 ) , whereas extensive involvement of the affected vascular territory on the dwi ( p = 0.51 ) and the combined use of iv thrombolytics and mechanical methods had no effect . although direct comparison is not possible because of the differences in the patient groups , the functional outcome in our series of patients was similar to that of other major thrombolysis trials ( 7 , 30 ) . among the factors that were found to influence the poor functional outcome were the old age , severe initial neurological symptoms , more extensive initial dwi lesions and involvement of basal ganglia on mr , failed recanalization , any contrast staining on ct immediately after the procedure and the presence of significant hemorrhagic transformation . other studies have found that these same factors were predictive of poor prognosis ( 8 , 31 , 32 ) . the primary limitations of this analysis were its retrospective design and the collection of data from several centers , all of which had different clinical and imaging indications for the procedure and different clinical settings . in addition , the data we collected were not representative of practice throughout korea because only a few active centers contributed to the study . due to our inability to collect some data from the participating centers , we were unable to perform appropriate multivariate analyses . this could be one of the possible reasons for our failure to determine any significant variables that affected the results of significant hemorrhagic transformation and poor functional outcomes . furthermore , our analysis of the imaging data was limited due to the between - center differences in the imaging protocols , including the use of contrast enhancement , mr pulse sequences and the angiographic filming techniques . nevertheless , the results presented here may be representative of the nation - wide data on current ia thrombolytic treatment , and they could serve as a basis for clinical practice guidelines , as well as for designing a prospective study on the basis of this protocol . in conclusion , our multicenter , retrospective analysis of ia thrombolysis showed an increased dependency on mr imaging during the initial patient evaluation and increased application of combined pharmacologic / mechanical thrombolysis in korea . both our imaging and clinical outcome results were comparable with those of the other major thrombolytic trials . our results could be used as the basis for clinical practice and for future prospective studies .

objectivethe purpose of the study was to review the current status of intra - arterial ( ia ) thrombolysis in korea by conducting a retrospective analysis of the data from multiple domestic centers.materials and methodsthe radiologists at each participating institution were asked to fill out case report forms on all patients who had undergone ia recanalization due to acute anterior circulation ischemia . these forms included clinical , imaging and procedure - related information . a central reader analyzed the ct / mr and angiographic results . the rates of successful recanalization , hemorrhagic transformation and functional outcome were obtained . the univariate analyses were performed together with the multivariate analysis.resultswe analyzed the data from 163 patients , and they had been treated at seven institutes . the initial imaging modalities were ct for 46 patients ( 28% ) , mr for 63 ( 39% ) , and both for 54 ( 33% ) . various mechanical treatment methods were applied together in 50% of the patients . radiologically significant hemorrhage was noted in 20/155 patients ( 13% ) . we found various factors that influenced the recanalization rate and the occurrence of significant hemorrhagic transformations . the favorable outcome rate , reported as modified rankin scale 2 , was 40% , and the mortality rate was 11% . the factors that predicted a poor functional outcome were old age ( p = 0.01 ) , initially severe neurological symptoms ( p < 0.0001 ) , mr findings of a wide distribution of lesions ( p = 0.001 ) , involvement of the basal ganglia ( p = 0.01 ) , performance of procedures after working hours ( p = 0.01 ) , failure of recanalization ( p = 0.003 ) , contrast extravasation after the procedure ( p = 0.007 ) and significant hemorrhagic transformation ( p = 0.002 ) . the subsequent multivariate analysis failed to show any statistically significant variable.conclusionthere was a trend toward increased dependency on mr imaging during the initial evaluation and increased usage of combined pharmacologic / mechanical thrombolysis . the imaging and clinical outcome results of this study were comparable to those of the previous major thrombolytic trials .

understanding the association between psychological affective disturbances and anthropometric parameters , including body mass index ( bmi ) , is important , because of the potential preventive effects of weight reduction on different aspects of women 's lifestyles such as psychopathological states . in this regard , the relationship between obesity and psychological disorders has been widely assessed , and recent evidence - based findings have consistently emphasized associations between eating behavior and both depression and anxiety . the relationship between weight and depression , suicidal ideation , or suicide attempts , has been investigated mainly in those classified as overweight or obese . moreover , both ends of the weight spectrum have been shown to increase the risk of future depression outcomes and suicide . these researches may be more important in women due to physiological and endocrinological fluctuations in different periods of their life including ; menstrual , pregnancy , and postmenopausal periods . in addition , the relationship between dietary restraint and bmi has been found in several longitudinal studies on adolescents , especially in women . in general , research has shown that women with higher socioeconomic status are more concerned about their weight and fitness indicating a probable association between restrained eating and anthropometric indices . several studies have done to find an association of adult weight gain and obesity with subscales of eating behavior characteristics in women . some of them found high levels of habitual disinhibiting were associated with substantial weight gain and obesity in older women european countries . but regarding to the difference in cultural and nutritional habits in different population and as to the best of our knowledge , no previous study have done among iranian woman to find relation of weight gain psychological factors among iranian employer women . the present study aimed to predict bmi based on psychological factors including ; depression , anxiety , dietary restraint , and nutritional habits and behaviors . in this regard , we considered women with sedentary jobs , in order to control for the confounding effects of dynamic active conditions . in this context , a reduction of bmi may effectively prevent the progression of psychological defects and vice versa . in a cross - sectional study , 200 women aged more than 25 years , working on sedentary and less standing jobs in isfahan , iran , including banks , social security organizations , were entered in the study . subjects with bmi higher than 30 kg / m were not included . to assess the severity of depression symptoms , the beck depression inventory ( bdi - ii ) was used . each answer was scored on a scale of 03 with the cut off points of 013 : minimal depression ; 1419 : mild depression ; 2028 : moderate depression ; and 2963 : severe depression . higher total scores indicated more severe depressive symptoms . according to previous population - based studies on the bdi - ii - persian version , this tool has a high internal consistency ( cronbach 's = 0.87 ) , and acceptable test - retest reliability ( r = 0.74 ) . anxiety levels were assessed with the state - trait anxiety inventory ( stai ) developed by charles spielberger . this psychological inventory is based on a 4-point likert scale and consists of 40 questions on a self - report basis . the stai measures two types of anxiety including ; state anxiety or anxiety about an event , and trait anxiety , or anxiety levels as a personal characteristic . the reliability and validity of the farsi adaptation of spielberger 's state - trait inventory were shown indicated to be high ( cronbach 's coefficient of 0.93 and content validity of 83.0% ) . to assess nutritional habits , a self - administered questionnaire was designed which included biological , behavioral , and cultural factors , as well as some nutritional - related items such as ; place of eating , eating time , speed of eating , and overeating . score equal to 15 or higher indicated proper nutritional habits , while lower scores showed the existence of poor nutritional habits that were not conducive for maintaining proper body weight and fitness . to assess dietary restraint status , the ruderman questionnaire was used ; it contained 10 items related to attitudes about ; eating , frequency of dieting , and weight fluctuations . the scores of this tool varied from 0 ( without response ) to 4 or 5 ( with the highest response ) . the reliability of this questionnaire in our study population was evaluated , and a cronbach 's coefficient of 0.68 was achieved . after obtaining permission from the relevant governmental organizations , a mechanical column scales ( seca weighing scale ; 700 , germany ) measured their body weight with an accuracy of 100 g and their height to the nearest 0.5 cm . bmi was calculated as weight in kilograms divided by the square of the height in meters ( kg / m ) . the results were presented as mean standard deviation for the quantitative variables , and they were summarized by absolute frequencies and percentages for the categorical variables . the pearson 's correlation coefficient test was applied to assess the correlation between the quantitative variables . multivariable linear regression analysis was used for predicting bmi on different psychological components . for the statistical analysis , a statistical software spss ( version 19.0 ) for windows ( spss inc . , chicago , il , usa ) was used . with respect to the baseline information ; 36.5% of the individuals were aged 2535 years , 43.5% were aged 3645 years , 18.0% were aged 4655 years , and 2% were aged more than 55 years . with regard to their marital status , the overall bmi mean in the studied women was 25.07 4.54 kg / m ( range : 1449 kg / m ) . in terms of their psychological states ; the mean score of depression severity was 10.9 7.4 ( range : 037 ) , the average score of trait anxiety was 44.77 9.99 ( range : 2272 ) , and the average score of state anxiety was 42.35 8.97 ( range : 1267 ) . the mean score of 3.08 10.16 ( range : 3121 ) was obtained for nutritional habits and a mean score of 11.02 4.49 ( range : 023 ) for dietary restraint . a pearson 's correlation coefficient [ table 1 ] showed a reverse correlation of depression severity as well as anxiety levels with nutritional habit means , indicating that severe symptoms of depression and trait anxiety were both significantly correlated to poor nutritional habits ( b = 0.18 , b = 0.16 , respectively ) . this analysis also found a reverse correlation between nutritional behavior and dietary restraint ( b = 0.015 ) . to assess the relationship between bmi and each of the psychological components using a multivariate regression model [ table 2 ] , only two components of nutritional habits ( b = 0.19 , p < 0.001 ) and dietary restraint ( b = 0.51 , p < 0.001 ) , could effectively predict bmi in women ; while depression and anxiety items had low predictive values for predicting bmi . in total , these four variables could predict 34% of the variance of the dependent indicator ( bmi ) . correlations between different psychological components and bmi multivariable linear regression analysis for predicting bmi on different psychological components the current study examined the value of a range of psychological components including ; depression , anxiety , nutritional behavior , and dietary restraint , in order to predict bmi as a main indicator of obesity among a sample of iranian women with sedentary jobs . our results demonstrated the hypothesis that there is an association between nutritional habits and dietary restraint with bmi in women aged higher than 25 years having sedentary jobs and working in some governmental organizations with a low workload . several studies have been conducted on girls and younger women in developed countries , which have revealed an association between restrained eating and higher bmi , however , those sub - groups generally contained women with healthier diets containing lower intakes of energy , carbohydrates , and proteins ; while in our study population , the main focus was directed at women with higher ages and considerably lower physical activity in their workplace , so in this sense , our population was different from other studied population . a previous systematic review has described employees with lower social positions as having higher stress levels and higher body weights , and these patterns were more apparent in women than in men . several studies have reported a link between stress and bmi levels . however , in a meta - analysis of longitudinal studies on stress and adiposity , it was established that stress promotes weight gain . work stress has also been associated with an increased bmi . in a follow - up study of a group of male and female employees , their findings showed increased alcohol consumption and decreased vegetable consumption in workers with low job control . on the other hand , in a group of low - income young mothers , longitudinal studies can provide insights into the direction and potential nature of associations among these variables . it is plausible that the obesity is a consequence of stress , for example reflecting the use of maladaptive coping strategies such as comfort eating or excessive sedentary behaviors . previous studies have reported that chronic stress is associated with binge or comfort type eating , reduced physical activity levels , and increased sedentary behaviors . preferences for more palatable , higher fat , energy dense foods have also been associated with stress . thus , it can be concluded that for women with sedentary jobs , who hold certain psychological attitudes , including ; inappropriate dietary restraint and poor dietary behavior , this combination can result in a high bmi , and as a consequence related complications such as cardiovascular disorders are significantly elevated . this is of particular importance when we know that the iranian women 's diet is currently undergoing negative changes , and fitness levels are falling . thus , the association between weight and bmi along with the previously mentioned psychological components will provide valuable information . on the other hand , improving dietary patterns and mental health in this sub - group of women , along with dietary regime modifications that result in improvements of bmi , are essential in order to prevent predictable health hazards . in this regard , some authors believed that lifestyle modification programs for prevention and treatment of adult - onset obesity currently focus on reducing situational and emotional overeating ; and hence a stronger emphasis on strategies that target habitual overeating can be warranted . this information could be valuable for identifying women in these types of conditions who might be at risk for obesity or its exacerbation due to underlying abnormal psychological attitudes . contrary to our expectations , the severity of anxiety and depression was not significantly correlated with bmi in this study . previous longitudinal studies have reported that depression in women is correlated with the development and persistence of high bmi levels . a recent review of eleven studies has suggested that depressive symptoms in women were associated with an approximately two- to three - fold increased risk of subsequent weight increase , when assessed after a period of 115 years . however , the results in these studies mainly focused on young women and consistent with our findings , little or poor association was found in the rates of obesity and mood disorders between the ages of 35 and 55 years . it seems that bmi can be potentially and widely affected by women 's hormonal fluctuations . furthermore , other confounders such as lifestyle and socioeconomic factors including ; education level and family income , may play a crucial role in the interaction between obesity and psychological factors . first , the study sample was composed of women employed in government organizations and completion of the questionnaire was done in their workplace . due to the special conditions of the workplace including ; time limits , job stress , responsibilities , and client referrals , it is possible that there was not enough attention paid to complete these questionnaires . in addition , the impact of nutritional habit , physical activity , and smoking was not considered in the study design . our study demonstrates the high value of nutritional habits and dietary restraint in predicting bmi status in women with sedentary jobs in governmental organizations in iran . however , predicting bmi through the use of depression and anxiety status assessments was not demonstrated in our survey .

background : understanding the association between psychological affective disturbances and anthropometric parameters , including body mass index ( bmi ) , is important . these issues may have potential preventive effects on weight reduction in relation to different aspects of women 's lifestyles and psychopathological states . the present study aimed to predict bmi based on psychological factors including ; depression , anxiety , dietary restraint , and nutritional habits , in a sample of women with sedentary jobs in several iranian governmental organizations.methods:two hundred consecutive women aged over 25 years , working on sedentary or low standing works such as banker , teachers , and employee in the social security organizations in isfahan , iran , were entered the study . to assess the severity of depression and anxiety symptoms , the beck depression inventory ii and the state - trait anxiety inventory were used , respectively . to assess nutritional habits , a self - administered questionnaire was designed , and to evaluate dietary restraint status , the ruderman questionnaire was used . to find the co - relationship between bmi and each of the psychological components , pearson 's correlation coefficient test was applied.results:to assess the relationship between bmi and each of the psychological components , a multivariate regression model was used . only two components of nutritional habits ( b = 0.19 , p < 0.001 ) and dietary restraint ( b = 0.51 , p < 0.001 ) could effectively predict bmi in iranian women ; while depression and anxiety components had low predictive values for predicting bmi . in total , these four variables could predict 34% of the variance of the dependent indicator ( bmi).conclusion : nutritional habits and dietary restraint have high value for predicting bmi status in women aged more than 25 years working in sedentary jobs , while bmi status could not be predicted by assessing depression or anxiety severity .

multiple myeloma is a malignant neoplasm that is characterized by a monoclonal proliferation of plasma cells . the clinical manifestations of the disease occur as a result of an expanding plasma cell mass in the bone marrow and other factors produced by these cells such as monoclonal immunoglobulin , bence - jones proteins and osteoclast activating factors . the common clinical signs and symptoms of multiple myeloma include pain in the bone , fatigue , anemia and infectious diseases . oral and maxillofacial manifestations as an initial sign or symptom of multiple myeloma are rare . in 12 - 15% of cases , oral involvement can be apparent as swelling , orofacial pain , mobility of teeth , numbness and paresthesia , hemorrhage , fracture and root resorption . we hereby present a case of multiple myeloma with first clinical manifestation as generalized gingival enlargement . a 58-year - old male patient was referred to our department because of the generalized enlargement of gingiva . the enlargement was first noted by the patient 6 months prior to the referral , and progressed steadily since then . the intraoral examination revealed soft , granular , friable , non - tender , and red / magenta enlargement that bled spontaneously . the enlargements were present on both buccal and lingual / palatal sides [ figure 1 ] . on physical examination , grade iii mobility was observed in 16 , 17 , 18 , 36 , 37 . the medical history of the patient included epilepsy for which he took prescribed medication with no occurrence of seizure for last 10 years . based on clinical presentation , a provisional diagnosis of inflammatory gingival enlargement routine blood investigations were done along with hiv and hepatitis b and sputum examination to rule out any leukemic infiltration and enlargement associated with tuberculosis . orthopantomography was advised , which revealed severe bone loss in 16 , 17 , 18 and 36 , 37 regions . after phase i therapy and consultation with the physician , gingivectomy was performed in anterior mandibular region and excised tissue was sent for histopathological examination . the patient was followed up every week [ figure 2 ] but after 1 month the clinical examination revealed recurrence of enlargement in the anterior mandibular region [ figure 3 ] . the subepithelial zone showed infiltration by sheets of plasma cells mainly mature with few being less differentiated [ figure 4 ] . based on these findings , serum protein electrophoresis and urine analysis for bence - jones proteins was carried out . subsequently , patient was advised to undergo cranial and pelvic radiography [ figures 6 and 7 ] . the patient was diagnosed as a case of multiple myeloma and chemotherapy was started with thalidomide . gingiva 10 days after excisional biopsy was taken recurrence after 1 month of biopsy areas of ulceration and sheets of plasma cells multinucleate and binucleate plasma cells pelvic radiograph showing osteolytic lesions skull radiograph showing osteolytic lesions normal gingiva 1 month after chemotherapy multiple myeloma is the most aggressive plasma cell neoplasia and most common primary malignancy of bone . it has a predilection for areas of active hematopoiesis such as the lumbar spine , ribs , and pelvic bones . jawbone involvement in multiple myeloma is common and often occurs in the advanced stages of the disease . jaw involvement in multiple myeloma was reported by bruce and royer to have a prevalence rate of 28.8% ( 17 of 59 total cases ) . epstein et al . , reported that 14.1% of 783 multiple myeloma cases had oral manifestations . lesions such as swelling , orofacial pain , mobility of teeth , numbness and paresthesia , hemorrhage , fracture and root resorption are more frequently found in the mandible than in the maxilla , especially in the posterior third and angle of the jaw , perhaps because of greater hematopoietic activity in these areas . a radiographic survey of patients with multiple myeloma reveals multiple well - defined punched out radiolucencies involving bone . these radiolucent areas of bone contain the abnormal plasma cell proliferations that characterize the disease . in our case , type 2 bone involvement was seen , which is by far , the most common presentation of multiple myeloma . in conclusion , dental surgeons can play an important role in the early recognition of oral lesions with underlying systemic disease , thus preventing the morbidity and mortality associated with such pathologies .

multiple myeloma is a malignant neoplasm that is characterized by a monoclonal proliferation of plasma cells . oral and maxillofacial manifestations as an initial sign or symptom of multiple myeloma are rare . a 58-year - old male patient presented with generalized gingival enlargement for last 6 months . based on clinical presentation , a diagnosis of gingival hyperplasia was made . after phase i therapy , excisional biopsy was taken in anterior mandibular region and excised tissue was sent for histopathological examination . the histopathology report revealed a lining of stratified squamous epithelium with foci of ulceration . the subepithelial zone showed infiltration by sheets of mainly binucleate and multinucleate plasma cells , few cells being less differentiated . rounded cytoplasmic inclusion bodies were identified in many of these cells . after a series of clinical investigations , a case of multiple myeloma was diagnosed . patient presenting with generalized gingival hyperplasia should be worked up for systemic disease like multiple myeloma .

cardiovascular disease ( cvd ) is a major cause of death and an important contributor to the wide and growing inequalities in mortality in high - income countries.1 as a result , the reduction of cvd has long been an objective of health policy in the uk and elsewhere , with a series of initiatives introduced with this as either a primary or secondary focus.2 recently , there has been interest in the potential of health checks ( a non - programmatic form of screening for cvd risk factors ) to contribute to reductions in overall cvd mortality and inequalities.3 4 the core theory for this approach is that it is possible to influence several of the factors ( such as smoking , high blood pressure and obesity ) that increase the risk of cvd , using primary prevention therapies such as statin and antihypertensive drugs and behavioural interventions to reduce cvd risk for those without cvd but currently at high risk . by systematically identifying and offering treatment to those at high risk , it is proposed that population health improvement ( and reductions in health inequality ) can be expected.3 however , there is controversy about whether health check programmes are effective in reducing the cvd risk of populations5 6 and whether an approach based on identifying individuals at high risk is appropriate.79 keep well ( kw ) was originally funded by the scottish government as a cvd prevention programme based on health checks in primary care.10 it was primarily designed to reduce cvd incidence and to narrow socioeconomic inequalities in cvd.11 previous evaluation work focused on implementation and reach of the programme , but questions surrounding the impact on health impacts could not be addressed.12 we aimed to assess whether trends in cvd - related mortality , hospitalisations and prescribing in those general practices involved in wave 1 of the kw programme changed after the implementation of the programme and whether they differed from the trends in non - kw practices . we used an interrupted time series design as the best available means of assessing its impact . the programme 's prespecified theory of change indicated that , if successful , there would be increased prescribing of preventive therapies for cvd risk factors in the short - to - medium term , and greater reductions in cvd hospitalisations and mortality in the medium - to - long term in the kw intervention practices compared with non - kw practices.11 the wave 1 kw model was introduced in 2006 using heath checks delivered in primary care for adults aged 4064 years living in areas of the greatest material deprivation.11 those eligible were offered a cvd - focused health check to identify modifiable risk factors ( using the assign score13 ) . those identified as having high cvd risk were offered a combination of advice , medical therapy and signposting or referral to other sources of support , including smoking cessation and weight loss programmes according to their identified risk factors.11 in wave 1 of kw , implementation occurred in the five community health partnerships ( chps ) with the highest proportion of their population living in the most deprived areas ; defined using the scottish index of multiple deprivation ( simd ) , which identifies small area concentrations of multiple deprivation across scotland.14 the five pilot sites were within four national health service ( nhs ) health boards ( nhs greater glasgow and clyde ( nhsggc ) , nhs lanarkshire , nhs lothian and nhs tayside ) . by 2013/2014 , nhs boards reported that a total of 251 734 health checks had been carried out , of which 85.5% were delivered to individuals residing in the two most deprived simd quintiles ( simd 1=70.8% , simd 2=14.7%).11 for the period january 1999 to august 2013 , we obtained monthly counts of deaths and incident hospitalisations for stroke and coronary heart disease ( chd ) among those aged 4065 years from the information services division ( isd ) of nhs national services scotland for two groups of general practices : ( 1 ) those taking part in wave 1 of kw and ( 2 ) all other scottish practices . incident cases were defined as admissions or deaths where there was no record of a hospitalisation for the same diagnosis in the 10 years prior to the index event . for each month , we obtained separate counts for kw wave 1 general practices and for all other scottish practices . we used corresponding population denominators for the kw and non - kw populations to calculate mortality and hospitalisation rates . chd was defined using international classification of diseases ( icd)9 codes 410 - 14 and icd10 codes i20 - 25 . stroke ( broadly defined as cerebrovascular disease ) was defined using icd9 codes 430 - 38 and icd10 codes i60 - 69 and g45 . prescribing data were obtained from isd for the period january 2002 to june 2011 for all general practices in scotland . we included prescriptions used in the primary prevention of cvd , categorised into statins , antihypertensives and antiplatelet drugs using classifications from the british national formulary . the data for each drug were provided as defined daily doses ( ddds ) by general practice and month of prescription . total general practice populations for each practice and each year were also provided by isd from the community health index ( chi ) database to serve as denominators . isd were provided with the date on which general practices started implementation of kw checks to allow coding of the prescriptions data as preimplementation or postimplementation without disclosure of the identity of individual practices . all data were provided at an aggregate level and the analytical approach described below reflects these data structure . we calculated monthly incidence rates for each health outcome for the kw and non - kw practice groups . we modelled the kw intervention effect using a covariate ( st ) coded as 0 at all time points before the intervention had started ( prior to october 2006 ) and coded as 1 at all time points after kw implementation ( after august 2010 ) . for time points between these dates , the covariate took a value between 0 and 1 , defined as the sum of the population size of practices that had started kw implementation at the given time point divided by the total population size of all kw practices by august 2010 . the intervention covariate ( st ) was therefore the proportion of the population size covered by kw at any given point in time as a fraction of the final kw population size . the interpretation of the exponential of the coefficient associated with st is a percentage change in the level of outcome after kw was implemented in all participating practices ( from september 2010 ) compared with before kw was implemented in any practice ( prior to october 2006 ) . mortality and hospitalisation rates were modelled using time series regression with autoregressive integrated moving average ( arima ) errors4 ( additional information in the online supplementary material s1 ) . our analytical strategy consisted of initially modelling the respective series without the intervention to obtain an adequate preliminary model that passed all diagnostic tests and then modelling and testing the effect of the intervention.15 the most appropriate and parsimonious model was selected using the akaike information criterion ( aic).16 web - table 1 provides further specifications of these models . to enable comparison with a control group , we employed the same analytical approach using data for non - kw practices . in addition , simultaneous time series of corresponding mortality and hospitalisation rates in non - kw practices were entered as a covariate in the arima error models for kw practices . this allowed us to control for factors external to the intervention which might affect mortality and hospitalisation rates , providing a net effect of the intervention.17 regression models were used to assess changes in prescribing after the introduction of kw for practices implementing wave 1 of the programme and for non - kw practices . arima error models were created for each drug category to account for autocorrelation and seasonality in the data.18 in further analyses , we examined the possibility of a variation of the intervention effect between different health boards by including interaction terms . data from 66 wave 1 kw practices were included for the additional analyses ( data from practices in nhs tayside did not include practice codes and could not be matched by isd to the practice - level data on prescription rates and so were excluded ) . generalised estimating equations ( gees ) were used to analyse prescribing data over time for practices in the remaining three health board areas . the quasi - likelihood under the independence model criterion ( qic ) statistic informed model selection.19 all analyses were undertaken using stata v.12.1 software ( stata corp , college station , texas , usa ; http://www.stata.com ) . the wave 1 kw model was introduced in 2006 using heath checks delivered in primary care for adults aged 4064 years living in areas of the greatest material deprivation.11 those eligible were offered a cvd - focused health check to identify modifiable risk factors ( using the assign score13 ) . those identified as having high cvd risk were offered a combination of advice , medical therapy and signposting or referral to other sources of support , including smoking cessation and weight loss programmes according to their identified risk factors.11 in wave 1 of kw , implementation occurred in the five community health partnerships ( chps ) with the highest proportion of their population living in the most deprived areas ; defined using the scottish index of multiple deprivation ( simd ) , which identifies small area concentrations of multiple deprivation across scotland.14 the five pilot sites were within four national health service ( nhs ) health boards ( nhs greater glasgow and clyde ( nhsggc ) , nhs lanarkshire , nhs lothian and nhs tayside ) . by 2013/2014 , nhs boards reported that a total of 251 734 health checks had been carried out , of which 85.5% were delivered to individuals residing in the two most deprived simd quintiles ( simd 1=70.8% , simd 2=14.7%).11 for the period january 1999 to august 2013 , we obtained monthly counts of deaths and incident hospitalisations for stroke and coronary heart disease ( chd ) among those aged 4065 years from the information services division ( isd ) of nhs national services scotland for two groups of general practices : ( 1 ) those taking part in wave 1 of kw and ( 2 ) all other scottish practices . incident cases were defined as admissions or deaths where there was no record of a hospitalisation for the same diagnosis in the 10 years prior to the index event . for each month , we obtained separate counts for kw wave 1 general practices and for all other scottish practices . we used corresponding population denominators for the kw and non - kw populations to calculate mortality and hospitalisation rates . chd was defined using international classification of diseases ( icd)9 codes 410 - 14 and icd10 codes i20 - 25 . stroke ( broadly defined as cerebrovascular disease ) was defined using icd9 codes 430 - 38 and icd10 codes i60 - 69 and g45 . prescribing data were obtained from isd for the period january 2002 to june 2011 for all general practices in scotland . we included prescriptions used in the primary prevention of cvd , categorised into statins , antihypertensives and antiplatelet drugs using classifications from the british national formulary . the data for each drug were provided as defined daily doses ( ddds ) by general practice and month of prescription . total general practice populations for each practice and each year were also provided by isd from the community health index ( chi ) database to serve as denominators . isd were provided with the date on which general practices started implementation of kw checks to allow coding of the prescriptions data as preimplementation or postimplementation without disclosure of the identity of individual practices . all data were provided at an aggregate level and the analytical approach described below reflects these data structure . we calculated monthly incidence rates for each health outcome for the kw and non - kw practice groups . we modelled the kw intervention effect using a covariate ( st ) coded as 0 at all time points before the intervention had started ( prior to october 2006 ) and coded as 1 at all time points after kw implementation ( after august 2010 ) . for time points between these dates , the covariate took a value between 0 and 1 , defined as the sum of the population size of practices that had started kw implementation at the given time point divided by the total population size of all kw practices by august 2010 . the intervention covariate ( st ) was therefore the proportion of the population size covered by kw at any given point in time as a fraction of the final kw population size . the interpretation of the exponential of the coefficient associated with st is a percentage change in the level of outcome after kw was implemented in all participating practices ( from september 2010 ) compared with before kw was implemented in any practice ( prior to october 2006 ) . mortality and hospitalisation rates were modelled using time series regression with autoregressive integrated moving average ( arima ) errors4 ( additional information in the online supplementary material s1 ) . our analytical strategy consisted of initially modelling the respective series without the intervention to obtain an adequate preliminary model that passed all diagnostic tests and then modelling and testing the effect of the intervention.15 the most appropriate and parsimonious model was selected using the akaike information criterion ( aic).16 web - table 1 provides further specifications of these models . to enable comparison with a control group , we employed the same analytical approach using data for non - kw practices . in addition , simultaneous time series of corresponding mortality and hospitalisation rates in non - kw practices were entered as a covariate in the arima error models for kw practices . this allowed us to control for factors external to the intervention which might affect mortality and hospitalisation rates , providing a net effect of the intervention.17 regression models were used to assess changes in prescribing after the introduction of kw for practices implementing wave 1 of the programme and for non - kw practices . arima error models were created for each drug category to account for autocorrelation and seasonality in the data.18 in further analyses , we examined the possibility of a variation of the intervention effect between different health boards by including interaction terms . data from 66 wave 1 kw practices were included for the additional analyses ( data from practices in nhs tayside did not include practice codes and could not be matched by isd to the practice - level data on prescription rates and so were excluded ) . generalised estimating equations ( gees ) were used to analyse prescribing data over time for practices in the remaining three health board areas . the quasi - likelihood under the independence model criterion ( qic ) statistic informed model selection.19 all analyses were undertaken using stata v.12.1 software ( stata corp , college station , texas , usa ; http://www.stata.com ) . we calculated monthly incidence rates for each health outcome for the kw and non - kw practice groups . we modelled the kw intervention effect using a covariate ( st ) coded as 0 at all time points before the intervention had started ( prior to october 2006 ) and coded as 1 at all time points after kw implementation ( after august 2010 ) . for time points between these dates , the covariate took a value between 0 and 1 , defined as the sum of the population size of practices that had started kw implementation at the given time point divided by the total population size of all kw practices by august 2010 . the intervention covariate ( st ) was therefore the proportion of the population size covered by kw at any given point in time as a fraction of the final kw population size . the interpretation of the exponential of the coefficient associated with st is a percentage change in the level of outcome after kw was implemented in all participating practices ( from september 2010 ) compared with before kw was implemented in any practice ( prior to october 2006 ) . mortality and hospitalisation rates were modelled using time series regression with autoregressive integrated moving average ( arima ) errors4 ( additional information in the online supplementary material s1 ) . our analytical strategy consisted of initially modelling the respective series without the intervention to obtain an adequate preliminary model that passed all diagnostic tests and then modelling and testing the effect of the intervention.15 the most appropriate and parsimonious model was selected using the akaike information criterion ( aic).16 web - table 1 provides further specifications of these models . to enable comparison with a control group , we employed the same analytical approach using data for non - kw practices . in addition , simultaneous time series of corresponding mortality and hospitalisation rates in non - kw practices were entered as a covariate in the arima error models for kw practices . this allowed us to control for factors external to the intervention which might affect mortality and hospitalisation rates , providing a regression models were used to assess changes in prescribing after the introduction of kw for practices implementing wave 1 of the programme and for non - kw practices . arima error models were created for each drug category to account for autocorrelation and seasonality in the data.18 in further analyses , we examined the possibility of a variation of the intervention effect between different health boards by including interaction terms . data from 66 wave 1 kw practices were included for the additional analyses ( data from practices in nhs tayside did not include practice codes and could not be matched by isd to the practice - level data on prescription rates and so were excluded ) . generalised estimating equations ( gees ) were used to analyse prescribing data over time for practices in the remaining three health board areas . the quasi - likelihood under the independence model criterion ( qic ) statistic informed model selection.19 all analyses were undertaken using stata v.12.1 software ( stata corp , college station , texas , usa ; http://www.stata.com ) . these show changes relative to the preintervention period for kw and non - kw practices . rates of incident hospitalisation and mortality for chd and stroke in kw and non - kw practices are shown in figure 1a d . chd mortality declined over time in kw and non - kw practices , with kw practices having consistently higher rates than non - kw practices ( figure 1a ) . all results refer to changes after adjusting for seasonal variation and the underlying temporal trends . following the introduction of kw health checks ( vertical line ) , a small increase of 0.4% in the chd mortality rate in the kw population was observed ( 95% ci 5.2% to 6.3% ; table 1 ) . in non - kw practices , chd mortality rates decreased by 0.3% ( 95% ci 2.7% to 2.2% ) over the same period . modelled changes in mortality , hospitalisation and prescriptions in kw and non - kw practices comparing preintervention and postintervention periods * percentage change in the mean level of outcome after kw was implemented in all participating practices ( from september 2010 ) compared with the mean level of outcome before kw was implemented in any practice ( prior to october 2006 ) . chd , coronary heart disease ; ddd , defined daily dose ; gee , generalised estimating equation ; ggc , greater glasgow and clyde ; kw , keep well ; nhs , national health service . ( a d ) mortality and incident hospitalisation rates ( coronary heart disease ( chd ) and stroke ) in keep well ( black line ) and non - keep well ( grey line ) practices ( vertical line indicates introduction of keep well intervention ) . for stroke mortality , a high degree of month - to - month variability was observed , with an overall downward trend for both groups , with higher rates in kw wave 1 practices ( figure 1b ) . comparing periods before and after the introduction of kw checks , stroke mortality increased by 6.7% ( 95% ci 2.6% to 16.9% ) in kw practices . the adjusted mortality rate in non - kw practices showed no appreciable change ( 0.2% ; 95% ci 6.7% to 6.7% ) during the same period . chd incident hospitalisation rates decreased by 1.1% ( 95% ci 3.4% to 1.3% ) in kw practices , comparing the periods before and after the introduction of health checks ( figure 1c ) . in non - kw practices , no appreciable change in chd incident hospitalisation rates was found ( 0.1% ; 95% ci 1.8% to 1.7% ) comparing the same two periods ( table 1 ) . stroke incident hospitalisations showed a decrease of 1.5% ( 95% ci 4.4% to 1.6% ) in kw practices and no appreciable change ( 0.1% ; 95% ci 1.5% to 1.3% ) in non - kw practices ( table 1 ) . the inclusion of the data series of non - kw practices did not alter results for kw practices appreciably ( table 1 ) . in kw and non - kw practices , prescription rates ( expressed as ddds per 100 patients ) for all drug classes increased over time , although rates for antiplatelets showed only a small increase . prescription rates for statins were consistently higher in kw than in non - kw practices ( figure 2a ) , while rates for antihypertensive drugs were similar ( figure 2b ) . for antiplatelet prescriptions , rates started to decline from 2009 onwards ( figure 2c ) . there was some degree of seasonality in rates , in particular for statins and antihypertensive drugs . ( a c ) mean rates of defined daily doses ( ddds ) prescribed by drug class in keep well ( black line ) and non - keep well ( grey line ) practices ( vertical line indicates introduction of keep well intervention ) . statin prescription rates showed a small increase of 0.4% ( 95% ci 10.4% to 12.5% ) in kw practices following the introduction of health checks ( table 1 ) . in non - kw practices , comparison of the corresponding periods showed a decrease in the statin prescription rate of 1.5% ( 95% ci 9.4% to 7.2% ) relative to the underlying trend . in kw practices , the antihypertensive prescription rate decreased by 2.5% ( 95% ci 12.3% to 8.4% ) following the introduction of the kw intervention , whereas in non - kw practices , a decrease of 1.6% ( 95% ci 7.1% to 4.3% ) was observed . the prescription rate for antiplatelets decreased by 0.9% ( 95% ci 6.5% to 5.0% ) in kw practices and by 2.4% in non - kw practices ( 95% ci 10.1% to 6.0% ) . we considered the possibility that the kw intervention might be more effective in some nhs boards than others , due to board - level variations in the degree of engagement with the programme by including interaction terms between health board and the intervention ( table 1 ) . for statins , a negative association was found between the intervention and prescription rates , suggesting a decrease in prescription rates after the introduction of kw . this decrease was modified by health board area , with nhsggc showing a larger decrease than the other two health boards ( 3.9% ( 95% ci 7.6% to 0.1% ) ) . prescription rates for antihypertensive drugs declined at a higher rate in lothian than in the remaining two health board areas ( 5.7% ; 95% ci 11.2% to 0.04% ) . trends in antiplatelet prescription rates were not altered by the introduction of the kw intervention ( figure 2c and table 1 ) . in nhsggc and lothian , the intervention was associated with an increase in rates of ddds , whereas in lanarkshire , a relative decrease was observed . the gee models without the interaction terms had a smaller qic value than those with interactions , partly supporting our conclusions that changes in prescription rates were not markedly modified by health board location . these show changes relative to the preintervention period for kw and non - kw practices . rates of incident hospitalisation and mortality for chd and stroke in kw and non - kw practices are shown in figure 1a d . chd mortality declined over time in kw and non - kw practices , with kw practices having consistently higher rates than non - kw practices ( figure 1a ) . all results refer to changes after adjusting for seasonal variation and the underlying temporal trends . following the introduction of kw health checks ( vertical line ) , a small increase of 0.4% in the chd mortality rate in the kw population was observed ( 95% ci 5.2% to 6.3% ; table 1 ) . in non - kw practices , chd mortality rates decreased by 0.3% ( 95% ci 2.7% to 2.2% ) over the same period . modelled changes in mortality , hospitalisation and prescriptions in kw and non - kw practices comparing preintervention and postintervention periods * percentage change in the mean level of outcome after kw was implemented in all participating practices ( from september 2010 ) compared with the mean level of outcome before kw was implemented in any practice ( prior to october 2006 ) . chd , coronary heart disease ; ddd , defined daily dose ; gee , generalised estimating equation ; ggc , greater glasgow and clyde ; kw , keep well ; nhs , national health service . ( a d ) mortality and incident hospitalisation rates ( coronary heart disease ( chd ) and stroke ) in keep well ( black line ) and non - keep well ( grey line ) practices ( vertical line indicates introduction of keep well intervention ) . for stroke mortality , a high degree of month - to - month variability was observed , with an overall downward trend for both groups , with higher rates in kw wave 1 practices ( figure 1b ) . comparing periods before and after the introduction of kw checks , stroke mortality increased by 6.7% ( 95% ci 2.6% to 16.9% ) in kw practices . the adjusted mortality rate in non - kw practices showed no appreciable change ( 0.2% ; 95% ci 6.7% to 6.7% ) during the same period . chd incident hospitalisation rates decreased by 1.1% ( 95% ci 3.4% to 1.3% ) in kw practices , comparing the periods before and after the introduction of health checks ( figure 1c ) . in non - kw practices , no appreciable change in chd incident hospitalisation rates was found ( 0.1% ; 95% ci 1.8% to 1.7% ) comparing the same two periods ( table 1 ) . stroke incident hospitalisations showed a decrease of 1.5% ( 95% ci 4.4% to 1.6% ) in kw practices and no appreciable change ( 0.1% ; 95% ci 1.5% to 1.3% ) in non - kw practices ( table 1 ) . the inclusion of the data series of non - kw practices did not alter results for kw practices appreciably ( table 1 ) . in kw and non - kw practices , prescription rates ( expressed as ddds per 100 patients ) for all drug classes increased over time , although rates for antiplatelets showed only a small increase . prescription rates for statins were consistently higher in kw than in non - kw practices ( figure 2a ) , while rates for antihypertensive drugs were similar ( figure 2b ) . for antiplatelet prescriptions , rates started to decline from 2009 onwards ( figure 2c ) . there was some degree of seasonality in rates , in particular for statins and antihypertensive drugs . ( a c ) mean rates of defined daily doses ( ddds ) prescribed by drug class in keep well ( black line ) and non - keep well ( grey line ) practices ( vertical line indicates introduction of keep well intervention ) . statin prescription rates showed a small increase of 0.4% ( 95% ci 10.4% to 12.5% ) in kw practices following the introduction of health checks ( table 1 ) . in non - kw practices , comparison of the corresponding periods showed a decrease in the statin prescription rate of 1.5% ( 95% ci 9.4% to 7.2% ) relative to the underlying trend . in kw practices , the antihypertensive prescription rate decreased by 2.5% ( 95% ci 12.3% to 8.4% ) following the introduction of the kw intervention , whereas in non - kw practices , a decrease of 1.6% ( 95% ci 7.1% to 4.3% ) was observed . the prescription rate for antiplatelets decreased by 0.9% ( 95% ci 6.5% to 5.0% ) in kw practices and by 2.4% in non - kw practices ( 95% ci 10.1% to 6.0% ) . we considered the possibility that the kw intervention might be more effective in some nhs boards than others , due to board - level variations in the degree of engagement with the programme by including interaction terms between health board and the intervention ( table 1 ) . for statins , a negative association was found between the intervention and prescription rates , suggesting a decrease in prescription rates after the introduction of kw . this decrease was modified by health board area , with nhsggc showing a larger decrease than the other two health boards ( 3.9% ( 95% ci 7.6% to 0.1% ) ) . prescription rates for antihypertensive drugs declined at a higher rate in lothian than in the remaining two health board areas ( 5.7% ; 95% ci 11.2% to 0.04% ) . trends in antiplatelet prescription rates were not altered by the introduction of the kw intervention ( figure 2c and table 1 ) . in nhsggc and lothian , the intervention was associated with an increase in rates of ddds , whereas in lanarkshire , a relative decrease was observed . the gee models without the interaction terms had a smaller qic value than those with interactions , partly supporting our conclusions that changes in prescription rates were not markedly modified by health board location . six years after the introduction of health checks in kw wave 1 practices , any effect of the intervention on trends in chd and stroke mortality , incident hospitalisations and prescription rates was likely to be very small . once the seasonal pattern and strong downward temporal trend are taken into account , our results are consistent with both increases and decreases in mortality and hospitalisation following the intervention , but based on the cis , we can fairly reliably exclude reductions of more than 12% in hospitalisations and of more than 36% in mortality ( because of the greater uncertainty due to the smaller number of deaths ) . comparing the periods before and after the start of kw health checks , we found small relative declines in prescribing of statins , antihypertensive and antiplatelet drugs . the data have complete national coverage and are likely to accurately estimate the incidence of cvd and cvd - related prescribing in scotland with a low likelihood of missing cases or misclassification . the arima models were supported by relatively long time series available both before and after the intervention and accounted for random variation , temporal trends and seasonality . the health outcomes were clearly linked to the original defined purpose of wave 1 of kw and allowed the theory to be evaluated at different points after implementation . wave 1 because subsequent waves had a less consistent focus on cvd , operated in more diverse settings where consistent monitoring data were not always available , and had a shorter duration of follow - up . we were not able to access individual - level data nationally because information sharing was not agreed with general practitioners ( gps ) . this meant that we had to define the eligible population at practice level ( for prescriptions ) or for specific age groups within practices ( for hospitalisations and mortality ) and no adjustments for patient characteristics was possible . however , any changes in practice configuration ( age structure , sex and deprivation ) would be similar in kw and in non - kw practices . since the analyses compare changes within kw practices and changes within non - kw practices , it is unlikely that aggregation would affect results . furthermore , aggregation meant a reduction in the power of the study to detect any impact of the intervention by misclassifying some untreated individuals as treated . similarly , it is likely that the comparison group ( the rest of the scottish population ) included some people who received a health check as part of subsequent waves of kw . the effect of both of these factors would be to dilute the measured impact of kw , creating a bias towards a null result . however , kw recipients other than wave 1 formed a very small proportion of the overall scottish population , so the impact of this bias is likely to be small . it is also important to recognise that analyses at individual level ( as performed in some local studies ) provide information only on efficacy ( ie , potential benefits for those who actually received checks ) rather than on real - life population impact . although aggregate mortality and hospitalisation data were used in our analyses , this approach is likely to give a more realistic estimate of the real - life effectiveness of the programme . information was available on the approximate dates on which each general practice started health checks as part of kw wave 1 but not on the speed or intensity with which the intervention was implemented . if some practices implemented the programme in a less vigorous way , this may have diluted the impact of kw . our analyses of prescription rates including interaction terms between health board and the intervention variable attempted to account for overall variations between practices in different health boards . clinical guidelines first published in 2007 recommended that cvd risk should be assessed in all individuals aged 40 years and above at least every 5 years , although this was based on low - quality evidence.20 guidelines also recommend ( for those at high risk ) prescription of antiplatelet therapy and statins , and antihypertensive therapy for those with hypertension.2123 however , the effectiveness of cvd screening programmes has been disputed for some time,24 25 with the latest cochrane reviews concluding that there is evidence for a reduction in risk factors but not mortality or morbidity and that universal health checks should therefore be abandoned.26 27 evaluation of health checks in england have found that , on average , there is no evidence of an increased identification of undiagnosed disease28 but some evidence of decreased cvd risk.29 30 a recent study reported an increased detection of hypercholesterolaemia but no increase in detection of obesity , smoking and hypertension.31 however , these studies do not account for secular trends ( eg , declining smoking rates ) and one is subject to a number of important biases including regression to the mean and selection bias.32 although these results are somewhat imprecise , they make it very unlikely that the implementation of the first wave of kw health checks in scotland was associated with substantial or important changes in cvd outcomes or prescribing . it is possible though that the overall level of health check coverage was not sufficient to produce a measurable effect or that participants did not receive all the interventions that were originally planned . this , however , reflects the nature of an effectiveness study ( rather than efficacy ) and is perhaps a more realistic assessment of the real - world impact . it is possible that while the individual components of the kw check ( drug therapy , lifestyle advice , etc ) are effective in trial situations , other factors including the challenges of programme delivery and the individual situations of people in deprived circumstances mean that their real - life effectiveness is much less . this may also include variations in care delivered by different healthcare professionals ; that is , nurses , gps and other staff . our analyses include a 6 years follow - up , but it is possible that clear benefits might only emerge later because of the length of time required for primary prevention to reduce risk factors and decrease morbidity and mortality at population level . given the change in prescription rates was found to be small , it seems unlikely that mortality and hospitalisation rates would change . the data have complete national coverage and are likely to accurately estimate the incidence of cvd and cvd - related prescribing in scotland with a low likelihood of missing cases or misclassification . the arima models were supported by relatively long time series available both before and after the intervention and accounted for random variation , temporal trends and seasonality . the health outcomes were clearly linked to the original defined purpose of wave 1 of kw and allowed the theory to be evaluated at different points after implementation . wave 1 because subsequent waves had a less consistent focus on cvd , operated in more diverse settings where consistent monitoring data were not always available , and had a shorter duration of follow - up . we were not able to access individual - level data nationally because information sharing was not agreed with general practitioners ( gps ) . this meant that we had to define the eligible population at practice level ( for prescriptions ) or for specific age groups within practices ( for hospitalisations and mortality ) and no adjustments for patient characteristics was possible . however , any changes in practice configuration ( age structure , sex and deprivation ) would be similar in kw and in non - kw practices . since the analyses compare changes within kw practices and changes within non - kw practices , it is unlikely that aggregation would affect results . furthermore , aggregation meant a reduction in the power of the study to detect any impact of the intervention by misclassifying some untreated individuals as treated . similarly , it is likely that the comparison group ( the rest of the scottish population ) included some people who received a health check as part of subsequent waves of kw . the effect of both of these factors would be to dilute the measured impact of kw , creating a bias towards a null result . however , kw recipients other than wave 1 formed a very small proportion of the overall scottish population , so the impact of this bias is likely to be small . it is also important to recognise that analyses at individual level ( as performed in some local studies ) provide information only on efficacy ( ie , potential benefits for those who actually received checks ) rather than on real - life population impact . although aggregate mortality and hospitalisation data were used in our analyses , this approach is likely to give a more realistic estimate of the real - life effectiveness of the programme . information was available on the approximate dates on which each general practice started health checks as part of kw wave 1 but not on the speed or intensity with which the intervention was implemented . if some practices implemented the programme in a less vigorous way , this may have diluted the impact of kw . our analyses of prescription rates including interaction terms between health board and the intervention variable attempted to account for overall variations between practices in different health boards . clinical guidelines first published in 2007 recommended that cvd risk should be assessed in all individuals aged 40 years and above at least every 5 years , although this was based on low - quality evidence.20 guidelines also recommend ( for those at high risk ) prescription of antiplatelet therapy and statins , and antihypertensive therapy for those with hypertension.2123 however , the effectiveness of cvd screening programmes has been disputed for some time,24 25 with the latest cochrane reviews concluding that there is evidence for a reduction in risk factors but not mortality or morbidity and that universal health checks should therefore be abandoned.26 27 evaluation of health checks in england have found that , on average , there is no evidence of an increased identification of undiagnosed disease28 but some evidence of decreased cvd risk.29 30 a recent study reported an increased detection of hypercholesterolaemia but no increase in detection of obesity , smoking and hypertension.31 however , these studies do not account for secular trends ( eg , declining smoking rates ) and one is subject to a number of important biases including regression to the mean and selection bias.32 although these results are somewhat imprecise , they make it very unlikely that the implementation of the first wave of kw health checks in scotland was associated with substantial or important changes in cvd outcomes or prescribing . it is possible though that the overall level of health check coverage was not sufficient to produce a measurable effect or that participants did not receive all the interventions that were originally planned . this , however , reflects the nature of an effectiveness study ( rather than efficacy ) and is perhaps a more realistic assessment of the real - world impact . it is possible that while the individual components of the kw check ( drug therapy , lifestyle advice , etc ) are effective in trial situations , other factors including the challenges of programme delivery and the individual situations of people in deprived circumstances mean that their real - life effectiveness is much less . this may also include variations in care delivered by different healthcare professionals ; that is , nurses , gps and other staff . our analyses include a 6 years follow - up , but it is possible that clear benefits might only emerge later because of the length of time required for primary prevention to reduce risk factors and decrease morbidity and mortality at population level . given the change in prescription rates was found to be small , it seems unlikely that mortality and hospitalisation rates would change . . however , our findings are consistent with systematic reviews suggesting that delivering these interventions in a systematic population wide way may not be effective . the kw approach ( like the english health check programme ) is essentially a screening programme , and more rigorous evidence of effectiveness is required before it is adopted . our interrupted time series approach could be criticised as a weaker design than cluster randomisation or stepped wedge approaches . we suggest that for large - scale population interventions , randomised approaches are more feasible than currently assumed by policymakers , and we think they should be more widely used . , these could prevent cardiovascular disease ( cvd ) in asymptomatic individuals and narrow existing socioeconomic inequalities in health . health check programmes have been implemented to specifically target those at high risk of cvd , but there is controversy about whether these are likely to be effective at the population level and about whether a screening approach is justified . a key early aim of the scottish keep well programme was to narrow socioeconomic inequalities in cvd by targeting individuals living in deprived areas , screening for cvd risk factors and then providing drug therapies ( eg , statins and antihypertensives ) and lifestyle advice for those at high risk . our study suggests that , after accounting for seasonal and secular trends , the effect of the keep well intervention on cvd mortality and incident hospitalisation was unlikely to be substantial .

backgroundeffective interventions are available to reduce cardiovascular risk . recently , health check programmes have been implemented to target those at high risk of cardiovascular disease ( cvd ) , but there is much debate whether these are likely to be effective at population level . this paper evaluates the impact of wave 1 of keep well , a scottish health check programme , on cardiovascular outcomes.methodsinterrupted time series analyses were employed , comparing trends in outcomes in participating and non - participating practices before and after the introduction of health checks . health outcomes are defined as cvd mortality , incident hospitalisations and prescribing of cardiovascular drugs.resultsafter accounting for secular trends and seasonal variation , coronary heart disease mortality and hospitalisations changed by 0.4% ( 95% ci 5.2% to 6.3% ) and 1.1% ( 3.4% to 1.3% ) in keep well practices and by 0.3% ( 2.7% to 2.2% ) and 0.1% ( 1.8% to 1.7% ) in non - keep well practices , respectively , following the intervention . adjusted changes in prescribing in keep well and non - keep well practices were 0.4% ( 10.4% to 12.5% ) and 1.5% ( 9.4% to 7.2% ) for statins ; 2.5% ( 12.3% to 8.4% ) and 1.6% ( 7.1% to 4.3% ) for antihypertensive drugs ; and 0.9% ( 6.5% to 5.0% ) and 2.4% ( 10.1% to 6.0% ) for antiplatelet drugs.conclusionsany impact of the keep well health check intervention on cvd outcomes and prescribing in scotland was very small . findings do not support the use of the screening approach used by current health check programmes to address cvd . we used an interrupted time series method , but evaluation methods based on randomisation are feasible and preferable and would have allowed more reliable conclusions . these should be considered more often by policymakers at an early stage in programme design when there is uncertainty regarding programme effectiveness .

we designed a cohort study in catalonia ( northeast spain ) that included two groups of patients aged 3074 years : consecutive first acute myocardial infarction patients without diabetes who survived at least 28 days after index myocardial infarction symptom onset and a random sample of type 2 diabetic patients without coronary heart disease . the study was approved by a local ethics committee ( institut municipal d'investigaci mdica ) and complied with all the laws and international ethics guidelines ( declaration of helsinki ) . acute myocardial infarction patients were recruited consecutively between 1990 and 2003 in the context of the population - based regicor ( registre giron del cor [ girona heart registry ] ) study ( 17 ) . all 10 public and private hospitals in the region participate in regicor , using the same standards to diagnose acute myocardial infarction : non q - wave and q - wave myocardial infarction , determined by a discharge electrocardiogram in patients who presented with chest pain lasting > 20 min on admission , followed by typical changes in serial electrocardiograms and an abnormal increase in the cardiac enzymes or troponin value curve . we excluded patients outside the selected age range and those meeting the national diabetes data group 1979 and american diabetes association 1997 criteria for diabetes ( 18,19 ) , including patients with two consecutive fasting plasma glucose values 7.8 or 7 mmol / l during admission , before and after 1998 , previous acute myocardial infarction , or other diseases that shortened life expectancy to < 1 year . type 2 diabetic patients were randomly recruited between 1993 and 1998 in the 53 primary health care centers in catalonia participating in the gedaps ( grupo de estudio de la diabetes en atencin primaria de salud [ primary health care diabetes study group ] ) network . diagnosis of type 2 diabetes was based on national diabetes data group 1979 criteria , i.e. , two fasting plasma glucose values 7.8 mmol / l or 2-h plasma glucose values 11.1 we excluded patients not within the age range of the study , with bmi < 22 kg / m , diseases that shortened life expectancy to < 1 year or history of any coronary heart event or ketoacidosis . for all participants , we recorded age , sex , follow - up ( in days ) , and , from medical records , their history of dyslipidemia and hypertension and smoking habit . for diabetic patients we also collected a1c concentration ( fructosamine levels were not considered ) , treatment ( diet , oral drugs , or insulin ) , and duration of diabetes in years . in acute myocardial infarction patients , , we completed up to 10 years follow - up by telephone , medical examination , or clinical record review . the individual end points considered were all - cause death , coronary death , stroke death , cardiovascular death , nonfatal acute myocardial infarction , and unstable angina . two composite end points were used in the analyses : cardiovascular mortality ( coronary , stroke , and other cardiovascular deaths ) and coronary heart disease incidence ( unstable angina or fatal or nonfatal acute myocardial infarction ) . the first cardiovascular event , regardless of its severity , was considered in the analysis . deaths were considered of coronary heart disease origin in cases of suggestive necropsy findings , clinical records of hospitalized patients , or the presence on death certificates of icd-9 codes 410412 , 414 , 429.9 , 798.1 , and 798.2 or icd-10 codes i210i214 , i219i229 , i236 , i240i249 , i250i259 , i46.1 , r960 , and r961 ) . stroke was defined by suggestive necropsy findings , clinical records for hospitalized patients , or icd-9 codes 430434 and 436438 ( excluding 437.4437.8 ) or icd-10 codes i619i639 , i64 , i670i679 , i688 , and i690i698 . other cardiovascular deaths were similarly defined . the applicable icd-9 codes were 401405 , 426428 , and 429.1429.9 and icd-10 codes were i10i110 , i50i52 , i440i499 , i500i509 , i250 , and i511i519 . nonfatal acute myocardial infarction was diagnosed when patients presented with chest pain lasting > 20 min on admission , followed by typical changes in serial electrocardiograms and an abnormal increase in the cardiac enzymes or troponin value curve . unstable angina during follow - up was diagnosed by the presence of angina symptoms without an abnormal increase in the cardiac enzymes or troponin and with electrocardiographic changes in serial electrocardiograms or when , with or without electrocardiographic changes , suggestive symptoms were recorded during the event and confirmed by a positive stress test , with or without isotopic stress gammagraphy , or a positive coronary angiogram ( stenosis > 70% ) . our study was sufficiently powered ( > 90% ) to identify a statistically significant hazard ratio ( hr ) 0.80 for type 2 diabetic patients compared with first acute myocardial infarction patients , assuming > 25% 10-year coronary heart disease incidence among the latter and a correlation < 0.3 of the type 2 diabetes variable with potential confounders . the two groups were approximately equally represented in the study : 51% diabetic and 49% myocardial infarction patients . differences between myocardial infarction and diabetic patients at 10 years were assessed by a test for categorical variables and by student 's t test for continuous variables or the nonparametric equivalents , as appropriate . cox proportional hazards models were fitted to estimate the adjusted hr of cardiovascular mortality and coronary heart disease incidence at 10 years . demographic , comorbidity , clinical , and severity variables that showed at least marginally significant differences ( p 0.10 ) between type 2 diabetes and acute myocardial infarction patients , as well as variables considered important based on clinical judgment , were included as potential confounders in the multivariate analyses . because the two cohorts were conducted at different time points , the results were adjusted for recruitment year , and a sensitivity analyses was performed in 2,260 diabetic patients and 828 acute myocardial infarction patients recruited in the same time periods ( 19931998 ) . we considered tertiles of diabetes duration , comparing the third tertile versus the first and second together and two groups of glycemic control ( a1c < 7% and 7% ) and therapy ( diet alone , only oral drugs , and insulin ) . survival curves were estimated with the kaplan - meier method and compared by mantel - cox statistics . calculations were made with r ( 2.6.2 package ; the r foundation for statistical computing , free software foundation , boston , ma ) . for all participants , we recorded age , sex , follow - up ( in days ) , and , from medical records , their history of dyslipidemia and hypertension and smoking habit . for diabetic patients we also collected a1c concentration ( fructosamine levels were not considered ) , treatment ( diet , oral drugs , or insulin ) , and duration of diabetes in years . in acute myocardial infarction patients , in 2008 , we completed up to 10 years follow - up by telephone , medical examination , or clinical record review . the individual end points considered were all - cause death , coronary death , stroke death , cardiovascular death , nonfatal acute myocardial infarction , and unstable angina . two composite end points were used in the analyses : cardiovascular mortality ( coronary , stroke , and other cardiovascular deaths ) and coronary heart disease incidence ( unstable angina or fatal or nonfatal acute myocardial infarction ) . the first cardiovascular event , regardless of its severity , was considered in the analysis . deaths were considered of coronary heart disease origin in cases of suggestive necropsy findings , clinical records of hospitalized patients , or the presence on death certificates of icd-9 codes 410412 , 414 , 429.9 , 798.1 , and 798.2 or icd-10 codes i210i214 , i219i229 , i236 , i240i249 , i250i259 , i46.1 , r960 , and r961 ) . stroke was defined by suggestive necropsy findings , clinical records for hospitalized patients , or icd-9 codes 430434 and 436438 ( excluding 437.4437.8 ) or icd-10 codes i619i639 , i64 , i670i679 , i688 , and i690i698 . other cardiovascular deaths were similarly defined . the applicable icd-9 codes were 401405 , 426428 , and 429.1429.9 and icd-10 codes were i10i110 , i50i52 , i440i499 , i500i509 , i250 , and i511i519 . nonfatal acute myocardial infarction was diagnosed when patients presented with chest pain lasting > 20 min on admission , followed by typical changes in serial electrocardiograms and an abnormal increase in the cardiac enzymes or troponin value curve . unstable angina during follow - up was diagnosed by the presence of angina symptoms without an abnormal increase in the cardiac enzymes or troponin and with electrocardiographic changes in serial electrocardiograms or when , with or without electrocardiographic changes , suggestive symptoms were recorded during the event and confirmed by a positive stress test , with or without isotopic stress gammagraphy , or a positive coronary angiogram ( stenosis > 70% ) . our study was sufficiently powered ( > 90% ) to identify a statistically significant hazard ratio ( hr ) 0.80 for type 2 diabetic patients compared with first acute myocardial infarction patients , assuming > 25% 10-year coronary heart disease incidence among the latter and a correlation < 0.3 of the type 2 diabetes variable with potential confounders . the two groups were approximately equally represented in the study : 51% diabetic and 49% myocardial infarction patients . differences between myocardial infarction and diabetic patients at 10 years were assessed by a test for categorical variables and by student 's t test for continuous variables or the nonparametric equivalents , as appropriate . cox proportional hazards models were fitted to estimate the adjusted hr of cardiovascular mortality and coronary heart disease incidence at 10 years . demographic , comorbidity , clinical , and severity variables that showed at least marginally significant differences ( p 0.10 ) between type 2 diabetes and acute myocardial infarction patients , as well as variables considered important based on clinical judgment , were included as potential confounders in the multivariate analyses . because the two cohorts were conducted at different time points , the results were adjusted for recruitment year , and a sensitivity analyses was performed in 2,260 diabetic patients and 828 acute myocardial infarction patients recruited in the same time periods ( 19931998 ) . we considered tertiles of diabetes duration , comparing the third tertile versus the first and second together and two groups of glycemic control ( a1c < 7% and 7% ) and therapy ( diet alone , only oral drugs , and insulin ) . survival curves were estimated with the kaplan - meier method and compared by mantel - cox statistics . calculations were made with r ( 2.6.2 package ; the r foundation for statistical computing , free software foundation , boston , ma ) . the study included 2,260 type 2 diabetic patients and 2,154 first acute myocardial infarction patients who survived 28 days after symptom onset . acute myocardial infarction patients were younger and less frequently than the diabetic participants were women , hypertensive , and dyslipidemic . baseline characteristics in non coronary heart disease type 2 diabetic patients compared with nondiabetic first acute myocardial infarction survivors data are n ( % ) or means sd unless specified otherwise . ami , acute myocardial infarction . * some missing values in these variables ( < 5% ) . evaluated in a sample of 499 patients at 6 months . included only patients with a1c : fructosamine alone was used in 497 ( 22% ) patients . the incidence rate for all event types was significantly worse among acute myocardial infarction patients , except for stroke death and unstable angina ( table 2 ) . these differences held after adjustment for sex , age , and baseline dyslipidemia , hypertension , and recruitment year . these findings were similar in both sexes , except for unstable angina : diabetic women had a significantly lower risk . incidence rate and adjusted hr of different cardiovascular end points at 10 years for initially non coronary heart disease diabetic patients compared with nondiabetic first acute myocardial infarction survivors in all participants and by sex data are n ( % ) or hr ( 95% ci ) . all models are adjusted for sex , age , recruitment year , and baseline dyslipidemia and hypertension . figure 1 shows the kaplan - meier curves of cardiovascular mortality and coronary heart disease incidence , respectively , comparing myocardial infarction patients with diabetic patients stratified by duration of type 2 diabetes , a1c , and therapy . all type 2 diabetes strata had significantly lower risk of both end points than acute myocardial infarction patients . free of cardiovascular mortality ( panel 1 ) and free of coronary heart disease ( panel 2 ) survival curves among initially non coronary heart disease diabetic patients compared with nondiabetic first acute myocardial infarction ( ami ) survivors . a : according to time of evolution of type 2 diabetes ( cut point 8 years ) . c : according to diabetes treatment ( diet alone , only oral drugs , or insulin ) . all subgroups of diabetic patients had significantly lower risk of both composite end points than their acute myocardial infarction counterparts ( table 3 ) . patients with type 2 diabetes receiving insulin therapy , with > 8 years of disease duration and a1c 7% were at significantly higher risk of coronary heart disease incidence or cardiovascular mortality than were those receiving dietary therapy alone or only oral drugs , with 8 years of evolution and a1c < 7% , respectively . other cutoff points of a1c ( < 6.5 vs. 6.5% and < 6.5 , 6.57.5 , and > 7.5% ) showed similar hrs for cardiovascular mortality and coronary heart disease incidence . at the cutoff point < 6.5 vs. 6.5% , the hr for cardiovascular mortality was 0.16 ( 95% ci 0.100.27 ) and 0.23 ( 0.170.32 ) and for coronary heart disease incidence was 0.34 ( 0.250.45 ) and 0.44 ( 0.360.53 ) , respectively . for the cutoff points < 6.5 , 6.57.5 , and > 7.5% , the hr for cardiovascular mortality was 0.16 ( 0.100.27 ) , 0.22 ( 0.140.36 ) , and 0.24 ( 0.160.35 ) and for coronary heart disease incidence was 0.34 ( 0.250.45 ) , 0.37 ( 0.280.50 ) , and 0.48 ( 0.380.60 ) , respectively . adjusted hr of 10-year end points for type 2 diabetes patients by baseline tertiles of duration , glycemic control , and therapy compared with nondiabetic first acute myocardial infarction survivors data are hr ( 95% ci ) . p < 0.05 compared with reference category ( myocardial infarction patients ) . p < 0.05 compared with immediately previous category . all models are adjusted for sex , age , recruitment year , and baseline dyslipidemia and hypertension . for example , the adjusted hr for 10-year coronary heart disease incidence and cardiovascular mortality was 0.53 ( 95% ci 0.420.67 ) and 0.30 ( 0.200.44 ) and 0.24 ( 0.170.34 ) and 0.23 ( 0.120.44 ) in men and women , respectively . the results of our study indicate that type 2 diabetic patients without previous coronary heart disease not only have lower 10-year cardiovascular mortality but also have lower coronary heart disease incidence than first acute myocardial infarction patients without diabetes . results of previous studies may have found similar cardiovascular event rates due to differences in prognosis . in some cases , the population - based diabetes samples included only patients receiving drug treatment , which would exclude up to 25% of the total diabetic population ( 35 ) . in several cohorts of diabetic patients from finland , scotland , and u.s . selected irrespective of treatment status , patients with myocardial infarction had more events than those with diabetes ( 610 ) . in our study the results suggest that diabetic patients receiving insulin or oral drug treatment may have a worse prognosis at 10 years than those treated with diet alone . however , diabetic patients treated with any of these modalities had significantly lower risk than myocardial infarction patients . duration of diabetes was a determinant of cardiovascular outcomes in our study , which concurs with other reports ( 7,11,16 ) . our cut point ( third tertile ) was 8 years ; other authors found that twice this evolution time ( 16 years ) was required to worsen prognosis at 25 years ( 11 ) . diabetic patients in our series had lower levels of mean a1c at baseline than those included in the intensive branch of the ukpds , 7.5 vs. 8.1% , respectively ( 14 ) . despite differences in study design , our results support the relationship reported by the ukpds between high levels of a1c and worse prognosis . the ukpds approach is probably more realistic and correct than the stricter targets ( a1c < 6.5% ) proposed in some intervention studies ( 20 ) that did not find significant differences or an increased number of cardiovascular events in the more intensive intervention arm . some studies with stricter targets have shown the important role of hypoglycemia episodes in the poor prognosis of patients randomly assigned to intensive treatment ( 21 ) . we found that incidence of unstable angina was similar in men with myocardial infarction and in men with diabetes , but lower in diabetic women than in their myocardial infarction counterparts . mortality due to stroke was similar in both groups of patients and in both sexes . this observation adds to the existing controversy , with some authors finding positive ( 3,11 ) and some negative ( 5,7 ) differences . the low number of events , due to the age range selection in our study and to the fact that only fatal events were considered , hampers a more conclusive result . our findings , taken together with the opposite observation in some high cardiovascular mortality countries ( 35 ) and intermediate observations in central - western europe ( 6 ) and the u.s . , the south - to - north gradient is persistently observed in myocardial infarction incidence and mortality rates . the paradox of high cardiovascular risk factor prevalence that contrasts with relatively low acute myocardial infarction incidence rates has been described in spain ( 17 ) . our findings also support such a gradient : the risk of fatal and nonfatal myocardial infarction in diabetic patients was 0.33 ( 95% ci 0.270.41 ) ; in similar cohorts , it was 0.42 ( 0.330.54 ) in the u.k . , primary prevention measures may need to be adapted to the particularities of cardiovascular and diabetes diseases by country or region . we have shown that lipid profile and blood pressure diagnosis and control improved between 1995 and 2005 in the region we studied ( 22 ) . our sample of myocardial infarction and diabetic patients is population - based in a region of northeast spain , where risk factor prevalence and myocardial infarction incidence and mortality are well studied by the regicor group ( 23 ) . over the follow - up period , many improvements occurred simultaneously in the management of both acute myocardial infarction and type 2 diabetes , which may have influenced the outcomes . in spain , a decrease in 28-day and 1-year mortality between 1995 and 2000 has been associated with increased use of reperfusion strategies and medical therapies ( 23 ) . these changes were paralleled by intensified management of patients ' cardiovascular risk factors and glycemic targets , following the ukpds results ( 14 ) and international recommendations ( american diabetes association and european association for the study of diabetes ) in primary care centers within the gedaps network . the proportion of diabetic patients who smoked was very similar to the proportion of smokers at 6 months after the index event that was reported in a sample of the acute myocardial infarction patients . finally , asymptomatic myocardial infarction is known to occur to a greater degree in patients with diabetes than in the general population . in our study type 2 diabetes is on the increase in developed countries , a trend related to the epidemics of obesity observed in the past two decades . between 19941995 and 20032004 , the annual incidence of diabetes increased by 23% and prevalence by 62% in individuals > 65 years ( 24 ) . the economic and clinical practice consequences of considering diabetic patients , who represent > 10% of the adult population in developed countries , for secondary prevention therapies are very important : benefits and effectiveness must be assessed and balanced , particularly in regions with low coronary heart disease incidence and mortality . type 2 diabetes is not a coronary heart disease equivalent for cardiovascular risk in the region studied . in fact , this equivalence also has not been found in countries with high cardiovascular risk ( 610 ) . although patients with diabetes are at higher risk than the general nondiabetic population ( 7 ) , individual cardiovascular risk scores are required before implementation of the level of treatment ( statins , antiplatelet therapy , and intensification of hyperglycemia treatment ) that has been shown to be useful in high - risk patients ( 25 ) . our study confirms that type 2 diabetic patients initially free of coronary heart disease are at lower adjusted 10-year cardiovascular mortality and coronary heart disease incidence risk than patients with a first acute myocardial infarction without diabetes . our findings also contribute to showing that length of diabetes , type of treatment , and glycemic control should be taken into account in future studies on prognosis of patients with type 2 diabetes initially free of coronary heart disease . we have shown that lipid profile and blood pressure diagnosis and control improved between 1995 and 2005 in the region we studied ( 22 ) . our sample of myocardial infarction and diabetic patients is population - based in a region of northeast spain , where risk factor prevalence and myocardial infarction incidence and mortality are well studied by the regicor group ( 23 ) . over the follow - up period , many improvements occurred simultaneously in the management of both acute myocardial infarction and type 2 diabetes , which may have influenced the outcomes . in spain , a decrease in 28-day and 1-year mortality between 1995 and 2000 has been associated with increased use of reperfusion strategies and medical therapies ( 23 ) . these changes were paralleled by intensified management of patients ' cardiovascular risk factors and glycemic targets , following the ukpds results ( 14 ) and international recommendations ( american diabetes association and european association for the study of diabetes ) in primary care centers within the gedaps network . the proportion of diabetic patients who smoked was very similar to the proportion of smokers at 6 months after the index event that was reported in a sample of the acute myocardial infarction patients . finally , asymptomatic myocardial infarction is known to occur to a greater degree in patients with diabetes than in the general population . in our study type 2 diabetes is on the increase in developed countries , a trend related to the epidemics of obesity observed in the past two decades . for example , in the u.s . between 19941995 and 20032004 , the annual incidence of diabetes increased by 23% and prevalence by 62% in individuals > 65 years ( 24 ) . the economic and clinical practice consequences of considering diabetic patients , who represent > 10% of the adult population in developed countries , for secondary prevention therapies are very important : benefits and effectiveness must be assessed and balanced , particularly in regions with low coronary heart disease incidence and mortality . type 2 diabetes is not a coronary heart disease equivalent for cardiovascular risk in the region studied . in fact , this equivalence also has not been found in countries with high cardiovascular risk ( 610 ) . although patients with diabetes are at higher risk than the general nondiabetic population ( 7 ) , individual cardiovascular risk scores are required before implementation of the level of treatment ( statins , antiplatelet therapy , and intensification of hyperglycemia treatment ) that has been shown to be useful in high - risk patients ( 25 ) . our study confirms that type 2 diabetic patients initially free of coronary heart disease are at lower adjusted 10-year cardiovascular mortality and coronary heart disease incidence risk than patients with a first acute myocardial infarction without diabetes . our findings also contribute to showing that length of diabetes , type of treatment , and glycemic control should be taken into account in future studies on prognosis of patients with type 2 diabetes initially free of coronary heart disease .

objectivethe aim of this study was to determine whether long - term cardiovascular risk differs in type 2 diabetic patients compared with first acute myocardial infarction patients in a mediterranean region , considering therapy , diabetes duration , and glycemic control.research design and methodsa prospective population - based cohort study with 10-year follow - up was performed in 4,410 patients aged 3074 years : 2,260 with type 2 diabetes without coronary heart disease recruited in 53 primary health care centers and 2,150 with first acute myocardial infarction without diabetes recruited in 10 hospitals . we compared coronary heart disease incidence and cardiovascular mortality rates in myocardial infarction patients and diabetic patients , including subgroups by diabetes treatment , duration , and a1c.resultsthe adjusted hazard ratios ( hrs ) for 10-year coronary heart disease incidence and for cardiovascular mortality were significantly lower in men and women with diabetes than in myocardial infarction patients : hr 0.54 ( 95% ci 0.450.66 ) and 0.28 ( 0.210.37 ) and 0.26 ( 0.190.36 ) and 0.16 ( 0.100.26 ) , respectively . all diabetic patient subgroups had significantly fewer events than myocardial infarction patients : the hr of cardiovascular mortality ranged from 0.15 ( 0.090.26 ) to 0.36 ( 0.240.54 ) and that of coronary heart disease incidence ranged from 0.34 ( 0.260.46 ) to 0.56 ( 0.430.72).conclusionslower long - term cardiovascular risk was found in type 2 diabetic and all subgroups analyzed compared with myocardial infarction patients . these results do not support equivalence in coronary disease risk for diabetic and myocardial infarction patients .

of the various behaviors and adaptations that have evolved in marine environments , bioluminescence stands out as one of the particular importance , given the fact that it has evolved independently more than 40 times ( haddock et al . 2010 ) . while it does not necessarily give specific advantage to species in cold environments , the long periods of continuous darkness that characterize winters at high latitudes create an environment , at least with respect to light , that is similar to the deep - sea . depending on which taxa these include defensive functions such as the counter - illumination , the burglar alarm and offensive mechanisms such as prey attraction and intraspecific communication other adaptations have evolved in both phytoplankton and zooplankton to survive in these harsh conditions that relate more to metabolic rates and the actual vertical space occupied in the water column within which a species spends the winter months . one such strategy is to enter a dormant state and overwinter at depth , seen for the copepods calanus glacialis and c. hyperboreus which are commonly reported to enter a state of diapause at depth during winter months ( fortier et al . they , then , return to shallower water in the summer to take advantage of the high productivity rates ( ashjian et al . 2009 ) for the first time provide acoustic evidence of active vertical migrations of zooplankton throughout the polar night in the high arctic . more recently , this phenomenon was corroborated in the southern hemisphere where diel vertical migration ( dvm ) was shown to continue through the austral winter in the lazarev sea , but ceased during the austral summer ( cisewski et al . the goal of the current study was to characterize plankton abundance and distribution patterns during a time of year that has rarely been studied by means of vertical net tows and autonomous underwater vehicle ( auv ) surveys . fitted on the auv were adcps , a ctd and a bathyphotometer designed to register bioluminescence potential in the water column . data were collected off the coast of ny lesund in kongsfjord , svalbard ( 7857 n , 1156 e ) in ~120 m of water from january 19 to 22 , 2010 . spatial and temporal dynamics of acoustic backscatter , salinity , temperature and bioluminescence were measured using a remus-100 auv ( moline et al . 2005 ) . the vehicle was equipped with upward and downward facing rd instruments 1,200-khz workhorse navigator acoustic doppler current profilers ( adcp ) , configured in this study to provide relative acoustic backscatter as an estimate of scattering volume , rather than current velocities , a neil - brown ctd and a bioluminescence bathyphotometer ( bp ; moline et al . the bp utilizes an impeller to continuously draw a measured volume of water into a chamber through the front of the nosecone where bioluminescence is measured by a photomultiplier tube at 60 hz [ see herren et al . ( 2005 ) for details ] . for statistical purposes , we restricted our analysis to the non - zero observations . the auv was deployed at 10:27 , 12:25 and at 13:40 ( local time 19th of january ) at depths of 15 , 45 and 75 m , respectively . the vehicle was also deployed at 21:30 ( 19th of january ) along the same transect as during the day but ran one mission surveying the transect at 15 , 45 and 75 m successively without breaks between each depth . adcp data were processed to remove noise and calculate relative backscatter coefficient ( sv ) according to deines ( 1999 ) for the data collected between 0.5 and 5.0 m away from either side of the vehicle . following the auv deployments , continuous bp observations were made from 15:00 on january 21 , 2010 until 09:00 on january 22 , 2010 at 1 m depth at the ny lesund harbor about 2 km from the study site . for logistical reasons ( power supply , stable holdfast for the instrument and weather conditions ) , it was not possible to carry out the continuous bp observations in the fjord at the same place as the auv deployments . however , given the short distance between the two locations and the absence of any physical barriers , the two sites are regarded as comparable for the examination of circadian rhythm ( see also " discussion " ) . concurrent with the remus deployments , vertical net hauls were conducted using a wp2 plankton net , 180-m mesh size with a 0.25 m opening . in order to collect vertical net hauls from the same depths surveyed by the remus adcp , two replicates from each depth were taken from 300 m , 600 m and from 900 m between 10:3013:00 lt . during the nighttime auv deployment , replicate hauls were taken between 300 m , but inclement weather resulted in only one sample from 600 m was collected and none between 900 m. to determine species composition and abundance for specific depth intervals of 6030 m and 9060 m , the results from 300 m were subtracted from those of 600 m , and results from 600 m from those of 900 m , respectively . additionally , the remus bp was equipped with cylindrical plankton nets ( 20-m mesh size ) , and these nets were fitted to each of the two exhausts of the bp ( moline et al . 2009 ) , which hence provided two replicate samples from each of the depths ( 15 , 45 and 75 m ) sampled using the auv . for all plankton samples , samples were later enumerated and identified to the lowest possible taxonomic unit using a leica stereomicroscope with 6.340 magnification . the temperature , salinity and density profiles were similar between night and day during the sample period ( fig . 1 ) and did hence not reveal any major advection events that would otherwise influence the measurements and results presented below . based upon willis et al . ( 2006 ) , physical parameters indicated that the water mass in kongsfjord was of artic water origin . bioluminescence was detected throughout the water column both night and day , with higher bioluminescence at depth during the day and increased surface bioluminscence at night ( fig . comparison of the bioluminescence at the specific depths also provided that this result with significantly greater bioluminescence intensity per flash was observed at 75 m during the day and at 15 m at night ( table 1 ) , suggesting that the organisms with more intense flashes , i.e. , larger zooplankton ( see moline et al . 1vertical profiles of a temperature ( c ) , b salinity ( ppt ) , c density ( kg m ) and d bioluminescence ( photons / l ) as a function of depth ( m ) taken from the remus auv during the final ascents from 75 m during the day and night deployments . daytime observations represented in gray and nighttime in black . for this profile , bioluminescence for the upper water column ( < 45 m ) was significantly less than that for the lower water column during the day ( mann whitney , p = 0.009 , n = 102 ) and higher bioluminescence in the upper water column at nighttable 1mean bioluminescence intensity per flash ( se ) surveyed by the auv at the three different depths during the daytime and nighttime deployments ( lt is local time)depth ( m)mean intensity / flash ( 10 ) daytime ( 10:3014 : 25 lt)mean intensity / flash ( 10 ) daytime ( 21:3022:30 lt)mean s v difference ( day night)159 2 * 160 142 16457 28 3357518 6 * 4 1 17significant differences were found between depths using mann whitney ( * p = 0.016 , n = 1,030 ; p = 0.025 , n = 286 ; p = 0.022 , n = 126 ) , and additionally , the difference in the mean acoustic backscatter coefficients ( s v ) between day and night is shown for each depth . the differences in s v between day and night were significant ( mann whitney , p < 0.001 ) at all depths vertical profiles of a temperature ( c ) , b salinity ( ppt ) , c density ( kg m ) and d bioluminescence ( photons / l ) as a function of depth ( m ) taken from the remus auv during the final ascents from 75 m during the day and night deployments . daytime observations represented in gray and nighttime in black . for this profile , bioluminescence for the upper water column ( < 45 m ) was significantly less than that for the lower water column during the day ( mann whitney , p = 0.009 , n = 102 ) and higher bioluminescence in the upper water column at night mean bioluminescence intensity per flash ( se ) surveyed by the auv at the three different depths during the daytime and nighttime deployments ( lt is local time ) significant differences were found between depths using mann whitney ( * p = 0.016 , n = 1,030 ; p = 0.025 , n = 286 ; p = 0.022 , n = 126 ) , and additionally , the difference in the mean acoustic backscatter coefficients ( s v ) between day and night is shown for each depth . the differences in s v between day and night were significant ( mann whitney , p < 0.001 ) at all depths it is important when interpreting changes in bioluminescence signals that the circadian rhythms in the bioluminescence potential of planktonic organisms be taken into account ( batchelder et al . the continuous bp observations at the ny lesund harbor showed no evidence of a circadian rhythm in the bioluminescence signal ( fig . 2 ) in a location ( sheltered by the pier and with a max depth of 5 m ) where vertical migration of zooplankton would be restricted . organisms collected by net hauls next to the moored bp and by nets connected to the bp during this period using identical methods described above for the auv showed > 80% similarity to the study transects ( data not shown ) , thus making these results applicable to the observations made by the auv.fig . 2hourly means of log bioluminescence potential ( solid black line ) with standard deviations ( dotted black lines ) collected at 1 m depth from 15:00 lt on january 21 to 09:00 lt on january 22 , 2010 , ( n = 9,545 , non - zero observations ) . flashes per unit time were also found to be consistent throughout the time series hourly means of log bioluminescence potential ( solid black line ) with standard deviations ( dotted black lines ) collected at 1 m depth from 15:00 lt on january 21 to 09:00 lt on january 22 , 2010 , ( n = 9,545 , non - zero observations ) . flashes per unit time were also found to be consistent throughout the time series estimates of relative backscatter coefficient as a relative measure of zooplankton biomass in a 10-m swath around the prescribed vehicle depths showed significantly higher intensity between 70 and 80 m during the day , and between 10 and 20 m and 4050 m at night ( table 1 ) . in combination with the changes in bioluminescence intensity , these data demonstrated a coordinated movement of biomass indicative of dvm . plankton enumerations from wp2 vertical net hauls show an increase above 60 m in the majority of the most abundant zooplankton taxa at night , including pseudocalanus spp . these genera have been reported to present throughout the year in this regions ( lischka and hagen 2005 ) . table 2 provides specific species classification and shows that this increase above 60 m at night is also apparent in the enumerations of other less abundant taxa including calanus finmarchicus , acartia longiremis , oncaea borealis and eukrohnia hamata . of these , metridia lucens , metridia longa , oncaea borealis , thysanoessa inermis and thysanoessa longicaudata most likely account for the increase in high - intensity bioluminescent flashes at 15 and 45 m during the night ( table 1).table 2concentrations of the plankton captured by the 180 m wp2 plankton net during the day and at night for depth intervals 300 , 6030 and 9060 mtaxadepth ( m)ind./m ( day)ind./m ( night)taxadepth ( m)ind./m ( day)ind./m ( night ) calanus finmarchicus 030 5082 heterorhabdus norvegicus*030 < 1<13060 411483060 < 166090 896090 4 calanus glacialis 030 410 harpacticus chelifer 030 9503060 31313060 543060901476090 < 1 calanus hyperboreus 030 < 11harpacticoida spp.030 2<13060 < 1<13060 11<16090 46090 < 1 pseudocalanus spp.030 3,10010,300 oithona atlantica 030 3887253060 < 116,8203060 < 15556090 7756090 < 1 microcalanus spp.030 5632,300 oithona similis 030 4008253060 1631,9003060 < 11,3356090 636090 131 metridia lucens*030 317appendicularia*030 17153060 4183060 336090 66090 96 metridia longa*030 < 1<1 limacina helicina 030 2<13060 < 133060 < 116090 116090 12 acartia longiremis 030 175375 sagitta elegans 030 < 123060 < 13853060 5116090 < 16090 12 paraeuchaeta norvegica 030 < 1<1 eukrohnia hamata 030 4303060 1<13060 2216090 < 16090 11 diastylis lucifera * 030 < 1<1 thysanoessa longicaudata*030 < 123060 1<13060 376090 < 16090 2 bradyidius similis 030 < 11 thysanoessa inermis*030 < 1<13060 < 133060 < 1226090 66090 < 1 oncaea borealis*030 12<13060 512006090 < 1asterisks indicate organisms known to be bioluminescent concentrations of the plankton captured by the 180 m wp2 plankton net during the day and at night for depth intervals 300 , 6030 and 9060 m asterisks indicate organisms known to be bioluminescent plankton enumerated from the > 20 m net collection of the bp exhaust suggests that during the day , the greatest biomass occurred at 45 m and was dominated by copepod nauplii , copepod eggs and the tintinnid acantostomella norvegica ( table 3 ) . the same three groups of organisms dominated the biomass at 15 and 75 m. other major contributors at each of these three depths were ceratium and protoperidinium spp . microcalanus spp . , oithona similis and oithona atlantica ( table 3 ) , consistent with the wp2 nets samples ( table 2).table 3concentrations of the plankton captured by the 20 m plankton nets covering the remus bp exhaust for daytime deployments at 15 , 45 and 75 mtaxadepth ( m)ind / m taxadepth ( m)ind / m ceratium arcticum 15229copepod nauplii*151,40945352452,72675246751,403 ceratium fusus * 159.6copepod eggs159034591.3451,6317580.3751,372 ceratium furca 15<1 oncaea borealis * 15545134552754754 protoperidinium spp.*15211 harpacticoida spp . microsetella norvegica 1574539145775387754diatom spp.151015545594577580754 acantostomella norvegica 151,252 oithona atlantica 1567452,02345117758277559 salpingella acuminata 15241 oithona similis 157245163451117512275108 heliocostomella subulata 152 eukrohnia hamata 15<1451345175<175<1 parafavella denticulata 157gastropoda larvae15234533453375457511 calanus finmarchicus 158appendicularia*1511458453975117521 paraeuchaeta norvegica ciii 15<1membranipora larvae1519451452075<17521 acartia longiremis 1529bivalve larvae15<1457457751875<1 pseudocalanus spp.15205 limacina helicina 15<14521545<175154754 microcalanus spp.15137 thysanoessa longicaudata * 15<14543745<175140751asterisks indicate organisms known to be bioluminescent concentrations of the plankton captured by the 20 m plankton nets covering the remus bp exhaust for daytime deployments at 15 , 45 and 75 m asterisks indicate organisms known to be bioluminescent evident from organisms collected from both net sampling approaches is that the day / night changes in the vertical distributions of bioluminescence and acoustic scattering resulted from the larger zooplankton . and appendicularia ) collected by the wp2 vertical net tows showed decreased abundance at the surface ( upper two depth layers ) during the day than during the night and the highest abundance of this size class was found at depth during the day ( table 2 ) . the smaller size class ( pseudocalanus spp , microcalanus spp . histograms of the bioluminescence intensity ( data not shown ) in conjunction with plankton enumerations from each depth suggest that an underlying low - to - intermediate - intensity bioluminescence was consistent between day and night and likely attributed to the dinoflagellates , from protoperidinium that occurred throughout the water column and to a lesser degree , ceratium furca and c.fusus that were present in significantly lower abundances ( table 3 ) . though ubiquitous in the world s oceans and important from an ecological and evolutionary perspective ( for review see haddock et al . 2010 ) , few studies have described bioluminescent communities and their distributions in the arctic , particularly during the winter darkness . buskey ( 1992 ) , and lapota et al . ( 1989 , 1992 ) examined bioluminescence distributions and community structure with the goal of developing methodology to use bioluminescence as a way to measure total biomass and light budgets of a given water mass during the spring in the greenland sea , during the fall in the beaufort sea and in summer in a norwegian fjord , respectively . in contrast , this study quantified the bioluminescent community during the polar night and demonstrated the absence of circadian rhythm in bioluminescence . furthermore , both the diurnal distribution of bioluminescence intensity and concurrent changes in acoustic backscattering provide independent evidence for an active dvm of the larger bioluminescent zooplankton ( and likely non - bioluminescent zooplankton ) within the upper 75 m of the water column . histograms of intensities showed the major differences between day and night occurring at the highest intensities , which is consistent with larger zooplankton ( lapota et al . 1992 ; moline et al . 2009 ) and with the enumerations in this study . bioluminscence and acoustic backscatting may in fact not be directly linked , but the circumstantial evidence provided herein suggests that the diurnal signal in bioluminescence is in fact caused by vertically migrating organisms . numerous studies have examined proximal triggers for dvm ( forward 1988 ; ringelberg 1995 ; ringelberg and van gool 2003 ; benoit - bird et al . 2009 ) , and many others have looked at triggers for the inhibition of bioluminescence as related to its circadian rhythm ( batchelder et al . 1992 ; kelly and katona 1966 ; raymond and devries 1976 ; swift et al . 1995 ) . interestingly all studies have in one way or another implicated a relative or absolute change in irradiance intensity , angle or daylength as a means of regulation for both dvm and the circadian rhythm of bioluminescence . although changes in light during the time of this study are not visible to the human eye , it is possible that they were sufficient to initiate dvm in the organisms present at the time of the study as was suggested in the study by berge et al . ( 2009 ) . however , while external light cues may play a role in regulating dvm , it might not be the only factor relevant to consider for understanding this behavior . it has been well established for photosynthetic dinoflagellates and for heterotrophic dinoflagellates of the protoperidinium genus that the bioluminescent inhibition occurs when light intensities are greater than the intensity of bioluminescence of the organisms themselves ( sweeney et al . this phenomenon could explain why no circadian rhythm existed in bioluminescence during december and january in antarctica ( raymond and devries 1976 ) and may also be related to the absence of a circadian rhythm in bioluminescence in the current study . previous studies have found that protoperidinium spp contributed between 20 and 90% of the total light budget from the surface to a depth of 100 m in the beaufort sea ( lapota et al . 1992 ) and that dinoflagellates were estimated to account for 96% of the total light budget in vestfjord , norway ( lapota et al . 1989 ) , so it is reasonable to assume that they were significant contributors to the overall light budget during this study . wherein the lack of sunlight facilitated an environment where the intensity of bioluminescence was not inhibited by light greater than the bioluminescence of the organisms themselves . while this regulatory factor has been established for dinoflagellates , it is possible that it plays a role in other bioluminescent taxa as well , such as copepods , appendicularians and arctic krill as in the case of this study . the most notable finding in this study is the detection of bioluminescent activity among zooplankton during the polar night , which may be an important ecological feature . while the ultimate and proximate explanations for both the bioluminescence and the dvm behavior detected during the campaign fall outside the data collected during this study , these results provide evidence for both endogenous and exogenous control of poorly understood or previously unknown processes . also , during this expedition , which took place during the darkest period of the polar night , we observed five different species of seabirds actively foraging at sea ; little auk ( alle alle ) , black - legged kittiwake ( rissa tridactyla ) , northern fulmar ( fulmarus glacialis ) , black guillemot ( cepphus grylle ) and brnnich s guillemot ( uria lomvia ) . these seabirds have , to the best of our knowledge , not been reported to overwinter at these latitudes . whether bioluminescence and/or dvm are playing roles in the foraging behavior of these visual predators is an exciting possibility , although still an open question . despite the limited scope of this study , results open new lines of enquiry regarding the function and process during a time of year when classical paradigms of arctic ecosystems postulate that organisms are predominately in a state of hibernation ( see berge et al . ultimately , these questions also have implications for human activities ( i.e. , oil exploration ) in the high arctic , which up until to now has been considered without life during the polar night .

this study examines the composition and activity of the planktonic community during the polar night in the high arctic kongsfjord , svalbard . our results are the first published evidence of bioluminescence among zooplankton during the arctic polar night . the observations were collected by a bathyphotometer detecting bioluminescence , integrated into an autonomous underwater vehicle , to determine the concentration and intensity of bioluminescent flashes as a function of time of day and depth . to further understand community dynamics and composition , plankton nets were used to collect organisms passing through the bathyphotometer along with traditional vertical net tows . additionally , using a moored bathyphotometer closed to the sampling site , the bioluminescence potential itself was shown not to have a diurnal or circadian rhythm . rather , our results provide evidence for a diel vertical migration of bioluminescent zooplankton that does not correspond to any externally detectable changes in illumination .

the baylisascaris genus of nematodes ( family : ascarididae ) is comprised of nine recognized species , each parasitizing distinct definitive hosts and a vast array of intermediate hosts ( bauer , 2013 , kazacos , 2001 , sorvillo et al . , 2002 ) . the most widely studied of these is b. procyonis , which primarily utilizes the common raccoon ( procyon lotor ) as its principal definitive host ( kazacos , 2001 ) . embryonated eggs of baylisascaris species are infectious to over 90 species of mammals and birds including humans . larval infections with baylisascaris species can lead to irreversible neural , optical and visceral damage , in both wild animals as well as humans ( gavin et al . the severity of pathology of baylisascaris infection in the intermediate host is known to vary depending on the species of the infecting parasite ( tiner , 1953 ) . understanding which species of baylisascaris a host is infected with may provide valuable information concerning prognosis and treatment , as well as the source of the infection . as with other nematodes , baylisascaris species have been traditionally identified through morphometric data . however , distinguishing larval nematodes to the species level is problematic ( graeff - teixeira et al . , 2016 ) . with the current widespread availability of molecular tools , the use of genetic analysis to rapidly and accurately identify organisms to the species level is increasingly standard . however , differentiation of closely related species such as b. procyonis and b. columnaris has proven difficult ( dangoudoubiyam et al . , 2009 , gatcombe et al . , 2010 ) . extensive previous research has documented the life - cycle , distribution and prevalence of b. procyonis ( graeff - teixeira et al . , 2016 , additionally , the complete mt genome of b. procyonis , has been sequenced ( xie et al . in contrast to the relative abundance of studies on b. procyonis , little is known of its closest relative , b. columnaris . previous research using copromicroscopic detection has shown b. columnaris infection prevalence of 25% in a population of eastern spotted skunks ( spilogale putorius ) ( lesmeister et al . , 2008 ) and 25% prevalence in captive striped skunks in europe ( d'ovidio et al . , 2017 ) . to our knowledge , the prevalence of b. columnaris in wild populations of striped skunks has not previously been reported . ( 2013 ) reported the cloning and sequencing of partial sequences of b. columnaris for the cox1 ( 413 bp ) and cox2 ( 483 bp ) genes . additionally , in this work franssen et al . identified three single nucleotide polymorphisms ( snps ) in the partial cox1 sequence and one snp found in the partial cox2 sequence which are useful in differentiating b. procyonis from b. columnaris . the partial sequence of these mitochondrial genes has provided a reference which has facilitated the differentiation of b. procyonis from b. columnaris by dna sequencing . in addition to the four snps which allow for the molecular differentiation of b. procyonis and b. columnaris , franssen et al . also reported a number of intragenic differences in b. columnaris mitochondrial sequences suggesting there is a high degree of genetic diversity in this species . in this study we sought to determine the prevalence of b. columnaris in a wild population of striped skunks ( mephitis mephitis ) in salt lake county , utah , usa . in addition to determining the prevalence of these parasites in a native skunk population we report the complete sequence of 11 mitochondrial genes ( cox1 , cox2 , nd2 and 8 trna genes ) comprising 3638 bp of the b. columnaris mitochondrial genome . results revealed several novel snps in these genes , which further facilitates and improves the molecular distinction between b. procyonis and b. columnaris . additionally , several intragenic snps were identified in b. columnaris worms . in summary , these results demonstrate the prevalence of b. columnaris in a wild population of skunks and extend the number of known genetic differences between b. procyonis and b. columnaris . skunks were acquired through nuisance animal calls from the public in salt lake county , utah , usa during the fall and winter of 2013 . institutional animal care and use committee ( iacuc ) or ethics committee approval was not necessary , as animals were not sacrificed for research purposes . skunks were captured in live traps ( tru - catch , belle fourche , south dakota , usa ) . skunks were euthanized by chemical immobilization with 5/1 ketamine / xylazine followed by intracardiac injection of potassium chloride . the presence of nematode parasites was determined by emptying the small intestinal contents through manual extrusion using finger / grip pressure and the visual examination of intestinal contents . species determination of collected worms was performed by extracting dna from one or more worms from each skunk and performing dna sequencing of the cox1 gene . sequences were aligned with the previously published cox1 to determine worm genus and species ( genbank : kc543474.1 ) ( https://blast.ncbi.nlm.nih.gov ) . a total of 34 worms were collected and the cox1 gene of 22 of these worms was sequenced . a single worm from several animals was then used for more extensive dna sequencing of a number of genes as described below . pcr primers were designed based on the mitochondrial genome of b. procyonis ( ac . no . primers were designed to flank the gene of interest by 100300 bp from the 5 and 3 ends . this primer design resulted in the amplification of segments of dna which contained the complete gene of interest ( cox1 , cox2 , nd2 ) as well as several small trna genes contained at the 5 and 3 ends of each amplicon ( table 1 ) . three separate amplicons were generated with amplicon 1 containing the sequences for the trna genes q , r , i , s , and l , as well as the complete nd2 gene . amplicon 2 contained the complete cox1 gene and amplicon 3 contained the trna genes d , g , and h as well as the complete cox2 gene . primer sequences for each amplicon are contained in table 1 . upon collection of parasites ( described above ) , individual specimens were preserved by immediately freezing each worm at 80 c. for genetic analysis , specimens were thawed and a 2.0 mm portion of each worm macerated with a razor blade . the macerated tissue samples were then placed in a 1.5 ml microcentrifuge tube and dna extracted using the qiagen dneasy blood & tissue kit ( qiagen inc . , pcr reactions were carried out in a final reaction volume of 50 l containing 2 l of dna , 25 l of onetaq dna polymerase ( new england biolabs , ipswich , ma usa ) , 0.5 l of each primer with 22 l of molecular grade water . polymerase chain reaction conditions used to amplify the cox1 and cox2 containing amplicons ( amplicons 2 and 3 ) , involved a 30 s initial denaturation at 94 c followed by 30 cycles of denaturation at 94 c for 30 s , annealing at 55 c for 30 s , and elongation at 68 c for 80 s , followed by a 10-min final extension at 68 c . the pcr amplification of the nd2 target region ( amplicon 1 ) was identical to the aforementioned profile , with the exception of the annealing temperature being 63 c . the pcr amplified products of interest for each amplicon were then extracted and gel purified using the wizard sv gel and pcr clean - up system ( promega madison , wi ) . the sequencing of each amplicon was performed using primers designed to bind approximately every 500 bp ( supplemental fig . 1 ) . cycle sequencing was performed by the brigham young university dna sequencing center using an abi 3730xl automated sequencer . primers were designed using the forward dna strand and sequencing was performed on a single strand . contigs for each sample were assembled by mapping sample reads to the mitochondrial genome of b. procyonis ( ac . sequence reads were determined using geneious software ( biomatters limited , san francisco , ca , usa ) . snp analysis , multiple alignments , and prediction of transmembrane and cytoplasmic regions were performed using geneious software . we first sought to determine the prevalence of b. columnaris in a wild population of skunks . the intestinal tracts of 16 skunks were collected and the prevalence of nematode infection determined by gross examination of the intestinal contents . ten of the 16 skunks examined were found to be infected with roundworms , with numbers of worms ranging from 1 to 10 in infected animals ( table 2 ) . all worms were identified as b. columnaris based on gross morphology and confirmed by detailed sequence homology to the published partial sequence of b. columnaris cox1 ( genbank : kc543474.1 ) . due to the essential function of cox1 , this gene is present in a wide variety of organisms yet has sufficient variation to distinguish closely related species ( hebert et al . , 2003 ) . consequently , the sequence of the cox1 gene is a frequently used marker for population genetic and phylogenetic studies ( ai et al . , 2011 , xie et al . , 2011b ) . in this study , the complete cox1 gene ( 1578 bp ) of eight baylisascaris worms isolated from eight different hosts our analysis revealed 11 novel loci , which consistently distinguished b. columnaris from b. procyonis , including the three previously reported by franssen et al . the majority of these species specific snps are found in the 3 portion of the gene with 8 of the 11 being found between nucleotides 1002 and 1506 of the cox1 gene . previously published work has shown a relatively high degree of intraspecies heterogeneity in the cox1 gene of b. columnaris ( franssen et al . , 2013 ) . in our analysis of cox1 , 11 total intraspecies snps were identified 10 of these having not previously been reported ( fig . 1 ) . previous sequencing of a partial sequence of the b. columnaris cox2 gene has shown one specific snp which was useful in differentiating b. procyonis from b. columnaris as well as three intragenic snps ( franssen et al . , 2013 ) . these previous studies were done in a european population of skunks . in our sequencing analysis of a north american population , we found six intragenic snps within the cox2 gene . importantly , our data showed that none of these nucleotide variations were species specific , whereas previous analysis had suggested the snp at position 168 was useful in differentiating b. columnaris from b. procyonis ( fig . 2 ) . there have been no previous reports of sequencing of the b. columnaris nd2 gene . in our sequencing of this gene and subsequent analysis , in addition , two intraspecies snp were identified in the nd2 gene of b. columnaris ( fig . alignment of eight of the b. columnaris concatenated sequences and homologous regions of b. procyonis were used to determine the sequence of several trna genes from b. columnaris . this analysis revealed two snps and an indel within mt - trna genes which differentiated b. procyonis from b. columnaris . in addition , five intragenic snps were identified ( one of them being in the same position as the indel of trna s ( fig . 4 ) ) . based on the high degree of similarity between these organisms , we were initially skeptical of this degree of variation in trna genes . therefore , we next compared the trna genes from all mt - dna genomes of sequenced baylisascaris species . this comparison demonstrated a relatively high degree of sequence variation in the trna genes of several closely related baylisascaris species ( fig . this level of heterogeneity in trna sequences from numerous baylisascaris species lends confidence in this unanticipated level of heterogeneity if trna genes . additionally , concatenated sequences were used to generate maximum likelihood relationships of b. columnaris with other ascarid species . results of these analyses demonstrated maximum likelihood relationships in agreement with previous findings ( franssen et al . , 2013 ) . both skunks as well as raccoons commonly live in urban areas facilitating human contact and potential ingestion of embryonated eggs which can cause visceral , ocular and neural larval migrans ( roussere et al . , 2003 , the molecular identification of b. columnaris was not possible due to the lack of any dna sequences in public databases . in 2009 , a partial ( 529 bp ) sequence of the b. columnaris cox2 gene was generated ( dangoudoubiyam et al . , 2009 ) . in 2013 work by franssen et al . resulted in partial sequences for b. columnaris cox1 ( 413 bp ) and cox2 ( 483 bp ) genes ( franssen et al . , the availability of these sequences has facilitated the molecular identification of these parasites for us as well as other researchers ( d'ovidio et al . , 2017 ) . in this study , we utilized gene sequencing to identify roundworms as b. columnaris . in this population of wild skunks we then extended earlier findings by sequencing a total of 3638 bp of the mitochondrial genome of b. columnaris . in so doing several novel snps were identified , facilitating the molecular discrimination of b. columnaris from b. procyonis . these snps provide additional species specific targets for the molecular differentiation of b. columnaris from b. procyonis . importantly this data demonstrate that three previously reported species specific snps do not accurately differentiate b. procyonis from b. columnaris ( 168 of cox2 and 804 and 834 of cox1 ) . all of these snps involved transitions between the purines g and a. this unique finding , compared to previously published work , is likely due to the previous study being done in the netherlands , while skunks ( and by extension parasites of skunks ) are native to the americas . parasites infecting european skunks are likely from a relatively small founder population of nematodes infecting skunks transported outside of north america . it is logical to assume that the nematodes ( as well as the skunks ) would have less genetic diversity than a native , free roaming population of animals . a recent study on the prevalence of b. columnaris in captive european skunks found that 25% of skunks tested were infected with b. columnaris ( d'ovidio et al . data indicates that in our study area there is a much higher infection rate of striped skunks than reported in studies of captive striped skunks in europe . this is likely due to wild animals encountering other wild skunks as well as infected intermediate hosts more commonly than captive skunks . as expected , more intragenic variation of parasites was observed from this population of native wild skunks compared to the imported non - native population studied previously ( franssen et al . , 2013 ) . the higher prevalence of parasites in our study compared to a previous study of spotted skunks ( lesmeister et al . , 2008 ) is likely due to the difficulty in accurately identifying infected animals through fecal analysis compared to our method of visual inspection of the small intestine . additionally , several of the animals in our study had very few worms infecting them . animals infected with a single male worm would not be detected by microscopic analysis of feces but would easily be identified through visual inspection on the intestinal contents . differences in the susceptibility to baylisascaris infection by these two types of skunks , as well as other environmental factors may also play a role in variations in infection prevalence . in summary , in this study several novel intragenic snps were identified . additionally , nine novel polymorphisms are identified which aid in the molecular differentiation of b. procyonis from b. columnaris . three polymorphisms , which were previously thought to differentiate these two species were shown to in fact be intragenic rather than species specific snps .

members of the genus baylisascaris utilize omnivores or carnivores as their definitive hosts . the best known member of this genus is baylisascaris procyonis , which is an intestinal parasite of raccoons . the closest relative of b. procyonis is b. columnaris , which utilizes the common skunk as its definitive host . although b. procyonis has been extensively studied , relatively little is known of b. columnaris . for example , the mitochondrial genome of b. procyonis has been sequenced in its entirety . conversely , the mitochondrial genome of b. columnaris remains largely unexplored . likewise , the prevalence of this parasite in its wild host has not been documented . in this study , we collected parasites from a wild population of skunks in the state of utah , united states . the cytochrome c oxidase subunit 1 and 2 genes , nadh dehydrogenase 2 and several trna genes were sequenced from the mitochondrial genome of these parasites . we also determined the prevalence of b. columnaris in a wild population of skunks . in this work we identify several novel polymorphic genetic loci between b. procyonis and b. columnaris . these findings provide additional molecular targets for the differentiation of baylisascaris species through clarification of genetic differences between b. columnaris and b. procyonis .

falls are one of the leading injury - related accidents involving older adults whose muscular strength , proprioceptive sense , and bodily coordination are compromised with aging1 . the elderly lack obstacle negotiation capacity and are prone to trip over commonly encountered environmental barriers such as door sills , pavement blocks , and safety bumps2 . in a local community , 33% of elderly people were reported to experience falls , of which 42.4% suffered falls3 . individuals with a history of falls have fears of repeated falls , depression , and anxiety , all of which lead to decreased physical activities4 . these negative experiences result in reduced physical capacity in terms of muscular strength , bodily flexibility , and coordination , rendering the affected elderly even more susceptible to falls in a vicious cycle5 . notably , elderly women are more at risk of secondary injury such as severe fractures after falling than elderly men because of their low bone density after menopause6 . interventions for fall prevention include exercise , education , environmental improvements , and medication7,8,9 , of which exercise has been used in fall prevention programs in many ways , as it takes less time and money to implement and programs are easy to set up . carter et al.10 reported in a bibliographical study on exercise intervention that exercise improved leg strength 44.4% and balance ability 37.5% in older adults and that as weakness of muscle strength in the trunk was more important than that in the lower extremities , strengthening of lumbar function can improve functional stability , leading to increased balance ability , gait ability , and prevention of falling11 , 12 . core stabilization exercise ( cse ) can improve balance control ability by reinforcing intersegmental muscles in the multifidus , transversus abdominis , and rotators13 and physicopsychological functions in a harmonious way by stimulating proprioception powerfully when it is accompanied by swiss ball exercises , improving balance sense , and maintenance ability14 . previous studies on falling in elderly adults involving cse have been limited to balance ability or gait ability15 , 16 , and there have been only a few studies on obstacles as an environmental factor or psychological factors . therefore , this study evaluated changes in the physicopsychological functions of elderly women with a fear of falling in negotiating obstacles such as a doorstep ( 5.2 cm ) of a bathroom , where falling occurs frequently , through a performance - oriented mobility assessment ( poma ) in order to understand whether cse is valid as an efficient exercise to prevent and control falling in consideration of the physicopsychological functions of elderly women . the subjects of the study were 20 elderly women above the age of 65 who were able to walk independently , did not have experience participating in regular balance training more than twice a week within the past 6 months , had a score higher than 24 on the mini - mental status examination - korea ( mmse - k ) , had no limits on exercise performance due to visual or musculoskeletal disorders and no previous experience of falling , and scored lower than 19 on the tinetti poma17 . the purpose and methods of this study were explained to all the participants , who read and signed an informed consent from that revealed all the details of the study protocol , which were approved by the ethics committee of kangwon national university ( no . the subjects selected were divided into an experimental group and a control group by drawing lots , and the average age , height , and weight in the experimental group were 73.203.46 years , 152.154.29 cm , and 57.787.95 kg , while those in the control group were 71.003.50 years , 149.836.45 cm , and 53.8011.04 kg . there were no significant differences among the groups with regard to age , height , or weight ( p>0.05 ) . the core stabilization exercise program proposed by jeffrey18 and hesari et al.19 was applied to the experimental group and cse was composed of three steps . level 1 consisted of abdominal hollowing in a supine position , abdominal hollowing in a prone position , abdominal hollowing in a quadruped position , abdominal hollowing in a supine position while curling the feet , abdominal hollowing in a prone position while curling the feet , and modified side bridging . level 2 consisted of bridging with abdominal hollowing , pelvic bridging , the dying bug with abdominal hollowing , and abdominal hollowing while seated on a swiss ball . level 3 consisted of pelvic bridging with a swiss ball , bird dog exercise , twists on a swiss ball , and bird dog exercises on a swiss ball . the experimental group performed a core stabilization program consisting of three levels for 30 min , three times per week on alternate days , for 6 weeks . this program consisted of three levels , and the subjects began at exercise level 1 and proceeded to the next level according to the protocol for the day . level 1 included static holds in a stable environment , level 2 included dynamic movements in a stable environment , and level 3 included dynamic movements in an unstable environment , such as on a swiss ball , and resisted dynamic movements in an unstable environment . before the program and 6 weeks after the program , the tinetti poma , crossing velocity ( cv ) , maximum vertical heel clearance ( mvhc ) and knee flexion angle , which are related to physical functions , were measured , and with respect to psychological functions , depression and fear of falling were measured . the tinetti poma is an instrument used to decide the degree of fear of falling and balance and mobility in elderly adults consisting of items scored on a 3-point scale . the maximum score is 28 , with 16 points for balance and 12 points for gait . if a subject scores below 19 , fear of falling is high , and scores of 19 to 24 indicate an intermediate degree for fear of falling ; scores of 25 to 28 indicate no fear of falling . for the crossing velocity ( cv ) , the horizontal distance from the point at which the leading limb leaves the ground to the point at which the heel returns to the ground again is divided by the time taken20 . for the maximum vertical heel clearance ( mvhc ) , the vertical distance from the height of the obstacle before obstacle negotiation to the ball of the leading limb was measured21 . the obstacle was 60 cm long , 10 cm wide , and 5.2 cm high . the subjects began their gait 5 m from the obstacle and continued on 3 m after obstacle negotiation . cv and mvhc were measured with the use of dartfish software ( dartfish , fribourg , switzerland)23 , and the results were output in a data table . to analyze depression , the ces - d ( center for epidemiological studies - depression scale ) was used . it is scored on a 4-point scale ( 03 ) , and includes a total of 20 questions . the higher the score , the higher the depression24 . for fear of falling , the fofq ( fear of falling questionnaire ) was used . it is scored on a 4-point scale ( 14 ) and includes a total of 11 questions . the higher the score , the higher the fear of falling25 . for statistical analysis of the results , spss 18.0 was used . to explain the differences in measurement variables according to the measurement period between exercise groups , a 2-way anova with repeated measure was used , and the level of statistical significance was =0.05 . repeated measure anova to analyze changes in the poma , cv , mvhc , kf , depression , and fof according to the measurement periods showed that there was a statistically significant difference in the interaction between time and the groups and that the changes in poma , cv , mvhc , kf , depression , and fof according to time differed ( p<0.001 ) ( table 1table 1.changes in physical and psychological function variablesgroupbeforeafterpoma ( score)eg ( n=10)17.601.3419.301.57**cg ( n=10)17.801.4017.601.43cv ( m / sec)eg ( n=10)1.560.211.600.21*cg ( n=10)1.560.101.550.09mvhc ( cm)eg ( n=10)7.421.387.021.33*cg ( n=10)7.361.627.311.74kf ( angle)eg ( n=10)111.003.16108.604.06*cg ( n=10)110.803.77110.303.16depression ( score)eg ( n=10)27.103.6724.503.34**cg ( n=10)26.002.5825.203.00fear of falling ( score)eg ( n=10)22.501.4319.801.69*cg ( n=10)22.603.2721.903.54meansd . eg , experimental group ; cg , control group ; poma , performance - oriented mobility assessment ; cv , crossing velocity ; mvhc , maximum vertical heel clearance ; kf , knee flexion . eg , experimental group ; cg , control group ; poma , performance - oriented mobility assessment ; cv , crossing velocity ; mvhc , maximum vertical heel clearance ; kf , knee flexion . * p<0.05 ; * * p<0.01 older adults have low balance and stability due to physiological and functional decreases that occur with ageing26 , and to compensate for balance and stability in gait , cadence and stride length decrease27 . in the case of obstacle negotiation , when the leading foot encounters obstacles , the center of the body moves forward and fear of falling increases28 . in this case , the mvhc , joint angle , and cv are important measures to assess the ability of elderly adults to safely cross obstacles during gait5 , 29 , 30 . although falling is not always accompanied by physical injury , fear of falling again can be used to predict falling in elderly adults5 . fear of falling leads to a decrease in activity and low self - esteem for independent behavior4 and has direct negative effects that result in decreased balance and gait disorder31 , 32 . cse is training to improve the stability of the trunk by inducing a correcting reaction and an equilibrium reaction through balance training of the flexors and extensors , and as it has been judged to have an effect on physicopsychological functions in obstacle negotiation , a clinical study was conducted . the results of the present study showed that the tinetti poma , cv , mvhc , and knee flexion angle were significantly improved . esculier et al.33 provided lumbar stability training through a wii fit program for parkinson s patients for 6 weeks and administered the tinetti poma . chou et al.20 reported that the cv was slow for elderly adults with lower balance ability . weerdesteyn et al.21 provided balance , gait , and coordination training for elderly adults and reported that foot clearance decreased , which matched with the results of the present study . park and lee22 reported that the maximum knee flexion angle of normal adults with lumbar stability was significantly lower in comparison with that of elderly adults with lumbar instability , which partially matched with the results of the present study . the study by lee et al.34 , who reported that a falling prevention program decreased the gds ( geriatric depression scale ) significantly , was in agreement with the study by duque et al.35 , who reported that fear of falling by elderly adults who experienced falling significantly decreased as a result of balance training using a virtual reality system . such results indicate that stability of the trunk was secured , as cse decreased the sway area of the center of mass . through harmonious exercise of the limbs based on physical stability , the subjects could negotiate obstacles precisely and easily , had higher self - esteem and had less fear of falling and depression . it is suggested that the 6-week cse decreased excessive obstacle gait with physical instability in elderly women and improved their physicopsychological functions involving obstacle gait .

[ purpose ] the aim of the present study was to investigate the effects of core stability exercise ( cse ) on the physical and psychological functions of elderly women while negotiating general obstacles . [ subjects and methods ] after allocating 10 elderly women each to the core stability training group and the control group , we carried out performance - oriented mobility assessment ( poma ) and measured crossing velocity ( cv ) , maximum vertical heel clearance ( mvhc ) , and knee flexion angle for assessing physical performances . we evaluated depression and fear of falling for assessing psychological functions . [ results ] relative to the control group , the core stability training group showed statistically significant overall changes after the training session : an increase in poma scores , faster cv , lower mvhc , and a decrease in knee flexion angle . furthermore , depression and fear of falling decreased significantly . [ conclusion ] cse can have a positive effect on the improvement of physical and psychological performances of older women who are vulnerable to falls as they negotiate everyday obstacles .

multiple myeloma ( mm ) is a b - cell neoplasia which is characterized by an malignant proliferation of aberrant plasma cells in the bone marrow . renal impairment and bone resorption are among the most important consequences of this disease [ 1 - 3 ] . novel treatment options are urgently needed and are being developed [ 4 - 6 ] . in mm malignant plasma cells usually produce excessive amounts of monoclonal proteins , which may involve intact immunoglobulins or serum free light chains ( sflc ) or both . in a study of 1027 patients with newly diagnosed mm , kyle et al . sflc play a crucial role in mm induced renal damage and are probably the most important cause of renal failure in these patients . in healthy individuals production of sflc this " physiologic " amount of sflc gets rapidly cleared and degraded by the kidneys . however , sflc production in mm can reach 30 g / day exceeding the clearance capacity of the kidneys . sflc are normally transported to the interstitium of the kidney via specific receptors in the proximal tubule . overload of these receptors by excessive amounts of sflc results in an overflow of them to the distal tubule . sflc entering the distal tubule usually bind to uromucoid ( tamm - horsfall protein ) and form casts , which obstruct tubular fluid flow , leading to disruption of the basement membrane and interstitial damage [ 8 - 12 ] . the amount of sflc necessary to cause renal impairment has recently been studied by nowrouisan et al . . the median serum concentration associated with overflow proteinuria and hence tubular damage were 113 mg / l for kappa and 278 mg / l for lambda light chain , respectively . thus , this level corresponds to a daily production of light chains about 5 g. reduction of sflc levels therefore represents a potential strategy to improve renal function in acute renal failure in mm . in this regard prompt initiation of an effective chemotherapy protocol is a precondition for success , but additional supportive treatment options are increasingly discussed in this regard . studies were initiated to investigate the use of plasma exchange in patients with mm and acute renal failure but failed to show any significant benefit on renal function . in the present manuscript we report on a new type of renal replacement therapy ( rrt ) , namely rrt using a protein leaking membrane . protein leaking membranes are characterized by an increased pore size of the dialyzer membrane promoting the elimination of middle sized molecules such as sflc . these membranes were initially developed to improve the clearance capacity for inflammatory mediators in critically ill septic patients . particularly one hemofilter , the hco1100 has been extensively studied in the regard . in this paper we analyzed the efficacy of the protein leaking membrane hco1100 to eliminate sflc in kappa light chain mm patients with acute renal failure necessitating rrt . the elimination capacity of the hco1100 was studied in the hemodialysis and hemodiafiltration mode and was compared to standard hemodialysis procedures as well as to plasma exchange . four consecutive patients with kappa light chain mm were included in this study . all patients necessitated rrt due to acute renal failure . baseline laboratory evaluation included blood creatinine , estimated creatinine clearance ( mdrd formula ) , urea , acid base and electrolyte household . before and after each renal replacement session sflc levels were measured using latex - enhanced nephelometric immunoassays ( the binding site , schwetzingen , germany ) adapted to the bn prospec nephelometer ( dade - behring , eschborn , germany ) . l for the sflc lambda assay with control sera ( n = 10 ) provided by the binding site . hemodialysis and hemodiafiltration procedures were performed with a dialysis device allowing the online preparation of ultrapure hemofiltration replacement fluids if required ( ak200s , gambro dialysatoren gmbh , hechingen , germany ) . polyamide high flux hemofilter ( polyflux 140h , polyflux 210h , steam sterilized , gambro dialysatoren gmbh , hechingen , germany ) were used for standard hemodialysis . for standard hemodiafiltration membrane specifications for the high flux hemofilter polyflux 140h ( pf 140h ) and polyflux 210h ( pf 210h ) are as follows : effective surface area 1.4 qm for pf 140h and 2.1 qm for pf 210h , wall thickness 50 m , inner diameter 215 m , membrane pore size ~ 5 nm , ultrafiltration coefficient 60 ( ml / h , mmhg ) for pf 140h and 85 ( ml / h , mmhg ) for pf 210h , cut off point 30 - 40 kd , sieving coefficient for vitamin b12 ( molecular weight 1.35 kd ) and inulin ( molecular weight 5.20 kd ) 1.0 , for 2 microglobulin ( molecular weight 11.8 kd ) 0.7 . for rrt ( either hemodialysis or hemodiafiltration ) with the protein leaking membrane a newly designed high cut off hemofilter , the polyamide hco 1100 hemofilter ( hco 1100 , effective surface area 1.1 qm , steam sterilized , gambro corporate research , hechingen , germany ) was used . the high cut off hemofilter is characterized by an increased pore diameter of ~10 nm with a membrane thickness of 50 m and an internal diameter of 215 m . the in vivo cut off point of the membrane is approximately 60 - 80 kd . the hemofilter has been designed to increase the in - vivo permeability for substances in the molecular weight range up to 60 kd . basic features of this new hemofilter have been recently described [ 18 - 21 ] . whenever the protein leaking membrane was applied regular controls of plasma albumin and total protein were performed ( pre - and post dialysis ) . in case of high transmembrane protein losses a balanced substitution of human albumin was uniformly set at 200 ml / h , dialysate flow rate was 500 ml / min . the ultrafiltration rate was based on the individual fluid balance of the patients . in a subgroup of patients sflc kappa - clearances in the initial phase ( 10 min after start of treatment ) and at the end of therapy were calculated as follows : clearance ( ml / min ) = dialysate concentration of flc / inlet serum concentration of flc dialysate flow rate . a roller blood pump device was used for plasma exchange ( bsm 22 , gambro hospal germany ) . a standard plasmafilter ( gambro pf 2000n , gambro , lund , sweden ) was chosen . 2500 ml of fresh frozen plasma were given in exchange during a 2.5 h session . paired data were compared using the wilcoxon rank sum test , unpaired data by the mann - whitney - u - test . patient 1 was a 73-year - old male who had previously received 6 cycles of chemotherapy with melphalan and prednisolone . at the time of admission he had acute renal failure with a creatinine level of 11.3 mg / dl ( egfr 5 ml / min ) . chemotherapy with adriamycin and dexamethasone was initiated . in order to reduce the amount of sflc two treatment sessions of plasma exchange were performed ( figure 1 ) . for uremia control rrt had to be started . six sessions of high flux hemodialysis ( figure 2 ) with a median ultrafiltration rate of 1680 ml ( range 900 - 2600 ml ) per session were applied . reduction rate ( % ) of sflc kappa with hemodialysis using the high flux membrane pf 140h . patient 2 was a 70-year - old female who had developed mm induced acute renal failure with a creatinine of 3.4 mg / dl and an estimated gfr of 14 ml / min at hospital admission . the patient was treated with hemodialysis thrice weekly using a standard high flux hemofilter as well as the protein leaking membrane on day 15 , 18 and 25 ( figure 3 ) . reduction rate ( % ) of sflc kappa with hemodialysis using the protein leaking membrane hco1100 . patient 3 was a 61-year - old woman with a long known history of light chain mm disease . previously , this patient had been treated with various chemotherapy protocols , including high - dose chemotherapy with autologous stem cell transplantation , thalidomide and dexamethasone , bortezomib , lenalidomide and dexamethasone . she was admitted to the hospital due to mm induced acute renal failure . at admission creatinine was 5.4 mg / dl ( egfr 9 ml / min ) . treatment was started with three sessions of hemodialysis using the protein leaking membrane ( figure 4 ) . the hemodialysis procedures were followed by 5 sessions of hemodiafiltration using the same hemofilter ( figure 5 ) . after hemodiafiltration 4 sessions of standard hemodialysis using a standard hemofilter ( pf 140h , gambro ) were performed ( figure 4 ) . reduction rate ( % ) of sflc kappa with hemodiafiltration applying the hco 1100 and pf210h hemofilter . although chemotherapy was promptly initiated , the patient developed progressive disease with an increase of sflc kappa from 5740 mg / a detailed sequence of the hemofilters used in this patient is given in figures 4 and 5 . this previously untreated patient was admitted to our hospital due to hypercalcaemia of 3.7 mmol / l and acute renal failure with a creatinine of 5.3 mg / dl ( egfr 12 ml / min ) . a total of 9 hemodialysis sessions with a median ultrafiltration rate of 415 ml ( range 0 - 1500 ml ) were performed . simultaneously furthermore the patient received 7 sessions of hemodiafiltration ( hc1100 ) with a median ultrafiltration of 550 ml ( range 0 - 1500 ml ) ( figure 7 ) . our data reveal that hemodiafiltration with the protein leaking membrane hco 1100 was superior to all other extracorporeal replacement strategies in eliminating sflc from circulating blood ( figure 8) . with the protein leaking membrane hco 1100 a median reduction rate of 40.8% ( range 13.9% -66.4% ) was achieved during hemodiafiltration and 23.7% ( range -32.9% -55.8% ) during hemodialysis ( p = 0.017 ) ( figure 8) . eliminating the extremes ( there were two protein leaking membrane hemodialysis sessions were the sflc increased during dialysis , probably due to an highly active disease ) significance still remained in favor for the hemodiafiltration mode ( p = 0.04 ) . of interest , the percentage decline of sflc kappa during plasma exchange was only 14.5% and 9.9% , respectively ( figure 1 ) . in the same patient high flux hemodialysis achieved a median reduction rate of 21.4% ( range -7.4% -32.7% , figure 2 ) . box - whiskers - plot : summary of median sflc kappa reduction rates by different renal replacement strategies . the median overall reduction rate for conventional high flux hemodialysis was 21.2% ( range -7,5% 41.5% ) . comparing the small ( pf 140h ) versus the large surface area membrane ( pf 210h ) revealed a slight , but statistically not significant advantage in favor for the large surface area membrane ( p = 0.66 ) . nevertheless , a significant difference was found between standard high flux hemodiafiltration ( pf 210h ) and hemodialysis ( pf 210h ) , in favor for the hemodiafiltration mode ( p = 0.005 ) ( figure 8) . in parallel best clearance rates were achieved during hemodiafiltration with the protein leaking membrane followed by hemodialysis with the protein leaking membrane . maximum clearances were achieved in the initial phase of the treatment with values reaching up to 25 ml / min during hco 1100 compared to 5 ml / min during conventional therapy ( pf 210h ) . however , even when the protein leaking membrane was used clearance rates declined to around 10 ml / min at 4 hours . blood pressure drops were equally distributed between standard high flux treatments and treatments performed with the protein leaking membrane . median albumin values before and after treatment with the protein leaking membrane were 3.3 ( range 3.1 - 3.7 ) and 3.4 ( range 3.3 - 3.89 ) , respectively . patient 1 was a 73-year - old male who had previously received 6 cycles of chemotherapy with melphalan and prednisolone . at the time of admission he had acute renal failure with a creatinine level of 11.3 mg / dl ( egfr 5 ml / min ) . chemotherapy with adriamycin and dexamethasone was initiated . in order to reduce the amount of sflc two treatment sessions of plasma exchange were performed ( figure 1 ) . for uremia control rrt had to be started . six sessions of high flux hemodialysis ( figure 2 ) with a median ultrafiltration rate of 1680 ml ( range 900 - 2600 ml ) per session were applied . reduction rate ( % ) of sflc kappa with hemodialysis using the high flux membrane pf 140h . patient 2 was a 70-year - old female who had developed mm induced acute renal failure with a creatinine of 3.4 mg / dl and an estimated gfr of 14 ml / min at hospital admission . the patient was treated with hemodialysis thrice weekly using a standard high flux hemofilter as well as the protein leaking membrane on day 15 , 18 and 25 ( figure 3 ) . reduction rate ( % ) of sflc kappa with hemodialysis using the protein leaking membrane hco1100 . patient 3 was a 61-year - old woman with a long known history of light chain mm disease . previously , this patient had been treated with various chemotherapy protocols , including high - dose chemotherapy with autologous stem cell transplantation , thalidomide and dexamethasone , bortezomib , lenalidomide and dexamethasone . she was admitted to the hospital due to mm induced acute renal failure . at admission creatinine was 5.4 mg / dl ( egfr 9 ml / min ) . treatment was started with three sessions of hemodialysis using the protein leaking membrane ( figure 4 ) . the hemodialysis procedures were followed by 5 sessions of hemodiafiltration using the same hemofilter ( figure 5 ) . after hemodiafiltration 4 sessions of standard hemodialysis using a standard hemofilter ( pf 140h , gambro ) were performed ( figure 4 ) . reduction rate ( % ) of sflc kappa with hemodiafiltration applying the hco 1100 and pf210h hemofilter . although chemotherapy was promptly initiated , the patient developed progressive disease with an increase of sflc kappa from 5740 mg / a detailed sequence of the hemofilters used in this patient is given in figures 4 and 5 . this previously untreated patient was admitted to our hospital due to hypercalcaemia of 3.7 mmol / l and acute renal failure with a creatinine of 5.3 mg / dl ( egfr 12 ml / min ) . a total of 9 hemodialysis sessions with a median ultrafiltration rate of 415 ml ( range 0 - 1500 ml ) were performed . simultaneously furthermore the patient received 7 sessions of hemodiafiltration ( hc1100 ) with a median ultrafiltration of 550 ml ( range 0 - 1500 ml ) ( figure 7 ) . our data reveal that hemodiafiltration with the protein leaking membrane hco 1100 was superior to all other extracorporeal replacement strategies in eliminating sflc from circulating blood ( figure 8) . with the protein leaking membrane hco 1100 a median reduction rate of 40.8% ( range 13.9% -66.4% ) was achieved during hemodiafiltration and 23.7% ( range -32.9% -55.8% ) during hemodialysis ( p = 0.017 ) ( figure 8) . eliminating the extremes ( there were two protein leaking membrane hemodialysis sessions were the sflc increased during dialysis , probably due to an highly active disease ) significance still remained in favor for the hemodiafiltration mode ( p = 0.04 ) . of interest , lowest sflc kappa elimination rates were achieved with plasma exchange . the percentage decline of sflc kappa during plasma exchange was only 14.5% and 9.9% , respectively ( figure 1 ) . in the same patient high flux hemodialysis achieved a median reduction rate of 21.4% ( range -7.4% -32.7% , figure 2 ) . box - whiskers - plot : summary of median sflc kappa reduction rates by different renal replacement strategies . the median overall reduction rate for conventional high flux hemodialysis was 21.2% ( range -7,5% 41.5% ) . comparing the small ( pf 140h ) versus the large surface area membrane ( pf 210h ) revealed a slight , but statistically not significant advantage in favor for the large surface area membrane ( p = 0.66 ) . nevertheless , a significant difference was found between standard high flux hemodiafiltration ( pf 210h ) and hemodialysis ( pf 210h ) , in favor for the hemodiafiltration mode ( p = 0.005 ) ( figure 8) . in parallel best clearance rates were achieved during hemodiafiltration with the protein leaking membrane followed by hemodialysis with the protein leaking membrane . maximum clearances were achieved in the initial phase of the treatment with values reaching up to 25 ml / min during hco 1100 compared to 5 ml / min during conventional therapy ( pf 210h ) . however , even when the protein leaking membrane was used clearance rates declined to around 10 ml / min at 4 hours . blood pressure drops were equally distributed between standard high flux treatments and treatments performed with the protein leaking membrane . median albumin values before and after treatment with the protein leaking membrane were 3.3 ( range 3.1 - 3.7 ) and 3.4 ( range 3.3 - 3.89 ) , respectively . in this study we analyzed different modes of extracorporeal elimination techniques for reducing sflc kappa in patients with kappa light chain mm associated nephropathy requiring dialysis . we compared a novel type of hemofilter , a so - called protein leaking membrane , applied in a hemodialysis and hemodiafiltration mode to standard procedures such as high flux hemodialysis and hemodiafiltration as well as plasma exchange . we found a superiority in favor for the protein leaking membrane particularly when applied in the hemodiafiltration mode . high flux hemofilters , particularly high flux membranes with a large surface area reached surprisingly good reduction rates for sflc kappa . however , when the clearance rates were analyzed , a significant advantage in favor for the protein leaking membrane was found . up to 10 fold higher clearance rates were achieved with the protein leaking membrane compared to the high flux hemofilter ( pf 210h ) . they are normally cleared in 2 - 4 hours at 40% of the glomerular filtration rate . however , removal may be significantly prolonged in patients with renal failure and can reach up to 2 - 3 days in mm patients in complete renal failure . free light chains are distributed in similar concentrations in serum , extra - vascular compartment and in tissue edema fluid . the intra - vascular compartment probably contains only 15 - 20% of the total amount . thus , redistribution from the interstitium into the vascular compartment occurs which makes a long extracorporeal treatment session necessary for an effective removal of sflc . clark et al . studied the effects of plasma exchange on clinical course of mm patients . they performed 5 to 7 plasma exchanges in their patients and found that it did not reduce a composite outcome of death , dialysis dependence , or glomerular filtration rate at 6 months . however , they initiated the study before the routine availability of the immunoglobulin free light chain assay , thus sflc were not investigated and subgroup analyses for light chain diseases could not be performed . very recently , cserti and co - workers studied sflc concentration in two patients and found that plasma exchange failed to effectively lower sflc levels . plasma exchange was performed using a continuous - flow cell separator . in our study plasma exchange the plasma exchange volume was 2.5 l , which is a standard dose for plasma exchange . based on this finding and having in mind the results from clark and co - workers we decided not to proceed with plasma exchange in light chain mm since the low serum reduction rates do not compensate for the potential risks ( allergic reaction , infections etc ) . hemodialysis with newly designed protein leaking membranes may represent an alternative strategy to clear sflc more effectively . due to an increased pore size of these membranes the elimination of middle sized molecules including sflc is eased . very recently hutchison and co - workers studied the impact of different protein leaking membranes on the capacity to clear sflc . excellent clearance rates for both kappa and lambda light chains were reported which translated in a significant removal of sflc in light chain mm patients with acute renal failure . , we analyzed the gambro hco 1100 membrane in the hemodialysis and hemodiafiltration mode extending the results of hutchison and co - workers . the maximum reduction rate for sflc kappa was 66% which is line with the data reported by hutchison . interestingly , we observed a high variability in the inter - and intra - patient reduction rate for sflc kappa during hco 1100 rrt which is best explained by a variable activity status of the underlying disease . while patient 2 and 4 had a more or less constant sflc kappa reduction rate of around 25 to 30% over time ( indicating a stable disease activity ) , patient 3 showed an increase in sflc kappa on day 1 of the first cycle of chemotherapy with adriamycin and dexamethasone from 4320 mg / l before starting hco 1100 dialysis to 5740 values increased even further in the dialysis free interval and reached 9739 mg / l on the consecutive dialysis day ( predialysis value ) . the missing fall in sflc kappa can be clearly interpreted as a sign of rapid disease progression . a switch in chemotherapy to bortezomib a highly effective chemotherapy is a precondition for treating mm associated nephropathy and must be initiated simultaneously to the rrt . after the first cycle of chemotherapy with adriamycin and dexamethasone sflc decreased from 6270 mg / l ( day1 ) to 3500 mg / l ( day14 ) , but then increased up to 6450 mg / l ( day 28 ) again . thus , we changed the regimen to bortezomib and dexamethasone . at day 16 after starting this chemotherapy regime sflc dropped to 48.8 mg / l . here , a combination of an effective chemotherapy and hemodialysis led to a very good result . importantly , the observed rebound phenomenon after rrt also results from the distribution of sflc into the different body compartments . only around 20% of the total amount of sflc are found in circulating blood whereas 80% lay in the so called " third compartment " . redistribution processes have to take place before these " trapped " sflc can be eliminated . hence , sustained elimination procedures are required . rrt with some protein leaking membranes can lead to substantial transmembrane protein losses particularly when used on a extended daily base as propagated by hutchison et al . . however , in our study population a severe protein depletion was not observed . in conclusion , extracorporeal elimination strategies with the protein leaking membrane hco 1100 may be a new and promising adjuvant treatment strategy for patients with sflc nephropathy requiring dialysis . hemodiafiltration and to lesser extend also hemodialysis with the hco 1100 hemofilter are able to eliminate substantial amounts of sflc kappa in mm patients . however , a rebound phenomenon is observed which not only demands a prolonged and repeated treatment strategy but also an effective chemotherapy protocol as a precondition for renal recovery .

multiple myeloma ( mm ) is a malignancy with excessive production of monoclonal proteins . at disease presentation 30% of mm patients have significant renal impairment which may progress to renal failure requiring dialysis . besides chemotherapy extracorporeal elimination procedures such as plasma exchange have been applied as adjuvant strategies to eliminate free light chains from circulating blood , however the efficacy was poor with older techniques . we report about a highly efficient method to eliminate serum free light chain ( sflc ) using a newly designed protein leaking membrane in patients suffering from sflc induced acute renal failure . the protein leaking membrane ( hco 1100 ) is characterized by increased pore size facilitating elimination of middle molecules such as sflc kappa ( 22.5 kd ) . the hco 1100 membrane was applied in a hemodialysis and hemodiafiltration mode and compared to standard procedures ( high flux hemodialysis , hemodiafiltration and plasma exchange ) . hemodiafiltration with the protein leaking membrane hco 1100 was superior to all other extracorporeal replacement strategies in eliminating sflc - kappa from circulating blood . a median blood reduction rate of 40.8% ( range 13.9% - 66.4% ) was achieved during hemodiafiltration . the corresponding peak clearance rate was 25 ml / min . importantly , the poorest elimination rate was achieved by plasma exchange followed by standard high flux hemodialysis . extracorporeal elimination strategies with the protein leaking membrane hco 1100 may be a promising adjuvant treatment strategy for patients with sflc nephropathy requiring dialysis . hemodiafiltration and to lesser extend also hemodialysis with the hco 1100 hemofilter are able to eliminate substantial amounts of sflc kappa in mm patients .

synchronous primary lung cancers are uncommon and the occurrence of synchronous non small cell lung carcinomas ( nsclc ) with different histological morphologies within the same lobe is rare . the aim of this report is to discuss surgical and oncological management of this entity together with a review of the current literature . a 61-year - old female smoker ( 42 pack years ) was found to have left upper zone shadowing on chest x ray following a history of weight loss . her performance status was 1 with a forced expiratory volume in one second ( fev1 ) of 1.94 litres . positron emission tomography in conjunction with computed tomography ( pet - ct ) showed two nodules in the left upper lobe . there was a 2 cm spiculated lesion in the anterior segment with a standardised uptake value ( suv ) max of 5.6 units ( fig 1a ) and a 2.2 cm cavitating lesion in the apico - posterior segment with a suv max of 2.7 units ( fig 1b ) . coronal view pet ct scan of a high uptake nodule in the left upper lobe coronal view of pet ct scan of moderate uptake synchronous nodule in the left upper lobe a left thoracotomy with upper lobectomy was performed and the intra - operative findings were consistent with the radiological appearances seen in the pet - ct scan . there was a 2 cm infiltrating squamous cell carcinoma in the anterior segment with vascular invasion ( pathological ( p ) t2a according to tnm 7th edition ) and a separate 2.4 cm adenocarcinoma with associated broncho - alveolar spread in the apical segment ( pt1b ) . an adjacent peribronchial lymph node was positive for tumour ( pn1 ) , however due to the poorly differentiated morphology of the tumour cells it was unclear as to which tumour it originated . cisplatin and vinorelbine adjuvant chemotherapy was administered with no overt complications ; however recurrence of the disease was discovered radiologically one year post - operatively in the form of a left hilar soft tissue mass with bony metastases . the patient subsequently received radiotherapy without complications but a staging ct chest / abdomen / pelvis performed three months later showed progressive disease with left lung collapse in addition to multiple , palpable subcutaneous nodules . these features were in keeping with the patient s symptoms of worsening breathlessness and wheeze . further investigations including bronchoscopy showed a tumour in left main bronchus which was treated with laser ablation and placement of a tracheo - bronchial stent . histological assessment of this nodule showed a poorly differentiated adenocarcinoma , which was confirmed with immunohistochemical techniques . specialist genetic analysis of the subcutaneous lesion was performed , which showed the presence of an egfr mutation sensitive to anti - egfr tyrosine kinase inhibitors . this enabled the oncologists to use monoclonal antibody therapies ( anti egfr tki ) and the patient was treated for one month with iressa ( gefitinib ) . she responded well with significant decrease in the size of the metastatic subcutaneous nodules . due to her progressive disease the patient deceased with an overall survival ( from the time of the lung resection ) of 20 months synchronous primary lung cancers ( splc ) were first described in 1924 by beyreuther h ( 1 ) . the true incidence of these remains uncertain but evidence has shown figures between 1% to 8% ( 2 ) . the criteria for the diagnosis of splc , which was proposed by martini and melamed in 1975 , are that synchronous tumours are physically distinct and that the histology is different surgical resection of splc was recommended by many of the reviewed authors ( 3 - 5 ) . rostad et al studied the outcome and characteristics of synchronous primary lung cancers . in 15,308 lung cancer resection cases , 94 patients were found to have synchronous non small cell lung cancers , nsclc , 9 patients had synchronous lung cancers with different histological morphologies and only 2 ( 0.01% ) patients had synchronous lung cancers with different histological morphologies within the same lobe . the relative survival rate in patients with different histological morphologies ( n=9 ) was 12.7% . patients with similar morphologies had a better outcome with a relative survival rate of 29.2% , although the difference was not statistically significant ( p=0.24 ) . the authors concluded that surgical resection should be offered to patients with synchronous lung cancers who are operable with respectable tumour . ( 3 ) lymph node metastases were found to be a statistically significant prognostic factor . in a study of 92 patients who had surgical resections for multiple synchronous primary lung cancers , the results showed the 5-year survival were 52.5% and 15.5% for patients without and with lymph node metastasis respectively ( p = 0.001 ) . ( 3 ) skin metastases from lung cancers are not uncommon , and their presence is a poor prognosis factor ( 6 ) . recently anti - egfr tki molecules have been introduced for the treatment of advanced malignancies including lung cancers , however long term treatment resistance remains a therapeutic challenge . our patient had a disease free survival of 12 months post surgery despite a positive lymph node , but she later developed recurrence with distant subcutaneous metastases and an endobronchial mass , which was treated palliatively . she received anti egfr1-tk inhibitors based on genetic analysis from the metastatic adenocarcinoma subcutaneous lesions and her initial response was good . patients with resectable synchronous primary non small cell lung carcinomas within the same lobe should be offered surgical resection after careful pre - operative staging . skin metastases should be biopsied and egfr testing should be requested in order to determine the originating the tumour and to assess the patients suitability for anti egfr - tki treatment .

we report the case of a sixty one year old female diagnosed with two synchronous primary lung cancers located within the same lobe . surgical resection was performed , followed by adjuvant chemotherapy . the patient developed distant bone and skin metastases one year post - surgical resection . in this report we discuss the multimodality therapy used to treat this patient .

all mice were housed and bred in a specific pathogen - free facility and used at 412 wk of age . tcr mice ( 21 ) were obtained from the jackson laboratory ( bar harbor , me ) . tcr mice ( 22 ) were mated with rag2 mice ( 23 ) to generate double - deficient rag2tcr mice . mice transgenic for a chimeric protein consisting of the extracellular and transmembrane domains of human cd25 and the cytosolic domain of murine tcr ( tt , line no . 35 ) ( 24 ) were also bred onto the rag2 background to make ragtt mice . rag2 and rag2 mice were injected intraperitoneally with 250 g of affinity - purified anti - cd3 mab ( 145 - 2c11 ) ( 25 ) or with the dose indicated . ragtt mice were injected with 250 g anti - tac mab ( 1ht4 - 4h3 ) . where indicated , rag2 mice were injected with 250 g of affinity - purified anti - cd3 mab on both day 0 and day 8 and then subjected to the coreceptor reexpression assay on day 12 . where indicated , rag2 mice were radiated with 400 cgy and analyzed 3 wk later ( 26 , 27 ) . performance of the coreceptor reexpression assay on electronically sorted thymocyte populations has been described previously ( 8) . in brief , single cell suspensions of thymocytes were stained with pe - conjugated anti - cd4 mab ( gk1.5 ; becton dickinson , san jose , ca ) and fitc - conjugated anti - cd8 mab ( 53 - 6 - 72 , becton dickinson ) . stained thymocytes were electronically sorted by a facstar plus according to the gates indicated in each figure . sorted cells were washed extensively with pbs and treated with 0.04% pronase ( calbiochem novabiochem , san diego , ca ) and 100 g / ml dnase i ( boehringer mannheim , indianapolis , in ) at 37c for 15 min , pelleted , and pronase treated for another 10 min at 37c . cells were placed in culture for 1216 h of culture at either 4 or 37c , after which harvested cells were restained with anti - cd4pe and anti - cd8fitc . for three - color analysis cells were also stained with anti - cd5 ( 53 - 7 - 3 ; pharmingen ) followed by cy-5 avidin ( caltag , san francisco , ca ) . dead cells were excluded by electronic gating on both forward light scatter and propidium iodide intensity . flow cytometry using three- or four - decade logarithmic amplification as indicated was performed on a facstar plus and data were analyzed using software designed by the division of computer research and technology at the national institutes of health . all mice were housed and bred in a specific pathogen - free facility and used at 412 wk of age . tcr mice ( 21 ) were obtained from the jackson laboratory ( bar harbor , me ) . tcr mice ( 22 ) were mated with rag2 mice ( 23 ) to generate double - deficient rag2tcr mice . mice transgenic for a chimeric protein consisting of the extracellular and transmembrane domains of human cd25 and the cytosolic domain of murine tcr ( tt , line no . 35 ) ( 24 ) were also bred onto the rag2 background to make ragtt mice . rag2 and rag2 mice were injected intraperitoneally with 250 g of affinity - purified anti - cd3 mab ( 145 - 2c11 ) ( 25 ) or with the dose indicated . ragtt mice were injected with 250 g anti - tac mab ( 1ht4 - 4h3 ) . where indicated , rag2 mice were injected with 250 g of affinity - purified anti - cd3 mab on both day 0 and day 8 and then subjected to the coreceptor reexpression assay on day 12 . where indicated , rag2 mice were radiated with 400 cgy and analyzed 3 wk later ( 26 , 27 ) . performance of the coreceptor reexpression assay on electronically sorted thymocyte populations has been described previously ( 8) . in brief , single cell suspensions of thymocytes were stained with pe - conjugated anti - cd4 mab ( gk1.5 ; becton dickinson , san jose , ca ) and fitc - conjugated anti - cd8 mab ( 53 - 6 - 72 , becton dickinson ) . stained thymocytes were electronically sorted by a facstar plus according to the gates indicated in each figure . sorted cells were washed extensively with pbs and treated with 0.04% pronase ( calbiochem novabiochem , san diego , ca ) and 100 g / ml dnase i ( boehringer mannheim , indianapolis , in ) at 37c for 15 min , pelleted , and pronase treated for another 10 min at 37c . cells were placed in culture for 1216 h of culture at either 4 or 37c , after which harvested cells were restained with anti - cd4pe and anti - cd8fitc . for three - color analysis cells were also stained with anti - cd5 ( 53 - 7 - 3 ; pharmingen ) followed by cy-5 avidin ( caltag , san francisco , ca ) . dead cells were excluded by electronic gating on both forward light scatter and propidium iodide intensity . flow cytometry using three- or four - decade logarithmic amplification as indicated was performed on a facstar plus and data were analyzed using software designed by the division of computer research and technology at the national institutes of health . to assess the possibility that dp thymocytes spontaneously terminated cd8 coreceptor synthesis even in the absence of tcr cd3 signals , we examined dp thymocytes from tcr mice by the coreceptor reexpression assay . tcr thymocytes can not express conventional tcr complexes and , consequently , can not differentiate beyond the dp stage of development ( 21 ) . to enrich for dp thymocytes that might have committed to the cd4 or cd8 t cell lineages , we electronically sorted for cd48 and cd48 transitional cell populations and utilized the coreceptor reexpression assay to determine the coreceptor molecules they were actively synthesizing ( fig . 1 , a and b ) . in the coreceptor reexpression assay , preexisting surface cd4 and cd8 coreceptor molecules are removed from the sorted cells by treatment with low doses of pronase , and the stripped cells then placed into single cell suspension cultures for 14 h. metabolic activity of cultured cells is inhibited at 4c , so that cell surface coreceptor reexpression does not occur ( fig . 1 a and b , middle columns ) . however , cells cultured at 37c do reexpress the cd4 and/or cd8 coreceptor molecules that they are actively synthesizing . indeed , we have previously demonstrated that coreceptor reexpression in this assay requires active coreceptor transcription and protein synthesis ( 8) . interestingly , virtually all cd48 sorted cells from tcr dp thymocytes reexpressed both cd4 and cd8 coreceptors and so reappeared as dp cells ( fig . identical results were obtained with cd48 sorted cells from tcr mice ( fig . 1 b , top ) . that is , none of the dp thymocytes present in tcr mice had selectively terminated either cd4 or cd8 coreceptor synthesis , indicating that none had undergone lineage commitment . thus , these results indicated that lineage commitment did not occur spontaneously in immature dp thymocytes but might be dependent upon signals transduced by surface tcr cd3 complexes . to assess directly the role of tcr cd3 signals in inducing lineage commitment in dp thymocytes , we assessed dp thymocytes from experimentally induced rag2 mice . rag2 mice fail to express any clonotypic tcr chain because they are unable to recombine any tcr gene locus . as a result , rag2 thymocytes are arrested at the cd48 ( double - negative , dn ) stage of development ( 23 ) . however , rag thymocytes can be induced to differentiate further into dp cells by either sublethal -irradiation ( 26 , 27 ) or by injection of anti - cd3 mab ( 28 , 29 ) ( fig . 1 , a and b , left column ) . induced rag dp thymocytes did not further differentiate into phenotypically mature t cells as thymocytes from stimulated rag mice that appeared cd48 or cd48 ( fig . 1 , a and b , left columns ) were predominantly precursor cells that spontaneously became cd48 in overnight culture ( data not shown ; reference 30 ) . even though sublethal -irradiation and anti - cd3 injection both induced generation of rag dp thymocytes ( fig . that is , sublethal -irradiation did not detectably stimulate cd3 signal transduction as revealed by the absence of cd5 upregulation , whereas injection of anti - cd3 mab did stimulate cd3 signal transduction and cd5 upregulation ( fig . 2 ) . assessment of cd48 and cd48 sorted cells from -irradiated rag2 mice by the coreceptor reexpression assay revealed that none had undergone lineage commitment ( fig . assessment of cd48 sorted cells from anti - cd3 induced rag2 mice revealed the presence of cd4-committed dp thymocytes that had selectively terminated cd8 coreceptor synthesis , as well as the presence of uncommitted dp thymocytes ( fig . the cd4-committed thymocytes that were present in anti - cd3induced rag2 mice expressed the phenotype of newly committed thymocytes in that they were hsa , thymic shared antigen ( tsa)-1 ( data not shown ) . in contrast , anti - cd3induced rag2 thymocytes had no cd8-committed cells among either cd48 or cd48 sorted cell populations ( fig . 1 , a and b ) . thus , these results ( a ) confirm that dp thymocytes do not undergo lineage commitment in the absence of tcr cd3 signals , and ( b ) demonstrate that cd3 signals stimulated by anti - cd3 mabs are sufficient to induce dp thymocytes to selectively terminate cd8 coreceptor synthesis and commit to the cd4 lineage , even in the absence of clonotypic tcr chains . to determine whether signals transduced by tcr chains are indispensable for induction of cd4 commitment , we generated double knockout rag2tcr mice . in vivo injection of anti - cd3 mabs into ragtcr double knockout mice induced the generation of dp thymocytes , as has been described ( 31 ) , and signaled these cells to upregulate cd5 expression ( fig . interestingly , we found that cd48 sorted thymocytes from these anti - cd3injected mice did contain cd4-committed cells that had selectively terminated cd8 coreceptor synthesis ( see fig . , no cd8-committed cells were detected in either cd48 or cd48 sorted cell populations ( fig . 1 , a and b ) . these results demonstrate that cd3-transduced signals can induce cd4 commitment in dp thymocytes in the absence of clonotypic tcr chains and in the absence of tcr chains . to determine whether signals transduced by tcr chains were able to induce cd4 commitment , we assessed lineage commitment in dp thymocytes from rag2 mice that expressed a chimeric transgenic protein consisting of the external and transmembrane domains of human cd25 and the cytosolic domains of tcr ( 24 ) . the extracellular domain of this transgenic protein is recognized by anti - tac mab . injection of anti - tac mab into ragtt mice induced the generation of dp thymocytes ( fig . 1 a ) , as previously reported ( 24 ) , and signaled them to upregulate cd5 expression ( fig . we found that cd48 sorted thymocytes from these mice did contain cd4-committed cells , but did not contain any cd8-committed cells in either cd48 or cd48 sorted cell populations ( see fig . 1 , a and b ) . these results demonstrate that signals transduced by the cytosolic portion of tcr chains are sufficient to induce dp thymocytes to selectively terminate cd8 coreceptor synthesis and to undergo cd4 commitment . next , we wished to evaluate the relationship between cd5 upregulation and cd4 commitment in anti - cd3 signaled rag2 thymocytes . in vivo injection of a single dose of either 10 or 250 g of anti - cd3 mab induced substantial numbers of dp thymocytes in rag mice when assayed 8 d later ( fig . while both injection doses induced differentiaton to the dp stage , we reasoned that only the high dose might persist long enough in vivo to stimulate a subsequent cd3 signal after dp thymocytes appeared . indeed , only dp thymocytes from high dose injected animals had upregulated cd5 expression , and only dp thymocytes from high dose injected animals contained cd4-committed thymocytes ( fig . 3 ) . dp thymocytes from low dose injected animals were cd5 and remained uncommitted ( fig . we conclude that cd4 commitment requires cd3 signals in dp thymocytes that are of sufficient intensity to upregulate surface cd5 expression . finally , having observed that a single injection of antibody was sufficient to stimulate cd3 or tcr chains to transduce signals that upregulated cd5 surface expression and induced cd4 commitment but not cd8 commitment , we assessed whether cd8-committed cells might appear in rag2 thymi upon antibody restimulation . in fig . 4 , rag2 mice were injected with anti - cd3 mab on both days 0 and 8 , and then assessed 4 d later on day 12 . thymocytes were sorted into cd48 and cd48 populations and then assessed for coreceptor synthesis by the coreceptor reexpression assay . we found that cd48 thymocytes contained cd4-committed cells that had selectively terminated cd8 coreceptor synthesis ( fig . 4 , middle row ) , but neither sorted population contained cd8-committed cells ( fig . , cd8-committed cells did not appear in rag2 thymi despite a second antibody injection and despite assessment on day 12 after the initial injection of antibody ( fig . , we also failed to detect cd8-committed thymocytes on days 28 and 35 after antibody injection ( data not shown ) . the present study demonstrates that immature dp thymocytes do not spontaneously terminate synthesis of either cd4 or cd8 coreceptor molecules . rather , selective termination of coreceptor synthesis by immature dp thymocytes requires signals transduced by either cd3 or tcr chains , and can occur in signaled dp thymocytes that lack clonotypic tcr chains . interestingly , cd3-signaled dp thymocytes upregulated cd5 expression and selectively terminated cd8 coreceptor synthesis , but did not selectively terminate cd4 coreceptor synthesis . thus , cd4 commitment is induced in dp thymocytes by cd3 signals that are of sufficient intensity to upregulate cd5 expression . the results of the present study are not readily compatible with either instructional ( 1 , 2 ) or stochastic / selection ( 37 ) models of lineage commitment . that is , the instructional model can not explain the presence of any lineage - committed rag2 thymocytes in response to lineage - neutral cd3 and tcr signals , whereas the stochastic / selection model can not explain why cd3 and tcr signals only induced rag2 thymocytes to become cd4 committed without inducing an equal number to become cd8 committed . in contrast with these two models of lineage commitment , the present results are concordant with the asymmetric commitment model of lineage commitment ( 8 , 13 ) . that is , our results are consistent with the concept that cd4 commitment , unlike cd8 commitment , can occur in the absence of lineage - specific signals . importantly , the present results extend the asymmetric commitment model by demonstrating that cd4 commitment does not occur spontaneously in unsignaled dp thymocytes , but rather is induced by lineage - neutral signals transduced by cd3 and/or tcr chains . importantly , we found that cd3 signaling did not induce all dp thymocytes in the present study to become cd4 committed , as only a small minority of cd3-signaled rag2 thymocytes terminated cd8 coreceptor synthesis , even though all dp thymocytes had upregulated cd5 surface expression . this observation is consistent with our recent finding that only a small fraction of cd5 dp thymocytes in normal mice have undergone lineage commitment , with most cd5 dp thymocytes remaining lineage uncommitted ( 20 ) . our current perspective is that cd3 signaling drives cd5 dp thymocytes to become cd5 , at which point they developmentally await the induction of lineage - specific signals . if lineage - specific signals are generated , perhaps by notch proteins ( 12 ) , cd5 dp thymocytes terminate cd4 coreceptor synthesis and become cd8 committed . but if lineage - specific signals are not generated , cd5 dp thymocytes terminate cd8 coreceptor synthesis and become cd4 committed . we do not know how long cd5 dp thymocytes await the appearance of lineage - specific signals before terminating cd8 coreceptor synthesis , and we do not know whether there are intrathymic signals that regulate the timing of this event . finally , the present results are remarkable in their demonstration that cd3-signaled dp thymocytes could undergo cd4 commitment even in the absence of clonotypic tcr chains . of course , dp thymocytes express surface molecules in addition to clonotypic tcr chains that can stimulate cd3 signaling , such as cd2 , cd5 , thy-1 , and ly6 ( 3234 ) . consequently , it is conceivable that engagement of such nonclonotypic molecules by intrathymic ligands can inefficiently mimic clonotypic tcr chains in their ability to stimulate cd3 signals that induce cd5 dp thymocytes to become cd5 , and so eventually to become cd4 committed . the absence of cd5 dp thymocytes and cd4-committed cells in tcr mice does not rule out intrathymic signaling by nonclonotypic molecules because surface cd3 expression is probably too low on tcr thymocytes to transduce such signals . indeed , stimulation of cd3 signals by nonclonotypic receptors may provide one explanation for the appearance of small numbers of cd4-committed dp thymocytes in mhc - deficient thymi ( 8) . purified populations of cd48 thymocytes ( a ) and cd48 thymocytes ( b ) were obtained by electronic cell sorting according to the indicated sorting gates superimposed on the starting thymocyte populations ( left columns ) . sorted thymocyte populations were stripped of surface coreceptor molecules by treatment with low doses of pronase , after which they were placed in suspension cultures at 4c ( middle panels ) or 37c ( right panels ) for 1216 h and restained for cd4 and cd8 surface expression . the coreceptor reexpression assay detects the coreceptor molecules that individual thymocytes synthesized during the 37c culture , and is dependent upon new transcription and new protein synthesis ( 8) . sorted thymocytes that reexpress both cd4 and cd8 coreceptor proteins are lineage - uncommitted cells ; those reexpressing only cd4 are cd4 committed ; and those reexpressing only cd8 are cd8 committed . cells cultured at 4c do not reexpress surface coreceptor molecules so that their cd4cd8 histograms reflect whatever coreceptor molecules that potentially remain after pronase treatment ( middle columns ) . as we have previously described ( 8) , the anti - cd4 mab used to prepare thymocytes for cell sorting minimally interferes with stripping of surface cd4 molecules by pronase , resulting in a small number of residual cd4 molecules remaining on the cell surface . consequently , to highlight changes in coreceptor reexpression during 37c cultures , histogram boxes were drawn based on the 4c profiles of each sorted and pronase - stripped cell population . the frequency of cells in each box is indicated . the number of the thymocytes obtained in these experimental mice were the following : tcr ( 9 10 cells ) , -irradiated rag2 ( 4 10 cells ) , anti - cd3 mab injected rag2 ( 1.1 10 cells ) , anti - cd3 mab injected ragtcr ( 8 10 cells ) , and anti - tac mab thymocytes from the indicated experimental mice were stained with anti - cd5 mab ( solid line ) or an irrelevant antibody ( shaded curve ) . cd5 expression on normal b6 thymocytes stained at the same time as a positive control is also shown for comparison ( dotted line ) . rag mice received one intraperitoneal injection of either 250 g or 10 g of affinity - purified anti - cd3 mab . 8 d later , thymocytes were sorted according to the indicated gates and assessed for lineage commitment by the coreceptor reexpression assay ( left panels ) . unsorted thymocytes were placed in culture for 12 h at 4c , at which temperature thymocyte phenotype is stable , and then assessed for surface cd5 expression ( right panels ) . the dotted line represents cd5 expression on normal b6 thymocytes stained at the same time as a positive control and is shown for comparison . the number of thymocytes obtained on day 8 were 11.5 10 cells and 34 10 cells with the injection of 250 g and 10 g , respectively . cd8-committed thymocytes do not appear in rag2 thymi even after two injections of anti - cd3 mab . rag2 mice were injected with 250 g of affinity - purified anti - cd3 mab on both day 0 and 8 . on day 12 , thymocytes were harvested , sorted into cd48 and cd48 populations , and assessed by the coreceptor reexpression assay for appearance of cd4-committed and cd8-committed cells .

as a consequence of positive selection in the thymus , immature cd4 + 8 + double - positive , [ dp ] thymocytes selectively terminate synthesis of one coreceptor molecule and , as a result , differentiate into either cd4 + or cd8 + t cells . the decision by individual dp thymocytes to terminate synthesis of one or the other coreceptor molecule is referred to as lineage commitment . previously , we reported that the intrathymic signals that induced commitment to the cd4 versus cd8 t cell lineages were markedly asymmetric . notably , cd8 commitment appeared to require lineage - specific signals , whereas cd4 commitment appeared to occur in the absence of lineage - specific signals by default . consequently , it was unclear whether cd4 commitment , as revealed by selective termination of cd8 coreceptor synthesis , occurred in all dp thymocytes , or whether cd4 commitment occurred only in t cell receptor ( tcr)cd3-signaled dp thymocytes . here , we report that selective termination of cd8 coreceptor synthesis does not occur in dp thymocytes spontaneously . rather , cd4 commitment in dp thymocytes requires signals transduced by either cd3 or chains , which can signal cd4 commitment even in the absence of clonotypic tcr chains .

acute intussusception is one of the most common causes of acute abdomen in infants . management includes both surgical and non - surgical methods , with gradually increasing importance recently being accorded to the latter . conservative measures , including hydrostatic reduction , are the methods of choice for initial management . though widely accepted , the expertise is still not widely available , especially in developing countries . a six - month - old child presented with complaints of repeated episodes of vomiting that contained only ingested milk . this was associated with three episodes of loose stools , initially watery and later with fresh red blood per rectum with jelly - like consistency . there was no history of fever , decreased urine output , lethargy , abnormal body movements , or difficulty in breathing . her immunizations were fully up to date with no history of rota virus immunization . on admission abdominal examination was remarkable for an ill - defined mass that could be felt in the right hypochondrium . she was initially kept nil per oral and started on conservative measures , including antibiotics . an urgent abdominal ultrasonography was done , which showed a round mass - like lesion giving a target appearance in the right hypochondrium ( suggestive of ileocolic intussusception ) . she was taken for immediate intervention , and as per surgical opinion , a hydrostatic reduction under radiological guidance was started . the child was first stabilized using intravenous fluids to treat dehydration , and a pre - procedure x - ray abdomen was performed to rule out intestinal perforation . the procedure was done in the ultrasonography room in the presence of a treating physician and a pediatric surgeon . without using any sedation , a 16 f foley catheter was inserted into the rectum in the supine position and the balloon was gently inflated while maintaining a tight anal seal . the patient s vitals , which included abdominal girth and distension , were monitored throughout the procedure . lactate solution , warmed to body temperature , was then slowly introduced into the catheter through a bag hanging at a height of approximately 100 cm . during reduction , the intussusceptum was observed under continuous ultrasound guidance as it proceeded to the cecum and then reduced across the ileocecal valve ( figures 1 - 4 ) . ileal loop ( star ) within ascending colon ( arrow ) , longitudinal view . this was associated with the patient s improved condition , which included less crying and irritability . the parents were told about the possibility of recurrence and asked to pay close attention for the onset of symptoms . acute intussusception is defined as the telescoping of the proximal bowel ( intussusceptum ) into the distal bowel ( intussuscipiens ) . the usual locations include the ileocolic region and ileocecal junction , which comprise the majority of cases . intussusception mostly presents between four months and two years , while the peak incidence is found around middle infancy . diagnosis usually needs a high suspicion on the part of the treating physician , as presentation can be quite varied , including irritability , vomiting , and poor feeding . characteristic manifestations include crying episodes , fresh red blood in stools ( hematochezia ) , and a mass in the abdomen . differential diagnoses include acute gastroenteritis , dysentery , sepsis syndrome , and volvulus , amongst others . the diagnosis is usually confirmed sonographically , which is highly accurate and has a reported sensitivity that exceeds 90% . various signs have been described in the literature , depicting intussusception as an abdominal mass with a target sign on the transverse section , a pseudokidney ( or sandwich sign ) on the longitudinal section , and a crescent - in - doughnut sign on axial images . the management of intussusception has seen a paradigm shift in recent years . in the past , the preferred non - surgical method involved use of a barium enema for reduction , followed by the air insufflation method . hydrostatic reduction using ultrasound - guided saline reduction has recently gained acceptance as the procedure of choice for initial nonsurgical management of intussusceptions in children . this popularity can be attributed to the avoidance of radiation exposure to the child and the treating team , as well as to the high success rates that have been achieved . there is still some controversy regarding the exact method of the procedure , which can be attributed to the poor training of physicians and to the general belief that intussusception is entirely a surgical problem , especially in developing countries . this procedure should be undertaken with a close liaison between pediatrics , pediatric surgery , and radiology teams to obtain the best results . the surgeon and physician should always be present at the time of reduction for clinical monitoring . after adequate optimization , including temperature controlled settings , the procedure should ideally be undertaken without any sedation . this is helpful for assessing a successful reduction using clinical criteria , as described later . we used ringer s lactate due to its near physiological constitution , avoiding the risk of chemical peritonitis due to a barium enema or tension pneumoperitoneum due to an air enema , as seen in cases of intestinal perforation . the risk of intestinal perforation following such a procedure is rarely encountered due to less hydrostatic pressure exerted and an even amount of pressure exerted on the bowel walls as compared to air insufflations . even if intestinal perforation occurs we chose 100 cm h2o pressure to lessen the risk of perforation , though different studies have used a wide range of pressures ( 75 to 125 cm h2o).6 , 9 the main ultrasonographical criteria of successful reduction is the flow of the fluid from the cecum into the terminal ileum . general improvement in the clinical profile of the patient including relief from crying , stable vitals , increasing abdominal distension during the procedure ( which indicates filling of the small bowel ) , and the disappearance of the abdominal mass , should also be given due consideration when deciding to terminate the procedure . a repeat ultrasound should always be done after the procedure as the chances of recurrence is high , especially in the immediate post - procedure period . , this method should be undertaken as teamwork , and not considered the work of a single specialty , which is the best way of treating the patients according to the standards of today .

we present a case of infantile intussusception treated successfully using hydrostatic reduction , highlighting the importance of non - surgical techniques as the first line of management for this entity . physicians should strive for mastery over such techniques by extensive training to prevent unwanted surgical procedures in such cases .

inhalation of particulate matter ( pm ) from fossil fuel combustion is associated with adverse health effects , including reduced lung function ( 1 ) and increased mortality ( 2 ) . although the mechanism for pm - induced health effects is not fully defined , animal models and in vitro studies suggest that pro - inflammatory cytokine release from airway cells is an important factor ( 3 ) . environmental agents associated with elevated lung cancer risk , such as ambient particulate matter , may damage the lung by inducing chronic inflammation . lung cancer risk is elevated in individuals with emphysema ( 4 , 5 ) , interstitial lung disease ( 6 ) , and asthma ( 7 ) , which could similarly reflect effects of the underlying inflammatory disorders . induction of pro - inflammatory mediators by alveolar macrophages exposed to ambient air particulate matter has been suggested to be a key factor in the pathogenesis of inflammatory and diseases in the lungs . different contributing physiological and psychosocial factors have been proposed ( 8) . a few prior studies have examined lung cancer risk in relation to polymorphisms in the genes coding for inflammation pathway signaling molecules , such as interleukin 1 ( il-1 ) ( 9 - 11 ) , il-1 receptor antagonist ( il-1rn ) ( 12 , 13 ) , il-6 ( 10 , 14 ) , il-10 ( 15 ) , cyclooxygenase 2 ( 14 ) , and tumor necrosis factor- ( 16 ) . these inflammatory cytokines are regulated by the pro - inflammatory transcription factor , nuclear factor nf-b ( 8) . given the close interaction between the external environment and the lung , tlrs have been implicated in lung - associated immune responses , including airway hyper responsiveness ( ahr ) and allergic asthma ( 17 ) . dysfunction and unregulated activation of the tlr pathway can contribute to decreased lung function and the pathogenesis of acute and chronic lung inflammatory diseases ( 18 ) . tlr activation , can occur via two pathways : 1- the myeloid differentiation primary - response protein 88 ( myd88)-dependent pathway , and 2- the myd88-independent pathway . these two pathways correspond to early and late - phase nf-b signaling and pathway - specific induction of pro - inflammatory cytokines and chemokines ( 19 , 20 ) . regular aerobic exercise results in multiple health benefits , including improvement of cardiorespiratory fitness and quality of life , reduction of obesity and blood pressure , and increased longevity ( 21 , 22 ) . when performed chronically on a regular basis , aerobic exercise also reduces oxidative stress systemically ( 23 ) in different diseases , such as heart diseases , type 2 diabetes , rheumatic arthritis , and alzheimer and parkinson diseases ( 23 ) , as well as in the airway epithelial cells of animals with long - term allergic lung inflammation ( 24 ) . chronic practice of regular exercise exerts a marked anti - inflammatory effect in different models of pulmonary diseases , such as in asthma models ( 25 - 28 ) , acute respiratory distress syndrome ( 29 , 30 ) , and chronic obstructive pulmonary disease ( 31 ) . studies that have investigated the effects of exposure to air pollutants during exercise have suggested that people exercising in polluted environments are at increased risk of respiratory and cardiovascular morbidity related to air pollution owing to an exercise - induced amplification in respiratory uptake , lung deposition , and toxicity of inhaled pollutants ( 32 - 35 ) . exercise may increase the likelihood of an adverse effect by increasing the dose of pollutants delivered to target sites in the lungs as ventilation increases to meet metabolic demands ( 36 ) . however , these studies do not take into account the potential anti - inflammatory and health effects of exercising in air pollution ( 37 ) , which could inhibit the pro - inflammatory events induced by air pollution . therefore , the aim of this study was to investigate the effects of 4 weeks of aerobic exercise performed in association with carbon black pm10 exposure on lung tissue inflammation and lung cancer . this study was approved by the tarbiat modares university of tehran ( code number : 62.2987 ) . twenty four adult male wistar rats aged 8 weeks were obtained from pasteur institute of iran and randomly divided into the 4 groups : a ; control ( without exposure carbon black pm10 and aerobic exercise ; n = 6 ) , b ; aerobic exercise ( five times per week for 4 weeks ; n = 6 ) , c ; exposure to carbon black pm10 ( 5 mg / m ; per rat ; n = 6 ) , d ; aerobic exercise concomitantly with exposure to carbon black pm10 ( n = 6 ) . rats were housed in cages under controlled environment ( 23c and 12 hour light - dark cycle ) with free access to normal chow and tap water . figure 1 shows the inhalation chamber at the laboratory of tarbiat modares university ( falonak ) where inhalation exposure was carried out . rats in groups of c and d were exposed to carbon black in the inhalation chamber at nominal concentrations of 5 mg / m for 2 h / day , 5 days per week for a total of 4 weeks . the control rats were exposed to clean , filtered air containing no carbon black for the same period . the concentrations , size and shape of cb particle were monitored once time weekly by grimm aerosol technique ( gmbh and co. kg . dorfstrae 9 - 83404 ainring - germany ( and light microscope ( acc.v - spot magn . 25.0 animals in b and d groups were adapted to the treadmill for rat ( will running treadmill , lafayette american ) training for 3 days ( 15 minutes , 20 m / min ) . on the fourth day , the individual maximal exercise capacity test was performed with a 5-minute warm - up ( 6 m / min ) and followed by an increase in treadmill speed ( 3 m / min every 3 minutes ) until animal exhaustion ( i.e. when they were not able to run voluntarily after 3 mechanical stimuli ) ( 24 , 27 , 28 ) . the maximal exercise capacity ( 100% ) the speed average of each group was calculated , and then the rats were submitted to treadmill training as a mean speed of the group workload . rats were trained at low intensity , corresponding to 50% of the initial maximal speed obtained in the exercise test , for 60 minutes , five times per week , as previously described ( 24 , 27 , 28 ) . after sacrificed rat and total lung tissue , rna was isolated using trizolereagent ( qiagen , germany ) , according to the manufacturer s instructions . the rna samples were subjected to reverse transcription using thermo scientific revert aid first strand cdna synthesis kit ( ferementase ) . in the subsequent step , the cdnas were used as templates to perform real - time pcr using sybr green pcr master mix ( sybr green i , ) by step one abi system ( applied biosystem ) . the crossing threshold values assessed by the real - time pcr were evaluated for the transcripts and normalized to the results for gapdh mrna . the corresponding primer pairs for tlr4 , nf-b , and tnf- and gapdh ( housekeeping gene ) were listed in table 1 . the threshold cycle ( ct ) for each specific gene , corresponding housekeeping gene ( gapdh ) and their differences ( ct ) were determined and then evaluated gene expression changes using 2 formula . differences in body weight between pre and post interventions were examined by 2-tailed t - test . in order to determine the significant differences between groups one way anova and lsd post hoc test and kruskal - vallis test after examined normal distribution of data by kolmogorov - smirnov test were used . twenty four adult male wistar rats aged 8 weeks were obtained from pasteur institute of iran and randomly divided into the 4 groups : a ; control ( without exposure carbon black pm10 and aerobic exercise ; n = 6 ) , b ; aerobic exercise ( five times per week for 4 weeks ; n = 6 ) , c ; exposure to carbon black pm10 ( 5 mg / m ; per rat ; n = 6 ) , d ; aerobic exercise concomitantly with exposure to carbon black pm10 ( n = 6 ) . rats were housed in cages under controlled environment ( 23c and 12 hour light - dark cycle ) with free access to normal chow and tap water . figure 1 shows the inhalation chamber at the laboratory of tarbiat modares university ( falonak ) where inhalation exposure was carried out . rats in groups of c and d were exposed to carbon black in the inhalation chamber at nominal concentrations of 5 mg / m for 2 h / day , 5 days per week for a total of 4 weeks . the control rats were exposed to clean , filtered air containing no carbon black for the same period . the concentrations , size and shape of cb particle were monitored once time weekly by grimm aerosol technique ( gmbh and co. kg . dorfstrae 9 - 83404 ainring - germany ( and light microscope ( acc.v - spot magn . animals in b and d groups were adapted to the treadmill for rat ( will running treadmill , lafayette american ) training for 3 days ( 15 minutes , 20 m / min ) . on the fourth day , the individual maximal exercise capacity test was performed with a 5-minute warm - up ( 6 m / min ) and followed by an increase in treadmill speed ( 3 m / min every 3 minutes ) until animal exhaustion ( i.e. when they were not able to run voluntarily after 3 mechanical stimuli ) ( 24 , 27 , 28 ) . the maximal exercise capacity ( 100% ) was defined as the maximum speed reached by each animal . the speed average of each group was calculated , and then the rats were submitted to treadmill training as a mean speed of the group workload . rats were trained at low intensity , corresponding to 50% of the initial maximal speed obtained in the exercise test , for 60 minutes , five times per week , as previously described ( 24 , 27 , 28 ) . after sacrificed rat and total lung tissue , rna was isolated using trizolereagent ( qiagen , germany ) , according to the manufacturer s instructions . the rna samples were subjected to reverse transcription using thermo scientific revert aid first strand cdna synthesis kit ( ferementase ) . in the subsequent step , the cdnas were used as templates to perform real - time pcr using sybr green pcr master mix ( sybr green i , ) by step one abi system ( applied biosystem ) . the crossing threshold values assessed by the real - time pcr were evaluated for the transcripts and normalized to the results for gapdh mrna . the corresponding primer pairs for tlr4 , nf-b , and tnf- and gapdh ( housekeeping gene ) were listed in table 1 . the threshold cycle ( ct ) for each specific gene , corresponding housekeeping gene ( gapdh ) and their differences ( ct ) were determined and then evaluated gene expression changes using 2 formula . results are expressed as mean sd . differences in body weight between pre and post interventions were examined by 2-tailed t - test . in order to determine the significant differences between groups one way anova and lsd post hoc test and kruskal - vallis test after examined normal distribution of data by kolmogorov - smirnov test were used . body weight : there were significant differences among the a ( control ) and c ( pm10 exposure only ) groups in body weight pre and post interventions . the mean of increase in body weight in c group was higher than a , b and d groups ( table 2 ) . data are presented as mean sd . significantly different at p 0.05 . presented data in figure 2 demonstrate that pm10 carbon black exposure increased the gene expression tlr4 , nf-b and tnf- in lung tissue compared with all of the other groups ( p 0.05 ) and that aerobic exercise in carbon black pm10 exposure decreased the expression of these cytokine compared with the carbon black pm10 exposure group ( p 0.05 ) . 0ne way anova also demonstrated that carbon black pm10 exposure presented a significant effect on the tnf- ( p = 0.047 ) and nf-b ( p = 0.014 ) . no significant effect was observed on the gene expression tlr4 by kruskal - vallis test ( p = 0.325 ) . asterisk indicates treatments that are significantly different at p 0.05 compared with all of the groups . presented data in figure 2 demonstrate that pm10 carbon black exposure increased the gene expression tlr4 , nf-b and tnf- in lung tissue compared with all of the other groups ( p 0.05 ) and that aerobic exercise in carbon black pm10 exposure decreased the expression of these cytokine compared with the carbon black pm10 exposure group ( p 0.05 ) . 0ne way anova also demonstrated that carbon black pm10 exposure presented a significant effect on the tnf- ( p = 0.047 ) and nf-b ( p = 0.014 ) . no significant effect was observed on the gene expression tlr4 by kruskal - vallis test ( p = 0.325 ) . asterisk indicates treatments that are significantly different at p 0.05 compared with all of the groups . in the present study , we demonstrated that aerobic exercise inhibits lung inflammation and pro - inflammatory cytokine release in lung tissue in an experimental model of pm10 carbon black - induced lung inflammation . considerable epidemiological and toxicological studies have established a clear link between exposure to air pollution particles and adverse pulmonary health effects . several experimental and human studies have demonstrated that increased levels of air pollution associated with pulmonary inflammation ( 37 , 38 ) . the association between ambient air pollution particles exposure and lung cancer risk has been investigated in prospective studies and the results are generally consistent , indicating that long - term exposure to air pollution may cause lung cancer . gene expression analysis identified the association with the effect of pm10 carbon black on the innate immune response . pm10 carbon black exposure increased the gene expression tlr4 , nf-b and tnf- in lung tissue . tlr4 activation is corresponded to nf-b signaling and pathway - specific induction of pro - inflammatory cytokines and chemokines or interferon signaling ( 39 ) . an extensive research in recent years has been indicated that chronic inflammation leads to various chronic disorders associated with cancer ( 40 - 42 ) . a central role in the induction of chronic inflammation is played by a set of genes encoding pro - inflammatory cytokines such as il-1 , il-2 , il-6 , and tnf- and monocyte chemotactic protein 1 that are regulated by the transcription factor ( nf-b ) ( 43 - 45 ) . these conditions are associated with derangements in the interplay between metabolic and immune processes and inflammation ( 47 ) . the authors observed significant increase in body weight with pm10 carbon black exposure which could explain the increased inflammation can be caused by pm10 exposure . studies have suggested that potential anti - inflammatory effects of aerobic exercise could inhibit the pro - inflammatory events induced by air pollution . the anti - inflammatory effects of exercise ( 48 - 50 ) have focused on three possible mechanisms : the reduction in visceral fat mass ; increased production and release of anti - inflammatory cytokines from contracting skeletal muscle ( such molecules are termed myokines ( 48 - 51 ) ; and reduced expression of toll - like receptors ( tlrs ) on monocytes and macrophages ( 52 ) . in the present study , aerobic exercise in pm10 carbon black exposure decreased the gene expression tlr4 , nf-b and tnf- in lung tissue and also body weight of animals . although this change in gene expression was slight , however this result can be clinically important . we conclude that low - intensity aerobic exercise presents protective effects from pm10 carbon black - induced lung inflammation . future studies should therefore be directed towards better defining the mechanism involved in the induction of symptoms by inflammatory molecules and risk of cancer in exposure to air pollution particles .

background : exposure to air pollution pm10 results in lung inflammation increased risk of lung cancer . regular aerobic exercise improves the inflammatory status in different lung diseases . however , the effects of long - term aerobic exercise on the pulmonary response to pm10 have not been investigated . objectives : the present study evaluated the effect of aerobic exercise on the lung inflammatory and risk of lung cancer of rat exposed to pm10 carbon black.materials and methods : twenty four adult male wistar rats were divided into 4 groups : a : control ( without exposure pm10 and aerobic exercise ; n = 6 ) , b : aerobic exercise ( five times per week for 4 weeks ; n = 6 ) , c : exposure to pm10 carbon black ( 5 mg / m3 ; per rat ; n = 6 ) , d : and aerobic exercise concomitantly with exposure to pm10 carbon black ( n = 6 ) . the gene expression of tlr4 , nf-b and tnf- were analyzed in lung tissue by real time - pcr . in order to determine the significant differences between groups , one way anova and lsd post hoc and kruskal - vallis test were used.results:aerobic exercise inhibited the pm10 -induced increase in the gene expression of tlr4 , nf-b and tnf-. but there was significant different only between b and c groups for tnf- and nf-b ( p = 0.047 , 0.014 , respectively).conclusions : we conclude that four week aerobic exercise presents protective effects in a rat model of pm10 carbon black - induced lung inflammation and risk of lung cancer . our results indicate a need for human studies that evaluate the lung responses to aerobic exercise chronically performed in polluted areas .

a total of 30 mabs that were either developed or in - licensed by companies are currently undergoing evaluation in late - stage studies ( tables 1 and 2 ) . five of these ( brodalumab , mabp1 , moxetumomab pasudotox , tildrakizumab , rilotumumab ) transitioned into phase 3 studies during the period from late 2012 to april 2013 . brodalumab ( amg827 , amgen ; khk4827 , kyowa hakko kirin ) , a human igg2 targeting the interleukin ( il)-17 receptor , is undergoing evaluation in three phase 3 studies ( amagine-1 , -2 , and -3 ) of patients with psoriasis . the amagine-1 study ( nct01708590 ) is evaluating the efficacy , safety , and effect of withdrawal and retreatment with brodalumab in patients with moderate - to - severe plaque psoriasis . in this study , either of two doses ( 140 mg or 210 mg ) or placebo is administered subcutaneously ( sc ) every two weeks until week 12 , when patients are rerandomized to placebo or continued treatment . the estimated primary completion date for the study is march 2014 . in the amagine-2 ( nct01708603 ) and amagine-3 ( nct01708603 ) studies , the efficacy and safety of induction and four maintenance regimens of brodalumab compared with placebo and ustekinumab in patients with moderate - to - severe plaque psoriasis is being evaluated . these studies have estimated primary completion dates of august and september 2014 , respectively . note : table compiled from information available as of april 25 , 2013 . abbreviations : cd , cluster of differentiation ; il , interleukin ; pcsk9 , proprotein convertase subtilisin / kexin type ; sle , systemic lupus erythematosus . note : table compiled from information available as of april 25 , 2013 . * in - licensed ; national cancer institute is sponsoring the phase 3 study in hairy cell leukemia patients . abbreviations : all , acute lymphoblastic leukemia ; cll , chronic lymphocytic leukemia ; hgf / sf , hepatocyte growth factor/ scatter factor ; nhl , non - hodgkin lymphoma ; nscl , non - small cell lung ; vegfr2 , vascular endothelial growth factor receptor 2 . a first phase 3 study ( nct01767857 ) of mabp1 ( xilonix , xbiotech , inc . ) , a human mab targeting il-1 , was recruiting patients as of march 2013 . the study will evaluate overall survival of metastatic colorectal cancer patients with cachexia who are administered the mab as a monotherapy . the effects of mabp1 administered intravenously ( iv ) every two weeks , plus best supportive care will be compared with those of megestrol acetate administered daily plus best supportive care . the difference between the study arms in median overall survival from baseline to 18 mo will be compared . the recombinant immunotoxin moxetumomab pasudotox ( cat-8015 , astrazeneca ) is undergoing evaluation in a phase 3 study ( nct01829711 ) of patients with relapsed / refractory hairy cell leukemia . the study is sponsored by the national cancer institute ( nci ) ; the mab is currently licensed to astrazeneca . in 2004 , nci licensed moxetumomab pasudotox to genencor , inc . the mab was then acquired in 2005 by cambridge antibody technology , which was itself acquired by astrazeneca in 2006 . moxetumomab pasudotox comprises a mouse disulfide stabilized variable fragment with the variable heavy domain fused with a 7-mer linker and the pseudomonas aeruginosa exotoxin a pe38 fragment . in the phase 3 study , patients will receive 40 g / kg of the immunotoxin administered iv over 30 min on days 1 , 3 , 5 of each 28 d cycle until complete response , progressive disease , initiation of alternate therapy or unacceptable toxicity is observed . initiated in march 2013 , the study s estimated primary completion date is december 2014 . two phase 3 studies of tildrakizumab ( sch900222/mk-3222 , merck ) , a humanized mab targeting the p19 subunit of il-23 , are recruiting patients . initiated in december 2012 , nct01722331 is a 64-week study to evaluate the efficacy and safety / tolerability of sc administration of tildrakizumab in patients with moderate - to - severe chronic plaque psoriasis . patients receive 100 mg or 200 mg tildrakizumab at week 0 and week 4 , and then every 12 weeks until study end or participant discontinuation . patients receive the same doses of tildrakizumab , but the study includes 50 mg etanercept as well as placebo as comparators . the two phase 3 studies have estimated primary completion dates of june and july 2015 , respectively . rilotumumab ( amgen ) is a human igg2 targeting human hepatocyte growth factor / scatter factor ( hgf / sf ) that blocks binding of hgf / sf to its receptor met , thereby inhibiting the met signaling pathways . the mab is undergoing evaluation in a phase 3 study ( nct01697072 ) of chemotherapy ( epirubicin , cisplatin and capecitabine ) with rilotumumab or placebo for untreated advanced met - positive gastric or gastresophageal junction adenocarcinoma . rilotumumab or placebo is administered iv at doses of 15 mg / kg every 21 d. the primary outcome measure is overall survival in a time - frame of three years . initiated in october 2012 , the estimated primary completion date for the study is december 2015 . recently announced results for three late - stage studies of two mabs for cancer ( farletuzumab , naptumomab estafenatox ) and tabalumab , a mab treatment for autoimmune disorders , indicate that clinical endpoints were not met . farletuzumab ( morab-003 , morphotek / eisai ) is a humanized igg1 mab that targets human folate receptor , which is overexpressed in most epithelial ovarian cancers , as well as some forms of endometrial , breast , renal , lung and colorectal cancers . the primary endpoint of progression - free survival ( pfs ) was not met in the phase 3 far-131 study ( nct00849667 ) of farletuzumab in combination with carboplatin and a taxane in patients with platinum - sensitive epithelial ovarian cancer in first relapse . patients received placebo or farletuzumab at either 1.25 mg / kg or 2.5 mg / kg weekly for ~six cycles . although the primary endpoint was not met , a trend toward improved pfs was observed in some patient subsets in a post - hoc analysis . the safety and efficacy of farletuzumab in combination with a platinum containing doublet in chemotherapy - nave subjects with stage iv adenocarcinoma of the lung are being evaluated in a phase 2 study ( nct01218516 ) . as announced in january 2013 by active biotech ab , initial results of a phase 2/3 study ( nct00420888 ) of naptumomab estafenatox ( abr-217620 , anyara ) in combination with interferon as a treatment for advanced renal cell carcinoma indicated that the study did not achieve its primary clinical endpoint of overall survival in the intention - to - treat cohort . a confounding factor was the presence of high levels of pre - formed anti - drug antibodies found in a majority of patients in this , but not previous , studies . a doubling of pfs and overall survival occurred in the 25% of patients who had low or normal levels of base - line il-6 and expected levels of anti - drug antibodies . naptumomab estafenatox is composed of an antigen - binding fragment ( fab ) that targets metastasis - associated 5t4 fused to a mutated form of staphylococcal enterotoxin a ( sea / e-120 ) . phase 3 studies of tabalumab ( ly2127399 , eli lilly and co. ) as a treatment for ra have been discontinued due to lack of efficacy . tabalumab , a human igg4 targeting b - lymphocyte stimulator , was being evaluated in five phase 3 studies of ra patients . tabalumab in combination with bortezomib and dexamethasone is also being evaluated in a phase 2/3 study ( nct01602224 ) of patients with previously treated multiple myeloma . historically , ~50% of mabs in the commercial clinical pipeline have been studied as cancer agents ; however , ~67% of the current cohort of mabs at phase 3 is in studies for non - cancer indications ( table 1 ) . these 20 mabs are human or humanized , with more than half ( 55% ) targeting an interleukin or an interleukin receptor . the mabs are being developed as treatments for immunological disorders such as ra , sle , ms and psoriasis , but also for hypercholesterolemia , cachexia and alzheimer disease . reflecting the overall greater challenge of demonstrating safety and efficacy in cancers , the molecular types of the 10 mabs in late - stage studies of cancer ( table 2 ) are more diverse than those for non - cancer indications . of particular note is the inclusion of a toxin in three molecules ( naptumomab estafenatox , moxetumomab pasudotox , inotuzumab ozogamicin ) and use of less than a full - length sequence for three molecules ( naptumomab estafenatox , moxetumomab pasudotox , onartuzumab ) . the majority ( 70% ) of the 10 product candidates are in studies of solid tumors , with the remainder undergoing evaluation as treatments for hematological cancers . the number of anti - cancer mabs at phase 3 is likely to increase in the next 35 y as more adcs move into and through the clinical pipeline . mabs will continue to track the progress of antibody therapeutics in clinical study throughout 2013 , and we look forward to reporting on results that may be released during the year in which are the antibodies to watch in 2014 ?

the transitions of antibody therapeutics to late - stage clinical development , regulatory review and the market are proceeding at a rapid pace in 2013 . since late 2012 , two monoclonal antibody ( mab ) therapeutics ( itolizumab , trastuzumab emtansine ) received their first approvals , first marketing applications for three mabs ( vedolizumab , ramucirumab , obinutuzumab ) were submitted to regulatory agencies , and five mabs ( brodalumab , mabp1 , moxetumomab pasudotox , tildrakizumab , rilotumumab ) entered their first phase 3 studies . the current total of commercially - sponsored antibody therapeutics undergoing evaluation in late - stage studies is 30 . recently announced study results for farletuzumab , naptumomab estafenatox , and tabalumab indicate that clinical endpoints were not met in some phase 3 studies of these product candidates .

the concept of this treatment started in the united states in the 1980s in randomized controlled studies in different cancers , resulting in reported significant increases in survival and disease - free period.13 its efficacy is enhanced by 20%30% when cell - based immunotherapy is combined with other conventional treatment methods . interferon ( ifn ) was one of the first cytokines found to have anticancer effects , and it was introduced into the combined modality regimens used to treat small cell lung cancer ( sclc ) in the early 1980s in an attempt to overcome the problem of early relapse . ifns are a group of proteins and glycoproteins produced by cells in response to viral stimulation.4 ifns have immunoregulatory effects on antibody production , natural killer ( nk ) and t cell activation , macrophage function , delayed - type hypersensitivity , and major histocompatibility complex molecule expression.57 they have also been shown to have antiproliferative effects and antiangiogenic properties.8 type 1 ifns ( the ifn- family and ifn- ) are known to inhibit tumor cell growth and stimulate the immune system.9 it has also been demonstrated that recombinant ifn- induces immunomodulation and has antiproliferative activity . combinations of ifn- and ifn- demonstrate synergistic antiviral and antiproliferative activity.1012 in addition , synergistic or additive interactions between ifn- and cytotoxic drugs , particularly cisplatin , have been demonstrated in the experimental setting1315 and in clinical phase ii studies.16 however , only a few randomized clinical trials have been conducted to evaluate the possible advantage of adding immunomodifiers to conventional induction therapy.17 in the past , our group has reported significant antitumor activity of ifn- and improved survival in patients with sclc.18 the aim of this study was to determine whether in patients with sclc the addition of either ifn- or ifn- or their combination with standard induction chemotherapy would improve response rates and survival at acceptable toxicity . furthermore , we evaluated the immunological responses in the peripheral blood by measuring the levels of nks and some subsets of t lymphocytes ( cd4 + t cells , cd8 + t cells , and nk t cells [ ctls ] ) and determining whether these immunoregulatory observations correlated with the progress of the disease . chemo - nave patients with histology - confirmed sclc ( limited or extensive disease ) , according to the veterans administration lung study group definition modified by the international association for the study of lung cancer , were eligible . exclusion criteria included age > 72 years , karnofsky index < 70 , central nervous system metastasis at the time of diagnosis , creatinine clearance lower than 60 ml/ min , and serious concomitant disease . from our department , 170 patients were initially recruited , from whom 164 patients were finally evaluable for assessment of response and toxicity . pretreatment investigations included : general medical examination ; chest radiography and computed tomography of the chest , upper abdomen , and brain ; tissue sampling by bronchoscopy or mediastinoscopy ; and a bone scan . all patients signed an informed consent form . a randomized , nonblinded trial was conducted . randomization was stratified for stage , and patients were divided into four groups as follows : group a , the control group , received conventional chemotherapy ; group b received chemotherapy and ifn- ( 3 million iu ) subcutaneously three times a week ; group c received ifn- ( 3 million iu ) subcutaneously three times a week ; and group d received ifn- and ifn- ( 1.5 million iu of each ) 3 times a week subcutaneously . chemotherapy was the same for all groups and consisted of eight cycles with carboplatin 5.5 mg / m intravenously on day 1 , ifosfamide 3.5 g / m intravenously ( with mesna ) on day 1 , and etoposide 200 mg / m total dose taken orally on days 1 through 3 , every 28 days , which has been shown to improve overall survival compared with standard chemotherapy in the lu21 trial.1921 after the completion of eight cycles of chemotherapy , patients were restaged by computed tomography scans of the brain , thorax , and upper abdomen . those found to have limited disease received primary site irradiation ( 4550 gy in 25 fractions ) and prophylactic cranial irradiation ( 25 gy in 12 fractions).22 immunotherapy was continued throughout these treatments and during the follow - up period as maintenance therapy . antiemetic treatment consisted of 5-hydroxytryptamine receptor antagonists , and acetaminophen 500 mg was given to treat ifn - related influenza - like symptoms . blood was taken before each course of chemotherapy and during follow - up to measure the total amount of t lymphocytes and their subgroups cd4 + t cells , cd8 + t cells , nkt ( natural killer t ) cells , and nk cells . patients who showed disease progression at any time were taken off study medication but were followed up for survival analysis . the study was initiated in may 2004 , and patient enrollment ended on february 2007 . the primary end - point of the study was survival ; the secondary end points were response after induction therapy and the fluctuation of the immunologic indices during the course of the disease ( figure 1 ) . statistical analysis was performed using the statistical package for the social sciences ( spss ; ibm corporation , armonk , n y , usa ) . differences in the four groups for clinical characteristics , response , and toxicity were assessed by chi - square statistics . survival was compared using kaplan meier statistics and log - rank test . a p - value of , 0.05 was considered significant . chemo - nave patients with histology - confirmed sclc ( limited or extensive disease ) , according to the veterans administration lung study group definition modified by the international association for the study of lung cancer , were eligible . exclusion criteria included age > 72 years , karnofsky index < 70 , central nervous system metastasis at the time of diagnosis , creatinine clearance lower than 60 ml/ min , and serious concomitant disease . from our department , 170 patients were initially recruited , from whom 164 patients were finally evaluable for assessment of response and toxicity . pretreatment investigations included : general medical examination ; chest radiography and computed tomography of the chest , upper abdomen , and brain ; tissue sampling by bronchoscopy or mediastinoscopy ; and a bone scan . randomization was stratified for stage , and patients were divided into four groups as follows : group a , the control group , received conventional chemotherapy ; group b received chemotherapy and ifn- ( 3 million iu ) subcutaneously three times a week ; group c received ifn- ( 3 million iu ) subcutaneously three times a week ; and group d received ifn- and ifn- ( 1.5 million iu of each ) 3 times a week subcutaneously . chemotherapy was the same for all groups and consisted of eight cycles with carboplatin 5.5 mg / m intravenously on day 1 , ifosfamide 3.5 g / m intravenously ( with mesna ) on day 1 , and etoposide 200 mg / m total dose taken orally on days 1 through 3 , every 28 days , which has been shown to improve overall survival compared with standard chemotherapy in the lu21 trial.1921 after the completion of eight cycles of chemotherapy , patients were restaged by computed tomography scans of the brain , thorax , and upper abdomen . those found to have limited disease received primary site irradiation ( 4550 gy in 25 fractions ) and prophylactic cranial irradiation ( 25 gy in 12 fractions).22 immunotherapy was continued throughout these treatments and during the follow - up period as maintenance therapy . antiemetic treatment consisted of 5-hydroxytryptamine receptor antagonists , and acetaminophen 500 mg was given to treat ifn - related influenza - like symptoms . blood was taken before each course of chemotherapy and during follow - up to measure the total amount of t lymphocytes and their subgroups cd4 + t cells , cd8 + t cells , nkt ( natural killer t ) cells , and nk cells . patients who showed disease progression at any time were taken off study medication but were followed up for survival analysis . the study was initiated in may 2004 , and patient enrollment ended on february 2007 . the primary end - point of the study was survival ; the secondary end points were response after induction therapy and the fluctuation of the immunologic indices during the course of the disease ( figure 1 ) . statistical analysis was performed using the statistical package for the social sciences ( spss ; ibm corporation , armonk , n y , usa ) . differences in the four groups for clinical characteristics , response , and toxicity were assessed by chi - square statistics . survival was compared using kaplan meier statistics and log - rank test . a p - value of , 0.05 was considered significant . from 170 patients initially recruited , 164 were finally eligible for assessment . from six patients who were not evaluated , four were excluded because of violation of protocol , and two patients in group c were lost because they did not attend . median survival was 10 months for group a ( 95% confidence interval [ ci ] , 9.310.6 months ) , 10.3 months for group b ( 95% ci , 7.1313.5 months ) , 8.3 months for group c ( 95% ci , 6.89.8 months ) , and 11 months ( 95% ci , 9.212.8 months ) for group d. if we compare all groups together , there is no significant difference in survival , even with pairwise comparison among four groups ( p . interestingly , when patients with only limited disease were considered , a significant survival benefit in group b was revealed compared with control group a ( p = 0.039 ) , group c ( p , 0.005 ) , and group d ( p = 0.038 ) . median survival for each group in all stages and for limited disease separately is shown in table 3 . ten patients in the ifn- group ( 25% ) and two in group d ( ifn- and ifn- ) , but none in the other groups , survived for 3 years or longer . assessment of fluctuations was based on three different time measurements : before treatment initiation ; before the fourth cycle of treatment ; and at the time of disease progression . the analysis of immunological measurements revealed improvement of immune markers accompanied by clinical improvement , whereas deterioration of all markers was accompanied , although not significantly , by disease progression ( time series analysis p < 0.05 ; figures 2 and 3 ) . significantly more patients in the immunotherapy groups than in the chemotherapy group experienced toxicity , but in groups b , c , and d , symptoms were mild and well manageable . assessment of fluctuations was based on three different time measurements : before treatment initiation ; before the fourth cycle of treatment ; and at the time of disease progression . the analysis of immunological measurements revealed improvement of immune markers accompanied by clinical improvement , whereas deterioration of all markers was accompanied , although not significantly , by disease progression ( time series analysis p < 0.05 ; figures 2 and 3 ) . significantly more patients in the immunotherapy groups than in the chemotherapy group experienced toxicity , but in groups b , c , and d , symptoms were mild and well manageable . the aim of this study was to investigate , in a randomized trial , the efficacy of adjunctive immunotherapy in sclc therapy . our previous pilot study17 has shown that combination therapy with ifn- is effective for sclc , particularly in those patients with limited disease . the present trial has confirmed that the addition of ifn- provides a good survival benefit in limited sclc , whereas administration of ifn- has no place as monotherapy in sclc treatment.23,24 immunomodulatory activity of ifn- and ifn- concerned in intensification of major histocompatibility complex i and ii molecule expression in cancer cells surface , thereby increasing the recognition and killing of these cells . ifns also directly activate other immune cells , such as macrophages and nk cells , with the difference being that ifn- protects cancer cells from the lytic activity of nk cells,25 which partly explains the absence of therapeutic effect in patients in group c , who received ifn- only . ifn- seems to have lower direct antitumor activity compared with either ifn- alone or ifn- and ifn- in combination , as shown in cancer cell lines.26 clinically , ifn- proved to be effective in the treatment of hairy cell leukemia and chronic myelogenous leukemia.27 in contrast , many other tumors , among them sclc , seem to resist cytokines , but they respond to ifns in their early stage.28 one hypothesis is that the reset of the immune system in limited disease is faster than in extensive disease ; this is also an explanation for the better response of limited disease in general . another possible explanation could be that despite chemotherapy affecting the immune system of the host negatively , ifns exert a positive influence on the immune system , and patients with limited disease have more reserve to respond to ifn stimuli . fischer et al29 found significant reduction of interleukin 2 , ifn- , ifn- , and tumor necrosis factor in patients with sclc , which improved after tumor control with chemotherapy . this immunomodulatory activity of chemotherapy seems to partly explain both the improvement of immune marker levels in patients who responded in chemotherapy and also the deterioration of them when patients experienced progressive disease in the control group . however , the lack of clinical effect is an indication of insufficient activation of cells produced or weakness of migration at the tumor site , evidenced by their appearance in large numbers in the peripheral blood . first , the total number of patients investigated was small ; this is the main limitation of the study . second , relatively low doses of cytotoxic agents were chosen to avoid a potentiation of the myelosuppressive effect by ifns.14 toxicity was characterized by fever and a higher grade of myelosuppression in the ifn groups , as could have been expected . increased neutropenia in group d shows increased toxicity when ifns are combined rather than received alone , even if their total dose remains the same . that is why sclc is uncommon in women and why the vast majority of patients enrolled in the study are men.30 in summary , among the cytokines used in this study , only ifn- appears to confer a survival benefit to patients with sclc who have limited disease . because of the low number of randomized patients , the clinical relevance of these findings remains to be defined . further clinical studies on the efficacy of ifn- as an adjunctive and/or maintenance treatment are warranted , as immunotherapy remains a challenge for the treatment of lung neoplasms .

purposeto evaluate the effect of immunotherapy on response , survival , and certain immune markers in patients with small cell lung cancer ( sclc ) who are receiving chemotherapy.patients and methodspatients with sclc ( n = 164 ) were assigned to receive either chemotherapy alone ( group a ) or a combination of chemotherapy and immunotherapy as follows : interferon ( ifn- ; 3 million iu ) 3 times per week ( group b ) ; ifn- ( 3 million iu ) 3 times per week ( group c ) ; and ifn- and ifn- ( 1.5 million iu of each ) 3 times per week ( group d ) . chemotherapy was the same for all groups and consisted of eight cycles with carboplatin 5.5 mg / m2 intravenously on day 1 , ifosfamide 3.5 mg / m2 intravenously on day 1 , and etoposide 200 mg / m2 total dose taken orally on days 1 through 3 , every 28 days . patients completing chemotherapy were restaged , and those who were found to have limited disease received primary site and prophylactic cranial irradiation . immunotherapy was continued throughout these treatments and during the follow - up period . blood was taken before each course of chemotherapy and during follow - up to measure cd3 + lymphocytes , cd3+cd4 + lymphocytes , cd3+cd8 + lymphocytes , natural killer cells , and natural killer t cells.resultsdifferences in response and survival were not significantly different when all patients were considered . however , among patients with limited disease , kaplan meier analysis disclosed a survival benefit for group b ( p , 0.05 ) . the analysis of immunologic measurements revealed that the improvement of immune markers was always accompanied by clinical improvement , whereas deterioration of all markers was accompanied by disease progression ( result not statistically significant except for group c ; p , 0.05).conclusionamong cytokines used in the study , only ifn- seems to confer a survival benefit to patients with sclc with limited disease . however , immunotherapy remains a challenge in the treatment of lung neoplasms and should be further explored .

dopa - responsive dystonia ( drd ) is characterized by childhood - onset dystonia and a dramatic and sustained response to administration of low - doses of oral levodopa . this disorder typically presents with gait disturbance , caused by lower limb dystonia with diurnal variation , a positive family history , and gradual progression to generalized dystonia . a ten - year - old girl was brought with complaints of difficulty in walking and stiffness of both lower limbs , for the past six months . she also had a history of frequent falls while walking and was unable to stand for prolonged periods . her antenatal , birth , and neonatal periods were uneventful and developmental milestones were appropriate for her age . her higher mental functions were normal , speech was hypophonic ; she had a gait disturbance characterized by leg stiffness , and a tendency to walk in an equinus posture , resulting in difficulty in balancing . her tone was slightly increased in all the four limbs , with cog - wheel rigidity . deep tendon reflexes in all the four limbs were exaggerated with extension of both great toes ( striatal toes ) . , it was found that the symptoms and signs were relatively mild in the morning during rounds , whereas , they gradually worsened as the day progressed , rendering the child almost unable to walk by evening . magnetic resonance imaging ( mri brain and spine were normal . in view of the typical diurnal variation of dystonia , a therapeutic challenge with levodopa / carbidopa was tried and there was a dramatic decrease in dystonia within two days and the child 's gait improved . the child was treated with a combination of levodopa , carbidopa , and trihexyphenidyl . on follow - up genetic studies were planned , but could not be done due to lack of resources . drd is an inherited disorder characterized by dystonia with diurnal variation and favorable response to levodopa / carbidopa . the inheritance is usually autosomal dominant ; however , autosomal recessive inheritance is also seen in some cases . the enzyme deficiency responsible for the manifestations is gtp cyclohydrolase 1 ( gch ) , which is a rate - limiting enzyme in the synthesis of dopamine . this disorder had been referred to as hereditary progressive basal ganglia disease , hereditary progressive dystonia with marked diurnal variation , segawa disease , and drd in the past . at present , segawa disease specifically denotes an autosomal dominantly inherited mutation in the gch 1 gene located on chromosome 14q22.1 to q22.2 . the disease usually manifests in school age children , however , adults with the disease have also been reported . initial manifestations of this disease include postural dystonia of the lower limbs with characteristic equino varus deformity of both feet . the dystonia gets worse as the day progresses , becomes maximal by evening , and decreases after sleep by morning . investigations characteristically reveal low levels of pteridine metabolites in the cerebrospinal fluid , normal neuroimaging , and increased blood phenylalanine levels after phenylalanine loading tests . assessing the therapeutic response to levodopa is a useful and recommended method of diagnosing drd , when the diagnosis is in doubt and when dystonia is not attributable to hypoxic ischemic encephalopathy . in one reported series , administration of low - dose levodopa had resulted in complete to near - complete recovery of symptoms in a cohort of chinese patients , with no significant long - term side effects . in our case , the child presented with gait disorder characterized by dystonic movements of the lower limbs of gradual onset , which disappeared during sleep , and reappeared after getting up from bed and progressively worsened throughout the day with symptoms , for the past six months . with the above - mentioned clinical findings and a dramatic response to the levodopa / carbidopa combination children with recent onset dystonia and gait abnormalities may pose a diagnostic challenge . a careful history and focused neurological examination , looking for diurnal variation in symptoms , holds the key in arriving at the diagnosis . in such children a therapeutic response to levodopa might be a safe and appropriate way of confirming drd .

children with recent onset dystonia and gait abnormalities may pose a diagnostic challenge . a ten - year - old , developmentally normal girl , presented with a six - month history of gait abnormality and dystonia . her complaint worsened as the day progressed . in view of typical diurnal variation of dystonia , a therapeutic challenge with levodopa / carbidopa was given and there was a dramatic response . hence , a diagnosis of dopa - responsive dystonia ( drd ) was made . drd is an inherited disorder characterized by dystonia with diurnal variation and favorable response to levodopa / carbidopa . the inheritance is usually autosomal dominant , however , in some cases , autosomal - recessive inheritance is also seen .

agrochemicals are widely used for decades , in an attempt to protect crops against insect pests . nevertheless , in the light of the increasing resistance , every year a vast array of new compounds is introduced into the market , with consequential negative side effects and increased costs in food production . more specifically , pesticides are poisons intentionally dispersed in the environment to control pests , and which subsequently persist in the soil , water and food , with toxicity related outcomes to both humans and animals ( schulz , 2004 ; carvalho , 2006 ; moraes et al . , 2009 ) . notwithstanding , the use of agrochemicals remains a common practice , especially in tropical regions ( carvalho , 2006 ) . brazil is the the third main consumer of pesticides worldwide ( agriculture and agri - food , canada , 2005 ) . numbers from the brazilian pesticide industry show that pesticide sales in brazil have risen from us$ 0.98 billion in 1992 to us$ 4.5 billion in 2004 , this representing an increase of 360% over the period ( brazil , 2008 ) . furthermore , discrepancies between brazilian regulation of the pesticide market , and those enforced in developed countries , have paved the way to the importation of vast amounts of pesticides that are highly toxic , severely restricted , or even banned in other nations ( carvalho , 2006 ; smith , 2001 ) . according to peres and moreira ( 2007 ) , the widespread and growing use of pesticides for raising crops and cattle in brazil has given rise to a number of environmental changes and problems , both by the contamination of human communities , as part of the environment , and by accumulation in biotic and abiotic segments of ecosystems . with year - round harvests , the low level of mechanization in the various phases of production , and the consequential exposure to multiple environmental contaminants ( pesticides ) , the vulnerability of brazilian workers and the environment itself to these genotoxic agents becomes evident . environmental quality control requires the monitoring of various indicators , including the assessment of pesticide residues . this is due to these animals living in direct contact with aquatic sediments adjacent to areas where pesticides are commonly used . in these water bodies , many poorly water - soluble compounds eventually settle , with the consequential increase in the degree of local contamination , when compared to the water column as a whole ( grisolia , 2005 ; umbuziero et al . , 2006 ) . . nevertheless , in recent studies , potentially genotoxic substances have been recognized and quantified , when using fish as experimental organisms ( andrade et al . , 2004 ) . nowadays , several tests have been well developed and standardized for assessing the genotoxic profile of a wide spectrum of substances . in comparison to other methods , the advantage of the simplicity of the micronucleus test facilitates counting the micronuclei in erythrocytes therefore , the test has been widely used to evaluate the mutagenic potential of new drugs and chemicals , being also indicated in routine screening and environmental monitoring ( al - sabti et al . , 1994 ; bcker et al . , 2006 ) . micronuclei are formed during the telophase of mitosis or meiosis , when the nuclear envelope is reconstituted around the chromosomes of daughter cells ( udroiu , 2006 ) . micronuclei are the result of chromosome acentric fragments ( clastogenic effect ) or whole chromosomes that , through incomplete migration , have been excluded from the main core ( aneugenic effect ) . thus , micronuclei represent a loss in chromatin as a result of damage to either chromosome structure ( fragmentation ) or the mitotic apparatus . there may also be the formation of bilobed nuclei , thereby indicating an early change in cellular metabolism ( fenech , 2000 ; grisolia and cordeiro , 2000 ; bombail et al . the aim was to survey the use of pesticides in guatambu , santa catarina state , brazil , and , through micronucleus testing , determine the risk and toxicological impact of pesticides contaminating regional dams . the study was undertaken in a farming community which included members of the lambedor river watershed association of guatambu , in the west santa catarina state , southern brazil , during april , 2009 . fish were collected in all the dams existing in agricultural rural properties ( 10 sampling areas ) , as shown in figure 1 . three fishes of similar size and weight ( between 700 g and 900 g ) were collected with cast nets in each dam . blood samples of approximately 40 l were taken from each by cardiac puncturing with sterile heparinized syringes and needles . samples were transferred to labeled eppendorf tubes containing edta , and then taken to the toxicology laboratory of the community university of the region of chapec for micronucleus testing . two blood smears per individual were prepared on clean microscope slides , to then be air - dried at room temperature . subsequently , they were first fixed with absolute methanol for 10 min and then air - dried for at least 24 h. after fixation , the slides were treated with 1n hcl for 11 min in a water - bath at 60 c . after washing with distilled water , they were then stained with a schiff solution in the dark . in sequence , the slides were removed and left to dry at room temperature , for subsequent microscopic examination . according to criteria already described by ayllon and garcia - varquez ( 2000 ) , only rounded non - refractive structures that had separated from the main nucleus were taken into account for micronuclei scoring . 3000 erythrocytes from each fish were counted ( 1000 erythrocytes on each slide ) at 1000 magnification under an oil - immersion objective , end examined for micronucleated cells . the study was undertaken in a farming community which included members of the lambedor river watershed association of guatambu , in the west santa catarina state , southern brazil , during april , 2009 . fish were collected in all the dams existing in agricultural rural properties ( 10 sampling areas ) , as shown in figure 1 . three fishes of similar size and weight ( between 700 g and 900 g ) were collected with cast nets in each dam . blood samples of approximately 40 l were taken from each by cardiac puncturing with sterile heparinized syringes and needles . samples were transferred to labeled eppendorf tubes containing edta , and then taken to the toxicology laboratory of the community university of the region of chapec for micronucleus testing . two blood smears per individual were prepared on clean microscope slides , to then be air - dried at room temperature . subsequently , they were first fixed with absolute methanol for 10 min and then air - dried for at least 24 h. after fixation , the slides were treated with 1n hcl for 11 min in a water - bath at 60 c . after washing with distilled water , they were then stained with a schiff solution in the dark . in sequence , the slides were removed and left to dry at room temperature , for subsequent microscopic examination . according to criteria already described by ayllon and garcia - varquez ( 2000 ) , only rounded non - refractive structures that had separated from the main nucleus were taken into account for micronuclei scoring . 3000 erythrocytes from each fish were counted ( 1000 erythrocytes on each slide ) at 1000 magnification under an oil - immersion objective , end examined for micronucleated cells . the responses from the questionnaires showed regular use of pesticides in 100% of sampling areas . as regards equipment washing , it was evident that 70% , of the water is normally returned to the farm itself , the remainder being discarded onto the soil . the responses also revealed that farmers were uncertain as to the correct destination of empty containers . the most widely used pesticides were roundup ( glyphosate ) , karate ( lambda cyhalothrin ) , herbimix ( atrasine and simazine ) and priori xtra ( azoxystrobin ) . cyprinus carpio , hypostomus punctatus , rhamdia quelen and oreochromis niloticus figured among the animals collected in this study . micronucleate erythrocytes ( figure 2 ) were found mat different frequencies among the captured fish species , as shown in figure 3 , the highest mean values being observed in cyprinus carpio ( 15.33 in weir 4 , 15.00 and 14.00 in weir 8 . figure 4 shows the means of micronuclei per dam , being especially perceptible in the larger sized sampling areas of dams 4 ( 12.44 ) and dam 8 ( 13.78 ) . however , they were close to the overall average in dam 5 ( 12.33 ) , where the species collected were rhamdia quelen and oreochromis niloticus . the significant contribution to the environmental impact in the region , through the paramount , current use and handling of pesticides in small and middle - sized farms in the community , was amply proven . induction of micronuclei was previously reported for fish collected in dams or rivers ( udroiu , 2006 ) and in situ quantification of micronuclei in piscine erythrocytes has been demonstrated to be an adequate biomarker in the evaluation of aquatic ecosystems quality ( aylln and garcia - vazquez , 2000 ; avas and ergene - gzkara , 2003 ; ergene et al . , 2007 ) . the water used both in the preparation of the pesticide solutions and in the washing of utensils , is a relevant factor and possible source of poisoning . as reported by respondents , in the past the whole washing procedure was carried out near water sources or streams . it is known that , depending on the characteristics of the soil where disposal occurs , the contamination of both surface and ground water is a possibility , thereby constituting a contributing factor to environmental contamination ( peres and moreira , 2007 ) . of late , the effects of pesticides on aquatic organisms , especially when using non - lethal doses , have been amply demonstrated ( crestani et al . , 2006 ; veiga et al . , 2006 ; glusczak et al . , 2007 ; marques et al . 2007 ; the processes of transport and impact on non - target organisms are governed by the rates of degradation and bioavailability of pesticides in soil or water , bioavailability itself depending on local physico - chemical and climatic conditions . several factors influence the effects of pesticides on fishes , among others , the fish - species studied , the class , dose and concentration of the pesticide , and exposure time ( glusczak et al . , 2007 ; fonseca et al . ; 2008 , cattaneo et al . , 2008 ) . ( 2005 ) , on examining differences in response between species by in situ analysis , noted the appropriateness of the species tilapia rendalli as a bioindicator of genotoxicity in a lake environment . according to grisolia ( 2002 ) , on exposure to different concentrations of glyphosate in the commercial formulation roundup , there was an increase in the frequency of micronuclei in erythrocytes . according to grisolia ( 2005 ) and udroiu ( 2006 ) , the prevailing , extensive farming procedures , on inducing considerable impacts on the environment , the subsequent increase in mutation rate would lead to an increase in genetic load and a reduction in adaptive potential , with the consequential elimination of susceptible genotypes . apparently the action of any chemical genotoxic agent may give rise to an increase in micronucleus frequency . consequently , based on the fact that spontaneous formation of micronuclei is normally low and nearly uniform among species ( siu et al . , 2004 ) , in environmental monitoring , micronucleus assaying has emerged as a simple , inexpensive and rapid method for detecting genotoxic effects . spontaneous formation of micronuclei in fish is normally very rare . in our study , however , significant frequencies were observed in the captured specimens from each dam , with micronucleus testing revealing the rate of micronucleated erythrocytes to be high , with a minimum of 6.21 and a maximum of 13.78 per 1,000 erythrocytes evaluated . when compared with the results obtained by rodriguez - cea et al . ( 2003 ) , with an average 3 micronuclei per 1000 erythrocytes examined , the above indices can be considered high . the data also further corroborated other studies in brazil which showed a high incidence of micronuclei and nuclear abnormalities in organisms exposed to various chemicals ( matsumoto et al . , it was proven through in vivo piscine micronucleus testing , that water from the lambedor water - shed can be considered genotoxic , with emphasis on the degree of genotoxicity from pollution in the area . this implies the possibility of pesticide effluents discharged into the river constituting a disease - hazard to local populations . it is recommended that the river water be analyzed chemically , with a mind to identifying additional classes of toxicants that may also be contributing to genotoxicity in this specific water - shed .

the aim of this study was to assess the use of pesticides on farms located in the lambedor river watershed in guatambu , state of santa catarina , as well as to determine , by micronucleus testing , the risk of genotoxic impact . samples from locally collected cyprinus carpio , hypostomus punctatus , rhamdia quelen and oreochromis niloticus gave evidence of a mean increase in micronuclei frequency from 6.21 to 13.78 in 1,000 erythrocytes , a clear indication of the genotoxic potenciality of pesticide residues in regional dams , and their significant contribution to local environmental contamination .

molecular diagnostics of cancer predisposition is very important for the medical treatment of the patient and persons belonging to the high risk group . molecular studies enable the detection of mutation carriers and release from unreasonable stress of persons from the group with increased risk of cancer occurrence . the mutation spectrums in the genes predisposing to colorectal cancer in the polish population have been described [ 1 - 4 ] . in the present work we focused on recurrent mutations of the apc gene causing fap . familial adenomatous polyposis ( fap ) is characterized by the appearance of numerous polyps in the large intestine . the correlation between mutations of the apc gene and the occurrence of familial adenomatous polyposis was described in 1991 and since then , mutations of the apc gene have been investigated in research centres leading to identification of various mutation types . mutations of the apc gene , in most cases , are small deletions or insertions with the most frequent mutations , in the greater part of the described populations , being the aaaga deletion at codon 1309 and the acaaa deletion at codon 1061 . clinical diagnoses of fap patients were conducted in collaborating genetic centres or gastroenterology clinics in the place of residence of patients . so far , samples of dna belonging to 280 polish fap families have been collected in the dna bank of polish fap families established in 1997 at the institute of human genetics , polish academy of science in poznan . dna was extracted from peripheral blood cells by the classical phenol purification method and entire coding sequence of apc gene was screened for mutations by pcr - hd and sscp methods in 280 probands . dna fragments showing heteroduplex or additional pattern in sscp analysis were sequenced by direct pcr product sequencing and analyzed using a megabace 500 sequencer according to the manufacturer 's specifications . we identified 72 mutations in 124 of our 280 fap families and observed eight types of recurrent mutations . the mutations and age of onset two of them were localized in exon 11 and the remaining six in the 3 ' part of exon 15 . the most frequent mutation , 39273931delaaaga , occurred in twenty - eight families ( 10% ) ; the second one was 31833187delacaaa , occurring in eight families ( 2.8% ) ; and the third most frequent mutation was 32023205deltcaa , detected in 5 families ( 1.7% ) . in our fap patients y500x occurred in four families ( 1.4% ) while q978x was detected in three families . each of the remaining four types of mutation occurred in two families and the frequencies of these mutations were below one percent . fifty - four recurrent mutations identified in apc gene and age of onset in a group of 124 diagnosed polish fap families nd no data available in the human mutations database at the institute of medical genetics in cardiff , considered the most representative population in the world , seven hundred mutations are listed for the apc gene . the mutation reports describe the frequency of this mutation from 0% in northwest spain , 2.4% in the australian population , 5% in dutch , 7% in israeli to 16% in the group of italian fap patients [ 6 - 9 ] . the second most frequent mutation , 31833187delacaaa , is reported with frequency ranging from 0% in northwest spain , 1.5% in israeli populations to 8.4% in australia [ 6 - 9 ] . a study of over 100 dutch families revealed equal frequency of those two most frequent mutations ( 39273931delaaaga and 31833187delacaaa ) . the latest published report in 2005 involved the analysis of over 1000 patients . in comparison to this study , the representative study of mutation frequency in the neighbouring population indicated two times higher frequency of 39273931delaaaga , whereas a difference in frequency of 31833187del - acaaa was not observed ( germany 3.8% , poland 2.7% ) . the frequency of 32023205deltcaa was equal ( 1.7% ) in both populations . in worldwide comparison differences in the frequency of mutations the polish population of fap patients belongs to the group where 39273931delaaaga occurred with higher frequency , whereas the frequency of mutation 31833187del - acaaa occurred with medium frequency in comparison with other populations . the two recurrent mutations localized on exon 11 were observed only in the polish population . in our two unrelated families with 14901491inst brain tumours were observed . another mutation ( q978x ) did not occur with higher frequency as described for other populations . in our fap patient group q283x , which occurs with frequency of 4.5% in uk fap patients , screening for these mutations permitted us to diagnose 19% of all families in our population but eight types of mutations constitute 43.5% of all our diagnosed families . the mutation study in our population should involve these eight mutations to improve molecular diagnostics of the apc gene . the study was financed by grant 2p05a10728 from the ministry of science and higher education .

the molecular diagnostics of genetically conditioned disorders is based on the identification of the mutations in the predisposing genes . hereditary cancer disorders of the gastrointestinal tracts are caused by mutations of the tumour suppressor genes or the dna repair genes . occurrence of recurrent mutation allows improvement of molecular diagnostics . the mutation spectrum in the genes causing hereditary forms of colorectal cancers in the polish population was previously described . in the present work an estimation of the frequency of the recurrent mutations of the apc gene was performed . eight types of mutations occurred in 19.4% of our fap families and these constitute 43% of all polish diagnosed families .

porokeratoses are a group of cutaneous entities of unknown etiology , characterized by disordered epidermal keratinization and , at histological examination , by the presence of coronoid lamella , a column of parakeratotic cells extending through the stratum corneum . disseminated superficial porokeratosis ( dsp ) is a distinct form of porokeratosis that , clinically , is characterized by numerous , small , superficial keratotic papules with verrucoid aspects , an atrophic center and peripheral keratotic border . only 7 cases of this association have been described in the literature ( table 1 ) . we report a eighth case of this type and an atypical immunohistochemical method used to characterize the amyloid substance . a 76-year - old woman was referred to our department in june 1999 . she reported a 20 years history of an erythematous and squamo - papular eruption located on the extremities , without itching or pain . the patient had noted a progressive extension of these lesions in the last year and an exacerbation of the lesions during the summer . upon physical examination , numerous papules with a size of less than 15 mm , an atrophic center and hyperkeratotic borders were distributed on the neck , chest and extremities . palms , soles , mucous membranes and nails were spared , and the patient denied any family history of similar skin lesions . we examined a skin biopsy that revealed atrophy of the epidermis with hyperorthokeratosis and presence of homogeneous and compact cornoid lamella of keratin , typically oriented sideways with respect to the epidermal tissue ( fig . 1 a ) . the superficial dermis showed a slight chronic inflammatory infiltrate with vascular ectasia and edema . moreover , in the papillary dermis a compact deposit of a cellular eosinophilic material suspicious for amyloid was observed ( fig . 1b ) . to demonstrate the epithelial origin of the amyloid , we performed two immunohistochemical stainings by using specific moabs to pan - cytokeratin ae1-ae3 . interestingly , there was a striking difference between the two results : in fact , the acellular deposits , mentioned above , were negative for ckae1-ae3 , whereas a strong immunoreactivity was demonstrated for ck5 , supporting a specific origin of the amyloid substance from the basal cell layer of the epidermis ( fig . in 1937 , andrews first described dsp and introduced this term to indicate a clinical variant of mibelli 's porokeratosis . later , chernoski and freeman proposed a possible actinic etiology of this dermatosis and coined the term disseminated superficial nowadays , this term is generally accepted in european dermatological literature , and this definition is based on clinical and histological findings . dermatological manifestations are typically confined to sun exposed areas , with actinic induction and exacerbations . in the literature , two types of localized cutaneous amyloidosis ( lca ) are described : primary lca ( macular amyloidosis and lichen amyloidosis ) , which is not associated with other dermatoses or systemic involvement , and secondary lca , which is associated with inflammatory , hamartomatous or neoplastic skin disorders . the mechanism by which dsp induces dermal amyloid deposits is not clear , but piamphongstant et al . first suggested that this process can derive from degenerated epidermal keratinocytes . we hypothesize that a mutant keratinocyte clone is responsible for induction of porokeratotic lesions , because these necrotic epidermal keratinocytes ( colloid bodies ) might be transformed into amyloid by dermal macrophages and fibroblasts . immunohistochemical staining has shown an overexpression of p53 protein in porokeratotic lesions ; this is a tumor suppressor protein , an important gatekeeper and effector of the cell cycle . mutations of the p53 gene in all forms of porokeratosis , also in dsap , create a permissive state of uncoordinated cell cycling , and predispose cells to death . in our case , the lack of systemic involvement led to our hypothesis of a secondary dermal deposition of amyloid proteins and the existence of a close relationship between these two processes . this hypothesis was confirmed by positive staining with congo red and immunohistochemical staining with the anticytokeratin moabs strongly positive for ck5 , just below the epidermal porokeratotic zone in close proximity to the cornoid lamella . this cytokeratin is , in fact , strongly represented in the basal cell layer and these results indicate that the dermal deposits were amyloid originating from the epidermis ( type ii keratin ) . nowadays , more studies are necessary to clarify the exact mechanism that leads to secondary deposition of dermal amyloid in porokeratotic disease and the frequency of this association . we also suggest utilization of moabs for ck5 as a first - line target in these conditions , encouraged by the immediate and strong positivity of this protein in immunohistochemical studies , whereas pan - cytokeratin at first produced negative results .

only 6 cases with an association of disseminated superficial porokeratosis with dermal amyloid deposits are reported in the literature . we present the case of a 76-year - old woman who presented with a disseminated superficial porokeratosis . histological examination revealed amyloid deposits in the upper dermis , which were typed with routine he stains , congo red stains and anticytokeratin antibodies ( ae1-ae3 and ck5 ) . positive staining with congo red and , moreover , with ck5 ( a cytokeratin strongly represented in the basal cell layer of the epidermis ) indicates an epidermal origin of this protein .

gastric volvulus , an entity seen in both pediatric and adult patients , occurs when the stomach twists upon itself . this event may be transient , producing few if any symptoms , or may lead to obstruction or even ischemia and necrosis . pare described the first case of gastric volvulus in 1579 in a patient with diaphragmatic injury from a sword wound . acute gastric volvulus in pediatric and adult patients has been reported but chronic organo - axial gastric volvulus with diaphragmatic eventration has not been reported . this report describes a rare case of gastric volvulus with a review of the literature . he had a feeling of fullness and discomfort in the upper abdomen for 2 months . twenty years ago , he was told that his chest radiography , performed due to routine survey for military service , was abnormal . but he had received no specific measures since then and he had been relatively healthy . a thorough review of symptoms was performed but did not disclose any respiratory or cardiovascular symptoms . on clinical examination , he appeared not acutely ill looking and his body temperature was 36c , blood pressure 120/70 mmhg , respiratory rate 24/min . auscultation of the chest showed diminished breathing sound at the left lung base and heart sound was normal . no abnormalities were observed in cbc , liver chemistry , serum amylase and stool examination . the chest radiograph showed the left hemidiaphragm to be located at an unusually high intercostal space with large air - fluid level ( figure 1 ) . upper gastrointestinal series demonstrated the typical appearance of an organo - axial volvulus of the stomach ( figure 2 ) . the barium - filled stomach was twisted on an axis from the pylorus to the esophagus . the patient refused surgical or endoscopic correction of the volvulus and has been followed in the outpatient clinic for more than 6 months without symptom aggravation . the stomach is relatively fixed at the esophageal hiatus and the pylorus by the four gastric ligaments . the gastrophrenic ligament and the retroperitoneal attachment of the second part of the duodenum provide the superior and inferior fixation . the gastrohepatic ligament tethers the lesser curve , the gastrocolic ligament connects the stomach to the transverse colon , and the gastrosplenic ligament tethers the greater curve . the clinical symptoms depend upon the extent or degree of rotation , obstruction and associated defect . borchard s triad of pain , violent retching and inability to pass a nasogastric tube6 should lead to a strong clinical suspicion of acute gastric volvulus . an acute volvulus is an emergency situation , with either obstruction or strangulation of the stomach and requires expeditious surgery . in comparison , this explains why the diagnosis is often delayed in the elderly or after complication has occurred . . gastro - esophageal reflux may give rise to epigastric pain , which is intermittent during the periods of engorgement or gastric emptying . gastric ulceration is caused by localized ischemia and acidity within the herniated stomach or mucosal congestion due to venous obstruction of the herniated stomach . angina - like pain and electrocardiographic abnormalities may make the differential diagnosis difficult in the elderly . the diagnosis is confirmed by the presence of a large , unusual gas - filled viscus in the chest or abdomen on plain radiographs . if necessary , a barium swallow study can define the anatomic changes more exactly . on barium examination , the characteristic findings are 1 ) esophagogastric junction lying lower than normal , 2 ) reversal of the greater and lesser curvatures , 3 ) pylorus pointing downward , 4 ) greater curvature crossing the esophagus , 5 ) two air - fluid levels and lowering of the gastric fundus . secondary gastric volvulus is more frequent than idiopathic volvulus , therefore the diagnosis of gastric volvulus can be made after a thorough search for possible causative factors . these conditions have been reported as follows ; para - esophageal hernia , traumatic rupture of the diaphragm , eventration of the diaphragm and phrenic nerve injury . in infants and children , 15 ( 33% ) among 46 patients with gastric volvulus had diaphragmatic eventration . organo - axial volvulus is commonly associated with diaphragmatic hernia and usually manifests as an acute event . diaphragmatic eventration is suggested when a part or all of the hemidiaphragm is located at an unusually high level in the thorax . it does not refer to a defect or hole in the diaphragm with discrete edges , but rather to a diffuse or localized bulging of the diaphragm itself . acquired lesions are usually related to phrenic nerve injury , which may be diverse in origin . radiological investigation combined with fluoroscopy of the diaphragm our patient showed no diaphragmatic movement during respiration , so he had complete left hemidiaphragmatic eventration . acute gastric volvulus and symptomatic chronic gastric volvulus require operative treatment . if the volvulus is secondary , definitive treatment must include correction of the associated abnormalities . this is done by advancing the endoscope just past the point of torsion , turning and locking the tip of the instrument , and rotating it 180 degrees . with rapid diagnosis and modern treatment , the rate of mortality from acute gastric volvulus is now about 15% to 20% . if the stomach is strangulated , the mortality rate of emergency surgery is 4060% . in recognized cases of chronic gastric volvulus

gastric volvulus occurs when the stomach rotates about its longitudinal axis ( organo - axial volvulus ) , or about an axis joining the lesser and greater curvatures ( mesentero - axial volvulus ) . primary gastric volvulus , making up one third of cases , occurs when the stabilizing ligaments are too lax as a result of congenital or acquired causes . secondary gastric volvulus , making up the remainder of cases , occurs in association with a paraesophageal hernia or other congenital or acquired diaphragmatic defects . while gastric volvulus may occur acutely , especially in children , it may not be clinically apparent and discovered incidentally . the authors present a case of chronic organo - axial volvulus of the stomach secondary to left hemidiaphragmatic eventration with a review of the relevant literature .

the shoulder joint is a joint that relies on interactions and balance among multiple joints1 , 2 . the shoulder joint relies more on muscles than on bones or ligaments to support the shoulder and maintain the stability of the shoulder3 . stabilization relates to the human ability to consciously or subconsciously control large or small movements occurring in the joints4 . the movements of the shoulder bone and the humeral bone are controlled by the coupling force of two muscles : the rotator - cuff muscle and the elevator muscle of the scapular . if this coupling force collapses , the muscle - coordination activity does not occur and the rotator cuff is compressed5 . in this case , the humeral head only partially maintains contact with the articular fovea , leading to many pathologic conditions , such as shoulder instability3 , 6 . the activity of the scapular stabilizer muscles is important in counteracting the instability that occurs due to the aforementioned structural problems . the scapular stabilizer muscles include the serratus anterior , the upper trapezius , and the lower trapezius7 . in particular , the activity of the serratus anterior muscle is very important for scapular movements and control8 , 9 . extensive electromyography ( emg ) studies of the stabilizer muscles that contribute to shoulder stabilization have been carried out . the muscle activity of the serratus anterior and that of the upper trapezius have been shown to be imbalanced in subacromial - impingement syndrome , subacromial bursitis , rotator - cuff laceration , and rotator - cuff and biceps - brachii tendinitis10 . closed - chain exercises are frequently used for shoulder - joint dysfunction prevention and in rehabilitation programs11 . closed - chain exercises are frequently used in therapeutic exercise programs to generate dynamic stability in the joints and to maintain posture , since these exercises not only improve strength and endurance , but also induce the co - contraction of many muscles through mechanical compression of articular surfaces eliciting larger proprioceptive responses by stimulating the afferent receptors around the joints12 . along with closed - chain exercises , sling exercises , which require performing exercises while hanging on swinging ropes , are one of the treatment methods that can be used to prevent injuries , since these exercises stimulate the proprioceptive senses to strengthen weakened muscles and give the necessary stimuli to maintain joint stability . kirkesola13 reported that if vibrations of the rope were given during sling exercises , instability would be increased , further stimulating the proprioceptive sensory organs in the joints , and incorrect neuromuscular control could be corrected , reducing muscle imbalance . therefore , the present study compared the muscle activity of the upper trapezius with those of the serratus anterior , and the lower trapezius when slings , unstable surfaces , were laterally vibrated , to examine the effects of vibration during sling exercises on the shoulder stabilization muscles . the subjects of the present study were 15 adult men without any history of damage to the shoulder girdle or shoulder pain . the subjects average age was 26.13 5.13 years , their average height was 174.00 5.44 cm , and their average weight was 73.47 13.46 kg . all subjects read and signed a written consent form . the subjects performed push - up exercises with knee flexion . al.1 advised that push - up exercises with knee flexion produce emgs similar to those produced during push - up exercises with knee extension , while eliciting less force than push - up exercises with knee extension . before emgs were measured , the subjects performed push - up exercises with their hands placed on the floor at shoulder width , their knees placed on the floor , and their elbow joints extended . the subject performed push - up exercises on an unstable surface ( sling ) and maintained isometric contraction for five seconds in a state of scapular protraction and elbow extension , in the final stage of the motion , and emg signals from the serratus anterior , the upper trapezius muscle , and the lower trapezius muscle were collected during state of isometric contraction . in the second exercise , the subject performed push - up exercises on a sling and maintained isometric contraction in the last stage , and the rope of the sling was manually vibrated during the isometric contraction . vibrations was given within a range of 10 cm for five seconds at a frequency of 1 hz , in time with a metronome . in the third and fourth exercises , vibrations of 3 hz and 3.5 hz , were respectively applied . usa ) was used to measure muscle activities during the exercises . for the measurement , electrodes were attached to the upper trapezius , the lower trapezius , and the serratus anterior . 18.0 ) , and one - way anova was used to compare muscle activities among the various vibration frequencies on the unstable surface . the upper trapezius and the lower trapezius did not show any significant differences among isometric contraction without vibration , isometric contraction with vibration of 1 hz , and isometric contraction with vibration of 3 hz , but showed significant differences at isometric contraction with vibrations of 3.5 hz . the serratus anterior showed a significant differences between isometric contraction with vibration of 3 hz and isometric contraction with vibration of 3.5 hz ( table 1table 1 . comparison of muscle activation among the various vibration frequencies ( unit : % rvc)ic1 hz3 hz3.5 hzut*0.034 0.0130.034 0.0130.035 0.0130.057 0.010lt*0.025 0.0080.027 0.0070.027 0.0070.053 0.009sa*0.051 0.0120.050 0.0110.083 0.0140.083 0.014*p<0.05 , mean sd , ic : isometric contraction , ut : upper trapezius , lt : lower trapezius , sa : serratus anterior ) . * p<0.05 , mean sd , ic : isometric contraction , ut : upper trapezius , lt : lower trapezius , sa : serratus anterior the coupling force of the upper trapezius , the serratus anterior , and the lower trapezius is the core of scapular movements14 . if this muscle recruitment is changed , or the motions of these muscles are limited by an antagonist , the movement pattern will collapse . it has been revealed that in shoulder impingement syndrome , patients shoulders are elevated , the muscle activity of the upper trapezius increases , and the muscle activity of the serratus anterior decreases . in particular , the serratus anterior , which is an important muscle scapular movement imbalance , has been studied by many researchers . fatigue of the serratus anterior decreases scapular rotation and protraction , and displaces the humeral head in a forward and upward direction1 , 10 , 11 . therefore , it may cause secondary impingement syndrome and rotator - cuff tear , decrease the range of scapular motion , or disturb the contraction timing of the serratus anterior , thereby inducing stress in the glenohumeral joint15 . if abnormal scapular movements appear , due to problems in the serratus anterior , the position of the fossa would be affected , and the force and alignment involved in movements around the glenohumeral joint would eventually be affected16 . kirkesola14 reported that when the rope used for sling exercises was vibrated , instability increased further stimulating the proprioceptive sensory organs in the joints , and incorrect neuromuscular control could be corrected , reducing muscle imbalance . as the load - bearing surface becomes more unstable , the muscle activities of the serratus anterior and the lower trapezius would increase , selectively strengthening the scapular - stabilizer muscles , reducing muscle imbalance . exercises on a sling elicit significantly higher muscle activities than exercises on a fixed surface . therefore , exercises on a sling should simultaneously strengthen many muscles that contribute to shoulder joint stability , increasing the stability of the shoulder complex . in the present study , to examine the effects of diverse vibration frequencies on the upper trapezius , the lower trapezius , and the serratus anterior during exercises on a sling , an unstable surface , vibration frequencies of 1 hz , 3 hz , and 3.5 hz were used . in the present study , all three muscles showed gradual activity increases as the frequency of vibration increased from 1 hz to 3 hz and 3.5 hz . the upper trapezius and the lower trapezius showed significant changes in muscle activity at 3.5 hz , and the serratus anterior showed prominent changes in muscle activity at 3 hz and 3.5 hz . therefore , as the vibration frequency increased , making the bearing surface more unstable , the recruitment of the upper trapezius , the lower trapezius , and the serratus anterior increased . to perform exercises that will selectively strengthen the serratus anterior ,

[ purpose ] the present study compared the muscle activity of the upper trapezius with those of the serratus anterior and the lower trapezius when slings , unstable surfaces , were laterally vibrated , to examine the effects of vibration during sling exercises on shoulder stabilization muscles . [ methods ] the subjects performed push - up exercises on a sling and maintained isometric contraction in the final stage , while vibration was manually administered to the rope of the sling during the isometric - contraction stage . vibration within a range of 10 cm was delivered for five seconds at a frequency of 1 hz in time with a metronome . vibrations were applied for five seconds at 3 hz and 3.5 hz , respectively . [ results ] the serratus anterior showed a significant differences between isometric contraction with vibration of 3 hz and isometric contraction with vibration of 3.5 hz . [ conclusion ] the upper trapezius and the lower trapezius showed prominent changes in muscle activity at 3.5 hz , and the serratus anterior showed prominent changes in muscle activity at 3 hz and 3.5 hz . therefore , as vibration frequency increased , making the load - bearing surface more unstable , the recruitment of the upper trapezius , the lower trapezius , and the serratus anterior increased . to perform exercises that selectively strengthen the serratus anterior , the exercises should be performed at a vibration frequency of 3 hz .

one disability that can sufficient to interfere with activities of daily living is non - syndromic hearing loss ( nshl ) ( 1 , 2 ) . people with even mild nshl have problems hearing speech when there is background noise and identifying the sounds sources ( 3 , 4 ) . nshl is the most common sensory deficit in humans , with an incidence of about 1 in 1,000 newborns . the prevalence increases during childhood , reaching a rate of 2.7 per 1,000 children before the age of 5 years and 3.5 per 1,000 adolescents . two thirds of people with nshl worldwide live in developing countries ( 5 , 6 ) . the transmembrane channel - like 1 ( tmc1 ) gene is considered a member of a gene family predicted to encode transmembrane proteins ( 7 , 8) . mutations in the tmc1 gene have been associated with profound prelingual deafness ( dfnb7/b11 ) and progressive postlingual hearing loss ( dfna36 ) ; thy have been reported in different populations ( 9 ) . the dfna36 and dfnb7/b11 loci are located on chromosome 9q13 - q21 ( 7 ) . tmc1 and tmc2 are members of a gene family predicted to encode transmembrane proteins and are located on p13 of chromosome 20 . the tmc1 gene encodes a sodium sensor and may function as ion transport channel or pump . tmc1 mrna is specifically expressed in neurosensory hair cells of the inner ear , and it is required for normal function of cochlear hair cells , although the molecular and cellular functions of the tmc1 protein are unknown ( 8) . since no report has yet determined the frequency of tmc1 gene mutations in the iranian population , the present study was performed to screen and identify the mutations of this gene associated with nshl using polymerase chain reaction single - stranded conformation polymorphism ( pcr - sscp ) and heteroduplex analysis ( ha ) . this experimental study was conducted at the cellular and molecular research center of shahrekord university of medical sciences from february 2011 to january 2012 . in the present study , we investigated the mutations of the tmc1 gene , locus dfnb7/11 , in a cohort of 100 patients with nshl in iran . the 890 blood samples of families with iranian origin was obtained in ethylene diamine tetra - acetic acid ( edta)-containing tubes ( sarstedt ) from 10 provinces of iran , namely semnan , sistan & baloochestan , fars , khozestan , kohgilooye va boyer ahmad , kordestan , chaharmahal & bakhtiari , booshehr , golestan , and gilan . finally , 100 patients ( one proband from each family ) were selected ( table 1 ) . nshl informational questionnaires were filled out for all families . in previous work , these patients had no mutations in the cx26 gene ( 10 ) . known environmental risk factors such as head trauma and use of ototoxic drugs could affect the study , so families with the possibility of exposure to these factors were excluded from the research . total genomic dna was extracted from peripheral blood samples of patients using the phenol and chloroform standard procedure ( 11 ) . the quality of extracted genomic dna was quantified by nano - drop 1000 spectrophotometer ( thermo scientific inc . , wilmington , de , usa ) at a wavelength of 260/280 nm according to the method described by sambrook and russell ( 12 ) for gene amplification of exons 7 and 13 of the tmc1 gene by pcr , two sets of overlapping primers were designed due to their length using the gene runner software version 3.0 ( hastings software , inc . , hastings , ny ) , and primer sequences were blasted in the national center for biotechnology information s ( ncbi s ) genbank . the details of the designed primers are shown in table 2 . mutant primers created by site - directed mutagenesis ( sdm ) as positive control , tmc1-m ( tmc1 mutant primer ) . site - directed mutagenesis ( sdm ) after gene amplification using the designed specific primers with changes in one nucleotide was used to generate positive control samples . standard pcr optimization was carried out in a total volume of 50 l reaction in 0.5 ml tubes for each amplicon in a gradient palm cycler ( corbett research , australia ) . the pcr reaction consisted of 0.2 pm of each primer , 2.5 mm mgcl2 , 200 m dntps mix , 5 m of 10 x pcr buffer ( 200 mm tris - hcl [ ph 8.4 ] , 500 mm kcl ) , 1 unit of taq dna polymerase ( all fermentas , germany ) , and 100 ng of template dna . pcr temperature programs involved an initial denaturation at 94c for 5 minutes followed by 32 cycles consisting of 50 seconds of denaturation at 94c , 50 seconds of annealing at 57.5c ( exon 7 ) or 60c ( exon 13 ) , 40 seconds of extension at 72c , and a final extension at 72c for 5 minutes . the pcr amplification products ( 2 l / lane ) were loaded on 8% polyacrylamide gel ( 29:1 acrylamide : bis - acrylamide ) electrophoresis ( page ) in 1 x tbe buffer ( 10.8 g of tris - base 89 mm , 5.5 g of boric acid 2 mm , edta ( ph = 8.0 ) 4 ml of 0.5 m edta ( ph = 8.0 ) , combined all components in sufficient h2o and were stirred to dissolve ) at 85 v for 30 minutes , and the gels were stained using the silver nitrate staining method . for sscp , microtubes containing pcr products were mixed with an equal volume of formamide loading dye , heated to 96c for 15 minutes , and chilled on ice for 5 minutes before loading on the polyacrylamide gel . the electrophoresis tank was filled by tbe buffer 0.6 x , and tbe 1 x was used in the gel ; electrophoresis was performed with 2.5 - 5% glycerol at 20 w at 4c for 6 - 8 hours . first , 3 l of edta ( 0.5 m ) was added to 2 l of pcr products . then , the pcr products were heated at 95c for 3 minutes and slowly cooled to 37c over 40 minutes . after mixing with 6 x triple dye loading buffer at a volume ratio of 1:5 , the pcr products were loaded on page with 10% urea and electrophoresis were performed at 320 v at 10 - 12c for 6 - 8 hours ( table 3 ) . finally , the heteroduplex fragments were visualized using standard silver nitrate staining . the samples containing shift bands on the sscp gel and after ha were subjected to direct dna sequencing of exons 7 and 13 of the tmc1 gene in an abi 3730xl automated sequencer ( applied biosystems ) by macrogen inc . for this study , the regional research ethical committee of shahrekord university of medical sciences ( grant number 91 - 3 - 2 , january 2011 ) approved the protocol and informed consent forms . informed consent was obtained from all hearing loss patients before enrollment in the study based on the declaration of helsinki ( doh ) . all data were collected in the statistical program for the social sciences software ( spss , inc . , the mean difference between groups was calculated using a t test . in this study , a p value of 0.05 was considered statistically significant . this experimental study was conducted at the cellular and molecular research center of shahrekord university of medical sciences from february 2011 to january 2012 . in the present study , we investigated the mutations of the tmc1 gene , locus dfnb7/11 , in a cohort of 100 patients with nshl in iran . the 890 blood samples of families with iranian origin was obtained in ethylene diamine tetra - acetic acid ( edta)-containing tubes ( sarstedt ) from 10 provinces of iran , namely semnan , sistan & baloochestan , fars , khozestan , kohgilooye va boyer ahmad , kordestan , chaharmahal & bakhtiari , booshehr , golestan , and gilan . finally , 100 patients ( one proband from each family ) were selected ( table 1 ) . nshl informational questionnaires were filled out for all families . in previous work , these patients had no mutations in the cx26 gene ( 10 ) . known environmental risk factors such as head trauma and use of ototoxic drugs could affect the study , so families with the possibility of exposure to these factors were excluded from the research . total genomic dna was extracted from peripheral blood samples of patients using the phenol and chloroform standard procedure ( 11 ) . the quality of extracted genomic dna was quantified by nano - drop 1000 spectrophotometer ( thermo scientific inc . , wilmington , de , usa ) at a wavelength of 260/280 nm according to the method described by sambrook and russell ( 12 ) for gene amplification of exons 7 and 13 of the tmc1 gene by pcr , two sets of overlapping primers were designed due to their length using the gene runner software version 3.0 ( hastings software , inc . , hastings , ny ) , and primer sequences were blasted in the national center for biotechnology information s ( ncbi s ) genbank . the details of the designed primers are shown in table 2 . mutant primers created by site - directed mutagenesis ( sdm ) as positive control , tmc1-m ( tmc1 mutant primer ) . site - directed mutagenesis ( sdm ) after gene amplification using the designed specific primers with changes in one nucleotide was used to generate positive control samples . standard pcr optimization was carried out in a total volume of 50 l reaction in 0.5 ml tubes for each amplicon in a gradient palm cycler ( corbett research , australia ) . the pcr reaction consisted of 0.2 pm of each primer , 2.5 mm mgcl2 , 200 m dntps mix , 5 m of 10 x pcr buffer ( 200 mm tris - hcl [ ph 8.4 ] , 500 mm kcl ) , 1 unit of taq dna polymerase ( all fermentas , germany ) , and 100 ng of template dna . pcr temperature programs involved an initial denaturation at 94c for 5 minutes followed by 32 cycles consisting of 50 seconds of denaturation at 94c , 50 seconds of annealing at 57.5c ( exon 7 ) or 60c ( exon 13 ) , 40 seconds of extension at 72c , and a final extension at 72c for 5 minutes . the pcr amplification products ( 2 l / lane ) were loaded on 8% polyacrylamide gel ( 29:1 acrylamide : bis - acrylamide ) electrophoresis ( page ) in 1 x tbe buffer ( 10.8 g of tris - base 89 mm , 5.5 g of boric acid 2 mm , edta ( ph = 8.0 ) 4 ml of 0.5 m edta ( ph = 8.0 ) , combined all components in sufficient h2o and were stirred to dissolve ) at 85 v for 30 minutes , and the gels were stained using the silver nitrate staining method . for sscp , microtubes containing pcr products were mixed with an equal volume of formamide loading dye , heated to 96c for 15 minutes , and chilled on ice for 5 minutes before loading on the polyacrylamide gel . the electrophoresis tank was filled by tbe buffer 0.6 x , and tbe 1 x was used in the gel ; electrophoresis was performed with 2.5 - 5% glycerol at 20 w at 4c for 6 - 8 hours . first , 3 l of edta ( 0.5 m ) was added to 2 l of pcr products . then , the pcr products were heated at 95c for 3 minutes and slowly cooled to 37c over 40 minutes . after mixing with 6 x triple dye loading buffer at a volume ratio of 1:5 , the pcr products were loaded on page with 10% urea and electrophoresis were performed at 320 v at 10 - 12c for 6 - 8 hours ( table 3 ) . the samples containing shift bands on the sscp gel and after ha were subjected to direct dna sequencing of exons 7 and 13 of the tmc1 gene in an abi 3730xl automated sequencer ( applied biosystems ) by macrogen inc . for this study , the regional research ethical committee of shahrekord university of medical sciences ( grant number 91 - 3 - 2 , january 2011 ) approved the protocol and informed consent forms . informed consent was obtained from all hearing loss patients before enrollment in the study based on the declaration of helsinki ( doh ) . all data were collected in the statistical program for the social sciences software ( spss , inc . , the mean difference between groups was calculated using a t test . in this study , in this study , blood samples of 100 patients with hearing loss ( mean age 16.5 2.01 years , 49.2% men and 50.8% women , 74.15% married ) were collected from 10 provinces in iran . the connexin 26 gene is responsible for a large proportion of deafness ( about 14.6% ) , and these patients had no mutations in this gene ( 10 , 13 ) . extracted dna with a 260/280 nm absorbance ratio of 1.8 - 2 was subjected to gene amplification . exons 7 and 13 of the tmc1 gene were amplified using the pcr technique with specific and mutated oligonucleotide primers . after page , fragments with a length of 187 and 250 bp were revealed for exons 7 and 13 , respectively , of the tmc1 gene . pcr products were used for sscp and ha for investigation of mutations in the sequences . neither pcr - sscp nor ha showed mutations in exons 7 and 13 of the tmc1 gene ( figures 1 and 2 ) . line m is a 100 bp dna ladder ( fermentas , germany ) , line 5 is a sample amplified by mutant primers with typical shift ( positive control ) , and lines 2 - 4 and 6 - 13 are deafness patients samples . all specimens have the same template bands without shifts in the sscp bands and after ha . line m is a 100 bp dna ladder ( fermentas , germany ) , line 1 is positive control , line 3 is a suspected sample containing different bands compared to other samples , and lines 2 and 4 - 15 are samples from deaf patients . in the present study , to increase the accuracy of the sscp reaction , ha and mutant control ( positive control created by sdm ) were performed . only a number of suspected fragments in exon 7 showed a different banding pattern , but after sequencing , the mutations were not confirmed ( figure 3 ) . in various human populations , different dominant ( e.g. , coch , dfna5 , and pou4f3 ) and recessive genes ( e.g. , gjb2 and slc26a4 ) have been reported as the cause of hearing loss worldwide ( 7 , 9 , 14 - 16 ) . the tmc1 gene is an autosomal recessive gene and a common cause of hearing loss in many countries , such as india , pakistan , algeria , iraq , lebanon , sudan , tunisia , and turkey ( 15 , 17 - 19 ) . the prevalence of non - syndromic hearing impairment due to tmc1 in the pakistani population is 4.4% , and one study has indicated that the tmc1 protein might have an important function in the k channels of the inner hair cells ( 16 ) . 2002 ) detected a 1.6 kb genomic deletion encompassing exon 14 of tmc1 in a recessive deafness mouse mutant , which lacks auditory responses and has hair - cell degeneration ( 7 ) . in the present study , the association of exons 7 and 13 mutations of tmc1 gene locus dfnb7/11 in deaf patients after gene amplification , neither sscp nor ha showed mutations in exons 7 and 13 of this gene related to hearing loss in these patients . only a suspected sample in exon 7 had a different banding pattern , but after sequencing , mutations in this exon were not identified . the strength of this study was the number of samples , as 890 blood samples were provided ; however , due to a lack of financial resources , we chose two exons of this gene . via the molecular analysis of the tmc1 gene in korean patients , one study showed that this gene was not the cause of nshl in the korean population ( 20 ) . in our study , as in the research in korea , a relation between the tmc1 gene and nshl in the iranian population was not observed . ( 2005 ) investigated four novel tmc1 ( dfnb7/dfnb11 ) mutations in turkish patients with congenital autosomal recessive nshl ( arnshl ) , and they indicated that tmc1 mutations account for at least 6% ( 4/65 ) of arnshl cases in gjb2-negative turkish families from the northeast and east of turkey ; however , in our study , mutation in exons 7 and 13 of tmc1 gene was not observed ( 14 ) . in another study in sudan , it was shown that tmc1 mutations contribute to deafness ; this confirmed and extended previous reports on the role of tmc1 in recessive non - syndromic deafness ( 20 , 21 ) . meanwhile , in the present study on the iranian population , no significant relation between exon 7 and 13 mutations of the tmc1 gene were observed . in pakistan , 2007 ) identified 10 new families segregating dfnb7/b11 deafness and tmc1 mutations , including three novel alleles ; moreover , they identified a c.100c > t mutation in exon 7 ( 16 ) . 2008 ) identified four new mutations in the tmc1 gene and suggested an additional deafness gene at loci dfna36 and dfnb7/11 in 51 familial of turkish patients with autosomal recessive hearing loss ; these results implied the presence of mutations outside the coding region of this gene , or alternatively , at least one additional deafness - causing gene in this region ( 9 ) . another study in turkey reported five novel mutations in the tmc1 gene related to arnshl ( 19 ) . in comparison with the other populations discussed , in a review study in iran by mahdieh et al . the frequencies and distributions of nshl included gjb2 , gjb6 ( large deletion ) , tecta , slc26a4 , and pejvk mutations . the researchers indicated that mutations in gjb2 , slc26 , tecta , and pjvk genes have an important role in deafness in iran , and a screening test should be generated for better intervention and diagnostic programs ( 22 ) . the study of hildebrand et al . in iranian families showed that two families are related to locus dfnb7/11 , and one of them had a c.776 + 1 g > a mutation in exon 7 ( 23 ) . in 2014 , lin et al . identified novel compound heterozygous mutant alleles of tmc1 responsible for arnshl in a tibetan chinese family ( 24 ) ; meanwhile , in the present work , we did not observe a relation with mutations of exons 7 and 13 of the tmc1 gene in iranian nshl . the findings of the current study indicate that mutations in exons 7 and 13 of tmc1 gene are not related to hearing loss in the iranian population . therefore , the tmc1 gene may not related to nshl , but further studies investigating related mutations in other parts of this gene in iranian population are necessary and could help in the genetic counseling of patients and design of practical strategies for the management of auditory disorder .

background : non - syndromic hearing loss ( nshl ) is the most common birth defect and occurs in approximately 1/1,000 newborns . nshl is a heterogeneous trait and can arise due to both genetic and environmental factors . mutations of the transmembrane channel - like 1 ( tmc1 ) gene cause non - syndromic deafness in humans and mice.objectives:the aim of the present study was to investigate the association of tmc1 gene mutations of the locus dfnb7/11 in exons 7 and 13 in a cohort of 100 patients with hearing loss in iran using polymerase chain reaction single - stranded conformation polymorphism ( pcr - sscp ) , heteroduplex analysis ( ha ) , and dna sequencing.patients and methods : in this experimental study , the blood samples of 100 nshl patients were collected from 10 provinces in iran . these patients had a mean age of 16.5 2.01 years and 74.15% of their parents had consanguinity . dna was extracted from specimens and mutations of exons 7 and 13 of the tmc1 gene were investigated using pcr - sscp . all samples were checked via ha reaction and suspected specimens with shift bands were subjected to dna sequencing for investigation of any gene variation.results:in this study , no mutation was found in the two exons of tmc1 gene . it was concluded from these results that mutations of the tmc1 gene s special exons 7 and 13 have a low contribution in patients and are not great of clinical importance in these iranian provinces.conclusions:more studies are needed to investigate the relationship between other parts of this gene with hearing loss in different populations through the country . more research could clarify the role of this gene and its relation with deafness and provide essential information for the prevention and management of auditory disorders caused by genetic factors in the iranian population .

technological advances in three - dimensional ( 3d ) imaging such as cone - beam computed tomography ( cbct ) appear to offer significant advantages in both quality and quantity of data representing true anatomy , . the increased use of computed tomography in dentistry has also spurred the improvement of existing software designed primarily for dental use , as well as the development of new software . however , it is necessary to select protocols and to assess the reliability and accuracy of landmark identification in 3d images . development of 3d imaging into a practical and usable cephalometric method was hindered for many years by technologic limitations , high costs , and high radiation doses . advances in cbct technology make its use feasible for specific indications , such as impacted teeth , craniofacial anomalies , airway and temporomandibular joint disorders . in these cases , cbct images could be used for routine cephalometric purposes beyond the diagnostic purposes for which they were primarily taken . this has been extensively done for cephalometric measurements , but to a much lesser extent for maxillofacial computed tomography imaging . whether cbct should be used routinely in all orthodontic patients the voxel is the smallest unit of ct images and its size has an influence on image spatial resolution . by decreasing the voxel size the image spatial resolution increases as there is a decrease in partial volume averaging . on the other hand , by decreasing the voxel size , the scan time increases , as does the probability of patient movement . this is especially of concern in the case of younger individuals , who constitute the overwhelming majority of orthodontic patients . this issue of increased radiation dose to children , and specifically for orthodontic purposes , has caused widespread concern and has been the source of much discussion even in the lay literature . in order to verify the clinical reliability of measurements on multiplanar sections and 3d reconstructions derived from cbct , it is also necessary to evaluate the measurements using anatomic landmarks without the use of metallic markers . the most recent studies involving cbct scans have shown reproducibility and accuracy of cephalometric measurements performed on lateral cephalograms reformatted from cbct images or in 3d - cbct images . however , most of the studies used radiopaque markers and these might have an influence on the accuracy and reproducibility of the measurements . the presence of metallic markers eliminates errors from landmark identification because metallic landmarks can be easily identified and located with a high degree of accuracy and precision . the aim of this study was to verify the influence of voxel size on the accuracy and reproducibility of linear measurements of the mandible performed without metallic markers on 3d cbct images , comparing multiplanar sections and 3d rendering images . approval for this study was received from the ethics committee at the university of so paulo , bauru , brazil . ten dry adult human mandibles , selected from the collection of dry skulls of the department of anatomy from the bauru school of dentistry were scanned . to fit the inclusion criteria , the mandibles had to be well preserved with no large metallic restorations on the teeth , in order to avoid scattering and artifacts . the sample size of each group was calculated based on an alpha significance level of 0.05 and beta of 0.2 to achieve 80% of power . to provide soft - tissue equivalent attenuation , the mandibles were positioned with the mandibular plane parallel and the midsagittal plane perpendicular to the floor , in accordance with the manufacturer s instructions for performing the scans in the clinical setting . the cbct images were acquired using the i - cat classic 3d dental imaging system ( i - cat classic , imaging sciences international , hatfield , pa , usa ) . each mandible was scanned twice : once with a 0.20 voxel resolution ( 36.12 mas , 40 sec , fov of 8 cm , 120 kv ) and once with a 0.40 voxel resolution ( 18.45 mas , 20 sec , fov of 8 cm , 120 kv ) . cbct data were exported in dicom ( digital imaging and communication in medicine ) multi - file format and imported into dolphin11.5 software ( dolphin imaging & management solutions , patterson technology , chatsworth , ca , usa ) . a computer a505-s6975 ( satellite , toshiba , tokyo , japan ) with a dedicated 512-mb video card on a 17-in crystalbrite lcd flat - panel color screen with a maximum resolution of 1440x900 pixels measurements were made separately on the multiplanar sections ( 2d ) and on 3d rendering images display window of dolphin 3d . the multiplanar window displays the sagittal , coronal and axial slices , as well as the 3d rendered image . it was possible to visualize the 3 projections and the 3d rendered image together or choose one of the four images in full screen . linear measurements of 3d coordinates were obtained using several craniometric anatomic landmarks ( figure 1 ) . for multiplanar groups , each landmark ( figure 2 ) was identified and marked on the three orthogonal slices simultaneously ( axial , sagittal and coronal ) . the corresponding linear measurements ( figure 3 ) were determined electronically with the measurement tool on the sectional images . for performing the linear measurements which landmarks were not located in the same sectional image , as c mf , the first mark of a line was performed in the sectional image showing the first landmark and the second mark was performed on a different section showing the second landmark . as the volume rendering was reoriented , the positions of the landmarks were verified in the axial , sagittal and coronal slices and , if necessary , relocated . figure 1frontal ( left ) and sagittal ( right ) views of mandible showing the points figure 2points . reference in the anatomical view and in the cone - beam computed tomography ( cbct ) images figure 3definitions of mandibular linear measurements for 3d rendering images , the landmarks were identified directly on the surface for 3d images . the measurements were performed twice by 2 blinded independent examiners ( tmff and ja ) . the physical measurements of the dry mandibles were obtained with a high - precision digital caliper ( mitutoyo , mitutoyo sul americana , suzano , sp , brazil ) , identical to those used in similar studies . in this initial study , we selected landmarks that are relatively easy to identify linear measurements were identified on two occasions , one month apart , by one operator ( mlp ) . the mean of the two measurements of each distance was calculated and used as the gold standard to evaluate the accuracy of the cbct 3d images . the measurements were grouped into five groups : physical measurements ( g1 ) ; measurements performed on multiplanar slices with 0.20 mm voxel size ( g2 ) ; measurements performed on multiplanar slices with 0.40 mm voxel size ( g3 ) ; measurements performed on 3d images with voxel size of 0.20 mm ( g4 ) and measurements performed on 3d images with voxel size of 0.40 mm ( g5 ) . intraclass correlation and bland - altman test was used to evaluate the intra- and interobserver reliability . analysis of variance for repeated measures ( anova ) and dunnett s test were used to compare the groups on cbct images and the physical measurements . all tests were performed with statistica software 6.0 ( statistica for windows ; statsoft , tulsa , ok , usa ) and spss version 10 ( spss , spss inc . measurements were made separately on the multiplanar sections ( 2d ) and on 3d rendering images display window of dolphin 3d . the multiplanar window displays the sagittal , coronal and axial slices , as well as the 3d rendered image . it was possible to visualize the 3 projections and the 3d rendered image together or choose one of the four images in full screen . linear measurements of 3d coordinates were obtained using several craniometric anatomic landmarks ( figure 1 ) . for multiplanar groups , each landmark ( figure 2 ) was identified and marked on the three orthogonal slices simultaneously ( axial , sagittal and coronal ) . the corresponding linear measurements ( figure 3 ) were determined electronically with the measurement tool on the sectional images . for performing the linear measurements which landmarks were not located in the same sectional image , as c mf , the first mark of a line was performed in the sectional image showing the first landmark and the second mark was performed on a different section showing the second landmark . as the volume rendering was reoriented , the positions of the landmarks were verified in the axial , sagittal and coronal slices and , if necessary , relocated . figure 1frontal ( left ) and sagittal ( right ) views of mandible showing the points figure 2points . reference in the anatomical view and in the cone - beam computed tomography ( cbct ) images figure 3definitions of mandibular linear measurements for 3d rendering images , the landmarks were identified directly on the surface for 3d images . the measurements were performed twice by 2 blinded independent examiners ( tmff and ja ) . the physical measurements of the dry mandibles were obtained with a high - precision digital caliper ( mitutoyo , mitutoyo sul americana , suzano , sp , brazil ) , identical to those used in similar studies . in this initial study , we selected landmarks that are relatively easy to identify . linear measurements were identified on two occasions , one month apart , by one operator ( mlp ) . the mean of the two measurements of each distance was calculated and used as the gold standard to evaluate the accuracy of the cbct 3d images . the measurements were grouped into five groups : physical measurements ( g1 ) ; measurements performed on multiplanar slices with 0.20 mm voxel size ( g2 ) ; measurements performed on multiplanar slices with 0.40 mm voxel size ( g3 ) ; measurements performed on 3d images with voxel size of 0.20 mm ( g4 ) and measurements performed on 3d images with voxel size of 0.40 mm ( g5 ) . intraclass correlation and bland - altman test was used to evaluate the intra- and interobserver reliability . analysis of variance for repeated measures ( anova ) and dunnett s test were used to compare the groups on cbct images and the physical measurements . all tests were performed with statistica software 6.0 ( statistica for windows ; statsoft , tulsa , ok , usa ) and spss version 10 ( spss , spss inc . , chicago , il , usa ) . the measurements for investigator 1 showed excellent reliability with intraclass correlation coefficients ( icc ) ranging from 0.93 to 1 ( icc average g2:0.97 ; g3:0.97 ; g4:0.98 and g5:0.98 ) . additionally , the mean absolute differences between the first and second measurements were less than 0.40 mm ( mean average difference g2:0.11 mm and g3:0.02 mm ) for multiplanar images and less than 0.64 mm ( mean average difference g4:0.14 mm and g5:0.03 mm ) for 3d model images . for investigator 2 , the results also showed excellent reliability , with icc coefficients ranging from 0.88 to 0.99 ( icc average - g2:0.96 ; g3:0.95 ; g4:0.97 and g5:0.97 ) . table 1intraobserver and interobserver variation for each group and analysis methods [ intraclass correlation coefficient ( icc ) and bland - altman ] multiplanar images3d model imagesvariable g2g3g4g5 0.2 voxel ( n=10)0.4 voxel ( n=10)0.2 voxel ( n=10)0.4 voxel ( n=10)intraobserver obser veradsd95% ciiccadsd95% ciiccadsd95% ciiccadsd95% ciiccc c 10.010.36 - 0.69 ; 0.711 - 0.010.15 - 0.31 ; 0.2910.120.67 - 1.20 ; 1.440.990.180.77 - 1.34 ; 1.700.98 2 - 0.410.75 - 1.89 ; 1.070.98 - 0.680.75 - 2.16 ; 0.800.970.170.87 - 1.54 ; 1.880.980.460.73 - 0.98 ; 1.900.98mf mf 1 - 0.130.46 - 1.03 ; 0.770.99 - 0.190.49 - 1.14 ; 0.760.980.170.72 - 1.25 ; 1.590.96 - 0.210.49 - 1.17 ; 0.750.98 2 - 0.301.18 - 2.16 ; 2.010.93 - 0.080.68 - 1.41 ; 1.250.97 - 0.210.82 - 1.82 ; 1.400.96 - 0.031.33 - 2.63 ; 2.570.92c mf 1 - 0.041.09 - 2.18 ; 2.100.970.310.67 - 0.99 ; 1.610.980.640.78 - 0.90 ; 2.180.980.260.81 - 1.32 ; 1.840.98 2 - 0.581.21 - 2.95 ; 1.790.95 - 0.920.97 - 2.81 ; 0.970.960.480.92 - 1.33 ; 2.290.970.420.75 - 1.04 ; 1.880.98c mf 10.190.84 - 1.46 ; 1.840.990.240.90 - 1.53 ; 2.010.970.390.67 - 0.93 ; 1.710.980.100.70 - 1.28 ; 1.480.99 2 - 0.660.81 - 2.24 ; 0.920.97 - 0.811.27 - 3.30 ; 1.680.940.100.73 - 1.34 ; 1.540.990.630.85 - 1.03 ; 2.290.97c mf 10.401.22 - 2.0 ; 2.800.950.051.40 - 2.68 ; 2.780.930.041.00 - 1.93 ; 2.010.960.261.06 - 1.83 ; 2.350.95 2 - 1.050.90 - 2.82 ; 0.720.91 - 0.291.01 - 2.27 ; 1.690.95 - 0.090.61 - 1.28 ; 1.100.990.410.68 - 0.92 ; 1.740.97c mf 10.121.40 - 2.62 ; 2.860.94 - 0.041.13 - 2.27 ; 2.190.950.090.90 - 1.67 ; 1.850.970.090.73 - 1.35 ; 1.530.98 2 - 0.650.88 - 2.36 ; 1.060.96 - 0.690.72 - 2.10 ; 0.720.96 - 0.020.99 - 1.96 ; 1.920.970.061.01 - 1.93 ; 2.050.97mco lco ( right)1 - 0.030.39 - 0.80 ; 0.740.98 - 0.070.40 - 0.84 ; 0.700.980.140.28 - 0.42 ; 0.700.99 - 0.030.45 - 0.91 ; 0.850.98 20.260.47 - 0.66 ; 1.180.970.180.71 - 1.22 ; 1.580.950.770.54 - 0.29 ; 1.830.900.680.55 - 0.40 ; 1.760.92mco lco ( left)10.390.44 - 0.47 ; 1.250.94 - 0.380.53 - 1.42 ; 0.660.930.010.35 - 0.67 ; 0.690.98 - 0.050.53 - 1.10 ; 1.000.95 2 - 0.050.53 - 1.08 ; 0.990.950.300.81 - 1.29 ; 1.890.88 - 0.080.43 - 0.93 ; 0.770.980.140.33 - 0.50 ; 0.780.98lco lco 10.300.38 - 0.34 ; 0.940.99 - 0.220.50 - 1.22 ; 0.780.99 - 0.030.31 - 0.65 ; 0.591 - 0.150.70 - 1.52 ; 1.220.99 2 - 0.300.70 - 1.09 ; 1.690.980.150.67 - 1.15 ; 1.450.990.770.53 - 0.28 ; 1.820.970.780.53 - 0.27 ; 1.830.97mco mco 1 - 0.070.35 - 0.76 ; 0.620.990.080.54 - 0.98 ; 1.140.98 - 0.220.35 - 0.91 ; 0.470.99 - 0.120.50 - 1.11 ; 0.870.99 2 - 0.330.51 - 1.34 ; 0.680.98 - 0.620.35 - 1.31 ; 0.070.97 - 0.080.35 - 0.78 ; 0.620.99 - 0.051.29 - 0.62 ; 0.520.99total10.11 0.970.02 0.970.14 0.980.03 0.98 2 - 0.41 0.96 - 0.35 0.950.18 0.970.35 0.97interobserverc c 1 x 2 - 0.540.98 - 2.46 ; 1.380.960.000.49 - 0.95 ; 0.960.99 - 0.110.88 - 1.84 ; 1.630.98 - 0.150.56 - 1.25 ; 0.950.99mf mf 1 x 21.000.61 - 0.20 ; 2.190.910.940.54 - 0.12 ; 1.990.923.591.490.66 ; 6.510.263.251.450.38 ; 6.090.45c mf 1 x 2 - 1.201.18 - 3.51 ; 1.150.93 - 1.491.09 - 3.63 ; 0.640.90 - 2.420.80 - 3.99 ; -0.840.84 - 2.560.66 - 3.86 ; -1.260.82c mf 1 x 20.631.05 - 1.45 ; 2.700.970.060.80 - 1.51 ; 1.640.980.071.20 - 2.30 ; 2.430.96 - 0.340.57 - 1.46 ; 0.790.99c mf 1 x 2 - 1.091.34 - 3.72 ; 1.530.89 - 1.070.94 - 2.91 ; 0.790.92 - 2.421.38 - 5.13 ; 0.300.70 - 2.471.06 - 4.56 ; -0.370.69c mf 1 x 20.400.60 - 0.78 ; 1.590.98 - 0.790.81 - 0.79 ; 2.380.950.070.68 - 1.28 ; 1.410.980.100.58 - 1.05 ; 1.240.99mco lco ( right)1 x 2 - 1.770.54 - 2.83 ; -0.710.69 - 1.840.50 - 2.83 ; -0.860.67 - 0.180.84 - 1.83 ; 1.460.92 - 0.490.46 - 1.40 ; 0.410.95mco lco ( left)1 x 2 - 1.430.54 - 2.49 ; -0.360.63 - 1.390.63 - 2.63 ; -0.140.63 - 1.130.84 - 2.79 ; 0.520.72 - 0.730.56 - 1.85 ; 0.380.86lco lco 1 x 2 - 1.420.55 - 2.50 ; -0.340.92 - 1.450.45 - 2.34 ; -0.550.93 - 0.091.16 - 2.35 ; 2.190.96 - 0.150.93 - 1.96 ; 1.670.97mco mco 1 x 21.990.461.08 ; 2.890.762.120.431.27 ; 2.960.761.340.610.14 ; 2.530.861.170.480.21 ; 2.130.90total -0.34 0.86 - 0.49 0.87 - 0.13 0.82 - 0.24 0.86ad : average difference ; sd = standard deviation ; ci : confidence interval ; icc : intraclass correlation coefficient ad : average difference ; sd = standard deviation ; ci : confidence interval ; icc : intraclass correlation coefficient cbct multiplanar reconstruction measurements showed good to high interobserver reliability with icc ranging from 0.63 to 0.99 ( mean average g2=0.86 and g3=0.87 ) with the absolute differences between the first and second measurements ranging for 0 to 2.12 mm ( mean average difference g2:-0.34 mm and g3:-0.49 mm , table 1 ) . for 3d reconstruction measurements , independently of the voxel size ( g4 or g5 ) , most of the variables presented good to high interobserver reliability , with the exception of inter - mental foramen width ( mf mf ) that had fair reliability on 0.2 mm voxel images ( g4=0.26 ) and moderate reliability for 0.4 voxel size ( g5=0.45 ) . the mean values and standard deviations of the physical measurements and of the cbct measurements for all groups are presented in table 2 . two variables ( mf mf and c mf ) at groups g2 and g3 were significantly smaller than physical measurements ( g1 ) . for 3d rendering images ( g4 and g5 ) , only two variables out of 10 were not significantly smaller than the physical measurements . no significant difference was found between the voxel size both for 3d rendering and multiplanar sections . table 2mean ( mm ) and standard deviation ( sd ) of linear measurements for dry mandibles compared to cone - beam computed tomography ( cbct ) images with different voxel sizes [ analysis of variance ( anova ) for repeated measures and dunnett s tests ] physical measurementmultiplanar images3d model images variableg1g2g3g4g5p 0.2 voxel0.4 voxel0.2 voxel0.4 voxel ( n=10)(n=10)(n=10)(n=10)(n=10 ) meansdmeansdmdmeansdmdmeansdmdmeansdmd c c 101.033.98101.154.150.11100.974.01 - 0,06101.124.060,09101.184.260.140.697mf mf 49.17 3.0347.91 2.98 - 1.2647.91 2.81 - 1,2647.16 2.64 - 2,0147.76 3.01 - 1.420.000*c mf 76.43 4.7176.50 4.420.0776.24 4.17 - 0,1875.59 4.60 - 0,8475.51 4.49 - 0.920.000*c mf 104.41 4.63103.82 4.79 - 0.58103.56 4.26 - 0,85102.76 4.38 - 1,64103.10 4.47 - 1.310.000*c mf 75.16 3.9975.66 3.640.5075.69 3.580,5374.48 3.46 - 0,6874.25 3.30 - 0.910.000*c mf 102.65 3.92102.44 4.02 - 0.21102.33 3.76 - 0,32101.29 3.90 - 1,36101.38 3.81 - 1.270.000*mco lco 21.42 2.4321.68 2.280.2621.69 2.280,2619.61 2.08 - 1,8120.07 2.19 - 1.350.000*mco lco 21.37 2.0521.28 1.67 - 0.0921.02 1.68 - 0,3419.31 1.82 - 2,0619.14 1.74 - 2.230.000*lco lco 124.25 4.05124.00 4.03 - 0.25123.78 4.10 - 0,47122.73 4.21 - 1,53122.74 4.04 - 1.510.000*mco mco 83.873.0984.072.860.2084.132.970,2684.072.810,283.762.98 - 0.110.251sd , standard deviation ; md , mean difference * statistically significantdifferent letters represent statistically significant differences ( dunnett s test ) sd , standard deviation ; md , mean difference statistically significant different letters represent statistically significant differences ( dunnett s test ) the availability of this technology is undoubtedly expanding the use and application of 3d imaging in the field of orthodontics . however , while cbct provides many advantages , patient radiation dose is still higher than conventional cephalometric and panoramic digital imaging modalities . this study was performed to evaluate the reliability and accuracy of linear measurements between common landmarks of the mandible taken from cbct multiplanar sections and 3d volumetric renderings with different voxel sizes . in addition , larger voxel sizes are consistent with the alara principle , keeping the dose as low as reasonably achievable for the intended purpose of the scan . most of the previous studies performed to validate cbct for 3d cephalometrics measured linear distances between anatomical landmarks in dry skulls in combination with radiopaque markers . cephalometric analyses are subject to the influence of the examiner , landmark identification and type of image analyzed ( 2d sections or 3d image ) . the major question of this study was if the difficulty in identifying the landmarks in 3d images can influence the reliability and accuracy of the measurements , and for this reason , metallic markers were not used . this method is close to the real clinical procedures and is more clinically relevant than making measurements between well - defined metallic landmarks . there was excellent intraobserver reliability for both examiners with the mean icc for all groups above 0.95 and mean average differences less than 0.41 mm , independently of the protocol analyzed ( table 1 ) . these differences are not clinically significant for orthodontic / surgical diagnosis and treatment planning , and acceptable for skeletal measurements . additionally , good to high interobserver precision was identified for all groups , with exception of measurement mf the main explanations for the good reliability found in this study are the use of landmarks which are easily identifiable , the calibration between observers and the high spatial definition of cbct images . the differences between multiplanar sections and 3d volume rendering images were probably because landmarks on multiplanar images were identified on the orthogonal slices simultaneously instead of being identified directly on the surface of 3d reformatted images . our study also evaluated the accuracy of measurements performed in cbct images ( table 2 ) . two variables at g2 and g3 and 8 variables at g4 and g5 were significantly smaller than physical measurements ( g1 ) . ( 2009 ) , who showed a tendency for the cbct measurements to slightly underestimate the gold standard . the consistency of the differences we found suggests a systematic error in the 3d model measurement method . one possible explanation is that this error might have been introduced by the measurement software , mainly building line . although few software systems currently have a 3d cephalometric module , most of them have not been tested or validated . the results of the validation measurements for a specific machine or software can not be extrapolated to the entire cbct machine and software . most of them are designed differently and software updates are constant and frequent . in addition , the difference ranged from 0.06 to 2.23 mm . in agreement with previous reports , the average difference in our study is below clinical significance . according to grauer , et al . ( 2009 ) , generating measurements in 3d volumetric images rather than simultaneously in sets of 2d multiplanar images introduces error because of the difficulty involved in locating landmarks in 3d space and the inaccuracies of the user entered threshold used for the construction of 3d virtual surface models . rendered for this reason , greater caution is suggested for linear measurements performed on 3d models . the spatial resolution of the image has an influence in the accuracy of the measurements . most studies on cbct with a large field of view used 0.4 mm voxel size , whereas others with a small field of view used a 0.2 mm or smaller voxel sizes . none of the measurements showed significant difference between the voxel sizes , independently of the method of measurement ( table 2 ) . ( 2010 ) , who have suggested that 0.4 mm voxel resolution is adequate for performing measurements of craniofacial structures . while small voxel sizes increase the image resolution , they also may increase the image noise . the benefits of a shorter scanning time , with its reduced likelihood of patient movement and especially the lower exposure dose compensates for the poorer resolution . besides , large measurements are less influenced by small differences in spatial resolution . on the other hand , the diagnostic ability of cbct images for evaluating small structures appears to be influenced by voxel size . the two most common voxel sizes used in orthodontics 0.3 and 0.4 mm provide lower spatial resolution than smaller voxel sizes . orthodontic scans with resolutions of 0.3 and 0.4 voxels are ideal for general treatment planning but should be used with caution if the goal is to assess small variations in bone thickness . ( 2009 ) investigated the usefulness of cbct for identifying artificial external root resorption in images with voxel sizes of 0.4 , 0.3 , and 0.2 mm . they concluded that , even though the results were the same for the different voxel sizes , diagnosis was easier at smaller voxel sizes of 0.3 and 0.2 mm . although the benefits of a shorter scanning time satisfy the as low as reasonably achievable principle , the risks of misdiagnosis and treatment complications must also be weighed . consequently , a scanning protocol with a 0.40 mm voxel size might not be suitable for every patient . voxel size may vary according to the size of the structures to be analyzed as well as to the level of detail desired . in this study , we measured distances that are in excess of the resolutions 0.2 and 0.4 mm of the scan , so that voxel size would not very likely influence the measurements . unlike in conventional cephalometrics , where all the landmarks are identified in one image , namely , the lateral cephalogram , with cbct each landmark must be identified in three different images ( axial , coronal and sagittal ) , making the process of performing the measurements in cbct images more time - consuming for the orthodontist . further , cbct demands a higher radiation dose than traditional cephalometric images . for these reasons , its use should be limited to specific indications , for example , patients with impacted teeth , or those with facial asymmetries or craniofacial anomalies where cbct is better able to quantify the differences between the right and the left side of craniofacial structures . evidence - based will tell whether future advances , especially in terms of dose reduction , will make cbct appropriate for routine use in all orthodontic patients . linear measurements obtained on multiplanar 2d cbct images with 0.2 and 0.4 voxel sizes using i - cat scanner and dolphin software are reliable and accurate for clinical diagnosis and treatment planning . caution should be taken in linear measurements on 3d rendering images , because the measurements were reliable , but not accurate . reducing the voxel size from 0.4 to 0.2 mm does not influence the accuracy and reliability of measurements of large craniofacial structures on cbct-3d images .

objective the purpose of this study was to determine the accuracy and reliability of two methods of measurements of linear distances ( multiplanar 2d and tridimensional reconstruction 3d ) obtained from cone - beam computed tomography ( cbct ) with different voxel sizes.material and methods ten dry human mandibles were scanned at voxel sizes of 0.2 and 0.4 mm . craniometric anatomical landmarks were identified twice by two independent operators on the multiplanar reconstructed and on volume rendering images that were generated by the software dolphin. subsequently , physical measurements were performed using a digital caliper . analysis of variance ( anova ) , intraclass correlation coefficient ( icc ) and bland - altman were used for evaluating accuracy and reliability ( p<0.05).results excellent intraobserver reliability and good to high precision interobserver reliability values were found for linear measurements from cbct 3d and multiplanar images . measurements performed on multiplanar reconstructed images were more accurate than measurements in volume rendering compared with the gold standard . no statistically significant difference was found between voxel protocols , independently of the measurement method.conclusions linear measurements on multiplanar images of 0.2 and 0.4 voxel are reliable and accurate when compared with direct caliper measurements . caution should be taken in the volume rendering measurements , because the measurements were reliable , but not accurate for all variables . an increased voxel resolution did not result in greater accuracy of mandible measurements and would potentially provide increased patient radiation exposure .

non - alcoholic fatty liver disease ( nafld ) is a relevant issue in public health . it represents the most common chronic liver disease in the general population and is expected to increase in the future , as a result of an ageing population , obesity and diabetes . nafld is the hepatic manifestation of metabolic syndrome and includes a spectrum of disease ranging from simple hepatic steatosis to non - alcoholic steatohepatitis ( nash ) , which can progress to cirrhosis . nafld occurs in 6095% of patients with obesity and in 2855% of patients with type 2 diabetes mellitus ( t2 dm ) . insulin resistance ( ir ) , with compensatory hyperinsulinemia , plays a pathogenetic key role in nafld progression . given its low cost , repeatability , safety and availability , ultrasound ( us ) is routinely the first - line imaging technique to detect liver steatosis . central obesity , defined as a presence of excess fat in the abdominal area , is frequently associated with nafld and their coexistence in the same subjects increases the likelihood of having more advanced forms of liver disease . liver steatosis progression is associated not only with the body mass index ( bmi ) , but also with visceral adiposity . however , fat mass and fat distribution can be different in subjects with the same bmi . for these reasons , the body composition ( bc ) evaluation is necessary for assess the nutritional status and the efficacy of nutritional strategies . a valid and precise tool to measuring bc , is the dual - energy x - ray absorptiometry ( dxa ) , an important method for studying not only osteoporosis but also the soft - tissue composition changes . the accuracy of dxa in measuring the fat mass is 98.8% , and combined with other standard anthropometric parameters , it is a reliable tool for the estimation of central fat . the adipose tissue , has been considered an energy storage organ , but over the last decade several findings have encouraged researches on its the endocrine role . furthermore , data highlighted that central adipose tissue is a metabolic and inflammatory organ that modulates the action and the metabolism of brain , liver , muscle and cardiovascular system . recently , the important role of adipokines , peptides synthesized from adipocytes , in the pathogenesis of ir and nafld has been discussed . in particular , adiponectin is considered an anti - inflammatory adipokine , able to reduce body fat , to improve hepatic and peripheral insulin sensitivity , and inversely associated with bmi , ir and hepatic fat accumulation . the aim of this observational study was to investigate , in overweight patients , the possible relationship between central fat mass , liver steatosis and the circulating levels of adiponectin . the study was carried out in overweight patients , referred to the division of clinical nutrition and nutrigenomics , university of rome tor vergata of rome ( italy ) , for a nutritional evaluation , in the period january the study was performed in accordance with good clinical practice and complying with the principles laid down of declaration of helsinki . the study was approved by the appropriate ethical committee of the centre , and all patients gave their written informed consent before the recruitment . other exclusion criteria were : insulin therapy , smoking habits , alcohol intake ( > 20 g / day ) , hepatic virus infection , auto - immune diseases , and use of drugs known to induce liver steatosis or nash . after the initial evaluation of 52 patients , 21 ( 40.4% ) were excluded : 5 for heart diseases , 4 for insulin therapy , 2 for hepatitis b virus infection , 6 for excessive daily alcohol intake and 4 for treatment with drugs - inducing steatosis or nash . body weight , height , bmi , and waist circumference were measured according to standard methods . all subjects underwent a biochemical examination of fasting glucose , fasting insulin and alanine aminotransferase ( alt ) . fasting glucose and insulin level were used also to calculate ir , according to the homeostasis homeostasis model assessment ( homa - ir ) . the enzyme - linked immunosorbent assay technique was used to measure fasting serum levels of adiponectin in all patients ( r&d systems inc . the patients underwent us liver by a toshiba eccocee , with a convex transducer of 3.7 mhz . the results were interpreted by an investigator ( la ) with experience in the field . the liver images were considered normal if the texture was homogenous , exhibited fine level echoes and isoechoic compared to the renal cortex and there was adequate visualization of the hepatic vessels and diaphragm ( grade 0 ) . criteria for determining the stage of liver steatosis , in according to the hamaguchi score , included : presence of bright echoes or increased hepato - renal contrast indicative of mild steatosis ( grade 1 ) ; presence of both bright echoes and increased hepato - renal contrast as well as vessel blurring indicative of moderate steatosis ( grade 2 ) ; severe steatosis was considered when in addition to the criteria for moderate steatosis , there was evidence of posterior beam attenuation and non - visualization of the diaphragm ( grade 3 ) . bc was assessed by dxa ( lunar dpx - iq ; ge medical systems , milwaukee , wi ) , according to the standardized described procedure . standard dxa quality control and calibration measures , were performed prior to each testing session . the test takes about twenty minutes , and the subjects remain in a supine position during the scanning . the results , were transmitted to a connected computer for further analysis , according to the manufacturer orientation . in order to show the relation between dxa parameters and us stage of nafld , we displayed graphically the evolution of these features among classes , and we assessed the statistical significance of clinical and laboratory features . statistical differences of laboratory and clinical variables between the four groups were evaluated by the anova test . body weight , height , bmi , and waist circumference were measured according to standard methods . all subjects underwent a biochemical examination of fasting glucose , fasting insulin and alanine aminotransferase ( alt ) . fasting glucose and insulin level were used also to calculate ir , according to the homeostasis homeostasis model assessment ( homa - ir ) . the enzyme - linked immunosorbent assay technique was used to measure fasting serum levels of adiponectin in all patients ( r&d systems inc . the patients underwent us liver by a toshiba eccocee , with a convex transducer of 3.7 mhz . the results were interpreted by an investigator ( la ) with experience in the field . the liver images were considered normal if the texture was homogenous , exhibited fine level echoes and isoechoic compared to the renal cortex and there was adequate visualization of the hepatic vessels and diaphragm ( grade 0 ) . criteria for determining the stage of liver steatosis , in according to the hamaguchi score , included : presence of bright echoes or increased hepato - renal contrast indicative of mild steatosis ( grade 1 ) ; presence of both bright echoes and increased hepato - renal contrast as well as vessel blurring indicative of moderate steatosis ( grade 2 ) ; severe steatosis was considered when in addition to the criteria for moderate steatosis , there was evidence of posterior beam attenuation and non - visualization of the diaphragm ( grade 3 ) . bc was assessed by dxa ( lunar dpx - iq ; ge medical systems , milwaukee , wi ) , according to the standardized described procedure . standard dxa quality control and calibration measures , were performed prior to each testing session . the test takes about twenty minutes , and the subjects remain in a supine position during the scanning . the results , were transmitted to a connected computer for further analysis , according to the manufacturer orientation . in order to show the relation between dxa parameters and us stage of nafld , we displayed graphically the evolution of these features among classes , and we assessed the statistical significance of clinical and laboratory features . statistical differences of laboratory and clinical variables between the four groups were evaluated by the anova test . clinical and laboratory characteristics of the patients , stratified on the basis of the us stage of nafld , are reported in table i. of the 31 patients included in the study , 13 were male ( 41.9% ) . the median value of anthropometric parameters assessed were : bmi 31.45.9 and waist circumference 97.216.2 cm , with 16 patients ( 7 males ) that presented central obesity , according to the definition of the national cholesterol education program adult treatment panel iii ( ncep atp iii ) . us evaluation of the liver showed that : in 8 patients ( 25.8% ) steatosis was absent , in 9 ( 29% ) mild ( grade 1 ) , in 6 ( 19.4% ) moderate ( grade 2 ) , and in 8 ( 25.8% ) severe ( grade 3 ) . the median value of alt was 33.627.3 ul-1 , with four patients that reported high alt value , one without , two with mild and one with severe us liver steatosis . of all dxa parameters analyzed , those that present a statistical significant difference between the us stages of nafld were ( figure 1 ) : abdominal fat ( af , % ) , mean value 42.611.9 ( p<0.05 ) ; abdominal tissue mass ( atm : fat plus lean mass , kg ) , ( mean value 72.5 , p<0.05 ) ; abdominal fat mass ( afm , kg ) , ( mean value 3.91 , p<0.05 ) . considering the only distribution of l2l5 fat tissue ( kg ) , ( mean value 5.21.2 ) , we observed that the value was higher , but reached no statistical significance , in patients with moderate and severe steatosis , compared to those with the mild type . the assessment of ir by homa - ir , showed a statistically significant increase of insulin level , related to steatosis progression ( p<0.05 ) . finally , the concentration of adiponectin ( 7.782.2 g / ml ) , was significantly lower in the advanced us stages of nafld ( p<0.05 ) . obesity , which is often associated with ir , represents a chronic low - grade inflammatory state , characterized by elevated circulating levels of cytokines and activation of pro - inflammatory signaling pathways . in particular , ir plays a key role in the pathogenesis of nafld , causing alterations in the uptake , degradation or secretion of lipid molecules , with consequent accumulation of lipid in the hepatocytes . nafld is usually prevalent in obese subjects , and several studies have reported that regional fat distribution associated with ir is an important factor for development and progression of liver steatosis . in our study , abdominal fat accumulation and anthropometric parameters , increased with us severity of liver steatosis and ir . in this context the first observation is that by using dxa , we quantified the regional distribution of adipose tissue and we found the association between increased central fat mass , and liver steatosis severity . this observation is in agreement with the progression of the values of bmi and circumference observed in our series . concerning the relationship between bc and nafld , this study has shown that central fat accumulation constitutes an important determinant of liver steatosis in overweight patients , independently to bmi . recent data document that the abdominal adipose tissue is an active endocrine organ capable to secrete a multitude of hormones , cytokines , chemokines , and enzymes , collectively known as adipokines . in particular adiponectin , leptin , resistin , visfatin and pro - inflammatory cytokines , such as tumor necrosis factor- , and interleukins , have been shown to be involved in pathogenesis and progression of nafld . the increased number of abdominal adipocytes produces a disequilibrium in adipokines secretion , which promotes liver fat accumulation , and subsequently the development of nafld . in particular , adiponectin improves muscular and hepatic insulin sensitivity , through its anti - inflammatory and anti - atherogenic activity , and its ability to decrease triglyceride synthesis and stimulate -oxidation . in fact adiponectin protects hepatocytes from triglycerides accumulation by increasing -oxidation of free fatty acid and/or decreasing de novo free fatty acid production in hepatocytes . adiponectin levels are reduced in obese patients , and t2 dm , and the plasma concentrations are inversely related to body weight , especially to visceral adiposity . moreover , adiponectin is inversely associated with other traditional cardiovascular risk factors , such as blood pressure , low - density lipoprotein cholesterol and triglyceride levels , and is positively related to high - density lipoprotein cholesterol levels . in our cohort , us liver steatosis progression is characterized by ir and low adiponectin serum levels , pathogenetic factors that can increase the concentrations of intra - cellular fatty acids , and may enhance oxidative stress that is the second stage in the pathogenesis of nash . bc assessed by anthropometry and dxa , may be used as indicator of nafld severity in overweight patients . however , further studies are required to better understand not only this correlation , but also to define the pathogenetic role of central fat distribution and the changes in adipokine levels in the progression of nafld .

aimnon - alcoholic fatty liver disease ( nafld ) is the most common chronic liver disease in the general population . overweight is a common condition in patients with nafld , and body composition ( bc ) assessment is useful to evaluate nutritional status and the efficacy of nutritional strategies . a valid tool for assessing bc is dual - energy x - ray absorptiometry ( dxa ) . adiponectin has been shown to be relevant to the pathogenesis of nafld . the aim of this observational study is to define the relationship between the severity of nafld , the central fat mass evaluated by dxa , and the circulating levels of adiponectin.methodsthe study was carried out in 31 overweight patients . the degree of liver steatosis was evaluated by ultrasound ( us ) examination . anthropometric parameters were measured according to standard methods . fasting glucose and insulin level were used also to calculate insulin resistance ( ir ) , according to the homeostasis model assessment - insulin resistance ( homa - ir ) . the enzyme - linked immunosorbent assay technique was performed to dose fasting serum levels of adiponectin.resultsnafld progression was signicantly associated with increased central fat ( p<0.05 ) . using dxa , we quantified the regional distribution of adipose tissue and found the expected association between central fat and the us severity of nafld . serum levels of adiponectin , were inversely related to nafld progression ( p<0.05).conclusionbc evaluated by anthropometry and dxa , may be used as indicator of nafld severity in overweight patients . the evaluation of bc in clinical practice , can improve the nutritional strategies and follow - up . in the clinical setting adiponectin may represent a potential marker for the staging of nafld .

development of human societies and industrialization as well as changes in stress sources has changed disease pattern in civilized societies . as a result , the disease pattern has changed from traditional diseases such as infectious diseases and malnutrition to diseases such as heart disease , diabetes , accidents and so forth . among them coronary artery disease is the most cardiovascular disorder as a health problem in developing and developed countries . in fact , this is not only a chronic disease associated with high mortality , but it causes limitations in life and disability in a large part of the productive forces of the country . it is also associated with reduced production and increased medical costs [ 3 - 5 ] . nowadays , cardiovascular diseases are among the most widespread chronic diseases in most countries . according to forecasts , the mortality rate in eastern mediterranean countries including iran will be 30 - 35% [ 6 , 7 ] . despite the emphasis on prevention and development of newtreatments , surgery is the only choice for many patients with cardiovascular disease . one of the surgical procedures for the treatment of cardiovascular patients is coronary artery bypass surgery . in fact , heart surgery significantly influences on the quality of life of patients with cardiovascular diseases . hence , heart surgery is an important event in the lives of patients causing collapse of economic , professional and personal life . on the other hand , each surgical procedure is associated with several psychological complications for patients . of the most important complications are anxiety and depression so that approximately 65% of cardiovascular patients experience them after surgical and medical interventions . in fact , anxiety and depression complicate the treatment process [ 7 , 8 ] . some scientists believe that anxiety is one of the essential elements of human life . however , acute and long - term anxiety is integral part of all psychological diseases . according to nemati and colleagues , in fact , anxiety is a common psychological response of cardiovascular patients which is associated with reduced quality of life and psychological morbidity [ 11 , 12 ] . on the other hand , anxiety causes increased heart rate and breathing as well as high blood pressure and even mortality . it is associated with increased risk of mortality , disability , increased medical care and functional impairment in daily activities [ 12 , 13 ] . clinical depression has been reported in 54% of patients after bypass surgery . according to world health organization ( who ) , depression is the fourth chronic disease where its disability is comparable with 8 major chronic disorders . the outcome of depression in patients with cardiovascular disease is deterioration of physical and emotional state . given the incidence of such complications in patients with cardiovascular disease the best practice in this area is appropriate communication with patient [ 13 , 15 ] . good and effective communication is very valuable from the perspective of patients . in general , communication is a set of learning skills . in fact , poor communication is the cause of most problems in treatment of patients . in fact , communication at the bedside is a therapeutic and professional communication . in health professionals , communication and communication skills play a very important role in satisfaction of patients and solving their problems . this is more important , especially in patients with chronic diseases or those requiring long - term care . the therapeutic communication is formed from the first encounter of the patient with the medical team [ 21 , 22 ] . however , studies have shown that the communication between patient and medical team is not efficient . the health personnel don not have adequate communication skills . in fact , nursing and medical team spend very little time to communicate with patients . accordingly , patients are not often satisfied with received information as well as the level and method of communication . hence , to achieve best therapeutic results , special attention should be paid to communication [ 16 , 19 , 22 ] . since nursing is a practical discipline based on professional knowledge , it is necessary to use knowledge infrastructure to develop new approaches in clinical practice . theories proposed by nursing scientists can be useful in this area [ 16 , 19 , 23 ] . for this purpose the nurse - patient communication is an essential element of this theory [ 21 , 24 ] . according to peplau theory , the purpose of nursing care is to achieve a common good nurse - patient communication . according to peplau , peplau refers to the importance of therapeutic communication with patients and its important role in reducing anxiety [ 21 , 23 , 24 ] . in fact , this theory provides a framework for nurse - patient communication . following this framework , the nurse will be able to respond communication needs of patients through establishing a good therapeutic communication with the patient [ 16 , 19 , 23 ] . hospitalization and a complex surgery such as heart surgery is a stressful process leading to depression in patients . furthermore , the need for security and psychological comfort is one of the basic needs of patients achieved through a proper structured therapeutic communication with low cost . accordingly , the objective of the present study is to examine the impact of peplau therapeutic communication model on anxiety and depression in patients who were candidate for coronary bypass surgery . this is a clinical trial with pre / post testing scheme examined the impact of independent variable ( peplau therapeutic communication ) on the dependent variables ( anxiety and depression ) . the population consisted of all patients who were candidates for coronary artery bypass referred to the research environment on non - emergency basis . the research environment was al - zahra heart hospital in shiraz due to the easy access to subjects and sufficient number of patients . data were collected using the hospital anxiety and depression scale ( kanter et al . ) . the reliability and validity of the scale have been studied by montazeri et al in 2003 . an internal consistency of 78% and 86% was respectively calculated for anxiety and depression using cronbach 's alpha . there are 14 four - option questions , each with a score of zero to 3 . odd and even questions are used to measure anxiety and depression , respectively ( seven questions to assess anxiety and seven questions to assess depression ) . a score of zero to 21 is given to each patient based on responses in each area ( a score of zero - seven : normal , eight-10 : moderate and > 10 : disease ) . due to the lack of access to error rate to calculate the sample size , 10 patients were selected as pilot ( with a depression and anxiety score of 10 or more based on the hospital anxiety and depression scale ) . after performing tests , the subjects were randomly divided into test and control groups ( using a coin ( toss ) ) . z12+z12s12+s22x1x22 the inclusion criteria were : being in a bypass list , having moderate to severe depression and anxiety scores , no history of mental illness , interesting in participating in the study ( i.e. the tendency of the patient and his family to participate in the intervention ) , lack of previous bypass surgery , aged between 35 and 70 years , ability to communicate verbally and ability to speak persian . the exclusion criteria included : lack of cooperation of patients and families during the intervention , failure to perform coronary artery bypass surgery for various reasons , mortality during the study , failure to attend therapeutic communication sessions ( at least the absence in two sessions ) . therapeutic communication sessions were held for the intervention group based on peplau 's model at four stages including : orientation , identification , exploitation and resolution . in total , seven sessions were held individually with the consent of the patient and his family at the hospital and patient 's home . it should be notedthat during therapeutic communication , duration of each session was variable given the location and patient 's needs . the place of each meeting was determined with the consent of the patient . at all meetings , the researcher used verbal and nonverbal communication skills to communicate with patients . to ensure the correct application of therapeutic communication skills , a self - control researcher - made tool approved by experts was used . the depression and anxiety of patients in both groups were assessed using the hospital anxiety and depression scale ( hads ) at baseline and then two and four months after surgery . the obtained data were analyzed using descriptive statistics as well as covariance analysis with the help of spss 16 . this study was approved by the ethics committee of the jondi shapoor medical university of ahvaz with rec.1392.58 code . the ethical issues considered in this study include an adequate explanation for the patients and their permission to participate in the study , the lack of any compulsion for patients to participate in the study , refusing to disclose secrets or private matters of patients and confidentiality of information , announcing the results without names and personal details . furthermore , the present study was registered with the irct2013072214110n1 code in clinical trial center ( irct.ir ) . this study was approved by the ethics committee of the jondi shapoor medical university of ahvaz with rec.1392.58 code . the ethical issues considered in this study include an adequate explanation for the patients and their permission to participate in the study , the lack of any compulsion for patients to participate in the study , refusing to disclose secrets or private matters of patients and confidentiality of information , announcing the results without names and personal details . furthermore , the present study was registered with the irct2013072214110n1 code in clinical trial center ( irct.ir ) . the percentage of male and female patients in the intervention group was 70.3% and 29.7% , respectively . the percentage of males and females in the control group was 51.4% and 48.6% , respectively . the status of the underlying disease was depicted in both intervention and control groups . according to results , the majority of patients were suffering from high blood pressure in both groups with a high frequency of 35.1% ( table 1 ) . as shown in table 2 , anxiety was reduced in the intervention group , while postoperative anxiety in the control group was increased . the hospital anxiety in the intervention group this indicates the role of peplau 's therapeutic communication in reducing hospital anxiety of patients . analysis of covariance was used to determine the effect of peplau 's therapeutic communication on anxiety level of the subjects . since hospital anxiety scale was used to measure anxiety , analysis of variance was performed using hospital anxiety scores . the results showed significant differences between experimental and control groups in terms of anxiety level immediately after surgery , two and four months after surgery after adjustment for post - test scores by eliminating the effect of pre - test . adjusted mean anxiety scores suggest that anxiety in the intervention group was lower than the control group . in fact , the mean anxiety scores of patients in the intervention group in pre - test and post - test stages represent the independent role of peplau 's therapeutic communication model in reducing anxiety in the intervention group ( f=174.02 , p=0.000 ) . moreover , the anxiety level in the intervention group two and four months after the intervention was decreased compared to the control group ( f=38.37 , p=0.000 ; f=11.58 , p=0.000 ) ( table 3 ) . as shown in table 4 , the mean depression in the intervention group was reduced after the surgery . the mean postoperative depression in the control group was increased . in fact , the hospital depression in the intervention group was reduced after the therapeutic communication . this indicates that the role of peplau 's therapeutic communication model in reducing hospital depression . analysis of covariance was used to determine the effect of peplau 's therapeutic communication on depression level of the subjects . since hospital depression scale was used to measure depression , analysis of variance was performed using hospital depression scores . the results showed significant differences between experimental and control groups in terms of depression level immediately after surgery , two and four months after surgery after adjustment for post - test scores by eliminating the effect of pre - test . adjusted mean depression scores suggest that anxiety in the intervention group was lower than the control group . in fact , the mean depression scores of patients in the intervention group in pre - test and post - test indicate the independent role of peplau 's therapeutic communication model in reducing depression in the intervention group ( f=163.27 , p=0.000 ) . such a difference is not observed in the control group . moreover , the depression level in the intervention group four months after the intervention was decreased compared to the control group ( f=20.58 , p=0.000 ) . however , no significant difference was found between two groups in terms of hospital depression two months after intervention ( p=0.61 ) ( table 5 ) . according to the results , peplau 's therapeutic communication is effective in reducing anxiety and depression in patients who were candidate for coronary artery bypass . it resulted in a dramatic decrease in anxiety and depression scores in the intervention group . depression and anxiety are the most important preventive factors in cardiovascular diseases causing disease rejection by the patient and reduce the incentive to treat and increase the likelihood of disease recurrence [ 25 - 27 ] . most patients with coronary heart disease who survive after surgery resume their normal life after several weeks or several months . previous studies show that patients suffer from anxiety and depression several months and sometimes several years after the surgery [ 26 , 27 , 29 ] . therefore , follow - up and strategies to deal with postoperative psychological complications are of utmost importance . it is not possible except through establishing a coherent and targeted therapeutic communication . indeed , effective communication enables the medical team to identify patients needs and take steps to solve problems and meet their needs . according to dadashi , hosseini and moghaddam ( 2009 ) , staff had close encounters with patients in 85.5% of cases . according to mastaneh and mouseli ( 2013 ) , the rate of respecting patient rights in terms of communication was 60% indicating a moderate level of communication skills . on the other hand , duration and how to communicate with patients , particularly in dealing with patients with chronic diseases are essential in the hope and positive attitude to treatment and follow - up after discharge [ 29 - 31 ] . in fact , the patient gains motivation to cope with illness and trust in nurses through proper therapeutic communication , because the care of patients with either physical or mental illness requires correct and consistent communication [ 30 , 31 ] . therefore , implementing a communication pattern will help medical team to establish a proper communication . peplau 's model is one of these communication patterns . to communicate with patients , especially patients with chronic diseases , peplau 's described four stages and defined roles for the nurse at every step as someone who is in close relationship with the patient [ 16 , 23 ] . peplau 's communication model helps nurses and patient to identify the disease , concerns and questions of the patient . through defining different roles for the nurse this model refers to nursing skills and abilities to establish a simple convenient therapeutic communication . the model enables nurses help the patient after discharge and to spend more time with patient [ 16 , 23 , 24 ] . for example , it has been used in communicating with cancer patients and solving systematic problems of families [ 32 , 33 ] . in this study , peplau 's communication models were employed as communication sessions for patients who were candidates for coronary artery bypass through applying nursing roles . the results showed that holding communication meetings with patients immediately after surgery dramatically reduced hospital anxiety and depression . accordingly , to improve the nurse - patient communication and establish more effective therapeutic communication processes which improves clinical basis and can have a positive impact on the treatment and discharge processes and rehabilitation of patients , especially in patients with chronic diseases , training courses should be included in in - service courses to train and introduce medical team with simple and inexpensive communication skills . furthermore , there is evidence that workshops on communication skills for health team can improve health outcomes and satisfaction of patients . another limitation of this study is the lower percentage of females ( 29.7% ) in the intervention group with respect to the control group ( 51.4% ) . the lower anxiety score in the intervention group could be due to therapeutic communication but also to gender differences . to write this article not contributed any financial resources and costs are the responsibility of the authors . all rights of this article reserved is owned by the ahvaz jundishapur university of medical sciences .

background and objective : anxiety and depression are among the psychological disorders in heart surgeries . establishing a simple communication is essential to reduce anxiety and depression . hence , the objective of the present studywas to examine the impact of peplau therapeutic communication model on anxiety and depression in patients , who were candidate for coronary artery bypass in al - zahra heart hospital , shiraz during 2012 - 2013 . methods : this is a clinical trial in which 74 patients were randomly divided into intervention and control groups , each consisted of 37 patients . anxiety and depression levels were assessed before , and two and four months after intervention using the hospital anxiety and depression scale ( hads ) . seven therapeutic communication sessions were held in four stages . data were analyzed with the spss ( version 16 ) using analysis of covariance . results : the mean anxiety and depression levels decreased in the intervention group after the therapeutic communication ( p<0.01 ) . anxiety scores in the intervention group before and after intervention were 10.23 and 9.38 , respectively . while the corresponding scores in the control group were 10.26 and 11.62 , respectively . depression scores in the intervention group before and after intervention were 11 and 9.13 , respectively . the corresponding scores in the control group were 11.30 and 12.08 , respectively . conclusion : the results demonstrated the positive role of therapeutic communication in reducing anxiety and depression of the patients . therefore , the therapeutic communication is recommended as a simple , cost effective and efficient method in this area .

the patient was a 33-year - old , 156 cm , and 62.5 kg primipara on the sixth day of the 40th week of pregnancy , and she was hospitalized on the day before the operation for vaginal delivery . the patient had not undergone any specific disease or operation according to her history , and the vital signs were normal at the time of hospitalization . the results of the blood test and urinalysis performed one month before the hospitalization were all normal . there was no cardiomegaly and abnormal finding in the pulmonary parenchyma by the chest radiography , and the electrocardiography results were all normal . the fetal heart rate measured at the time of hospitalization was 154 bpm , and the fetus showed normal reactivity . although induced labor was carried out by injecting oxytocin during the afternoon on the day of hospitalization , the fetal heart rate was reduced , and thus , the oxytocin injection was stopped . then , the fetal heart rate returned to normality and natural labor began . when oxytocin was injected again on the day of the operation , the fetal heart rate was reduced . since meconium staining was found following the amniotic membrane rupture , an emergent cesarean section was decided . the vital signs that were measured by the electrocardiography , pulse oximetry , and the automated blood pressure device at the operation room were 150 mmhg of systolic blood pressure , 100 mmhg of diastolic blood pressure , 90 of heart rate , and 94% of oxygen saturation . because there was no specific finding by the inquiry and auscultation , the low oxygen saturation was considered as an error by the low body temperature since the hands and feet of the parturient woman were cold . spinal anesthesia was decided because a little dry cough was found . after having the parturient woman take the right lateral decubitus position , 0.5% hyperbaric bupivacaine 10 mg mixed with fentanyl 20 ug was injected with a 26 g spinal needle between the l3 and l4 spines . the cold sense check was performed with an alcohol swab to verify the sensory block level to the fourth thoracic spinal segment . five minutes after the spinal anesthesia , blood pressure was decreased to 90/50 mmhg , and the pulse oxygen saturation to 88% . the parturient woman complained of severe dyspnea and a continued cough and a large amount of sputum mixed with pink bubbles were observed . after ephedrine was immediately injected 3 times , 8 mg each , the blood pressure was recovered to 110/80 mmhg , the heart rate to 90/min , and the oxygen saturation to 92% , and then the operation was initiated . the results of the arterial blood gas study at that moment were ph 7.39 , oxygen partial pressure ( pao2 ) 77.7 mmhg , carbon dioxide partial pressure ( paco2 ) 34.5 mmhg , and oxygen saturation 95% . just after the start of the operation , the spontaneous breathing seemed no longer impossible due to the severe dyspnea and excessive airway secretion . thus , after injecting propofol 60 mg and succunycholine 70 mg , endotracheal intubation was carried out with the endotracheal tube of the inner diameter 7.0 mm . after the endotracheal intubation , a large amount of pink discharge was continuously aspirated through the tube to the extent that it blocked the tube , and the oxygen saturation of the patient was decreased down to 70% . after the fetus was delivered , the blood pressure was decreased to 60/40 mmhg , the oxygen saturation to 65% , and the heart rate to 60/min . although dobutamine and norepinephrine were injected since heart failure was suspected , pulseless electrical activity ( pea ) was found and the operation was suspended temporarily to carry out cardiopulmonary resuscitation . after the injection of epinephrine 1 mg and atropine 0.5 mg with continued thoracic compression , the sinus rhythm was recovered and the vital signs were increased to the blood pressure of 150/80 mmhg , oxygen saturation of 95% , and heart rate of 120/min . with the continuous intravenous injection of norepinephrine , as well as dopamine and dobutamine , a central vein catheter was inserted through the right internal jugular vein and an arterial catheter through the left femoral artery . although the operation was then resumed , a severe hemorrhage was found and the blood pressure was reduced to 60/40 mmhg and the oxygen saturation to 80% at the end of the operation . the results of the arterial blood gas study at that moment were ph 7.154 , pao2 63.5 mmhg , paco2 57.5 mmhg , bicarbonate 19.8 mmol / l , and oxygen saturation 85.1% , which indicated a serious respiratory and metabolic acidosis and hypoxia . bicarbonate was injected to correct the acidosis and the 50% dextrose in water ( d / w ) solution to which cacl2 600 mg and insulin were added was also injected . since the possibility of hemorrhage by coagulation disorder , not the surgical hemorrhage , could not be excluded , the packed red blood cells and the fresh frozen plasma were transfused , too . although the blood test related with disseminated intravascular coagulation ( dic ) was carried out during the operation ( bleeding time , prothrombin time , activated prothromin time , antithrombin iii , fibrinogen , d - dimer , and fibrinogen degradation product ) , the results could not be obtained because of coagulation of the blood sample . even after the finish of the operation , the low blood pressure was maintained and the patient returned to the pea state . cardiopulmonary resuscitation was performed and epinephrine 1 mg and atropine 0.5 mg were injected 2 times for each . after the patient was recovered to the sinus rhythm , she was moved to the intensive care unit . the total operation duration was 1 hour ; the urination was 150 ml ; the blood loss was 1,500 ml ; the injected fluid was 1,850 ml ; and the transfused blood was 1,250 ml . more than 3,000 ml of secretion was discharged through the endotracheal tube and it spouted out continuously even in the intensive care unit . however , the blood pressure was dropped to 53/30 - 40/20 mmhg , the heart rate to 20 - 30/min , and the oxygen saturation to 50 - 60% . the complete heart failure was continued as the patient 's status did not become any better , and the patient showed no response to the medications . though the cardiac compression was continually carried out , the patient died after 3 hours . the autopsy was performed by the national institute of scientific investigation and the cause of death was proved to be amniotic fluid embolism . amniotic fluid embolism is a fatal syndrome that takes place during pregnancy or during or after delivery . amniotic fluid embolism was firstly described in 1926 , and it was recognized as a syndrome by steiner and lushburgh as fetal debris were found within the pulmonary blood vessels of the parturient women who died during labor by similar clinical characteristics . although it was reported that the incident rate was 1 out of 8,000 - 15,200 live births and the mortality was in the range of 61 - 86% , the mortality was decreased to 13.3 - 44.0% according to a recent study . it is thought that this trend may be because the diagnosis and treatment have been advanced and only fatal cases were reported selectively . amniotic fluid embolism holds 5 - 15% of the overall causes of maternity deaths , and it leaves permanent neurological damage to 61% of the parturient women and 50% of the newborn children . many have been known as the risk factors of amniotic fluid embolism , but parturient women over the age of 35 , cesarean section , forceps and fetal suction , placenta previa , abruptio placentae , eclampsia , and fetal distress syndrome were verified as the risk factors by a large scale research that was carried out recently although the correlation among them has not been understood clearly . the major clinical characteristics include cardiovascular collapse accompanied by severe hypotension and arrhythmia , cyanosis , respiratory distress , pulmonary edema or acute respiratory failure syndrome , respiratory arrest , consciousness fluctuation , and massive hemorrhage by dic . among these , the major causes of maternal death are cardiac arrest , massive hemorrhage by dic , acute respiratory failure syndrome , and multiple organs dysfunction . according to the report by lewis , 11 of the 17 parturient women who had experienced amniotic fluid embolism complained of prodromal symptoms such as dyspnea , chest pain , chilliness , restlessness , the feeling of being pierced by a pin , nausea and vomiting . the time interval between the observations of these symptoms to the women 's collapse varied , ranging from several minutes to 4 hours . in this case report , even though the parturient woman showed the symptom of dry coughing from the morning of the operation day onwards , it was not very severe and no other specific symptom was observed . the causes of the prophase hypoxia , which is the most representative clinical characteristic of amniotic fluid embolism , are severe ventilation - perfusion mismatch by pulmonary vasoconstriction and bronchospasm , and those of the anaphase hypoxia include pulmonary edema by left ventricular failure and nonpsychogenic pulmonary edema that is related to the increase of the capillary permeability . the parturient woman in this case report showed an oxygen saturation of 94% at the time of entering the operation room , which might have been the indication of hypoxia that occurred already , rather than the error by hypothermia . in addition , from the chest radiograph taken in the operation room after the fetus was delivered ( fig . 1 ) , severe pulmonary edema at both lungs and normal central venous pressure were found , though the pulmonary arterial pressure was not measured . thus , it is assumed that the hypoxia might have been caused by pulmonary edema due to the left ventricular failure or the increase of the pulmonary capillary permeability , rather than by the pulmonary vasoconstriction at the initial stage . right ventricular failure can take place at the initial stage by the pulmonary vasoconstriction and pulmonary hypertension due to the secretion of endogenous mediators , which was proved in a number of reports . reported that acute right ventricular failure accompanied by severe pulmonary hypertension , left deviation of the atrial , and ventricular septum was found from the transesophageal echocardiography performed within 30 minutes after the occurrence of amniotic fluid embolism . a large amount of ascites was also found after the laparotomy in our case , which is thought to be the secondary result of the right ventricular failure . as the operation moved on to the anaphase , the pulmonary hypertension did not continue , but it was shifted to left ventricular failure , which might have been caused mainly by the decreased filling of the left ventricle following the enlargement of the right ventricular enlargement . other causes of myocardial failure that are known include myocardial ischemia due to hypoxia , decreased blood flow in coronary artery due to decreased cardiac output , and direct myocardial depression by the substances in amniotic fluid such as endothelin . the blood pressure of the parturient woman in our report was decreased after the spinal anesthesia , which could have resulted from the high level of sensory block to the t4 spine , but it could be probably because of the decrease of the general vascular resistance following the left ventricular failure . dic is also one of the factors that can cause hypovolemic shock , and it is found in 50% of the parturient women with amniotic fluid embolism . the mechanism of dic is not clear , but it is known by a previous animal experiment that amniotic fluid is related to the thromboplastin - like effect , platelet aggregation , and the activation of complement reaction . in addition , lockwood et al . discovered a large amount of tissue factors in amniotic fluid and explained the triggering of blood coagulation and consumptive coagulopathy caused by the activation of extrinsic pathway and factor x. dic , which is clinically characterized by continuous blood loss , can take place in any stage of amniotic fluid embolism , from the initial stage to the terminal stage . in this case report , the heart was temporarily recovered by the cardiopulmonary resuscitation after the cardiac arrest , but the prognosis could become worse as hypovolemic shock took place due to the dic afterward . in conclusion , the main causes of the maternal death of the parturient woman are thought to be the hypotension by the combined effect of the left ventricular failure , the decrease of the general vascular resistance due to the spinal anesthesia and continued blood loss by dic , and pulmonary failure by the severe pulmonary edema . the pathway of amniotic fluid influx to the maternal circulatory system includes the uterine cervical vein , damaged uterine site , and placental site . in early studies , pulmonary vascular occlusion was considered as the main etiology of amniotic fluid embolism , but various clinical characteristics of amniotic fluid embolism were hardly explained by the mechanism and it was not verified by animal experiment , implying that there might be another mechanism rather than the mechanical occlusion . now , the secretion of primary or secondary endogenous mediators following the amniotic fluid inflow to the maternal circulatory system is considered as the major etiology of amniotic fluid embolism based on several reports . the known mediators include histamine , bradykinin , endothelin , leukotriene , and arachidonic acid metabolites . these immunological factors can be supported by the fact that amniotic fluid embolism is found more frequently among the parturient women who have conceived a male fetus and with the history of drug allergy . in other words , amniotic fluid embolism can be strongly suspected in a pregnant woman or a puerperal woman , immediately after the delivery , with cardiovascular collapse as well as respiratory failure , dic , and convulsion , excluding anaphylaxis , septisemia , pulmonary embolism , myocardial infarction , perinatal cardiomyopathy , and hemorrhagic shock ( atony , uterine rupture , and abruptio placentae ) . although in many studies amniotic fluid tissue was found in the blood aspirated at the terminal of the pulmonary artery catheter installed in parturient women who were diagnosed as having amniotic fluid embolism , the amniotic fluid tissue was found in only about 50% of the parturient women in the study of clark et al . , as well as in other studies . moreover , the component of amniotic fluid is also found among the women who do not have amniotic fluid embolism or the women who are not pregnant . hence , that a component of amniotic fluid is found in a maternal circulatory system does not necessarily mean that amniotic fluid embolism has occurred . however , the probability of amniotic fluid embolism is increased in the case where there are the clinical characteristics that suggest amniotic fluid embolism . also , in the parturient woman in this case , the diagnosability was increased since a great amount of amniotic fluid component was found in the pulmonary blood vessels , as well as the clinical characteristics of amniotic fluid embolism . beside this , another known method of amniotic fluid embolism diagnosis is to measure zinc coproporphrin , sialyl tn antigen , tryptase , and complement factors in the peripheral blood of a parturient woman , but more research is required for it . the treatment is performed symptomatically depending on the symptoms of the patient and the basic direction of the treatment is maintenance of the appropriate oxygenation level and blood pressure , and correction of the coagulopathy . firstly , for the hypoxia , tracheal intubation should be immediately carried out and positive - pressure ventilation with oxygen of high concentration should be performed so as to maintain the oxygen saturation higher than 90% . for the hypotension and the shock , since an overdose of fluid can worsen the heart failure at this time , it is helpful to monitor by means of pulmonary catheter or electrocardiography . in the case of a serious hypotension which does not respond to a fluid treatment , vasopressors such as norepinephrine or cardiac inotropic agents such as dopamine , dobutamine , and milrinone can be used . when a large amount of hemorrhage occurs due to dic , packed red blood cells should be primarily transfused to supply oxygen to the tissue appropriately . platelets , fresh frozen plasma , and cryoprecipitate also need to be transfused . among them , cryoprecipitate has been known to help the patient to recover the cardiopulmonary and hematological state rapidly by enhancing the removal of antigenic and toxic substances such as amniotic fluid since it contains fibronectin . when cardiopulmonary arrest takes places during the clinical course , cardiopulmonary resuscitation should be immediately carried out and cesarean section should be performed as early as possible in order to improve the prognosis of the parturient woman and the fetus . since the gravid uterus represses the venous return by the aortocaval compression , quick delivery of the fetus makes the cardiopulmonary resuscitation more effective . in addition to these , other treatments are available including aprotinin , serine proteinase inhibitor , cardiopulmonary bypass , pulmonary embolectomy , hemofiltration , and inhalation of nitric oxide gas , etc . recently , a case where extracorporeal membrane oxygenation showed a good result in a parturient woman with amniotic fluid embolism who had a cardiac arrest was reported , and it was newly suggested as a treatment . in conclusion , as experienced in this case , amniotic fluid embolism is rare , but it is a rapidly developing , fatal disease . however , this disease is hard to diagnose in its onset , and the treatment is still difficult . thus , a prompt and positive treatment should be carried out if there is a parturient woman in whom a sudden cardiopulmonary collapse , respiratory failure , and hemorrhage are found . particularly when cardiac arrest takes place , cardiopulmonary resuscitation should be carried out immediately and the fetus should be delivered at the same time in order to improve the prognosis of the parturient woman and the fetus . it must be noted that even after the parturient woman has recovered from the cardiac arrest , neurologic damage caused by hypoxia can still take place .

amniotic fluid embolism ( afe ) is a rare but fatal obstetric emergency , characterized by sudden cardiovascular collapse , dyspnea or respiratory arrest and altered mentality , disseminated intravascular coagulation ( dic ) . it can lead to severe maternal morbidity and mortality , but the prediction of its occurrence and treatment are very difficult . we experienced a case of afe during emergent cesarean section in a 40 + 6 weeks healthy pregnant woman , age 33 . sudden dyspnea , hypotension , signs of pulmonary edema and dic were developed during cesarean section , and cardiac arrest followed after these events . the course of these events was so rapid and catastrophic , which was consistent with afe . thus , we report this case precisely and review pathophysiology , diagnosis , treatment of afe by referring to up - to - date literatures .

bone marrow cells from all 13 mice transplanted with single cd150 hscs gave rise to hematopoiesis after secondary transplantation ( morita et al . , 2010 ) . in contrast , only a minority of single cd150 or cd150 hscs gave rise to significant hematopoiesis in secondary recipients ( morita et al . these findings dovetail with data demonstrating ( with an entirely different hsc enrichment strategy ) that cd150 expression predicts successful repopulation of secondary recipients ( kent et al . , 2009 ) . 2008 ) about whether cd150 hscs exist into perspective and suggest that the answer is to some degree arbitrary . a proportion of hscs lacking cd150 meet the conventional criteria for hsc activity in primary recipients , but these cells largely lack durable self - renewal in secondary transplant assays . morita et al . ( 2010 ) further demonstrate that single cd150 hscs display robust myeloid reconstitution potential upon transplantation , whereas cd150 hscs mediate only faint myeloid , but superior lymphoid , reconstitution ; these patterns appeared stable in secondary transplantations . recently , three other groups independently reported similar findings , even though the enrichment strategies for hscs were quite dissimilar except for the use of cd150 ( kent et al . , 2009 ; 2009 ) examined lymphoid versus myeloid reconstitution patterns associated with the absence or presence of cd150 on hscs ( defined as cd45epcrcd48 ; kiel et al . 2006 ) and report a strong predominance of myeloid or lymphoid reconstitution after transplantation of cd150 or cd150 cells , respectively . challen et al . ( 2010 ) demonstrated that myeloid and lymphoid - biased hscs can be purified based on hoechst dye efflux in combination with the absence of lineage markers and the presence of sca-1 and c - kit ; further separation of these hscs based on cd150 expression enhanced discrimination of the lineage bias . ( 2010 ) used an hsc isolation strategy similar to that used by nakauchi s group and detected the same lineage bias associated with cd150 expression . interestingly , this study revealed that the proportion of cd150 hscs strikingly increases with aging , whereas the proportion of cd150 or cd150 hscs strikingly diminishes with aging , suggesting that the well documented aging - related compromise of lymphopoiesis ( sudo et al . , 2000 ; rossi et al . , 2005 ) is not caused by loss of lymphoid potential by a homogeneous population of stem cells , but rather by expansion of hsc populations with predominantly myeloid potential ( beerman et al . , 2010 ) . this model is also supported by recent experiments using limiting dilution transplantation of total bone marrow ( cho et al . , 2008 ) . collectively , these data suggest that the output of different blood cell types by the hematopoietic system may be influenced by a balance of coexisting stem cell populations with different lineage propensities . the proportions of different stem cells appear to impact the final composition of differentiated blood cell populations even before the separation of distinct lineages occurs ( fig . if different hsc populations coexist , an important question is whether they exist in a hierarchy . in principle , the different populations might be able to convert into each other , or they may derive from a parental stem cell that may or may not persist into adulthood ( challen et al . , 2010 ) . ( 2010 ) demonstrate that cd150 hscs can give rise to cd150 hscs ( themselves ) , as well as to cd150 and cd150 hscs after transplantation , but that cd150 and cd150 hscs fail to give rise to cd150 hscs . thus , cd150 hscs represent the top of the hierarchy . yet , it is unclear at this point whether cd150 hscs continuously replenish cd150 and cd150 hscs during the steady state in adult bone marrow . ( 2009 ) demonstrated that cd150 hscs ( lineage markerc - kitsca-1cd48 ) turn over almost as slowly as cd150 hscs . hence , it is possible that this population can be maintained for long periods of time , even with limited self - renewal capacity . during normal aging , cd150 hsc populations expand while cd150 hsc populations diminish , suggesting that they are differentially regulated ( beerman et al . , 2010 ) . more directly , this notion is supported by the demonstration that lineage - biased hsc subtypes respond differently to transforming growth factor-1 ( challen et al . , 2010 ) . deciphering the regulation of different hsc populations and investigating their potential cross talk is an important task for the future . cd150 hscs harbor an impressive capacity to reconstitute the lymphoid system ( kent et al . , 2009 ; however , their partially preserved self - renewal and the absence of expression of flt-3 clearly distinguish cd150 hscs from the first known major stage of lymphoid differentiation , the lymphoid - primed multipotent progenitor ( lmpp ; adolfsson et al . , 2005 ) . supporting a close developmental relationship between cd150 hscs and lmpps , cd150 hscs give rise to lmpps after transplantation much more efficiently than cd150 hscs ( beerman et al . in addition , cd150 hscs harbor diminished erythroid - megakaryocytic potential in vitro ( morita et al . , 2010 ) . this is intriguing because loss of erythroid - megakaryocytic potential , even if controversial ( forsberg et al . , 2006 ) , was described as a hallmark trait of lmpps ( adolfsson et al . thus , it appears that self - renewal is not always entirely extinguished during the initial steps toward lineage differentiation . ( 2010 ) suggests that the cd150 hsc population is still heterogeneous . in the future , first , some stem cells appear to exclusively give rise to myeloid cells and , nevertheless , are capable of repopulating secondary hosts . second , up to 10% of cd150 hscs represent latent stem cells , which did not give rise to detectable progeny for at least 3 mo after transplantation , and then produced only low levels of myeloid reconstitution in the primary host . the authors speculate that latent hscs may overlap with recently described highly quiescent label - retaining hscs ( wilson et al . such label - retaining hscs , like latent hscs , exhibited much stronger reconstitution activity after secondary transplantation ( wilson et al . single - cell transplantation of label - retaining hscs will be necessary to definitively clarify their overlap with latent hscs . whether or not they are quiescent , the existence of the latent reconstitution patterns demonstrates that not all hscs contribute to blood formation at all times . this observation is reminiscent of retroviral marking experiments , which suggested that hematopoiesis at any given time is sustained by only very few clones ( lemischka et al . , 1986 these experiments support a clonal succession model , in which most of the cells within the hsc pool remain quiescent , periodically releasing active clones to sustain blood production , as previously active clones exhaust their proliferative capacity . however , a clonal succession model was difficult to reconcile with data from chimeric mice made with different embryonic stem cell founders ( harrison et al . , 1987 ) that showed that hematopoiesis was sustained simultaneously by large numbers of hscs derived from different founder cells . nevertheless , the latter observations do not prove that all or even the majority of hscs produce blood at all times . it is tempting to speculate that latent stem cell behavior may be even more common in the nontransplanted host in the absence of stimulation from cytopenias resulting from myeloablative irradiation . it will likely be possible in the future to directly investigate such issues using conditional clonal genetic marking strategies that do not involve transplantation . at least in primary transplant recipients , latent hscs do not meet widely accepted operational criteria for hsc activity , which set the bar at a > 1% contribution to myeloid and lymphoid progeny in the peripheral blood for 16 wk ( morrison and weissman , 1994 ; miller and eaves , 1997 ; ema et al . , 2005 ) . as ( 2010 ) reasonably emphasize that current criteria for defining hscs may need to be reexamined . in fact , defining hsc activity based on peripheral blood analysis alone is also problematic for biological reasons . although granulocytes are short - lived so that their presence in the blood , indeed , indicates ongoing active cell production , the situation is much more complicated with blood b and t lymphocytes . conditional ablation of b and t cell production by time - controlled disruption of the recombination - activating gene-2 gene has shown that peripheral b and t lymphocytes are maintained for very long time periods , even in the absence of any new production ( hao and rajewsky , 2001 ; bourgeois et al . , 2008 ) . for example , naive follicular b cells are only gradually lost , with a half - life of 4.5 mo , and some smaller b cell subsets remain stable indefinitely ( hao and rajewsky , 2001 ) . as a result , using 1% lymphoid reconstitution as a criterion may lead to considerable overestimates of hsc properties , particularly for hsc populations with predominantly lymphoid output . it is emerging that heterogeneous hsc populations in the bone marrow coexist and coordinately give rise to hematopoiesis . in combination with other markers , the slam family member cd150 allows for the prospective enrichment of hsc populations with distinct characteristics . high expression of cd150 appears to identify cells at the very top of the hematopoietic hierarchy , as it is correlated with high self - renewal and marks the only cells with the potential to give rise to themselves , as well as to all other stem cell populations after transplantation . absence or low expression of cd150 on hscs is associated with the acquisition of powerful lymphoid reconstitution potential . in the future , it will be important to further define the precise regulation , developmental relationships , and cross talk between different hsc populations , as well as to explore their role in disease .

hematopoietic stem cells ( hscs ) save lives in routine clinical practice every day , as they are the key element in transplantation - based therapies for hematologic malignancies . the success of clinical stem cell transplantation critically relies on the ability of stem cells to reconstitute the hematopoietic system for many decades after the administration of the powerful chemotherapy and/or irradiation that is required to eradicate malignant cells , but also irreversibly ablates patients own blood forming capacity . surprisingly , despite enormous efforts and continuous progress in the field , our understanding of the basic biology of hscs is still rather incomplete . several recent studies substantially refine our understanding of the cells at the very top of the hematopoietic hierarchy , and suggest that we may need to revise the criteria we typically use to identify and define hscs .

the etiology of psoriasis is unknown , but genetic , metabolic , and immunologic mechanisms have been proposed . it is known that psoriasis can occur due to abnormalities in essential fatty acid metabolism , lymphokine release , free radical generation , and lipid peroxidation . alterations in plasma lipid and lipoprotein composition including a tendency toward an increase in total cholesterol ( tc ) and triglyceride ( tg ) and decrease in high - density lipoprotein cholesterol ( hdl - c ) levels suggest that psoriasis may associate with the disorders of lipid metabolism [ 2 , 3 ] . healthy skin secretes 85 mg of cholesterol within 24 hours whereas a psoriatic patient loses 12 grams of cholesterol with scales during that time . the morphology of psoriatic skin is characterized by epidermal thickness and parakeratosis , a pronounced dermal vascular plexus , and the presence inflammatory cells in the superficial dermis and epidermis . increased polymorph nuclear leukocyte levels damage surrounding tissue by releasing reactive oxygen species produced via nadph oxidase / myeloperoxidase and proteolytic enzymes . increased production of oxygen metabolites is a common feature of most human diseases including psoriasis and it usually triggers an upregulation of the antioxidant capacity , which is overwhelmed . when the oxidative stress develops , it leads to the oxidative damage of lipids and proteins [ 2 , 4 , 5 ] . oxidation of the low - density lipoproteins ( ldl ) results in the production of modified ldl . one of the major and early lipid peroxidation products is oxidized low - density lipoprotein ( ox - ldl ) . high titers of autoantibodies against ox - ldl have been reported in patients with psoriasis . the level of autoantibodies against ox - ldl has been suggested to reflect the in vivo oxidation of ldl [ 7 , 8 ] . the presence of ox - ldl accumulation in psoriatic skin sample has not been shown before . the current study has been designed to evaluate the presence of ox - ldl accumulation in skin biopsy materials of psoriatic patients . this prospective study was performed in the psoriatic patients attending the dermatology outpatient clinic of zonguldak karaelmas university hospital . eighty four psoriatic patients who were diagnosed clinically and histopathologically by the department of dermatology and a total of 40 age - and sex - matched healthy controls recruited from the general population as a control group were enrolled in the study . the patients with secondary hyperlipidemia such as chronic renal insufficiency , nephrotic syndrome , hypothyroidism , diabetes mellitus , obstructive liver disease , and the connective tissue disease were excluded . all patients and controls were included in the study after giving an informed consent . the height and weight of all subjects were recorded and their body mass indexes were calculated as weight ( kg)/height ( m ) . blood samples were taken after a 12-hour overnight fast and sera were separated by low - speed centrifugation for 15 min . the levels of serum tc , hdl - c , ldl cholesterol ( ldl - c ) , and tg were determined by enzymatic methods using a roche cobas integra 800 autoanalyzer . the skin punch biopsy specimens were collected from both lesional and nonlesional skin of 84 psoriatic patients . the presence of ox - ldl in biopsy materials of psoriatic individuals was evaluated using an immune - fluorescent staining method . the slides were prepared from biopsy sections , which were cut at 7-micron thickness . slides were further divided into two pieces ; one was used for the test and the other was used for negative control . thirty l anti - oxidized ldl igg solution ( mouse igg2 antibodies , antibodyshop , copenhagen , denmark ) as primary antibody was added only on test slides , and the control slides were manipulated only with the same amount of phosphate buffered saline solution ( pbs ) . after 30 minutes of incubation in a humid chamber at room temperature , both the control and test slides were washed with ( pbs ) , and 30 l fitc ( fluorescent isothiocyanate)-labeled goat anti - mouse igg ( chemicon international , california , usa ) was administered as a conjugate substance . for a further 30 minutes , the slides were kept and incubated at room temperature , and then washed with the standard pbs solution . after open - air drying , slides were examined under fluorescent microscopy at 100x magnification ( leica dmrx , wetzlar , germany ) . differences in continuous variables between the patient and the control groups were analyzed using the student t - test . mann - whitney u test was used to compare nonparametric variables between two groups . all values were expressed as mean standard deviation ( sd ) unless otherwise stated . table 1 illustrates the clinical and demographic characteristics of the study population . there was no statistically significant difference between psoriatic patients and the controls considering age , sex , weight , height , or bmi ( p > .05 ) . tc , tg , and ldl - c levels were significantly higher , but hdl - c levels were lower in the psoriatic patients than in the control subjects . hdl - c levels were significantly lower in the female psoriatic patients than in the female control subjects ( p < .05 ) . there was no statistically difference for tc , tg , and ldl - c levels between the psoriatic and the control subjects in the female group ( table 3 ) . tc , tg , and ldl - c levels were significantly higher in the male psoriatic patients than in the male control subjects ( p < .05 ) . there was no statistically difference in hdl - c levels between the psoriatic and the control subjects in male group ( table 4 ) . we did not observe any positive immune - fluorescent staining in the nonlesional skin biopsy materials of the psoriatic patients . significant positive immune - fluorescent staining was observed in the psoriatic skin biopsy materials . the dense cellular staining among the many studies on serum lipid values in psoriasis , conflicting results have been reported . in studies on serum tc levels in psoriatic patients , high [ 2 , 9 , 10 ] , low [ 1 , 11 ] , and normal [ 12 , 13 ] values have all been reported . in our study , we found significantly higher levels of tc values in the psoriatic patients ( p < .05 ) . as for serum ldl - c levels , high or normal [ 1 , 10 , 12 ] values have also been reported in psoriasis . we found that ldl - c values in the patients with psoriasis were significantly higher than the control group ( p < .05 ) . normal [ 9 , 10 , 13 ] and low [ 1 , 2 ] serum levels of hdl - c have been detected . in our study hdl - c levels in psoriatic patients were significantly lower than the control group ( p < .01 ) . the same controversy exists regarding serum tg levels , high [ 1 , 2 ] , low , and normal [ 9 , 10 , 12 ] values have also been reported in psoriasis . we found that tg values in the psoriatic patients were significantly higher than the control group ( p < .001 ) . the variety of data presented may be due to the fact that the patients included in statistical analyses suffer from different forms of psoriasis such as erythroderma and they undergo various treatments . tc , tg , and ldl - c levels were significantly higher in the male psoriatic patients than in the male control subjects . there was no statistically difference in the female groups . only for the females , hdl - c levels were lower in the psoriatic patients than in the control subjects . considering the results of our research we would suggest analyzing the lipid profiles separately in males and females . native ldl is only taken up modestly by macrophages , whereas modified ldl is rapidly taken up via scavenger receptors . the major modification of ldl particles in vivo is believed to be the oxidation of both its lipid and protein components . oxidized or modified the oxidized ldl hypothesis is discovered by goldstein et al . and modified by steinberg et al . . the current oxidative modification or stress hypothesis of atherosclerosis predicts that ldl oxidation is an early , essential event in atherosclerosis and that ox - ldl does contribute to both initiation and progression of atherosclerosis . the oxidative modification hypothesis focuses on the concept that ldl in its native form is not atherogenic . the presence of ox - ldl in atherosclerotic lesions has been studied using antibodies that recognize specific epitopes on ox - ldl , which are not present in its native , nonoxidized , form . these antibodies avidly stain atherosclerotic lesions in humans with no demonstrable staining in normal arteries . a typical feature of atherosclerosis is the accumulation of oxidatively modified ldls within plaques . also these lipoproteins are considered to contribute to the inflammatory state of atherosclerosis and to play a key role in its pathogenesis . the cellular uptake of ox - ldl leads to the generation of reactive oxygen species ( ros ) . ros are potentially very harmful substances , because they can react with proteins , dna , or lipids . in other words , the importance of this manuscript is to show the existence of ox - ldl in psoriatic skin . this study shows for the first time the accumulation of oxidized low - density lipoprotein in psoriatic skin lesions by direct immune - fluorescent method . ox - ldl or anti - ox - ldl antibody levels can be measured in blood samples or body fluids by conventional biochemical and immunological methods . especially , the level of anti - ox - ldl has been suggested to reflect the in vivo oxidation of ldl . the studies of vanizor kural and rem have demonstrate the existence of these antibodies in psoriasis . the level of anti - ox - ldl antibody was positively correlated with tc and negatively correlated with hdl - c in their studies . in our study , we detected accumulation of ox - ldl in psoriatic skin . this accumulation is markedly increased especially in upper epidermis . the upper - epidermal cells had dens cellular staining with anti - ox - ldl antibodies . we did not observe any positive immune - fluorescent staining in the nonlesional skin biopsy materials of the psoriatic patients . have observed that tissue - cultered skin fibroblasts from psoriatic patients have reduced ldl receptor activity . their study has no difference in ldl receptor activity between involved and uninvolved skin from our psoriasis patients . in our study , we only focused on the epidermal layer of involved and uninvolved skin from psoriatic patients . we detected accumulation of ox - ldl in the upper epidermis of the involved skin from the psoriatic patients . ox - ldl can use native ldl receptor . however , it has a high - affinity advantage than its native form . in conclusion , ox - ldl is an important marker of oxidative stress and lipid peroxidation process . this capability may directly affect psoriatic epidermis and we believe that the accumulation of ox - ldl in the psoriatic skin may have an important role in pathogenesis of psoriasis .

background . psoriasis is a chronic inflammatory skin disease characterized by an accelerated turnover of epidermal cells and an incomplete differentiation in epidermis with lesion . however , the exact etiology of psoriasis is unknown . abnormalities in essential fatty acid metabolism , free radical generation , lipid peroxidation , and release of lymphokines have been proposed . objective . our purpose was to evaluate the plasma lipids and oxidized low - density lipoprotein accumulation in psoriatic skin lesion in order to ascertain the possible participation of oxidative stress and oxidative modification of lipids in pathogenesis of psoriasis . methods . the study group included 84 patients with psoriasis , and 40 sex- and age - matched healthy volunteers . blood lipid profile was determined . psoriatic and nonlesional skin samples of psoriatic patients were evaluated for the presence of oxidized low - density lipoprotein by using an immune - fluorescent staining method . results . the mean levels of lipids ( total cholesterol , triglyceride , and ldl cholesterol ) in patients with psoriasis were found to be significantly higher than those of healthy subjects . psoriatic skins were shown positive oxidized low - density lipoprotein staining . there was no staining in nonlesional skin samples of the same individuals . conclusion . lipid peroxidation mediated by free radicals is believed to be one of the important causes of cell membrane destruction and cell damage . this study shows for the first time the accumulation of oxidized low - density lipoprotein in psoriatic skin lesion . we believe that accumulation of ox - ldl in psoriatic skin may have an important role in the immune - inflammatory events that result in progressive skin damage .

kcm is itself very rare and distinct type of keratoacanthoma which usually occurs over extremities and scalp is unusual site for development of lesion . treatment is difficult as lesion of kcm present with large raised , rolled borders with peripheral extension . keratoacanthoma ( ka ) is a rapidly evolving tumor , composed of keratinizing squamous cells originating in pilosebaceous follicles and resolving spontaneously if left untreated . it is relatively common , especially in whites occurring in middle age while being uncommon in dark - skinned . . it presents as firm , rounded , flesh - colored or reddish papule ; with a rapid growth phase becoming 10 - 20 mm and then spontaneous healing taking place over three months . there are three rare clinical variants of solitary ka , namely giant ka , keratoacanthoma centrifugum marginatum ( kcm ) and subungual ka . in kcm , lesions are large , reaching upto 20 cms . the most common locations are dorsa of hands and legs , lesions on scalp being rare . we report a rare case of kcm occurring on the scalp which is an unusual site . a 62-year - old male , watchman by occupation presented with asymptomatic raised lesion on scalp since one year . lesion had developed de novo , as a pea sized lesion with gradual increase to cover entire vertex . cutaneous examination revealed a single , irregular , 12 15 cm , yellowish plaque on vertex of scalp with nodular surface and a central crater [ figure 1 ] . on palpation , the plaque was firm , non - tender , non - indurated and not attached to underlying structure . ultrasonography of lesion showed ill defined hypo echoic mass involving epidermis , dermis and subcutaneous tissue . a single , irregular , 12 15 cm , yellowish plaque on vertex of scalp with nodular surface and a central crater . deep punch biopsy revealed exoendophytic , globular , well circumscribed central cup shaped crater , epidermal invagination with marked hyperkeratosis and horn cyst with rich keratin filled crater imparting glassy appearance and well demarcated regular base . . marked epidermal proliferation with crater formation with horn pearls and inflammatory infiltrate at the base of the lesion was suggestive of a fully developed lesion of kcm . complete surgical excision with grafting was done successfully with dramatic improvement with no recurrence at 9 months [ figure 5 ] . exoendophytic , globular , well circumscribed central cup shaped crater , epidermal invagination with marked hyperkeratosis and horn cyst with rich keratin filled crater imparting glassy appearance and well demarcated regular base epidermis forming buttress over the pseudhorn cyst dense nodular lymphocytic infiltrate in the upper dermis surgical excision with grafting the etiology is multifactorial that includes chronic ultraviolet ray exposure , smoking , contact with chemical carcinogens like pitch , mineral oil , tar , trauma and vaccination . the role of human papilloma virus remains inconclusive but in one study , hpv type 6 and 11 were detected within the lesion . it can be localized to any region of the body but is more frequently seen on dorsum of hands and legs . progressive peripheral extension with a raised rolled - out margin and atrophy at the centre is a characteristic feature of kcm . the margin of the lesion showing multiple comedonal orifices giving rise to a cribriform pattern may represent a unique phenomenon of kcm . it is hypothesized that this typical appearance may arise as a result of sequential involvement of multiple adjacent hair follicles in a centrifugal fashion . kcm does not show tendency for spontaneous regression , a feature also seen in giant ka , which grows rapidly reaching a size of 5 cm or more and occurring commonly on nose and eyelids . kcm is differentiated from giant ka by absence of downward vertical spread and destruction of underlying tissue . nevertheless , in giant ka spontaneous involution takes place after several months , often accompanied by detachment of a large keratotic plaque . the other differential diagnoses include squamous cell carcinoma , lupus vulgaris , botryomycosis , blastomycosis - like pyoderma and pseudoepitheliomatous hyperplasia , hypertrophic lupus erythematosus , atypical mycobacterial infections , or deep fungal infections . there are reports that kcm has been treated successfully with oral retinoids ( acitretin , etretinate , or isotretinoin 0.5 - 1 mg / kg / day ) which should be given until complete clearance of lesion . surgical intervention is a preferred mode of therapy keeping in mind that a wide excision is performed to prevent subsequent recurrence . other treatment modalities successfully used include topical 5-fluorouracil , intralesional injections of interferon alpha , methotrexate , or bleomycin , or mohs micrographic surgery . in kcm , lesions are usually too large for excisional biopsy for diagnosis , but certain characteristic features like clinical behavior , appearance of lesions , histological features ( hyperkeratosis and hyperplasia ) , and cytological features ( tumor cells with eosinophilic and glassy cytoplasm , few mitoses ) help clinch the correct diagnosis . first time from india we are reporting case of kcm presenting over scalp which is successfully treated with surgical excision without any recurrence .

keratoacanthoma ( ka ) is a rapidly evolving tumor , composed of keratinizing squamous cells originating in pilosebaceous follicles and resolving spontaneously if left untreated . it is relatively uncommon in dark - skinned and occurs in middle aged individuals . males are three times more affected than females . it presents as firm , rounded , flesh - colored or reddish papule ; with a rapid growth phase followed by spontaneous healing over three months . two types of ka exist i.e. , solitary and multiple . there are three rare clinical variants of solitary ka , namely giant ka , keratoacanthoma centrifugum marginatum ( kcm ) and subungual ka . in kcm , lesions are large , reaching upto 20cms . there is peripheral extension with raised , rolled border and atrophy in the center . there is no tendency toward spontaneous involution . the most common locations are dorsa of hands and legs , lesions on scalp being rare . a rare case of kcm occurring on scalp which is an unusual site is reported .

an unfortunate consequence of the childhood and adolescent obesity epidemic is the emergence of metabolic syndrome , a condition that was historically seen in adults . thirty percent of obese adolescents in the united states meet criteria for metabolic syndrome and the prevalence is increasing . consistent with adult rates , prevalence of metabolic syndrome in adolescents is highest among hispanics , followed by african americans when compared to non - hispanic whites , while type 2 diabetes is increasing among american indian adolescents more than any other racial / ethnic group in the united states . weight loss through behavioral modification is an appropriate first step in the primary care treatment of metabolic syndrome . nevertheless , almost 80% of pediatricians report frustration with their ability to make an impact on obesity and many providers feel they do not have the tools to effectively address lifestyle modification for weight loss . schools , where children and adolescents spend the majority of their time , are promising venues for intervention work . a recently released institute of medicine report strongly endorsed this perspective , citing schools as a national focal point in efforts to address obesity in children and adolescents . as several systematic reviews demonstrate , however , there is a paucity of obesity intervention research in high school settings targeting older adolescents and even less in school - based health centers ( sbhcs ) [ 1012 ] . located in the school setting , sbhcs have several compelling features for a successful lifestyle intervention program to treat overweight / obesity and prevent development of metabolic syndrome : ( 1 ) sbhc clinicians have more access to adolescents than community health care providers ; ( 2 ) adolescents have better compliance and followup in school - based clinics ( both of which are needed for weight loss and maintenance ) ; and ( 3 ) sbhcs focus on early identification of high - risk problems [ 1318 ] . in response to the obesity epidemic and adolescents ' interest in promoting physical activity and healthy eating , our research was built on a long - standing partnership between the university of new mexico school - based health center program and an urban school community . through previous engagement with school - based clinicians and adolescent students , we reached consensus on collective interest to create a dvd to help adolescents achieve weight loss and a clinician tool kit to assist providers in their motivational interviewing efforts . this partnership led to the creation of a culturally and developmentally appropriate overweight / obesity intervention program , adolescents committed to improvement of nutrition and physical activity ( action ) . consistent with american academy of pediatric expert committee recommendations regarding the treatment of adolescent overweight / obesity , the action intervention featured eight sessions with a health care provider using motivational interviewing to encourage behavior change toward active living and healthful eating . this paper describes the use of an adaptive community based participatory research ( cbpr ) process through which formative research data were collected for development of an sbhc intervention for prevention of metabolic syndrome in multiethnic high school students . formative research is used to understand people 's beliefs , perceptions , and behaviors for the purpose of developing culturally appropriate behavioral change programs . health researchers typically include a formative stage of research in their study designs to ensure that the identification of such cultural attributes and contextual factors can be integrated into intervention activities [ 2224 ] . given our interest in developing intervention tools ( dvd and toolkit ) to help school - based providers and multiethnic adolescents , we designed this study to ground formative assessment through a cbpr partnership [ 25 , 26 ] . our review of the literature revealed that use of cbpr in formative research is quite limited and we have not found examples of other manuscripts reporting this type of approach in the context of an sbhc study . we provide further detail showing the link between cbpr principles and our research steps in table 1 . to maximize time for development and completion of the dvd and toolkit in one year , recruitment for semistructured interviews with overweight / obese adolescents and parents of overweight / obese adolescents was concurrent with recruitment of overweight / obese adolescents and their parents to the community advisory council ( cac ) . adolescents and parents were recruited from the two participating urban high schools with long - standing school - based health centers . interviews with participants unknown to the cac were conducted to obtain fuller disclosure of sensitive information about living with obesity that the university research team felt may have been too sensitive to discuss openly among the cac members . in the second year of the study , the intervention was implemented using these materials . the study protocol was approved by the university of new mexico human research protections office and the urban schools ' research , development , and accountability department . a purposeful sampling strategy that reflected the culturally diverse school population was used to recruit overweight / obese adolescent and parent dyads . given the intent to understand perceptions of obesity and barriers / facilitators to weight loss , we focused recruitment efforts on overweight / obese adolescents . we also conducted extreme case sampling to identify adolescents who self - reported success in achieving weight loss ( table 2 ) . we believed that these more unusual cases would lead to insights relevant to our intervention development . all of the adolescents were referred to the research team by participating school - based health center providers and partnering school staff ( e.g. , counselors , school nurse , and teachers ) . the research team coordinated recruitment efforts with collaborators at the participating high schools including the activity directors , school health advisory council members , student senate members , and sbhc clinicians . adolescent and parent interview guides were designed to assess concordance of perspectives in the following areas : media use and information seeking strategies , definitions of health , health concerns ( weight related ) , strategies / approaches to weight loss , barriers / facilitators to health in the school and home environment , and suggested content and style for the dvd . the interview guide was designed to be neutral to potentially stigmatizing perspectives about adolescent obesity and contributing factors . adolescents and parents were interviewed separately and interviews were conducted at times and locations convenient for the family , mostly in their homes and some at the university . consent / assent forms were mailed in advance for review and respondents were formally consented at the start of each interview . most interviews lasted approximately one hour and each respondent received a $ 20 reimbursement for their time . following an iterative analytic process , the multidisciplinary research team including a medical anthropologist , adolescent medicine physician , primary care nurse practitioner , health communications researcher , and community based participatory research practitioner reviewed sets of 3 to 4 transcripts independently , identifying key themes relevant to the subsequent creation of the intervention materials . we specifically focused on responses between adolescents and parents , seeking both complementary and divergent perspectives on key elements for the dvd and provider toolkit materials including content , messaging , and context of use . ongoing data collection and analysis continued until 15 interviews had been conducted ( 7 students , 8 parents ; the sample consisted of 6 parent - child pairs ) , at which point the research team reached consensus that the full range of interpretive themes had been identified . at that point , the medical anthropologist ( als ) coded each interview in the qualitative data analysis software program nvivo 8 to facilitate text retrieval and create summary reports . further demographic details of the student and parent interviewees are presented in table 2 . following the intervention and as part of a broader process evaluation effort designed to examine a range of implementation issues , we assessed adolescent and parent views of acceptability and satisfaction with the materials developed in the formative phase . intervention students were each asked to fill out questionnaires with likert scale ratings of the action dvd , toolkit handouts , and overall satisfaction with the intervention . parent questionnaires focused on materials they received as part of the action intervention , including a parent newsletter as well as views of the toolkit handouts and overall satisfaction with their child 's participation in action . descriptive summaries of the questionnaires for each group were assessed . once the interviews and a preliminary analytic summary had been developed , we convened the community advisory council ( cac ) as a forum in which to translate themes from the interviews into specific strategies and materials to be used in the intervention . our goal was to form a group with similar demographic characteristics to the targeted study population : hispanic , african american , and american indian overweight / obese students and their parents . given our expectation of attrition , we recruited a total of 16 participants ( divided equally between students and parents , including four parent - child pairs ) . we began a series of six consecutive monthly meetings from december 2008 to may 2009 at the university of new mexico during which the dvd and toolkit were developed . the cac met on a less frequent basis throughout the second year of the study ( august 2009 to may 2010 ) to provide implementation guidance and feedback . we provided a full dinner , free parking , and reimbursement for travel expenses ( $ 10 ) for all participants immediately following each meeting . cac members were also informed that all would receive the dvd resulting from their efforts as well as a personal dvd player . sessions were typically comoderated by the dvd producer and study coinvestigator ( als ) . however , when meetings were focused on topics of nutrition and/or physical activity , sessions were led by the research team registered dietician and fitness expert . this approach addressed a central cbpr principle in fostering information exchange between community members and the research team . the meetings were designed to be cyclical and iterative as a way to reach consensus and tailor evidence - based strategies to ensure the cultural and age appropriateness of the dvd and toolkit materials as well as stylistic elements ( e.g. , music and images ) . given the flexible , participatory nature of these meetings , cac members shaped the process by volunteering to test potential data collection methods ( e.g. , maintaining a food diary ) and requesting further information of the research team to facilitate colearning ( connections between obesity and health ) . overview . we have organized the presentation of results to reflect the sequential process of data collection leading to the creation of the action intervention materials . we first present the thematic findings from key informant interviews that provided guidance to the research team and the cac . we then describe how the cac translated this input into the dvd and provider toolkit . lastly , we include process evaluation measures reflecting adolescent and parent views of these materials following the action intervention . adolescents consistently indicated use of the internet as a primary source for both entertainment and information seeking . a lot of teenagers i know use the internet and look up information that way . if it 's something that looks interesting they 'll go look at it . a lot of teenagers i know use the internet and look up information that way . if it 's something that looks interesting they 'll go look at it . further , most adolescents described using the internet as a way to gather health information about a range of issues , including diet and exercise . they viewed the internet as a reliable source of information that could be accessed in private . another female adolescent described using the internet for health information and as a basis for discussion with her doctor : they have that pyramid , i 'm not sure what it 's called . and you can look at how many calories you 're supposed to take in a day and i looked at those and i just started doing that last month with my doctor . they have that pyramid , i 'm not sure what it 's called . and you can look at how many calories you 're supposed to take in a day and i looked at those ( 2 ) functional definition of health . in each of the interviews , we asked adolescents how they define health and what that means to them . in some cases , they identified the association between being overweight / obese and the future development of health problems such as diabetes and heart disease . several of these teens were aware of these risks given family members dealing with these conditions . however , the most striking theme to result from these interviews related to how being overweight / obese limited adolescents activities in the present . overall , they were less concerned with the long - term consequences and referred to a functional definition of health that focused on the degree to which their weight impacted the ability to engage in activities of interest . one male adolescent , echoing the response of several others , responded to the question what does it mean to be healthy ? by stating ( 3 ) barriers to weight loss in the school . not surprisingly , both teens and parents reported a wide range of challenges related to their struggles with food choices and physical activity in their weight control efforts . teens consistently noted that healthful foods / drinks were not available in the school setting . adolescents regularly purchased foods from vendor carts but were frustrated by the lack of healthful options such as water rather than soda . in addition to expressing a need for healthier snacks from vendors , the teens also pointed to unhealthful meal choices at school . as a potential way to address this problem , one teen stated mostly not sell as much pizza , cause like everywhere you go they sell pizza and burritos , like breakfast burritos and sometimes even regular burritos at lunch . even if they change the menu up a little bit that 'd be better because it would n't be the same thing every day just burritos , burritos or pizza , pizza , pizza . mostly not sell as much pizza , cause like everywhere you go they sell pizza and burritos , like breakfast burritos and sometimes even regular burritos at lunch . even if they change the menu up a little bit that 'd be better because it would n't be the same thing every day just burritos , burritos or pizza , pizza , pizza . parents consistently agreed with adolescents about the need for schools to offer foods that are both appealing and offer higher nutritional value . another challenge identified by both the teens and the parents is the lack of opportunity for exercise in the school setting . teens emphasized the need for more physical activities not related to formal participation on sports teams . parents focused their concern on curricular issues such as the need to require a gym class for all four years of high school rather than just one semester . several adolescents interviewed reported success in their efforts to lose weight . although a range of strategies were described mostly involving diets and increases in physical activity adolescents expressed the importance of internal motivation as the essential element in weight loss initiation efforts . consistent with the functional definition described above , students identified personal goals such as making a sports team or improving their appearance for a significant school event such as the prom as a reason to make physical activity and nutritional changes . similarly , there was consensus among the parents that it was their obligation to serve as a principal source of motivation . as one parent saidi would have to say it ( motivation ) would have to be left on the parent 's shoulders . i think if they see the parents motivated to want to exercise and take care of their body and eat healthy then the children are going to see that and that 's going to help them . mom 's doing this , maybe we should do this , too . ' i would have to say it ( motivation ) would have to be left on the parent 's shoulders . i think if they see the parents motivated to want to exercise and take care of their body and eat healthy then the children are going to see that and that 's going to help them . okay , mom 's doing this , maybe we should do this , too . ' ( 5 ) parent views on changing home environment . while we mostly focused these interviews on the school environment , we specifically engaged parents about the challenges they confront in weight loss efforts at home . the goal of these discussions was to elicit strategies that could be included in the home - based component of the action intervention . parents echoed the adolescents in recognizing the broad importance of motivation as a way to initiate health behavior changes . as one mother indicated some ideas on how to motivate your kids ideas on how to keep them wanting to live a good lifestyle , you know , healthy lifestyle and full of exercise . and then just trying to change their mentality , change their way of thinking so that it 's stuck in their head . my goodness , it 's hard . some ideas on how to motivate your kids ideas on how to keep them wanting to live a good lifestyle , you know , healthy lifestyle and full of exercise . and then just trying to change their mentality , change their way of thinking so that it 's stuck in their head . we also asked parents for suggestions of informational needs about nutrition and/or physical activity that would be useful to them at home . another mother described interest in having a list of practical comparisons of food choices : i always find it interesting when they do comparisons like which is actually the healthier ( food ) and you 're going , i do n't know , they 're actually both kind of bad , but which is actually the better choice ? ' i always find it interesting when they do comparisons like which is actually the healthier ( food ) and you 're going , i do n't know , they 're actually both kind of bad , but which is actually the better choice ? ' ( 6 ) input on dvd content . lastly , the interviews provided a rich opportunity to gather input on content and stylistic elements of the proposed dvd . consistent with the theme of internal motivation , most adolescents requested that the dvd content did not scare or threaten by presenting adverse health consequences of overweight / obesity . instead , they emphasized the importance of featuring teens in the dvd that were currently overweight / obese so that adolescents would relate more to the content : include overweight people and show that they 're really working hard and then show them successfully doing it and get in a normal ( weight ) range . include overweight people and show that they 're really working hard and then show them successfully doing it and get in a normal ( weight ) range . reflecting common elements of teen culture , most of the teens suggested using popular activities as a vehicle through which the messages could be promoted . this included differing forms of dance , kickboxing , use of appropriate music , and presenting these activities with careful attention to style through rapid cutting to different camera angles and shots . when asked what should be in the dvd , one male adolescent quickly responded dancing , i 'm pretty sure cause everyone loves to dance ; say if you want to learn how to fight and get in shape and defend yourself there 's kickboxing , so pretty much those things . dancing , i 'm pretty sure cause everyone loves to dance ; say if you want to learn how to fight and get in shape and defend yourself there 's kickboxing , so pretty much those things . as the research team finalized the analysis of the parent and adolescent interviews , we convened the cac and held the first of six consecutive monthly sessions . the first session was primarily an orientation to the project and subsequent meetings were led by content expert moderators from the research team to identify and refine intervention content . below , we provide a list of the major themes and the resulting strategies that were integrated into the dvd and toolkit . cac participants confirmed this and agreed with a proposal to include websites in the intervention materials for adolescents to access nutritional and physical activity information . during one of the sessions , we reviewed potential websites for inclusion ( ranging from purely informational to highly interactive ) with the cac and our final selection was based on feedback pertaining to ease of use and clarity of presentation . functional definition of health articulated during the adolescent interviews and the cac concurred with the preference to encourage individual autonomy and goal setting rather than emphasize negative outcomes associated with poor health . the cac adolescent participants were clear that the kids in the dvd needed to be real teens ( both overweight / obese and not overweight / obese teens ) dealing with challenges of weight . this strategy was incorporated into the dvd by having cac participants and friends appear in the dvd and briefly describe their struggles as well as the internal sources of motivation that inspired them to make changes . the group advocated that the dvd contain practical nutritional information to help them make healthier choices amongst likely food offerings ( e.g. , pizza , burritos , bagels , etc . ) . websites containing further information were also referenced in both the dvd and health care provider toolkit . as described above , teens indicated that any effort to achieve weight loss had to begin with an individual identifying some type of internal motivation and , ideally , having a supportive network of friends and family to sustain the effort . while the cac agreed with this perspective and specific strategies were included in the dvd to encourage recognition of this internal motivation , the group also advocated for the inclusion of physical activities to jumpstart the process . therefore , following the input of both the interviewees and the cac , brief instructional segments featuring dance , kickboxing , and weight lifting were added to the dvd . ( adolescents and parents in the cac agreed that it was not sufficient to only focus on the school environment . the group offered a series of strategies for involving parents in a practical and appropriate way during the action intervention . this included having the school health provider call the parent to provide regular updates , distribution of a short parent newsletter , and ideas for affordable and quick healthy recipes for busy families . ( 6 ) dvd content . toward the conclusion of the six cac sessions in the first year of the study , the group reached consensus on three sections to feature on the dvd : ( 1 ) adolescent motivation for change , ( 2 ) strategies targeting energy balance and nutritional quality , and ( 3 ) physical aerobic dance and strength / resistance training instructional segments . the cac reviewed each of the elements to be included in the dvd as well as offered stylistic suggestions relating to background music and video editing techniques to appeal to adolescents . ( iii ) use of the action dvd and toolkit . at the conclusion of the six cac sessions , coordination and filming of the dvd were led by the video producer . many of the cac members teens and parents participated in this process and appear in the final dvd either sharing their own personal stories or presenting the nutrition or physical activity strategies . the action toolkit contained a parent newsletter and adolescent session tools ( e.g. , goal setting , internet resources , activity / food journal , etc . ) . ( iv ) action outcomes and satisfaction results . while the focus of this paper is to report on the process of developing the action intervention materials , we are also able to offer a brief overview of process evaluation findings related to participant satisfaction with these components as well as the primary outcome measure . health outcome measures for the action study showed that students receiving the intervention had greater prepostimprovements in bmi percentile ( p = 0.04 ) and waist circumference ( p = 0.04 ) as compared to the standard care control group . bmi median percentile decreased 0.3% in the intervention group while the standard care control group 's bmi median percentile increased by 0.2% . mean waist circumference in the intervention group remained unchanged and there was a 1.7 cm increase in the standard care group . while these outcome measures are important , we also conducted process evaluation aimed at elucidating factors that may help us to better understand how the use of this adaptive cbpr approach relates to these results . students reported high levels of satisfaction with the materials used in the intervention . on a scale of 0 ( not at all useful ) to 5 ( very useful ) , students were asked to rate the usefulness of the dvd ( n = 26 ; mean score 3.1 ) , the clinician toolkit handouts ( n = 27 ; mean score 4.0 ) , and their overall satisfaction with the intervention ( n = 28 ; mean score 4.4 ) . parents favorably rated the usefulness of the parent publication ( n = 18 ; mean score 3.6 ) , the usefulness of the clinician toolkit handouts ( n = 23 ; mean score 3.7 ) , and their overall satisfaction with the intervention ( n = 25 ; mean score 4.4 ) . lastly , process evaluation findings reveal high rates of retention among participants in both the intervention group ( 90% ) and standard care control group ( 79% ) . we used formative assessment research guided by an adaptive cbpr approach to create an sbhc obesity intervention program . the student and parent interviews generated initial thematic findings that were used to guide discussions in the subsequent cac sessions toward the development of the intervention dvd and clinician toolkit . this approach enabled us to accomplish the following goals in the first year of this study : ( 1 ) gain a better understanding of the school setting with regard to barriers to physical activity and healthful food choices , ( 2 ) create strategies / materials to address these barriers , and ( 3 ) develop culturally appropriate intervention materials and approaches based on input from study participants . we identified several elements that may be useful in subsequent efforts to develop and/or further refine obesity intervention materials in school - based settings . adolescents emphasized the importance of media , particularly use of the internet , as a primary source for most informational needs . we also observed that these culturally diverse ( hispanic , american indian , and african american ) adolescents expressed mostly similar views about the types of barriers and proposed solutions to addressing overweight / obesity both at home and in the school . there was strong consensus regarding a functional definition of health that focused on identifying internal sources of motivation to achieve goals and engagement in activities as opposed to one based on more standard biomedical measures of health ( e.g. , blood pressure or cholesterol levels ) . partnering with these teens and adolescents was essential in order to ensure not only the appropriate content in the action intervention but also the stylistic elements that maximize appeal and likelihood of use . we believe that the high levels of satisfaction reported by action participants , as well as the promising results in the primary outcome measures , are attributable to this adaptive cbpr process . over the past several years , increasing attention has been directed toward schools settings as promising venues for obesity prevention and treatment interventions . the recently released institute of medicine report identified schools as catalysts in our efforts to accelerate progress in obesity prevention . a growing body of literature supports the value of directing programmatic and research resources to school - based interventions . prior research has demonstrated the efficacy of a quality improvement initiative aimed at enhancing sbhc provider implementation of obesity treatment guidelines . another recently published study reports findings from an adolescent obesity screening program conducted through a school - based health center . in this study , following a medical evaluation , adolescents determined to be outside while these types of quality improvement and screening and referral projects are consistent with the need to engage schools in obesity prevention efforts , there is still a gap in the development and implementation of obesity interventional research through sbhcs . we believe the action study is the first to actually create and test such an intervention through a partnership with key stakeholders located in the school community . conducting research in partnership with the sbhcs , the school administration , the students , and their families poses a set of logistical and study design challenges . the action study involved each of these key stakeholders in different ways and at different stages to both maintain relationships that preceded this project and establish new ones to sustain our efforts going forward . the adaptive cbpr design employed in the action study may offer useful guidance to other researchers conducting research in school settings . while much cbpr is oppositely structured forming the community advisory group first in this two - year study it was imperative that the formative research and production of the intervention materials ( dvd and clinician toolkit ) are completed by the start of the intervention , coinciding with the school calendar . our goal was to incorporate the core principles of cbpr while operating in a compressed timeframe imposed by the research funding . as evidenced from both the process evaluation findings and health outcome measures , we believe that these adaptations were essential to create an authentic partnership while adhering to a timeline that may not have been feasible using a more traditional cbpr approach . while use of cbpr appears to enhance the effectiveness of interventions , there exists a lack of clarity regarding how different types of partnership configurations and processes contribute to these outcomes . the first relates to the limited generalizability of these findings to other populations and school - based settings . our purpose in this paper , however , is to demonstrate the applicability of an adaptive cbpr process through which researchers can identify locally and contextually relevant factors in their school - based obesity interventions . the second limitation involves the relatively small sample size of interviews in the formative assessment phase . the iterative process of data collection and analysis led to data saturation at an early stage and the consistency of thematic responses enhanced the confidence of findings from ethnoracially diverse students and parents . cbpr partnerships with school - based communities represent an important opportunity for researchers seeking to address a broad spectrum of adolescent health problems . there is an urgent need to find innovative , cost - effective , and sustainable strategies to reach underserved communities . we found that conducting formative assessment using an adaptive cbpr process with a range of school - based stakeholders was an effective way to develop culturally appropriate and tailored intervention materials and approaches in these multiethnic school settings . we demonstrated that researchers can adapt their study designs in ways that best maximize limited resources and time constraints without compromising the core principles of cbpr .

purpose . from our previous clinical work with overweight / obese youth , we identified the need for research to create an effective weight management intervention to address the growing prevalence of adolescent metabolic syndrome . formative assessment through an adaptive community - based participatory research ( cbpr ) approach was conducted toward the development of a nutritional and physical activity ( dvd ) and clinician toolkit for a school - based health center ( sbhc ) weight management intervention . methods . we first conducted parent and adolescent interviews on views and experiences about obesity while convening a community advisory council ( cac ) recruited from two participating urban new mexico high schools . thematic findings from the interviews were analyzed with the cac to develop culturally and developmentally appropriate intervention materials . results . themes from the parent and adolescent interviews included general barriers / challenges , factors influencing motivation , and change facilitators . the cac and university - based research team reached consensus on the final content of nutrition and physical activity topics to produce a dvd and clinician toolkit through six monthly sessions . these materials used in the sbhc intervention resulted in a greater reduction of body mass index when compared to adolescents receiving standard care . conclusions . formative assessment using an adaptive cbpr approach resulted in the creation of culturally and age appropriate weight reduction materials that were acceptable to study participants . this trial is registered with clinicaltrials.gov nct00841334 .

elastosonography is an ultrasound based diagnosis which measures the elasticity or stiffness of the tissue . compressibility of the tissue is a parameter evaluated by an elastogram and it is expressed as the change in tissue displacement as a function of its distance from the probe which is recognised as strain . among the 400450 lymph nodes ( lns ) of the human body , 6070 are located in the head and neck area . lns display reactive enlargement in infection and also when they are primarily or secondarily involved in malignancies of head and neck . an important criterion is the hardness of the ln , because lns might demonstrate increased stiffness on the elastogram if affected by a malignant process [ 24 ] . the purpose of this study is to determine the reference sonoelastography values of cervical lns . after obtaining the approval of the ethical committee , patients referred to the radiology department for cervical vascular doppler ultrasonography examination from january to march 2013 were enrolled in this prospective study . 97 cervical lns of 89 consecutive patients ( 70 lns in 64 females , 27 lns in 25 males ; mean age 54 years old ; age range , 2184 years ) who were referred to our clinic for the examination of cervical vasculature were analysed by elastosonography . one radiologist with 20 years of conventional sonography and 5 years of elastosonography experience performed the examination . the scanner used was esaote gold platform mylab 60 ( italy ) equipped with elasto software ( elaxto ) . b - mode images were first obtained for each ln . afterwards elastosonography was performed by applying light vertical pressure followed by decompression until a good quality image was obtained . elaxto software also made the sonographer aware of the compression degree by the help of its unique optimal compression scale for measurement . real time b - mode and elastosonographic images were demonstrated next to each other on the screen . a region of interest ( roi ) box was placed in the ln taking the adjacent subcutaneous fat tissue as the reference . depth , length , width , length to width ratio , hilar thickness , and cortical thickness of the lns were measured . lns with cortical thickness less than half of its hilar thickness were categorised as group a , and lns with cortical thickness equal to or more than half of the hilar thickness were categorised as group b. first strain ratio ( sr ) of the lns was measured . afterwards , elastographic images were given 1 of 4 elasticity scores ( ess ) based on the percentage of stiff areas modified from choi et al . which was introduced for the differential diagnosis of axillary lns in breast cancer . es 1 ( figure 1 ) , an absent or a very small red area ( i.e. , stiff ) ; es 2 ( figure 2 ) , small , scattered red areas , total red area < 45% ; and es 3 ( figure 3 ) , large red area , total red area 45% ; and es 4 ( figure 4 ) , red area occupying the entire ln , were the classifications used in the study . 64 ( 66% ) of the lns were found on the right side and 33 ( 34% ) were found on the left side of the neck . the mean size of the lns was 17 mm with a range of 628 mm . the mean depth of the lns was 11 mm , with a range of 519 mm . 18 lns ( 18.6% ) were categorised as group a. 79 lymph nodes ( 81.4% ) were categorised as group b. 67 lns ( 69.1% ) had sr less than 3 and were categorised as group 1 . 30 lns ( 30.9% ) with sr equal to or more than 3 were categorised as group 2 . 32 lns ( 33% ) displayed es 1 ; 30 lns ( 30.9% ) demonstrated es 2 ; 22 lns ( 22.7% ) showed es 3 ; 13 lns ( 13.4% ) had es 4 . 62 lns ( 63.9% ) with scores 1 and 2 were assigned as es grade 1 ; 35 lns ( 36.1% ) having scores 3 and 4 were assigned as es grade 2 . there was a statistically significant correlation between thickness ratio groups and elasticity score grades ( p : 0.011 ) . a statistically significant correlation was also demonstrated between sr groups and elasticity score grades ( p : 0.000 ) . elastosonography , a noninvasive imaging modality , can potentially help the differentiation of benign and malignant lns by reducing the number of unnecessary biopsies . in the literature , several studies have been published for the sonographic diagnosis of metastatic cervical lns by b - mode [ 610 ] . b - mode criteria for evaluating superficial lns are size , shape , the presence or the absence of the hilum , ratio of the cortex to hilum , borders , echogenicity , and homogeneity of internal structures . however no single ultrasonography criterion for malignant lns had high sensitivity and specificity [ 11 , 12 ] . specific criteria to differentiate reactive and metastatic cervical lns are still not well established . a simple , reproducible , noninvasive imaging technique for diagnosis of malignant lns is necessary . sohn et al . investigated the b - mode criteria to differentiate metastatic lns from benign lns taking fine needle aspiration biopsy as the gold standard . they evaluated the diagnostic performance of each ultrasound feature ( loss of fatty hilum , presence of cystic change or calcification , hyperechogenicity , and round shape ) . they concluded that the most accurate ultrasound criterion to differentiate metastatic from benign lymph nodes was the presence of one of these malignant ultrasound findings , excluding the loss of fatty hilum . the 5-point elasticity scoring system was introduced by itoh et al . for breast lesions . published their results in the diagnosis of cervical ln metastasis using gray - scale sonographic elastography . they utilized a 4-point scale including visibility , relative brightness , margin regularity , and margin definition of the ln . they measured strain index of the lns by comparing the absolute strain values of the lymph nodes with the absolute strain values values of the nearby muscles . strain index greater than 1.5 was the most useful criteria in metastatic ln classification , having 98% specificity , 85% sensitivity , and 92% accuracy . alam et al . used a 5-pattern color scoring system based on distribution and percentage of area with high elasticity in the cervical lns . the cut - off line for reactive versus metastatic lns was set between patterns 2 and 3 ; patterns 35 were considered metastatic . they evaluated the sum of scores of five criteria , that is , short - axis diameter , shape , border ( regular or irregular ) , echogenicity ( homogeneous or inhomogeneous ) , and presence or absence of hilum for b - mode diagnosis . the cut - off line for reactive versus metastatic was set between scores 6 and 7 . sensitivity , specificity , and accuracy of b - mode sonography were 98% , 59% , and 84% , respectively ; 83% , 100% , and 89% for elastography ; and 92% , 94% , and 93% for the combined evaluation . they concluded that the combination of elastography and b - mode sonography has the potential to improve the diagnosis of metastatic enlarged cervical lns . concluded that elastosonography had 93.8% sensitivity and 89.5% specificity in the differentiation of benign and malignant cervical lymph nodes . in a meta - analysis of the real - time elastography for the differentiation of benign and malignant superficial lns ying et al . concluded that even though es measurement had a good diagnostic accuracy , it had significant interobserver variability thus , a quantitative method such as strain ratio measurement was needed for analysis of elasticity of the ln . although the accuracy of strain ratio measurement was similar to es measurement , it improved the diagnostic sensitivity value . their meta - analysis suggested that real - time elastography could be used as a good identification tool for malignant lns . nevertheless only a limited number of studies have been carried out to determine the elasticity of various tissues . levinson et al . evaluated the elasticity of skeletal muscles and arda et al . conducted a study to determine the elasticity values of normal soft tissue including thyroid , parotid , and submandibular glands , gastrocnemius and masseter muscles , supraspinatus and achilles tendons , renal cortex , and pelvis , spleen , and pancreas of healthy volunteers . estimation of tissue elasticity is useful for characterization of normal tissue . to our knowledge , the normal elasticity values of cervical lymph nodes have not been studied so far . in our study 36.1% of the evaluated cervical lns the aim of elastography is not necessarily to diagnose malignancies but to help reduce the number of unnecessary biopsies for benign processes by determining soft versus hard tissues due to its high specificity [ 2 , 3 , 19 ] . elastosonography is a user dependent technique closely related to experience . due to the shape of the neck , the pulsations from the neighboring great vessels may also make it difficult to obtain good quality elastograms . the primary aim of this research was to determine the reference values of cervical lns to guide elastographic studies not to establish the benign versus malignant nature of the lymph nodes .

background . it is crucial to differentiate between reactive and malignant lymphadenopathies . elastosonography measures the elasticity of the tissue . having a reference value for benign lymph nodes ( lns ) is important in interpretation . the purpose of this study is to determine the reference elastosonography values of cervical lns . methods . 97 lns were evaluated by b - mode and elastosonography . depth , length , width , length to width ratio , hilar - cortical thickness , strain ratio , and elasticity scores were measured . results . in 18.6% of the cervical lns cortical thickness was less than the hilar thickness ( group a ) and in 81.4% it was equal or more ( group b ) . 69.1% of lns showed strain ratio ( sr ) less than 3 ( group 1 ) and 30.9% equal to or more than 3 ( group 2 ) . 33% of lns displayed elasticity score ( es ) 1 ; 30.9% es 2 ; 22.7% es 3 , and 13.4% es 4 . there was a significant correlation between thickness ratios and elasticity scores ( p : 0.011 ) . a significant correlation was also demonstrated between sr groups and elasticity scores . conclusion . a simple , reproducible , noninvasive imaging technique for diagnosis of malignant lns is necessary . elastosonography can aid in the differentiation of benign versus malignant cervical lns , thus help reduce the number of unnecessary biopsies for benign processes .

a 9-month - old , 10-kg girl without any relevant medical history was presented at our emergency room because she had aspirated a peanut 5 hours previously . chest computed tomography showed a foreign body at the carina and the proximal right main bronchus , and emphysema in both lung fields ( fig . anesthesia was induced intravenously with thiopental 60 mg and rocuronium 6 mg after administering atropine 0.2 mg . approximately 25 minutes after the start of the operation , spo2 gradually decreased to 78% from 100% . accordingly , the surgeon was asked to stop the procedure , and an uncuffed endotracheal tube ( i d 4.0 , euromedical , denmark ) was intubated and fixed at 10 cm . left lung sounds were heard clearly , but no right lung sounds were evident on auscultation . spo2 did not improve despite manual ventilation with 100% o2 and an increased inspiratory pressure airway was detected . accordingly , a simple chest radiograph was taken . while waiting for the result of the chest radiograph , the otolaryngologist extubated the endotracheal tube and attempted to re - insert the rigid bronchoscope in order to check for possible causes of the airway obstruction , such as , residual foreign body . meanwhile , the heart rate decreased to 50 beats / min , and spo2 decreased to 57% ( blood pressure was not measured ) . atropine 0.2 mg and epinephrine 1 g were injected intravenously , but vital signs did not improve and no carotid artery pulse was palpable . external cardiac compression was performed and epinephrine 10 g was injected intravenously three times . heart rate and blood pressure then recovered , and thus , cardiac compression was stopped . a chest tube was immediately inserted into the right thorax and a massive air leak was observed . at this time , the sounds of both lungs were clearly heard and spo2 improved to 100% . follow up chest radiography showed a significant reduction of the right tension pneumothorax and a scattering of subcutaneous emphysema at the left upper chest and around the neck ( fig . the tension pneumothorax suggested major tracheobronchial injury and a subsequent bronchoscopic examination revealed three major sites of laceration , one in the mid trachea of length 1.5 cm , a second in the distal trachea of length 2 cm ( immediately above the carina ) ( fig . 4 ) , and a third in the right main bronchus of length 1 cm . an arterial line was placed in the left radial artery to monitor blood pressure continuously . arterial blood gas analysis showed ; ph 7.10 , paco2 63 mmhg , pao2 77 mmhg , hco3 19.6 mmol / l at fio2 1.0 , etco2 24 mmhg , and spo2 100% . after a rigid bronchoscope examination confirmed the absence of remaining foreign body , an uncuffed i d 4.5 endotracheal tube was inserted into the left main bronchus for single lung ventilation under fiberoptic bronchoscopic guidance . however , the patient did not tolerate single lung ventilation well and her spo2 decreased to 79% . in view of the facilitation of surgical approach and the risks of hypoxemia and hypoventilation , after 1,000 units of heparin was injected as a bolus , a 10 fr catheter ( fem - flex ii femoral cannula , edwards lifesciences , irvine , ca , usa ) was placed in the right internal jugular vein for drainage , and a 12 fr catheter was placed in the right femoral vein for perfusion using a sono - guided seldinger technique ( fig . 5 ) . ecmo flow was maintained at 0.7 - 0.8 l / min , and spo2 remained at 98 - 100% without mechanical ventilation . at the initiation of ecmo , anesthesia was maintained with continuous intravenous infusions of sufentanil 5 g / kg / h , midazolam 0.1 mg / kg / h , and vecuronium 0.1 mg / kg / h during ecmo . dopamine was infused at 5 g / kg / min and vital signs were stabilized . an activated clotting time ( act ) of approximately 200 seconds was maintained and arterial blood gas analysis under maximal ecmo support showed ; ph 7.34 , paco2 41 mmhg , pao2 81 mmhg , and hco3 22.1 mmol / l at fio2 1.0 . when the ecmo flow rate reduced , it was recovered by pulling the cannula in the internal jugular vein slightly . body temperature , measured using an oral thermometer , fell to as low as 35.0 after ecmo was initiated , and thus , a forced air warmer was applied to prevent further hypothermia . total anesthesia time was 8 hr 25 min , estimated blood loss was 300 ml , and urine output was 120 ml ; 250 ml of crystalloid solution and 1 unit of packed red blood cells were administered . ecmo was maintained postoperatively to minimize airway pressure . in the intensive care unit ( icu ) , the mechanical ventilation was performed using pressure control mode ( inspiratory airway pressure 10 cmh2o , pressure support 11 cmh2o , respiratory rate 20/min , fio2 0.5 and peep 3 cmh2o ) , which resulted in the expiratory tidal volume of 49 ml . vital signs were stable in the icu , and the patient awoke with no signs of respiratory or neurologic deficit on the first postoperative day . the simple chest radiograph taken in the icu showed reduced subcutaneous emphysema and no residual pneumothorax . ventilating bronchoscopy has the risk of severe complications , such as , infection , laryngeal edema , laryngospasm , bronchospasm , bleeding , hypoxia , pneumomediastinum , and pneumothorax . rothmann and boeckman reported that the probability of pneumothorax during rigid bronchoscopy for the removal of a foreign body is approximately 1% . therefore , the possibility of pneumothorax should be suspected when ventilation worsens during ventilating bronchoscopy . however , an early diagnosis of pneumothorax during anesthesia is not easy because the symptom is masked by anesthesia . tension pneumothorax during surgery may manifest as a reduction in oxygen saturation , a rapid increase in airway pressure , hypotension , and/or tachycardia . in this case , the reasons for the delayed diagnosis in the present case were that the reduction in oxygen saturation and the diminished right lung sounds were mistakenly attributed to airway obstruction by a foreign body . in addition , if lung sounds had been checked more carefully , it might have been easier to determine whether the respiratory failure was due to foreign body obstruction or tension pneumothorax . furthermore , it would have been safer to evaluate airway with a fiberoptic bronchoscope inserted through the endotracheal tube under ventilation rather than attempt airway assessment using a ventilating bronchoscope after extubation . during surgery on the trachea and bronchus , a double lumen endotracheal tube or however , in small children , because conventional single lung ventilation methods are likely to fail to maintain adequate ventilation and oxygenation , the elective application of ecmo may be a good option . ecmo was also useful during the postoperative course in the described case because it can minimize inspiratory airway pressure during mechanical ventilation . during ecmo , heparin was administered as a single bolus of 100 unit / kg followed by continuous intravenous infusion at 20 - 60 unit / kg / min . anticoagulation was accomplished by titrating heparin administration to maintain an act of between 180 and 220 seconds . furthermore , levels of blood anticoagulation were monitored hourly , and the amount of heparin administered was carefully controlled to prevent bleeding complications . in addition , because the ecmo machine was not equipped with a sevoflurane vaporizer and almost no mechanical ventilation was performed during ecmo , intravenous opioids and midazolam were added for anesthesia . summarizing , the tension pneumothorax should be considered when inspiratory pressure is increased and oxygen saturation is decreased during ventilating bronchoscopy . effective communication between the operator and anesthesiologist is essential when unexpected complications are encountered to facilitate the prompt planning of coping strategies .

tension pneumothorax during ventilating bronchoscopy for foreign body removal is a rare but life - threatening complication . the authors present a case of cardiac arrest caused by tension pneumothorax in a 9-month - old girl who underwent ventilating bronchoscopy for foreign body ( peanut ) removal . tension pneumothorax was due to tracheobronchial lacerations caused by a bronchoscope . the patient was successfully resuscitated by cardiopulmonary resuscitation and chest tube insertion . the airway injury was effectively repaired by thoracotomy under extracorporeal membrane oxygenation .

micropolar fluids are those fluids consisting of randomly oriented particles suspended in a viscous medium , which can undergo a rotation that can affect the hydrodynamics of the flow , making it a distinctly non - newtonian fluid . they constitute an important branch of non - newtonian fluid dynamics where microrotation effects as well as microinertia are exhibited . modelling and analysis of the dynamics of micropolar fluids have been the field of very active research due to their application in a number of processes that occur in chemical , pharmaceutical , and food industry . such applications include the extrusion of polymer fluids , solidification of liquid crystals , cooling of a metallic plate in a bath , animal bloods , exotic lubricants , and colloidal and suspension solutions , for example , for which the classical navier - stokes theory is inadequate . the essence of the theory of micropolar fluids lies in the extension of the constitutive equations for newtonian fluids so that more complex fluids can be described by this theory . in this theory , rigid particles contained in a small fluid volume element are limited to rotation about the centre of the volume elements described by microrotation vector . it is well known that heterogeneous mixtures , such as ferro liquids , colloidal fluids , most slurries , and suspensions , are some liquids with polymer activities which behave differently from newtonian fluids . the main difference is that these types of fluids have a microstructure and exhibit microrotational effects and can support surface and body couples which are not present in the theory of newtonian fluids . in order to study such types of fluids eringen developed the theory of microfluids which include the effect of local rotary inertia , the couple stress , and inertial spin . this theory is expected to be successful in analyzing the behavior of non - newtonian fluids . eringen also developed the theory of micropolar fluids for the case where only microrotational effects and microrotational inertia exist . he extended the theory of thermomicropolar fluids and derived the constitutive law for fluids with microstructure . an excellent review of micropolar fluids and their applications was given by ariman et al . . in view of lukaszewicz , micropolar fluids represent those fluids which consist of randomly oriented particles suspended in a viscous medium . several authors have studied the characteristic of the boundary layer flow of micropolar fluid under different boundary conditions . takhar and soundalgekar [ 6 , 7 ] studied the flow and heat transfer of micropolar fluid past a porous plate . further , they [ 8 , 9 ] discussed these problems past a continuously moving porous plate . often experimental and analytical investigations of free convection flows are carried out by the researchers , since in many situations in technology and nature , one continually encounters masses of fluid arising freely in an extensive medium due to the buoyancy effects . [ 10 , 11 ] investigated the natural convection from a heated vertical plate in micropolar fluid . the problem of flow and heat transfer for a micropolar fluid past a porous plate embedded in a porous medium has been of great use in engineering studies such as oil exploration and thermal insulation . raptis and takhar and kim have considered the micropolar fluid through a porous medium . all the above mentioned studies are limited only to applications where radiative heat transfer is negligible . the role of thermal radiation in the flow heat transfer process is of great relevance in the design of many advanced energy conversion systems operating at higher temperatures . thermal radiation within these systems is usually the result of emission by hot walls and the working fluid . nuclear power plants , gas turbines , and the various propulsion devices for aircraft , missiles , satellites , and space vehicles are examples of such engineering areas . perdikis and raptis illustrated the heat transfer of a micropolar fluid in the presence of radiation . raptis studied the effect of radiation on the flow of a micropolar fluid past a continuously moving plate . recently , elbashbeshy and bazid and kim and fedorov have reported on the radiation effects on the mixed convection flow of micropolar fluid . makinde examined the transient free convection interaction with thermal radiation of an absorbing emitting fluid along moving vertical permeable plate . rahman and sattar studied transient convective flow of micropolar fluid past a continuous moving porous plate in the presence of radiation . moreover , when the radiative heat transfer takes place , the fluid involved can be electrically conducting in the sense that it is ionized owing to high operating temperature . accordingly , it is of interest to examine the effect of the magnetic field on the flow . thermal radiation effects on hydromagnetic natural convection flow with heat and mass transfer play an important role in manufacturing processes taking place in industries for the design of fins , glass production , steel rolling , casting and levitation , furnace design , and so forth . the process of fusing of metals in an electrical furnace by applying a magnetic field and the process of cooling of the first wall inside a nuclear reactor containment vessel where the hot plasma is isolated from the wall by applying a magnetic field are examples of such fields where thermal radiation and magnetohydrodynamics ( mhd ) are correlative . this fact was taken into consideration by abd - el aziz in his study on micropolar fluids . raptis and massalas studied magnetohydrodynamic flow past a plate by the presence of radiation . the rotating flow of an electrically conducting fluid in presence of magnetic field has got its importance in geophysical problems . investigation of hydromagnetic natural convection flow in a rotating medium is of considerable importance due to its application in various areas of geophysics , astrophysics , and fluid engineering , namely , maintenance and secular variations in earth 's magnetic field due to motion of earth 's liquid core , internal rotation rate of the sun , structure of the magnetic stars , solar and planetary dynamo problems , turbo machines , rotating mhd generators , rotating drum separators for liquid metal mhd applications , and so forth . it may be noted that coriolis and magnetic forces are comparable in magnitude and coriolis force induces secondary flow in the flow - field . changes that take place in the rotation suggest the possible importance of hydromagnetic spin - up . taking into consideration the importance of such study , unsteady hydromagnetic natural convection flow past a moving plate in a rotating medium mention maybe made of research studies of singh , raptist and singh , tokis , nanousis , and singh et al . . this problem of spin - up in magnetohydrodynamic rotating fluids has been discussed under varied conditions by takhar et al . . the study of heat and mass transfer due to chemical reaction is also very importance because of its occurrence in most of the branches of science and technology . the processes involving mass transfer effects are important in chemical processing equipment which is designed to draw high value products from cheaper raw materials with the involvement of chemical reaction . ibrahim and makinde investigated radiation effect on chemically reactive mhd boundary layer flow of heat and mass transfer past a porous vertical flat plate . babu and satya narayan examined chemical reaction and thermal radiation effects on mhd convective flow in a porous medium in the presence of suction . das and sivaiah investigated studied the effect of chemical reaction and thermal radiation on heat and mass transfer flow of mhd micropolar aid in a rotating frame of reference . convection problems associated with heat sources within fluid - saturated porous media are of great practical significance in geophysics and energy - related problems , such as recovery of petroleum resources , cooling of underground electric cables , storage of nuclear waste materials groundwater pollution , fiber and granular insulations , chemical catalytic reactors , and environmental impact of buried heat generating waste . [ 32 , 33 ] presented an analysis on mhd free convection and mass transfer adjacent to moving vertical plate for micropolar fluid in a rotating frame of reference in presence of heat generation / absorption and a chemical reaction using perturbation technique . babu and narayana analyzed unsteady free convection with heat and mass transfer flow for a micropolar fluid through a porous medium with a variable permeability bounded by a semi - infinite vertical plate in the presence of heat generation , thermal radiation and first - order chemical reaction . the current development of magnetohydrodynamics application is toward a strong magnetic field ( so that the influence of electromagnetic force is noticeable ) and toward a low density of the gas ( such as in space flight and in nuclear fusion research ) . under this condition the rotating flow of an electrically conducting fluid in the presence of a magnetic field is encountered in cosmic fluid dynamics , medicine and biology . mhd was pioneered cowling and he emphasized that when the strength of the applied magnetic field is sufficiently large , ohm 's law needs to be modified to include hall current . the hall effect is merely due to the sideways magnetic force on the drafting free charges . the electric field has to have a component transverse to the direction of the current density to balance this force . in many works of plasma physics , much attention is not paid to the effect caused due to hall current . however , the hall effect can not be completely ignored if the strength of the magnetic field is high and the number of density of electrons is small as it is responsible for the change of the flow pattern of an ionized gas . hall effect results in a development of an additional potential difference between opposite surfaces of a conductor for which a current is induced perpendicular to both the electric and magnetic field . , saha et al . , and ahmed et al . have presented some model studies on the effect of hall current on mhd convection flow because of its possible application in the problem of mhd generators and hall current . preeti and chaudhary analyzed an unsteady hydromagnetic flow of a viscoelastic fluid from a radiative vertical porous plate , taking the effects of hall current and mass transfer into account . studied the heat and mass transfer in unsteady free convection flow with radiation absorption past an impulsively started infinite vertical porous plate subjected to strong magnetic field including the hall effect . investigated the simultaneous effects of hall current and free stream velocity on the magneto hydrodynamic flow over a moving plate in a rotating fluid . recently , seth et al . investigated the problem of an unsteady mhd free convective flow past an impulsively started vertical plate with ramped temperature immersed in a porous medium with rotation and heat absorption taken into account the hall effect . when heat and mass transfer occur simultaneously in a moving fluid , the relations between the fluxes and the driven potential are important . it has been found that an energy flux can be generated not only by temperature gradients but by composition gradient as well . the energy caused by a composition gradient is called the dufour or the diffusion - thermo effect , also the mass fluxes can also be caused by the temperature gradient and this is called the soret or thermal diffusion effect ; that is , if two regions in a mixture are maintained at different temperatures so that there is a flux of heat , it has been found that a concentration gradient is set up and in a binary mixture , one kind of a molecule tends to travel toward the hot region and the other kind toward the cold region . the dufour effect is neglected in this study because it is of a smaller order of magnitude than the magnitude of thermal radiation which exerts a stronger effect on the energy flux . soret or thermal diffusion effect has been utilized for isotope separation in mixtures between gases with very light molecular weight ( h2 , he ) and medium molecular weight ( n2 , air ) and it was found to be of a magnitude that it can not be neglected due to its practical applications in engineering and sciences . soret effects due to natural convection between heated inclined plates have been investigated by raju et al . . m. g. reddy and n. b. reddy investigated soret and dufour effects on steady mhd free convective flow past an infinite plate . mohamed studied unsteady mhd flow over a vertical moving porous plate with heat generation and soret effect . practically , in many engineering applications , the particle adjacent to a solid surface no longer takes the velocity of the surface . the particle at the surface has a finite tangential velocity ; it slips along the surface . this flow regime is called the slip - flow regime and this effect can not be neglected . the fluid slippage phenomenon at the solid boundaries appear in many applications such as microchannels or nanochannels and in application where a thin film of light oils is attached to the moving plates or when the surface is coated with special coating such as thick monolayer of hydrophobic octadecyltrichlosilane , that is , lubrication of mechanical device , where a thin film of lubricant is attached to the surface slipping over one another or when the surfaces are coated with special coating to minimize the friction between them . chaudhary and jain examined the effects of radiation on the hydromagnetic free convection flow set up due to temperature as well as species concentration of an electrically conducting micropolar fluid past a vertical porous plate through porous medium in slip - flow regime . chaudhary and sharma [ 49 , 50 ] studied the free convection flow past a vertical porous plate with variable suction in slip - flow regime . have considered the magnetohydrodynamic unsteady flow of a viscous stratified fluid through a porous medium past a porous flat moving plate in the slip flow regime with heat source . singh and kumar presented the fluctuating heat and mass transfer on unsteady free convection flow of radiating and reacting fid past a vertical porous plate in slip flow regime using perturbation analysis . kumar and chand have studied the effect of slip conditions and the hall current on unsteady mhd flow of a viscoelastic fluid past an infinite vertical porous plate through porous medium . recently , oahimire et al . investigated the effects of thermal - diffusion and thermal radiation on unsteady heat and mass transfer by free convective mhd micropolar fluid flow bounded by a semi - infinite vertical plate in a slip - flow regime under the action of transverse magnetic field with suction . to the best of our knowledge , considerably less work has been done concerning the combined effect of hall current and soret effect on chemically reactive magnetomicropolar fluid flow incorporating the effect of rotation in slip flow regime in the presence of radiation and heat absorption . the results are in accordance with the physical realities which validate the correctness of our work presented here . consider an unsteady hydromagnetic flow of an incompressible , viscous , and electrically conducting micropolar fluid past an infinite vertical permeable plate embedded in a uniform porous medium in slip - flow regime and in a rotating system taking hall current , thermal radiation , soret effect , and chemical reaction into account . the coordinate system is chosen in such a way that x - axis is considered along the porous plate in vertically upward direction , y - axis is taken along the width of the plate , and z - axis normal to the plane of the plate in the fluid as shown in figure configuration ( figure 1 ) . since the plate is infinite in extent in x- and y- directions , hence all physical quantities will be independent of x and y and they are functions of z and t only ; that is , u/x = u/y = v/x = v/y = 0 , and so forth . a magnetic field of uniform strength b0 is applied in a direction parallel to z - axis which is perpendicular to the flow direction . it is assumed that the induced magnetic field generated by fluid motion is negligible in comparison to the applied one . this assumption is justified because magnetic reynolds number is very small for liquid metals and partially ionized fluids which are commonly used in industrial applications . it is assumed that there is no applied or polarized voltage so the effect of polarization of fluid is negligible . this corresponds to the case where no energy is added or extracted from the fluid by electrical means . the entire system is rotating with an angular velocity about the normal to the plate . it is assumed here that the hole size of the porous plate is significantly larger than the characteristic microscopic length scale of the porous medium . the fluid is considered to be a gray , absorbing - emitting but nonscattering medium and the rosseland approximation is used to describe the radiative heat flux . the radiative heat flux in the x - direction is considered negligible in comparison with that of z - direction . when the strength of the magnetic field is very large , the generalized ohm 's law in the absence of electric field takes the following form : ( 1)j+eeb0jh=evh+1enepe . under the assumption that the electron pressure ( for weakly ionized gas ) , the thermoelectric pressure and ion - slip conditions are negligible ; now the above equation becomes ( 2)jx=eh01+m2mvu , jz=eh01+m2mu+v , where u is the x - component of v , v is the y - component of v , and m ( = ee ) is hall parameter . the suction velocity is assumed to be w = w0(1 + aet with these foregoing assumptions , the governing equations under boussinesq approximation can be written in a cartesian frame of reference as follows consider the following : ( 4)ut+wuz2v = +r2uz2+gttt+gccc ukr2z+e2h02mvu1+m2,vt+wvz+2u = +r2vz2uk+r1ze2h02mu+v1+m2 . consider the following : ( 7)ct+wcz=dm2cz2+dmkttm2tz2rccc. the initial and boundary conditions suggested by the physics of the problem are ( 8)u=v=0 , 1=2=0,t = t , c=ckkkkkkkkkkkkkkikkfor t0,(9)u=ur+luz , v=0,1=12vz , 2=12uz,t = tw , c=cwikkkkkkkkkkkkkkkkkkkat z=0u0 , v0 , 10,20 , tt , ccikkkkkkkkkkkkkkkkkkkkkias zikkkkkkkkkkkkkkkkkkkkkkikfor t>0 . the boundary condition for microrotation components 1 and 2 describes its relationship with the surface stress . in the above boundary condition ( 9 ) the plate is in uniform motion and subjected to variable suction and slip boundary condition . in the parameter l = ( ( 2 m1)/m1)l , l is the molecular mean free path and m1 is the tangential momentum accommodation coefficient . integration of continuity equation ( 3 ) for variable suction velocity normal to the plate gives ( 10)w=w01+aet , where w0 represents the normal velocity at the plate which is positive for suction and negative for blowing . the radiative heat flux term by using rosseland approximation is given by ( 11)qr=43art4z. we assume that the temperature differences within the flow are such that t may be expressed as a linear function of the temperature t. this is accomplished by expanding t in a taylor series about t and , neglecting higher - order terms , we have ( 12)t44t3t3t4 . by using ( 11 ) and proceeding with analysis , we introduce the following dimensionless variables : ( 14)u = uur , v = vur , z = zur,t = tur2 , =ur2 , 1=1ur2 , 2=2ur2,gr=gttwtur3 , gc=gccwcur3,r=2ur2 , s = w0ur , =r,=tttwt , c = cccwc , k = kur22,m=eh0ur , =j , pr=cpk , sc=dm , f=4t3kar , q = q2ur2k , sr = dmkttwttmcwc , =rcur2 , h = lur. in view of ( 14 ) , the governing equations ( 4)(7 ) and ( 13 ) reduce to the following dimensionless form : ( 15)uts1+aetuzrv = 1+2uz2+gr+gmm21+m2 + 1ku 2z+mm21+m2v,(16)vts1+aetvz+ru = 1+2vz2m21+m2 + 1kv+1zmm21+m2u,(17)1ts1+aet1z=21z2,(18)2ts1+aet2z=22z2,(19)ts1+aetz=1pr1 + 4f32z2qpr,(20)cts1+aetcz=1sc2cz2+sr2cz2c . the boundary conditions ( 8)-(9 ) in view of ( 14 ) are then given by the following dimensionless form : ( 21)u = v=0 , 1=2=0 , =0 , c=0kkkkkkkkkkkkkkkkkkkkkkkkkkkkkfor t0u=1+huz , v=0 , 1=12vz,2=12uz , =1 , c=1kkkkkkkkkkkkkkkkkkkkkkkkkat z=0u0 , 10 , 20,0 , c0kkkkkkkkkkkkkkkkkkkkas zkkkkkkkkkkkkkkkkkkkkkkkfor t>0 . to simplify ( 15)(18 ) , we substitute the fluid velocity and angular velocity in the complex form as v = u + iv , = 1 + i2 and we get ( 22)vts1+aetvz+irv = 1+2vz2+gr+gmm21+m2 + 1kv izimm21+m2v,ts1+aetz=2z2 . the associated boundary conditions ( 21 ) become ( 23)v=0 , =0 , =0 , c=0kkkkkkkkkkkkikkkkkkkkkkfor t0v=1+huz , =i2vz , =1 , c=1kkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkiat z=0v0 , 0 , 0 , c0kkkkkkkkkkkkkkkkkkikkkkkkkkkkas zkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkfor t>0 . in order to reduce the above system of partial differential equations to a system of ordinary differential equations in dimensionless form , we represent the translational velocity v , microrotation velocity , temperature , and concentration c as ( 24)vz , t = v0z+etv1z+o2,z , t=0z+et1z+o2,z , t=0z+et1z+o2,cz , t = c0z+etc1z+o2 . by substituting the above equations ( 24 ) into ( 19 ) , ( 20 ) , ( 22)-(23 ) and equating the harmonic and nonharmonic terms and neglecting the higher - order terms of o( ) , we obtain the following pairs of equations for ( v0 , 0 , 0 , c0 ) and ( v1 , 1 , 1 , c1 ) . zero - order equations are : ( 25)1+v0+sv0a1v0+gr0+gmc0+i0=0,0+s0=0,3 + 4f0+3prs03q0=0,c0+sscc0scc0=sr0. first - order equations are : ( 26)1+v1+sv1a2v1+gr1+gmc1 + av0+i1=0,1+s11=sa0,3 + 4f1+3prs13q+pr1 = 3prsa0,c1+sscc1sc+c1=sscac0srsc1. the prime denotes differentiation with respect to y. the corresponding boundary conditions can be written as ( 27)v0=1+hv0z , v1=hv1z,0=i2v0z , 1=i2v1z,0=1 , 1=0 , c0=1 , c1=0 at z=0v00 , v10 , 00,10 , 00 , 10,c00 , c10kkkkkkkkkkkas z. solving ( 25)-(26 ) satisfying the boundary conditions ( 27 ) we obtain the expression for translational velocity v , microrotation velocity , temperature , and concentration c as ( 28)vz , t = b11er5z+b8er1z+b9er3z+b10es/z+etb20er7z+b13er1z+b17er5z+b18es/z+b19er6z + b14er2z+b15er4z+b16er3z + b17er5z+b18es/z+b19er6z,(29)z , t = d1es/z+etd2er6z+b12es/z,(30)z , t = er1z+etb1er1zer2z,(31)cz , t = b3er3z+b2er1z+etb4er3z+b5er1z + b6er2z+b7er4z . the exponential indices and the coefficients appearing in ( 28)(31 ) are given in the appendix . in technological applications , the wall shear stress , the wall couple stress , and the heat and mass transfer rate are often of great interest . the skin friction coefficient ( cf ) at the wall in dimensionless form is given by ( 32 ) cf=wz=0ur2=1+1+i2vzz=0(33 ) = 1+1+i2 b11r5+b8r1+b9r3+b10s+b17r5+b18s+b19r6 + etb20r7+b13r1+b17r5+b18s+b19r6 + b14r2+b15r4+b16r3 + b17r5+b18s+b19r6 . the couple stress coefficient ( cm ) at the plate is defined by ( 34)mw=zz=0 and in the dimensionless form it is given by ( 35)cm = mwjur=1+2zz=0=1+21zz=0+i2zz=0=1+2d1s+etd2r6+b12s. knowing the temperature field , it is interesting to study the effect of the free convection and thermal radiation on the rate of heat transfer and this is given by ( 36)qw=ktz43art4zz=0 . using t4tt 3t the above equation becomes ( 37)qw=ktwtur1 + 4f3zz=0 . the rate of heat transfer between the fluid and the plate is studied in terms of nondimensional nusselt number , which is given by ( 38)nu = xqwktwt=rex1 + 4f3zz=0 , where rex = urx/ is the local reynolds number ( 39)nurex1=1 + 4f3zz=0=1 + 4f3r1+etb1r1r2 . the definitions of the local mass flux and the local sherwood number are , respectively , given by ( 40)jw=dmczz=0,shx = jwxdmcwc=rexczz=0,shxrex1=czz=0=b3r3+b2r1+etb4r3+b5r1+b6r2+b7r4 . in the preceding sections , the governing equations along with the boundary conditions are solved analytically employing the perturbation techniques . the effects of main controlling parameters as they appear in the governing equations are discussed on the temperature , concentration c , translational velocity v , microrotation , skin - friction cf , nusselt number , and sherwood number . in order to get a physical insight of the problem the above physical quantities are compiled numerically and displayed graphically . in the entire calculations we have chosen = 0.01 , = 0.1 , t = 1 and a = 1 while pr , s , f , q , sr , sc , m , m , gr , gm , r , h , k , , and are varied over the range which are listed in the figure legends . the numerical values of fluid temperature computed from the analytical solutions ( 29 ) are illustrated graphically versus boundary layer coordinate z in figure 2 for various values of prandtl number ( pr ) , suction parameter ( s ) , heat absorption parameter ( q ) , and radiation parameter ( f ) . the values of prandtl number are chosen as pr = 0.71 , 0.025 , and 7.0 which physically correspond to air , mercury , and water at 25 temperature and one atmospheric pressure . it is inferred that the temperature falls more rapidly for water in comparison to air which is physically true thus the thermal boundary layer falls quickly for large value of prandtl number . the thickness of thermal boundary layer is greatest for pr = 0.025 ( mercury ) than for pr = 0.71 ( air ) , thereafter for pr = 7 ( water ) and finally the lowest for pr = 11.62 ( water at 4c ) ; that is , an increase in prandtl number results in a decrease of temperature . the reason underlying such a behavior is that pr signifies the relative effects of viscosity to thermal conductivity and smaller values of prandtl number possess high thermal conductivity and therefore heat is able to diffuse away from the surface faster than at higher values of pr . the fluid temperature also decreases with an increase of heat absorption parameter ( q ) and suction parameter ( s ) . the temperature decreases with an increase in the heat absorption parameter because when heat is absorbed the buoyancy forces decrease the temperature profiles . the effect of thermal radiation parameter ( f ) is to enhance the fluid temperature throughout the boundary layer region . this is consistent with the fact that thermal radiation provides an additional means to diffuse energy because thermal radiation parameter f = 4t/kar and therefore an increase in f implies a decrease in rosseland mean absorption coefficient ar for fixed values of t and k. thus it is pointed out that radiation should be minimized to have the cooling process at a faster rate . the temperature profiles attain their maximum value at the wall and decrease exponentially with z and finally tend to zero as z . hence the accuracy is checked and it validates that the analytical results for temperature is correct . graphical results of concentration profiles c for different values of schmidt number ( sc ) and chemical reaction parameter ( ) are displayed in figure 3(a ) . the values of schmidt number are chosen to represent the most common diffusing chemical species which are of interest and they are sc = 0.22 ( hydrogen ) , sc = 0.3 ( helium ) , sc = 0.6 ( water vapor ) , sc = 0.94 ( carbon dioxide ) and sc = 2.62 ( propylbenzene ) at 25c temperature and one atmospheric pressure . a comparison of curves in the figure show the concentration distribution decreases at all points in the flow field with an increase in schmidt number because smaller values of sc are equivalent to increasing chemical molecular diffusivity ( d ) . this shows that the heavier diffusing species have a greater retarding effect on the concentration distribution . furthermore , it is interesting to note that concentration profiles fall slowly and steadily for hydrogen ( sc = 0.22 ) and helium ( sc = 0.30 ) but falls very rapidly for water vapor ( sc = 0.6 ) and propylbenzene ( sc = 2.62 ) . physically this is true because of the fact that the water vapor can be used for maintaining normal concentration field whereas hydrogen can be used for maintaining effective concentration field . similar effects are seen in the case when chemical reaction parameter ( ) is increased . further , this figure clearly demonstrates that the concentration profiles decrease rapidly when chemical reaction parameter is increased this is due to the fact that boundary layer decreases with an increase in the value of in this system , results in the consumption of the chemical and hence result in decreasing concentration profile . the effects of heat absorption parameter ( q ) and soret number ( sr ) on concentration profiles across the boundary layer are displayed in figure 3(b ) . the results show that concentration boundary layer suppresses with an increase in heat absorption parameter and soret number . the profiles fall rapidly with an increase of soret number and thereafter increase and tend to zero as z . figure 3(c ) is plotted to show the effects of radiation parameter ( f ) and suction parameter ( s ) on the species concentration profiles . it is revealed that the presence of suction parameter diminishes the concentration distribution whereas reverse phenomena are observed with increasing values of radiation parameter . in figures 3(a)3(c ) the concentration profiles attain their maximum value at the wall and decrease exponentially with z and finally tend to zero as z . hence it is found to be in good agreement with boundary condition given in ( 23 ) . moreover these figures provide a check of our analytical solution for the concentration field . the microrotation profiles ( ) against span wise coordinate z incorporating the effect of various parameters influencing the flow field are demonstrated in figures 4(a)4(h ) . it is revealed from figures 4(a)4(h ) that these profiles attain a distinctive maximum value near surface of the plate and decrease properly on increasing boundary layer coordinate z to approach free stream value . figure 4(a ) shows the influence of prandtl number ( pr ) , suction parameter ( s ) and radiation parameter ( f ) on microrotation profiles . physically , it is true due to the fact that an increase in prandtl number increase the viscosity of the fluid , so the fluid becomes thick and consequently leads to a decrease in velocity . this figure further indicates that the microrotation profiles decrease with an increase in suction parameter ( s ) because sucking decelerates the fluid particles through the porous wall and hence reduce the growth of the fluid boundary layer as well as thermal and concentration boundary layers . indicating the usual fact that suction stabilizes the boundary layer growth . this is because when the intensity of heat generated through thermal radiation increases , the bond holding the components of the fluid particle is easily broken and the fluid velocity will increase . from figure 4(b ) it is perceived that microrotation profiles decrease with an increase in heat absorption parameter ( q ) . figure 4(c ) elucidates the influence of magnetic parameter ( m ) and hall parameter ( m ) on microrotation profiles ( ) ; it is clear from these curves that these profiles increase when magnetic parameter and hall current parameter are increased . the profiles corresponding to m = 0 reveals that microelements close to the wall are unable to rotate ; hence , is very small . figure 4(d ) demonstrates the effect of thermal and concentration buoyancy forces , that is , grashof number ( gr ) and modified grashof number ( gm ) on the microrotation profiles . here the negative value of grashof number ( gr < 0 ) , physically , corresponds to heating of the plate while the positive value ( gr > 0 ) represents cooling of the plate . hence , it is observed from the comparison of the curves that an increase in thermal grashof number leads to an increase in the velocity due to an enhancement in buoyancy forces . an increase in grashof number indicates small viscous effects in the momentum equation and consequently causes an increase in the velocity profiles . furthermore , the comparison of the curves illustrates that velocity increases with increasing gm . the modified grashof number ( gm ) represents the relative strength of concentration buoyancy forces to viscous hydrodynamic force . as expected , the fluid velocity increases and the peak value is more distinctive due to an increase in the species buoyancy force . the profiles attain a maximum value near the wall and then decrease rapidly to approach the free stream value . hence we are confident at the accuracy of our solution given by ( 30 ) . for various values of rotational parameter ( r ) , figure 4(f ) presents the effect of viscosity ratio ( ) and material parameter ( ) on . the magnitude of microrotation is greater for a newtonian fluid ( = 0 ) with given parameters as compared with micropolar fluids ( 0 ) . also , it is observed that the magnitude of microrotation profiles decrease with an increase in material parameter ( ) and viscosity ratio ( ) . rarefaction effects that give rise to slip flow become significant when the molecular mean free path is comparable to characteristic length of the system . the microrotation profiles presented in figure 4(g ) incorporate the influence of rarefaction parameter ( h ) and permeability parameter ( k ) . it is noticed that an increase in the value of rarefaction parameter decreases the magnitude of microrotation profiles while the comparison of curves for different values of permeability parameter ( k ) reflects that profiles increase with increasing values of k. a similar behavior is also expected because when we increase the permeability it increases the size of the pores inside the porous medium due to which the drag force decreases and hence the magnitude of microrotation profiles increases . microrotation profiles showing the variation of soret parameter ( sr ) , schmidt number ( sc ) , and generative chemical reaction ( ) are presented in figure 4(h ) . it is analyzed that the influence of sr , sc , and is to reduce the magnitude of microrotation profiles . comparison of the curves in this figure indicate that the magnitude of microrotation profiles is the greatest for helium ( he : sc = 0.3 ) and then for carbon dioxide ( co2 : sc = 0.94 ) and the lowest for propylbenzene ( c9h10 : sc = 2.62 ) . physically it is justified this figure also displays the fact that these profiles decrease during the destructive reaction ( > 0 ) . figures 5(a)5(h ) illustrate graphically the behavior of translational velocity ( v ) versus boundary layer coordinate z for various involved parameters governing the flow field . for various values of prandtl number ( pr ) , suction parameter ( s ) , and radiation parameter ( f ) , it is clearly evident that translational velocity decreases on increasing pr because since prandtl number is the ratio of kinematic viscosity to thermal diffusivity , so as pr increases , the kinematic viscosity of the fluid dominates the thermal diffusivity of the fluid which leads to decreasing of the velocity of the flow field . moreover , it is noticed that velocity first increases in the region adjacent to the plate and then decreases on moving away from the plate with increase in the suction parameter ( s ) showing the suction has a stabilizing effect on the flow field . this figure also incorporates the fact that radiation ( f ) tends to accelerate the translational velocity throughout the boundary layer region . physically , it is true , as higher radiation occurs when temperature is higher and ultimately the velocity rises . the velocity distribution attains maximum value in the neighborhood of the wall and then decrease to approach the free stream value . the effect of heat absorption parameter on translational velocity ( v ) is depicted in figure 5(b ) and it is found that velocity reduces due to the presence of heat absorption parameter ( q ) . figure 5(c ) incorporates the influence of magnetic parameter ( m ) and hall parameter ( m ) on the translational velocity profiles ( v ) . this phenomenon has an excellent agreement with the physical fact that the presence of transverse magnetic field in an electrically conducting fluid always generates a resistive type of force called lorentz force which is similar to drag force and hence serves to decelerate the flow . form this figure it is also found that hall currents ( m ) tends to accelerate the fluid velocity throughout the boundary layer region which is consistent with the fact that hall currents induces flow in the flow field . the combined effect of thermal and concentration buoyancy forces on the translational velocity are depicted in figure 5(d ) . it is evident from this figure that with an increase in grashof number ( gr ) and modified grashof number ( gm ) , which is a measure of thermal and concentration buoyancy forces , there is a substantial growth in the momentum boundary layer for the same reasons as explained earlier in this section . figure 5(e ) depicts the effect of rotational parameter ( r ) on the fluid velocity and it is perceived that rotation tends to retard fluid velocity throughout the flow field . this is due to the reason that coriolis force is dominant in the region near to the axis of rotation . variation of translational velocity profiles for different values of soret parameter ( sr ) , schmidt number ( sc ) , and chemical reaction ( ) are displayed in figure 5(f ) . the comparison of the curves shows that the velocity of the flow field decreases due to an increase in schimdt number and soret number . it is also observed from this figure that velocity decreases during the destructive reaction ( < 0 ) . figure 5(g ) depicts the influence of viscosity ratio ( ) and permeability parameter ( k ) on the translational velocity ( v ) . for different values of permeability parameter this figure shows that velocity increases with increasing values of k while an increasing viscosity ratio ( ) results in an enhancement of the total viscosity in fluid flow because is directly proportional to vortex viscosity which makes the fluid more viscous and so weakens the convection currents and hence the velocity decreases . figure 5(h ) incorporates the effect of slip or rarefaction parameter ( h ) and material parameter ( ) on the translational velocity ( v ) . it is observed that an increase in the values of rarefaction parameter result in an enhancement of the flow field inside the boundary layer . this behavior is readily understood from the velocity slip condition at the surface ( 23 ) . the case when h = 0 corresponds to the no slip condition and in the present case it reduces to the case when the plate moves with constant velocity in the longitudinal direction . the effects are more visible in the region near to the plate and afterwards it fall slowly and steadily to its free stream value as z . lastly the velocity decreases with increasing material parameter ( ) . in figures 5(a)5(h ) we observe that the velocity become maximum in the vicinity of the plate and then decreases away from the plate and finally takes asymptotic values far away from the plate . the numerical values of nusselt number computed from the analytical solution given in ( 39 ) are presented graphically versus time ( t ) in figure 6 for various values of prandtl number ( pr ) , suction parameter ( s ) , heat absorption parameter ( q ) , and radiation parameter ( f ) . it is noteworthy that the prandtl number , suction parameter , heat absorption parameter , and radiation parameter enhance the rate of heat transfer at the surface of the plate . the reason behind this phenomenon is explained earlier in the text . the rate of heat transfer is more for water ( pr = 7.0 ) than that of air ( pr = 0.71 ) . c(0 ) at the porous plate versus time ( t ) . from all these figures it is analyzed that sherwood number increase with an increase in schmidt number ( sc ) , chemical reaction parameter ( ) , soret number ( sr ) , suction parameter ( s ) , heat absorption parameter ( q ) , and radiation parameter ( f ) . the variation of couple stress coefficient ( cm ) for various involved parameters is displayed in figures 8(a)8(h ) versus time ( t ) . figure 8(a ) exhibits that couple stress coefficient decreases with increasing values of radiation parameter ( f ) and suction parameter ( s ) it increases with increasing values of prandtl number ( pr ) . the effect of heat absorption parameter ( q ) on cm is shown in figure 8(b ) and it is found that couple stress coefficient enhances with the rise in the values of q. from figures 8(c)8(f ) it is apparent that the effect of increasing values of magnetic parameter ( m ) , hall parameter ( m ) , grashof number ( gr ) , modified grashof number ( gm ) , rotational parameter ( r ) and viscosity ratio ( ) are to decrease the values of couple stress coefficient whereas reverse effect is found on increasing the values of material parameter ( ) . figure 8(g ) shows a substantial growth in couple stress coefficient with increasing values of slip parameter ( h ) while reverse happen for increasing values of permeability parameter ( k ) . finally , the schmidt number ( sc ) , soret number ( sr ) , and chemical reaction parameter ( ) have the tendency to increase couple stress coefficient and this is clearly visible in figure 8(h ) . from all these figures from figures 8(a)to 8(h ) it is understandable that as time progresses couple stress coefficient ( cm ) is getting enhanced whereas from figures 9(a)to 9(h ) it is visible that skin friction coefficient ( cf ) is getting suppressed for increasing values of time ( t ) . the skin friction is an important phenomenon which characterizes the frictional drag at the solid surface , so the numerical values of skin friction coefficient ( cf ) computed from ( 33 ) is presented in figures 9(a)9(h ) taking different values of f , s , pr , q , m , m , gr , gm , r , sc , sr , , , , k , and h. the skin friction coefficient increases with increasing values of radiation parameter while it decreases with increase in suction parameter , prandtl number , and heat absorption parameter and this fact is depicted in figures 9(a ) and 9(b ) . it is noticed from figure 9(c ) that the skin friction coefficient is reduced due to an increase in magnetic field strength as expected , since the applied magnetic field tends to impede the flow motion and thus reduces the surface friction force while the hall parameter tends to increase the skin friction . figure 9(d ) demonstrates the growth in skin friction for increasing values of thermal buoyancy parameter ( gr ) and modified grashof number ( gm ) because an increase in buoyancy effect in mixed convection flow leads to an acceleration of the fluid flow which increases the friction factor . an opposite trend is observed for increasing values of rotational parameter ; that is , cf decreases with an increase in r. the influence of schmidt number ( sc ) , soret number ( sr ) , and chemical reaction parameter ( ) on skin friction coefficient is exhibited in figure 9(f ) and all these parameters tend to retard the surface friction forces . finally , figures 9(g ) and 9(h ) exhibit a significant growth in cf with increasing values of viscosity ratio , permeability parameter , and slip parameter while reverse happens with increasing material parameter . the effects of various parameters on the temperature , concentration c , translational velocity v , microrotation , skin - friction cf , nusselt number , and sherwood number are examined . from the present calculations , we arrive at the following findings.thermal radiation tends to enhance fluid temperature whereas there is a decrement in fluid temperature with an increase of prandtl number , suction parameter , and heat absorption parameter.the species concentration profiles decrease at all points in the flow field with an increase in schmidt number , chemical reaction parameter , heat generation parameter , soret number , and suction parameter but are enhanced with an increase in radiation parameter while these physical quantities show reverse trend for sherwood number.thermal radiation , magnetic parameter , hall parameter , and permeability parameter tend to enhance the microrotation distribution whereas these physical quantities have reverse effect on couple stress coefficient.microrotation profiles decrease with an increase in prandtl number , material parameter , slip parameter , soret number , schmidt number , and chemical reaction parameter whereas these physical quantities have reverse effect on couple stress coefficient.microrotation profiles and couple stress coefficient decrease with an increase in suction parameter , rotation parameter , and viscosity ratio.thermal radiation parameter , permeability parameter , and slip parameter tend to enhance the translational velocity profiles throughout the boundary layer region and the skin - friction coefficient.prandtl number , magnetic parameter , suction parameter , rotation parameter , soret number , schmidt number , chemical reaction parameter , viscosity ratio , and material parameter tend to enhance both translational velocity profiles and skin - friction coefficient.slip parameter increases the translational velocity profiles but decreases the skin - friction coefficient.thermal radiation parameter , prandtl number , suction parameter , and heat absorption parameter tend to enhance dimensionless rate of heat transfer , that is , nusselt number . thermal radiation tends to enhance fluid temperature whereas there is a decrement in fluid temperature with an increase of prandtl number , suction parameter , and heat absorption parameter . the species concentration profiles decrease at all points in the flow field with an increase in schmidt number , chemical reaction parameter , heat generation parameter , soret number , and suction parameter but are enhanced with an increase in radiation parameter while these physical quantities show reverse trend for sherwood number . thermal radiation , magnetic parameter , hall parameter , and permeability parameter tend to enhance the microrotation distribution whereas these physical quantities have reverse effect on couple stress coefficient . microrotation profiles decrease with an increase in prandtl number , material parameter , slip parameter , soret number , schmidt number , and chemical reaction parameter whereas these physical quantities have reverse effect on couple stress coefficient . microrotation profiles and couple stress coefficient decrease with an increase in suction parameter , rotation parameter , and viscosity ratio . thermal radiation parameter , permeability parameter , and slip parameter tend to enhance the translational velocity profiles throughout the boundary layer region and the skin - friction coefficient . prandtl number , magnetic parameter , suction parameter , rotation parameter , soret number , schmidt number , chemical reaction parameter , viscosity ratio , and material parameter tend to enhance both translational velocity profiles and skin - friction coefficient . thermal radiation parameter , prandtl number , suction parameter , and heat absorption parameter tend to enhance dimensionless rate of heat transfer , that is , nusselt number .

an analysis study is presented to study the effects of hall current and soret effect on unsteady hydromagnetic natural convection of a micropolar fluid in a rotating frame of reference with slip - flow regime . a uniform magnetic field acts perpendicularly to the porous surface which absorbs the micropolar fluid with variable suction velocity . the effects of heat absorption , chemical reaction , and thermal radiation are discussed and for this rosseland approximation is used to describe the radiative heat flux in energy equation . the entire system rotates with uniform angular velocity about an axis normal to the plate . the nonlinear coupled partial differential equations are solved by perturbation techniques . in order to get physical insight , the numerical results of translational velocity , microrotation , fluid temperature , and species concentration for different physical parameters entering into the analysis are discussed and explained graphically . also , the results of the skin - friction coefficient , the couple stress coefficient , nusselt number , and sherwood number are discussed with the help of figures for various values of flow pertinent flow parameters .

as the silver tsunami continues through the us and globally , the number of drivers over the age of 75 will increase significantly . as of 2012 , there were 36 million older americans licensed to drive , which was a 34% increase from 1999 . accidents or unintentional injuries are the 7th leading cause of death in persons 65 and older and are often the result of impairment . in the 64 to 75 age group , injuries from motor vehicle accidents ( mvas ) are the leading cause of injury related death and the second leading cause in the 75 to 84 age group . drivers who are 80 years old and older have 9 times greater fatality rate than drivers aged 25 to 69 . assessment of driving capability in the older adult is becoming an essential aspect of the comprehensive geriatric exam , even though many primary care providers ( pcps ) do not view themselves as experts in this area and may defer driving assessment and capability to the local department of motor vehicles ( dmv ) office . however , a survey conducted by the american association of retired persons ( aarp ) showed that 40% of people surveyed aged 50 and older believed that their pcp could best determine their ability to drive safely [ 5 , p. 92 ] . earlier assessment and intervention lead to prolonged health maintenance and independence , which is an essential aspect that the pcp must consider for the older adult driver . according to the american association of nurse practitioners ( aanp ) ( 2012 ) , 80% of nurse practitioners ( nps ) practice in a primary care setting . currently , there are three np graduates to every one primary care physician graduate in the united states ( us ) , and nps are improving access to care , especially in rural areas where alternative transportation methods may not be available . driver safety is considered a public and personal safety issue and the np has a duty to routinely assess and intervene when appropriate . variations across state laws and regulations regarding drivers make it challenging to have a nationally accepted clinical guideline or standard of care specific to assessment of safe driving . driving regulations , laws , and reporting policies vary according to state and many providers are not fully aware of these laws and policies within the state where they practice . in one state , a healthcare provider can be sued for alerting the dmv of a person 's medical diagnosis , whereas , in another state , the healthcare provider is negligible if they do not report it . these extremes in variability make it confusing and difficult for nps to know what to do and what to follow . report a lack of specific guidelines for primary care providers to use when caring for an older adult driver in the office setting . nps caring for older adults do not have an efficient evidenced - based screening instrument for use during a routine 15-minute office visit . the current guidelines published by the american medical association in collaboration with the national highway traffic safety administration ( nhtsa ) are considered opinion - based according to one author ( [ 5 , 9 ] ) . another source describes the guidelines as comprehensive but lengthy and cumbersome to use during a routine office visit and they are not supported by evidenced - based research . the purpose of this critical incident study was to identify strategies utilized by nps in the assessment and intervention of older adult drivers in southeastern north carolina . the aims of this study were to ( 1 ) determine current practice of np 's assessment of and intervention strategies with older adult drivers in southeastern north carolina ; ( 2 ) assess the nps knowledge deficits in assessing safe driving capability of the older adults ; and ( 3 ) assess nps current knowledge of resources available for older adult drivers and their families . assessment and intervention of safe driving in the older adult healthcare providers have standards of care or published guidelines to assess , treat , and evaluate chronic diseases such as the jnc guidelines for hypertension . a similar guideline for assessment , intervention , and evaluation of driving safety in the older adult would prove beneficial . the aging baby boomer generation started turning 65 in 2011 and , by 2030 , 1 in every 5 persons will be 65 years old or older in the united states . persons 85 years old and older are growing at four times the rate of the us population . american 's average life expectancy has increased at the same time death rates for the 65 to 84 age group have decreased . as our population ages , there is an increased need for specialized geriatric care that includes a comprehensive , holistic approach . personal safety behaviors , including safe driving habits , are part of the shuler nurse practitioner model . this theoretical framework guides the np to focus on prevention of disease and disability through maintenance of maximal function , improvement in healthcare status , rapidly responding to declines , and periodic routine assessments . the schuler model assesses the physiological and the psychological needs and status of the older adult . through this holistic approach , the np assesses various areas of functional capability that affect the older adult 's ability to drive safely . a literature review was performed using these databases : ebsco host , pubmed , nursing @ ovid , proaquest nursing and allied health , science direct , and web of science . keywords used were as follows : older adult , elderly , geriatric , driving , safety , assessment , and nurse practitioner . johnson interviewed 25 nps on their perceptions of their role in assessing driving ability in the older adult driver who lives in an urban region . johnson found nps to be comfortable performing the physical assessment as it relates to driving ability such as vision , hearing , and cognitive status , but they reported being uncomfortable approaching or discussing the topic of driving safety and/or driving termination . johnson also concluded that early assessment , discussions , and intervention of safe driving were preferred by the older adult , rather than a discussion of driving termination . even though this study focused on nps , many other healthcare disciplines such as physicians , social workers , and occupational therapists have a vested interest in driving safety of the older adult . a qualitative research method developed by flanagan , the critical incident technique , was selected as the design for this study . the critical incident technique is a tool used to create a functional description of behavioral activity . according to flanagan , obtaining a description of the activity can be achieved by asking the persons who actually perform the work a series of brief , precise questions aimed at identifying behaviors . flanagan stressed that , in a critical incident study , the sample size is not determined by the number of participants but rather by the number of critical incidents observed or reported and whether the incidents represent adequate coverage of the activity being studied . the primary objective of the critical incident technique is the development of a behavioral classification system or taxonomy to find solutions to practical problems or to determine the prevalence and distribution of critical behaviors . critical incident studies have been widely used by health service researchers to identify and categorize behavioral responses of healthcare providers in significant and decisive situations [ 17 , 18 ] . critical incident interviews aimed at identifying specific behaviors and strategies used by nps when assessing and intervening with older drivers were conducted with a sample of 21 nps recruited through the north carolina nurses association ( ncna ) council of nurse practitioners - coastal region . the open - ended self - administered critical incident survey consisted of the following questions : ( 1 ) think of the last time you cared for an older adult driver in your practice . what assessment strategies or parameters did you use to determine if the driver was safe or not ? ( 2 ) what strategies did you use to approach that driver about the issue of driving safety ? ( 3 ) how did you make the determination to intervene if you felt that the older adult was not safe to drive ? permission was obtained through the institutional review board ( irb ) at an appropriate institution prior to initiating the study . data analysis was performed using statistical package for the social sciences ( spss , version 20.0 ) to assess the demographic characteristics of respondents . the narrative data obtained through the critical incident surveys was analyzed through an inductive classification process developed by flanagan . the critical incidents were initially reviewed by an advanced practice nurse with a specialty in gerontology and an expert in using critical incident methodology to determine which incidents should be included in the analysis . incidents that were judged to be nearly identical or very similar were grouped together to form subcategories of behaviors . the incidents were resorted and discussed by the research team to refine and determine the final set of categories . percentages of agreement between the researchers were calculated for each critical incident analysis . as an estimate of the importance of each category , the number of incidents sorted by the researchers was counted and placed into a hierarchical structure or taxonomy that identified categories and their related frequencies . a total of 21 nurse practitioners in the southeast region of the united states ( us ) participated in this study . the predominantly female sample ( n = 20 , 95% ) had a mean age of 48.3 years ( sd 11.3 years ; range 2968 years ) . the average length of employment as a nurse practitioner was 9.1 years ( sd 8.4 years ) . the majority of the participants earned master 's degree ( n = 18 , 85.7% ) . one held bachelor 's degree with a family nurse practitioner certificate , one had a doctorate of nursing practice ( dnp ) , and one received ph.d . approximately one - half of the participants ( 47.6% ) achieved specialty certifications reflecting widely varied areas of advanced nursing practice including : adult , geriatrics , psychiatric mental health , diabetes , oncology , hospice , and wound , ostomy , and continence nursing . none of the study participants had reported attendance at a continuing education program on dealing with older drivers . ninety - five percent ( n = 20 ) reported that they see older adults ( > 65 years ) who are still driving at least once a week or more often in clinical practice settings . when asked what age they usually start screening for driving ability , 76% ( n = 16 ) indicated that there was no specific age level . two participants ( 10% ) reported initiating driver 's safety screening between the ages of 50 and 65 years , while three ( n = 14% ) began screening at the age of 66 years and above . ninety percent ( n = 19 ) involved family members in discussions about driving safety . six categories of driver behavior prompted nps to consider performing an evaluation for driving safety . examples of the circumstances that trigger an assessment of driving capabilities are presented in table 1 . the participants ' self - rating of competence in assessing older driver 's safety reflected a moderate level of success at 6.6 ( sd 1.3 , 110 scale with 1 very poor and 10 excellent ) . self - ratings of comfort in approaching older adults about their driving safety were higher at 8.0 ( sd 2.3 , 110 scale ) . a total of 89 incidents described strategies that nurse practitioners used in assessing an older driver 's safety . when the incidents were analyzed , 11 major categories emerged with an interrater agreement of 95% . a listing of assessment categories and circumstances that trigger an assessment of driving capabilities is presented in table 1 . the largest category , assessing diminished short - term memory , forgetfulness , or dementia , accounted for 30 incidents ( 34% ) . the use of the mental status exam or the montreal cognitive assessment ( moca ) was mentioned most frequently . nurse practitioners shared the following:i ask them about how many times they are getting lost [ while driving ] . i also ask them how many times do you beep ' your horn at other people , and how many times do other people beep their horns at you ? that 's a big red flag to me . if they ca n't tell me how they drove here from their house , that 's another red flag.i had one patient whose daughter mentioned a concern about driving . i asked her [ the patient ] to draw a clock [ i used the clock drawing test to assess cognition ] and this was really abnormal even though the geriatrician had just done a mental status exam 8 months prior . there was a significant change.i [ conducted a ] mental status exam . the trigger for me was having to repeat the same conversation over and over again to a patient who was asking the same questions over and over again , day to day , and week to week . i ask them about how many times they are getting lost [ while driving ] . i also ask them how many times do you beep ' your horn at other people , and how many times do other people beep their horns at you ? that 's a big red flag to me . if they ca n't tell me how they drove here from their house , that 's another red flag . i asked her [ the patient ] to draw a clock [ i used the clock drawing test to assess cognition ] and this was really abnormal even though the geriatrician had just done a mental status exam 8 months prior . there was a significant change . the trigger for me was having to repeat the same conversation over and over again to a patient who was asking the same questions over and over again , day to day , and week to week . the second largest category , identifying physical frailty , included 21 or 55% of the incidents ( n = 21 ) . specific elements included conducting a fall assessment , assessing [ the patient 's ] ability to turn from side to side , including limited range of motion in the neck , how well they can feel their feet , and assessing whether or not the patient is able to use the dominate foot to control the pedals of the car . participants added the following.i assess their physical ability on walking by watching them walk in ' [ to the office ] and also look at their ability to get up on the exam table and also their balance.i [ look for ] patients who describe a poor balance and i assess vertigo or syncope . i also look for people who are losing visual field related to macular degeneration or other visual changes . most people with this are already limiting their own driving.i observe my patients getting out of and then back into their car . i assess their physical ability on walking by watching them walk in ' [ to the office ] and also look at their ability to get up on the exam table and also their balance . i [ look for ] patients who describe a poor balance and i assess vertigo or syncope . i also look for people who are losing visual field related to macular degeneration or other visual changes . i observe their gait , walking , balance , flexibility , and overall mobility . behavioral incidents in the third largest category , assessing changes in sensory impairment , i ask every patient when they had a last eye exam and will [ evaluate their vision ] with the snellen chart . if a person can not seem to hear me in a normal conversation , i 'll check their ears for impaction and the whisper test . a fourth category , obtaining collateral information from family members , accounted for 11% ( n = 10 ) of the total number of incidents . as one participant stated that a trigger is when an adult child makes a comment such as i wo n't ride with him ' or that they will not allow their grandchildren to ride with the patient . a fifth category , assessing for impulsivity / lack of judgment , accounted for 6% ( n = 5 ) of the total number of incidents , while a sixth category contained incidents that described evaluating medications for drowsiness side - effects ( n = 3 , 3% ) . to a far lesser extent , nurse practitioners described retrieving a self - report of impaired driving or motor vehicle accident ( n = 2 , 2% ) , evaluating symptoms of new onset neurologic disease ( n = 2 , 2% ) , assessing changes in functional status ( n = 2 , 2% ) , assessing history of drugs and/or alcohol abuse ( n = 1 , 1% ) , and evaluating postmyocardial infarction status ( n = 1 , 1% ) . the two most common strategies were using therapeutic communication to initiate a nonthreatening conversation about driving safety and expressing concern for the older adult 's driving safely . a total of 66 critical incidents were reported which described strategies that nurse practitioners use in practice to increase safety in older adults . when the incidents were analyzed , nine categories of behaviors were determined with an interrater agreement of 100% . categories included the following : involving family members , using therapeutic communication , referring patient to occupational / physical therapy , conducting a vision examination , using a step approach to driving termination , using objective data , referring patient to the department of motor vehicles , referring patient to a medical specialist , and limiting medications on the beers list . one study has shown that clinicians and older adult drivers are open to the idea of using a tiered approach to driving assessment in the older adult . the largest category , involving family members , accounted for 24% ( n = 16 ) of the incidents . as one nurse practitioner stated the following.bring the family into it . ultimately , they are the ones to take the keys if the patient is resistant.others added the following . sometimes i will voice safety concerns that have been brought up by the family . if the family is present , i try to talk to them and coach them to do the right thing like take the keys or put the care in another place.i tell families to stop being the caregiver and go back to being a family member and make me the bad guy ' . i do n't want to ride with you ' and that is a pretty strong message.it's important to work with families and ask them , why are you letting the most unable person in the room drive down the street ? ' it usually has to do with the way this family has made decisions in the past as well as cultural issues , and family dynamics . when i brought up the situation to the family , they agreed and the patient stopped driving until his medical situation improved . he 's doing a lot better now.the second largest category involved the use of therapeutic communication strategies ( n = 14 , 21% ) . one participant stated ask the older adult about how they feel about their ability to drive . i show empathy toward him and his loss of independence and having to rely on family for transportation , and i try to be honest about safety concerns.i used therapeutic communication . i told him the concerns such as him not being able to recall the names of the roads that he just drove on to get here.i talked to her conversationally about how she 's been doing and how her family has been doing . i started asking about driving when i asked her about her vision and eye glasses . this made her feel comfortable first so then i asked about this [ the driving ] . bring the family into it . ultimately , they are the ones to take the keys if the patient is resistant . sometimes i will voice safety concerns that have been brought up by the family . if the family is present , i try to talk to them and coach them to do the right thing like take the keys or put the care in another place . i tell families to stop being the caregiver and go back to being a family member and make me the bad guy ' . i do n't want to ride with you ' and that is a pretty strong message . it 's important to work with families and ask them , why are you letting the most unable person in the room drive down the street ? ' it usually has to do with the way this family has made decisions in the past as well as cultural issues , and family dynamics . when i brought up the situation to the family , they agreed and the patient stopped driving until his medical situation improved . i show empathy toward him and his loss of independence and having to rely on family for transportation , and i try to be honest about safety concerns . i told him the concerns such as him not being able to recall the names of the roads that he just drove on to get here . i talked to her conversationally about how she 's been doing and how her family has been doing . i started asking about driving when i asked her about her vision and eye glasses . this made her feel comfortable first so then i asked about this [ the driving ] . a third largest category contained incidents that described incidents where the nurse practitioner referred the patient to occupational or physical therapy ( n = 10 , 15% ) , while 12% ( n = 8) involved conducting a vision screening examination . eleven percent of the incidents ( n = 7 ) described using a step approach to driving termination . first thing is to eliminate driving at night and then i try to find out what 's the most important thing they want to drive to . i have one patient who only wants to drive 2 miles from his home to the gas station where he goes into buy his coffee and newspaper and then he drives home . for the longest time this is the only driving he would do and then came the day when i did n't feel he was safe to do that anymore and we had him stop driving i do n't think you can just take it all away from them unless it 's really imminent that they are going to hurt themselves.work with patient to drive only at certain times of the day and to certain areas that are familiar . i had one patient who had very slow response times so we placed a restriction that he could only drive on certain roads and stay off main roads . first thing is to eliminate driving at night and then i try to find out what 's the most important thing they want to drive to . i have one patient who only wants to drive 2 miles from his home to the gas station where he goes into buy his coffee and newspaper and then he drives home . for the longest time this is the only driving he would do and then came the day when i did n't feel he was safe to do that anymore and we had him stop driving i do n't think you can just take it all away from them unless it 's really imminent that they are going to hurt themselves . work with patient to drive only at certain times of the day and to certain areas that are familiar . i had one patient who had very slow response times so we placed a restriction that he could only drive on certain roads and stay off main roads . additional categories reflected the importance of using objective data when discussing driving safety with older adults ( n = 5 , 7% ) and referring the patient to the department of motor vehicles ( n = 3 , 5% ) . two incidents ( 3% ) described referring the patient to a medical specialist , while one incident ( 2% ) described the importance of avoiding the use of inappropriate , high - risk medications with older adult drivers . when participants ( nurse practitioners ) were asked to list perceived barriers for driving cessation , the most frequently mentioned barriers included resistance from the patient with denial , lack of alternative transportation , spousal denial , and increased isolation . a listing of the most frequent barriers that nurse practitioners face in working with older adult drivers is presented in table 4 . the participants in this study closely represent the national np demographics . compared to the 2012 national aanp sample survey , the demographics of nps nationally were a mean age of 51 years , white ( 85.4% ) , black / african american ( 3% ) , female ( 9.4% ) , having a graduate level degree ( 97.2% ) , and having practiced as a np for 11 years . this study identified strategies used by nps in the assessment and intervention of older adults in one geographical region . obvious limitations of this study are the limitation to one geographical area . to the author 's knowledge , this is one of the first studies to examine self - report behaviors and strategies of nps on this topic . clinical practice for assessing and intervening with older adult drivers varies significantly from provider to provider . nps are educated to practice holistically and place great importance on building rapport and trust with their patients and their families . this trusting relationship will prove beneficial when a difficult conversation such as driving cessation is needed . impaired cognition was the most common reason a np determined a need to intervene . assessing cognitive status , physical frailty , and sensory impairments were the most common physical exam strategies used . obtaining collateral information from family members ranked just below obtaining physical exam or objective data . using therapeutic communication techniques and a concern of safety for the older adult were the most common strategies used in approaching the older adult about driving . nps ranked the most helpful intervention was to involve family members and the least helpful intervention was the lack of family guidance from the provider . dealing with patient concerns about lack of transportation , especially in rural areas , and resistance to driving termination were the two most common perceived barriers . the wide variation in ways which nps make the determination to address driving in older adults and the lack of consistent guidelines for dealing with this issue should be noted . as the supply of primary care physicians is expected to decrease , the number of nurse practitioners ( nps ) is expected to increase 94% between 2008 and 2025 . nps are trained to provide a holistic approach to patient care and approximately half of all nps practice in the primary care setting . standardized curriculum for nurse practitioner students does not require faculty to address concerns of older adult drivers ; therefore , few nps graduate with this training . this could be due to lack of clinical guidelines , and faculty are unclear as to what should be taught . as this is a public safety concern having difficult conversations with older adults such as driving cessation is time consuming and possibly threatens the patient - provider relationship . there is a need for the development of clinical guidelines that nps can use efficiently during a routine office visit . nps feel comfortable using evidenced - based clinical guidelines to guide their clinical practice with common chronic disease management . development of an evidenced - based clinical guideline for assessment and intervention with older adult drivers would be used similarly . our social work and occupational therapist colleagues have been working on this issue for quite some time . occupational therapists have developed guidelines for them to use in assessment of older adult drivers . nps and np students should also be aware of community resources available in their areas of clinical practice . simply making themselves aware of resources available to them and the older adult , such as the online aarp driver safety program that can be found at http://www.aarp.org/home-garden/transportation/driver_safety/ would prove beneficial . this study shows the varied and inconsistent clinical practice of nps assessing and intervening with older adults drivers . who is to say that one strategy is better than the other ? it is clear that nps value the relationship they have with their patients and they value the need to intervene when public safety is at risk . development of evidenced - based clinical guidelines would prove beneficial for nps to use in clinical practice , as long as the guidelines were able to be used quickly and efficiently .

as the silver tsunami continues , assessing and intervening with older adult drivers are becoming an essential aspect of the comprehensive geriatric exam . the current lack of time efficient clinical guidelines is a concern and barrier for nps . the purpose of this study was to identify strategies currently used by nps . the critical incident technique was used to obtain data from a convenience sample of nps . a total of 89 incidents were collected . the perspective of the np can provide important information for developing clinical guidelines to promote individual and community safety .

a stratified , multistage probability cluster sampling design was used in this survey ( 13 ) . based on socioeconomic characteristics , in the first stage of sampling , 22 counties were randomly selected from each of the 6 regions in china . in the second stage , three townships were randomly selected from each of the selected counties . from each of the townships , 2 residential villages were randomly selected ; and 90 households were then randomly sampled from each village for physical examination . one - third of the households were selected to participate in the dietary survey and blood draw . for the present study , we used residents who were living in rural areas and were born between october 1 , 1952 , and september 30 , 1964 , as our analytic population . to minimize misclassification of the exposure periods , subjects who were born between october 1 , 1958 , and september 30 , 1959 , and between october 1 , 1961 , and september 30 , 1962 , were excluded since the exact dates of the start and the end of the chinese famine were not available and not the same across regions . flow chart on the sampling method in each region * of the 2002 china national nutrition and health survey . * the mainland of china is classified into 6 regions defined by the chinese bureau of statistics according to their socioeconomic development . they are metropolis , general city , type i rural site , type ii rural site , type iii rural site , and type iv rural site . subjects were categorized into five exposure cohorts : nonexposed cohort , fetal - exposed cohort , early childhood exposed cohort , mid childhood exposed cohort , and late childhood exposed cohort . subjects who were born between october 1 , 1962 , and september 30 , 1964 , were classified as the nonexposed cohort ; and subjects who were born between october 1 , 1959 , and september 30 , 1961 , were classified as fetal - exposed cohort . subjects who were born between october 1 , 1952 , and september 30 , 1958 , were grouped by every 2 years and were classified into one of the three childhood - exposed cohorts . mean ages for subjects in nonexposed cohort , fetal - exposed cohort , early childhood exposed cohort , mid childhood exposed cohort , and late childhood exposed cohort were 39 , 42 , 45 , 47 , and 49 years , respectively . the chinese famine affected the entire mainland of china , but the severity varied across regions due to different weather conditions , population density , and local policies regarding food shortage ( 7 ) . as previously described , we used the excess death rate of each province to determine the severity of the famine ( 7 ) . the excess death rate was calculated as the percentage change in mortality rate from the mean level in 19561958 to the highest value during the period 19591961 ( 7 ) . an excess death rate of 50% was used as the threshold : regions that had an equal or higher rate than this cutoff were categorized as severely affected famine areas , and otherwise as less severely affected famine areas . we split all five cohorts into severely affected famine areas and less severely affected famine areas . this enabled us to test the hypothesis that the famine effect is stronger in the severely affected famine areas than that in the less severely affected famine areas and to consider both birth cohort effects and regional differences . all subjects were invited for blood collection after an 10 to 14 h overnight fast . the plasma was separated by centrifugation at 3,200 rpm for 1015 min within 1 h of collection , and kept at room temperature without sunshine . fasting plasma glucose ( fpg ) concentration was measured using the glucose oxidize enzymatic method within 3 h of plasma preparation . every tenth sample was measured twice ( the correlation coefficient of duplicate measurements was 0.98 ) . we used criteria proposed by the who expert committee on diabetes mellitus ( 14 ) . mmol / l , including impaired fasting glucose , impaired glucose tolerance , and type 2 diabetes . in addition , subjects who had been previously diagnosed with type 2 diabetes were added as cases of hyperglycemia and type 2 diabetes . dietary patterns , economic status , and bmi measured in 2002 were used as measures of the nutritional environment in adulthood and to examine the mismatch between fetal nutrition and adult nutrition . the method for assessing dietary patterns has been described in detail elsewhere ( 15 ) . briefly , four dietary patterns were derived through cluster analysis , which were labeled as green water , yellow earth , new affluence , and western adopter . green water and yellow earth patterns represent the traditional chinese diets in the south and the north , respectively , whereas the other two represent westernized dietary patterns . in this study , we combined the clusters of green water and yellow earth as the traditional dietary pattern , and we combined the clusters of new affluence and western adopter as the affluent / western pattern . current economic status was assessed by the mean annual income in the year prior to the 2002 cnnhs , which was treated as a dichotomous variable . the mean level of the current sample ( 2,000 chinese yuan per person per year ) was used as a cutoff point for economic status . we used the criteria recommended for chinese adults and classified subjects as overweight if bmi 24 kg / m , or otherwise normal ( 16 ) . the protocol of the 2002 cnnhs was approved by the ethical committee of the national institute for nutrition and food safety , chinese center for disease control and prevention . signed consent forms were obtained from all participants . we performed survey analyses with sas 9.2 for windows ( sas institute , cary , nc ) to estimate statistics for this complex , multistage - designed survey sample . survey weights were derived from the 2000 china national population census and associated administrative data . mean fpg differences between the exposed cohorts and the nonexposed cohort were tested by generalized least squares estimation ( 17 ) . risks of hyperglycemia and type 2 diabetes among fetal and childhood - exposed subjects , compared with nonexposed subject , were examined with the method of maximum likelihood by using the survey logistic regression model . interaction between famine exposure cohort ( fetal- or childhood - exposed vs. nonexposed ) and area ( severely affected and less severely affected ) was tested by adding a multiplicative factor in the survey logistic regression model . analyses were adjusted for sex , family history of diabetes , educational level , current smoking , alcohol use , and physical activity level , all assessed in 2002 . to explore whether the associations between fetal exposure to severe famine and hyperglycemia were affected by an improved nutritional environment in later life , we subsequently stratified the analyses by dietary patterns , economic status and bmi in adulthood . the odds ratio of hyperglycemia in the fetal - exposed cohort compared with the nonexposed cohort was calculated within each category of the stratified factor . to distinguish severely and less severely affected famine areas more appropriately , we performed sensitivity analysis by using a more stringent cutoff point , i.e. , we used an excess death rate 100% to define the severity of famine . in addition , we performed analyses by using the cohort born during october 1 , 1962 , to september 30 , 1968 , as a nonexposed cohort for association analyses , or by excluding participants with a family history of diabetes . subjects were categorized into five exposure cohorts : nonexposed cohort , fetal - exposed cohort , early childhood exposed cohort , mid childhood exposed cohort , and late childhood exposed cohort . subjects who were born between october 1 , 1962 , and september 30 , 1964 , were classified as the nonexposed cohort ; and subjects who were born between october 1 , 1959 , and september 30 , 1961 , were classified as fetal - exposed cohort . subjects who were born between october 1 , 1952 , and september 30 , 1958 , were grouped by every 2 years and were classified into one of the three childhood - exposed cohorts . mean ages for subjects in nonexposed cohort , fetal - exposed cohort , early childhood exposed cohort , mid childhood exposed cohort , and late childhood exposed cohort were 39 , 42 , 45 , 47 , and 49 years , respectively . the chinese famine affected the entire mainland of china , but the severity varied across regions due to different weather conditions , population density , and local policies regarding food shortage ( 7 ) . as previously described , we used the excess death rate of each province to determine the severity of the famine ( 7 ) . the excess death rate was calculated as the percentage change in mortality rate from the mean level in 19561958 to the highest value during the period 19591961 ( 7 ) . an excess death rate of 50% was used as the threshold : regions that had an equal or higher rate than this cutoff were categorized as severely affected famine areas , and otherwise as less severely affected famine areas . we split all five cohorts into severely affected famine areas and less severely affected famine areas . this enabled us to test the hypothesis that the famine effect is stronger in the severely affected famine areas than that in the less severely affected famine areas and to consider both birth cohort effects and regional differences . all subjects were invited for blood collection after an 10 to 14 h overnight fast . the plasma was separated by centrifugation at 3,200 rpm for 1015 min within 1 h of collection , and kept at room temperature without sunshine . fasting plasma glucose ( fpg ) concentration was measured using the glucose oxidize enzymatic method within 3 h of plasma preparation . every tenth sample was measured twice ( the correlation coefficient of duplicate measurements was 0.98 ) . we used criteria proposed by the who expert committee on diabetes mellitus ( 14 ) . mmol / l , including impaired fasting glucose , impaired glucose tolerance , and type 2 diabetes . in addition , subjects who had been previously diagnosed with type 2 diabetes were added as cases of hyperglycemia and type 2 diabetes . dietary patterns , economic status , and bmi measured in 2002 were used as measures of the nutritional environment in adulthood and to examine the mismatch between fetal nutrition and adult nutrition . the method for assessing dietary patterns has been described in detail elsewhere ( 15 ) . briefly , four dietary patterns were derived through cluster analysis , which were labeled as green water , yellow earth , new affluence , and western adopter . green water and yellow earth patterns represent the traditional chinese diets in the south and the north , respectively , whereas the other two represent westernized dietary patterns . in this study , we combined the clusters of green water and yellow earth as the traditional dietary pattern , and we combined the clusters of new affluence and western adopter as the affluent / western pattern . current economic status was assessed by the mean annual income in the year prior to the 2002 cnnhs , which was treated as a dichotomous variable . the mean level of the current sample ( 2,000 chinese yuan per person per year ) was used as a cutoff point for economic status . we used the criteria recommended for chinese adults and classified subjects as overweight if bmi 24 kg / m , or otherwise normal ( 16 ) . the protocol of the 2002 cnnhs was approved by the ethical committee of the national institute for nutrition and food safety , chinese center for disease control and prevention . signed consent forms were obtained from all participants . we performed survey analyses with sas 9.2 for windows ( sas institute , cary , nc ) to estimate statistics for this complex , multistage - designed survey sample . survey weights were derived from the 2000 china national population census and associated administrative data . mean fpg differences between the exposed cohorts and the nonexposed cohort were tested by generalized least squares estimation ( 17 ) . risks of hyperglycemia and type 2 diabetes among fetal and childhood - exposed subjects , compared with nonexposed subject , were examined with the method of maximum likelihood by using the survey logistic regression model . interaction between famine exposure cohort ( fetal- or childhood - exposed vs. nonexposed ) and area ( severely affected and less severely affected ) was tested by adding a multiplicative factor in the survey logistic regression model . analyses were adjusted for sex , family history of diabetes , educational level , current smoking , alcohol use , and physical activity level , all assessed in 2002 . to explore whether the associations between fetal exposure to severe famine and hyperglycemia were affected by an improved nutritional environment in later life , we subsequently stratified the analyses by dietary patterns , economic status and bmi in adulthood . the odds ratio of hyperglycemia in the fetal - exposed cohort compared with the nonexposed cohort was calculated within each category of the stratified factor . to distinguish severely and less severely affected famine areas more appropriately , we performed sensitivity analysis by using a more stringent cutoff point , i.e. , we used an excess death rate 100% to define the severity of famine . in addition , we performed analyses by using the cohort born during october 1 , 1962 , to september 30 , 1968 , as a nonexposed cohort for association analyses , or by excluding participants with a family history of diabetes . basic characteristics of the study population are shown in table 1 . in our main study population ( n = 7,874 ) , 1,005 ( 12.8% ) subjects had been exposed to the chinese famine during fetal life , and 4,915 ( 62.4% ) subjects had been exposed during childhood . as compared with the nonexposed individuals , fetal - exposed subjects were 0.9 cm shorter as adults , and childhood - exposed subjects were 1.5 cm shorter ( table 1 ) . the prevalence of hyperglycemia among adults in the nonexposed , fetal - exposed , early childhood , mid childhood , and late childhood exposed birth cohorts was 2.4% , 5.7% , 3.9% , 3.4% , and 5.9% , respectively . basic characteristics of study population according to chinese famine exposure * * data are adjusted means ( se ) . adjusted factors included sex , educational level , family history of diabetes ( only for glucose ) , current smoking , alcohol use , and physical activity level . compared with the nonexposed cohort , p < 0.05 . in severely affected famine areas , fpg concentration was significantly higher in the fetal - exposed cohort than in the nonexposed cohort with a mean difference of 0.20 no significant difference was observed in the less severely affected famine areas ( p for interaction = 0.001 , table 2 ) . compared with nonexposed subjects , fpg was higher in the late childhood exposed cohort in both the severely affected famine areas and less severely affected famine areas . a significant interaction between the exposed cohort and areas was found only for the fetal - exposed cohort ( table 2 ) . concentrations of fasting plasma glucose and prevalence rates of hyperglycemia and type 2 diabetes by birth cohort and severity of the chinese famine area * data are adjusted means ( se ) for fasting plasma glucose , sex standard prevalence , and odds ratio for hyperglycemia and diabetes . adjusted factors included sex , education level , family history of diabetes , and current smoking , alcohol use , and physical activity level . subjects exposed to famine during fetal life in severely affected famine areas had a higher prevalence of hyperglycemia than the nonexposed cohort . the odds ratios were significantly different between the severe and less severe famine areas ( table 2 ) , suggesting a stronger famine effect in the severely affected famine areas . compared with the nonexposed cohort , subjects in the late childhood exposed cohort had a higher risk of hyperglycemia in both severely and less severely affected famine areas , but the odds ratios were not significantly different between the severe and less severe famine areas ( table 2 ) . a significantly higher prevalence of type 2 diabetes was observed among subjects exposed in late childhood as compared with the nonexposed cohort ( table 1 ) . however , table 2 shows that after stratification of this group by severity of famine exposure , no significant difference of type 2 diabetes risk was observed anymore between different famine cohorts . stratified analyses by dietary pattern , economic status , and bmi for severely affected famine areas are shown in fig . figure 2a1 shows that the prevalence of hyperglycemia was highest ( 18.9% ) in subjects in the fetal - exposed cohort and who consumed an affluent / western diet . as compared with the relatively nonexposed cohort , the odds ratio of hyperglycemia in the fetal - exposed cohort was 7.63 ( 95% ci : 2.4124.1 , p = 0.0005 ) for those who had an affluent / western dietary pattern , and 2.34 ( 95% ci : 0.826.70 , p = 0.112 ) for those with a traditional dietary pattern . prevalence of hyperglycemia among birth cohorts according to early life famine exposure and later life dietary patterns ( a1 and b1 ) , socioeconomic status ( a2 and b2 ) , and bmi ( a3 and b3 ) in severely ( column a ) and less severely affected famine areas ( column b ) . figure 2a2 shows that as compared with nonexposed subjects , the odds ratio of hyperglycemia in the fetal - exposed cohort was 6.20 ( 95% ci : 2.0818.5 , p = 0.001 ) in subjects with a higher adult economic status , and 1.68 ( 95% ci : 0.505.71 , p = 0.404 ) in subjects with a lower adult economic status . figure 2a3 shows that overweight subjects in the fetal - exposed cohort had the highest prevalence of hyperglycemia ( 13.9% ) . however , the risks of hyperglycemia were largely comparable in these two groups ; the odds ratio of hyperglycemia in the fetal - exposed cohort was 3.71 ( 95% ci : 1.1312.2 , p = 0.031 ) in overweight subjects and 4.37 ( 95% ci : 1.1516.5 , p = 0.030 ) in normal weight subjects , respectively , compared with the nonexposed cohort . similar analyses were performed in subjects exposed to less severely affected famine areas during fetal life and childhood ( fig . 2 , right column , graphs b1 , b2 , and b3 ) , but did not show consistent associations . when we defined the severely affected famine areas as those with an excess death rate 100% , the prevalence of hyperglycemia among the fetal - exposed cohort in severely affected famine areas increased to 8.1% , but this did not change the associations between fetal exposure to famine and risk of hyperglycemia in adulthood . in addition , neither using subjects who were born between october 1 , 1962 , and september 30 , 1968 , as a nonexposed cohort nor excluding subjects with a family history of diabetes materially changed the associations ( table 3 ) . prevalence rate of hyperglycemia by birth cohorts and severity of famine areas : sensitivity analyses all odds ratios used the nonexposed cohort as the reference cohort . adjusted factors include sex , educational level , family history of diabetes , and current smoking , alcohol use , and physical activity level in 2002 . in this study of a large sample of chinese adults , we found a significant association between severe famine exposure during the fetal period and an increased risk of hyperglycemia in adulthood . this association was stronger in subjects with a western dietary pattern or higher economic status in adulthood . several mechanisms might explain the associations between fetal famine exposure and risk of diabetes in later life . exposure to extreme starvation in rats led to poor development of pancreatic -cell mass and function and insulin resistance , which might persist in later life ( 18 ) . a poor intrauterine environment may also reduce skeletal muscle development ( 19 ) , which may subsequently lead to insulin resistance in peripheral tissues ( 20 ) . it has also been suggested that stress suffering from fetal famine exposure could change the setpoint of the hypothalamic - pituitary - adrenal ( hpa ) axis , which could result in long - term changes in secretion of neuroendocrine mediators of the stress response , and predispose to cardiovascular and metabolic disease in later life ( 21,22 ) . to our knowledge , thus far three studies have assessed the associations of exposure to famine with measures of glucose intolerance . these studies were performed in the netherlands ( the dutch famine study ) ( 4,5 ) , russia ( the leningrad siege study ) ( 6 ) , and china ( our chinese famine study ) . the dutch famine study reported higher 2-h glucose and insulin levels among subjects who were exposed to famine during fetal life ( 4,5 ) , but this association was not observed in the leningrad siege study ( 6).the inconsistent results might be due to differences in postnatal environmental life exposures . although the dutch population rapidly developed into a wealthy and rich population after the famine , the leningrad people remained relatively poor . in our study , we observed that fetal exposure to the severe chinese famine increases the risk of hyperglycemia in adulthood , which was exacerbated by an unhealthy adult diet and higher economic status . our results support the hypothesis that exposure to a nutritionally rich environment modifies the association between fetal famine exposure and disease in later life ( 1,20,23 ) . the association between fetal famine exposure and hyperglycemia was stronger in participants with an affluent / western dietary pattern . these subjects were , to a large extent , less poor and more highly educated ( 15 ) , and they have benefited most from dramatically enhanced economic opportunities and have broken away from traditional chinese food patterns ( 15 ) . their diet is characterized by a high intake of meat , eggs , dairy , sugary beverages , edible oils , and a low vegetable use ( 15 ) . apparently , this nutrition rich environment did not match the fetal starvation environment that people of fetal exposed cohort experienced , which in turn increased the risk of hyperglycemia in later life ( 1,20,23 ) . our study used annual mean income as the cutoff to categorize economic status ( 2,000 chinese yuan / person / year ) . subjects in the lower economic group might consume mostly traditional plant foods with little meat . therefore , the discrepancy between the nutritional environment in adulthood and fetal undernutrition conditions may be less evident for those with a higher economic status . in other words , there was probably greater mismatch between in utero and adulthood environments in the higher economic group , which triggered an increased prevalence of hyperglycemia in the fetal - exposed cohort . similar results were described in the dutch famine study ( 4 ) , showing that 2-h glucose concentrations were especially high among people exposed to the famine during fetal life and who became obese in later life . however , the relative risk of hyperglycemia in overweight subjects was not different from that in normal weight subjects . this may be partly due to the increased prevalence of hyperglycemia in the nonexposed cohort in overweight subset . these results therefore indicate that both improving fetal nutritional environmental and controlling bmi in later life are important for prevention of a disturbed glucose metabolism . childhood nutritional status , particularly during infancy , is another key factor in influencing the propensity to develop disease in adulthood ( 23 ) . animal studies have shown that postnatal caloric restriction might hamper -cell development ( 24 ) and might disturb glucose metabolism in later life in rats ( 25 ) . our study found significantly increased fpg in the early childhood exposed cohort in the severely affected famine areas , but no significant differences in fpg in the less severely affected famine areas . we also observed a higher risk of hyperglycemia among subjects exposed in late childhood in both severely and less severely affected famine areas . these results suggest that famine exposure during childhood may increase the risk of hyperglycemia in later life . however , we can not exclude a potential cohort effect , such as aging ( 26 ) . similar risks of hyperglycemia among subjects exposed during childhood in both famine - exposed areas and nonfamine - exposed areas suggest rather a cohort ( older age ) effect than a famine effect . however , since almost all rural regions in china were affected by the famine during 19591961 , no valid nonfamine - exposed cohort comprising subjects born in the same time period was available . thus , the association between childhood exposure to famine and risk of hyperglycemia needs to be studied in more detail . first , we assumed that the residents we investigated at the time of the survey were born in the same province and in a similar rural area . however , severe restrictions on migration and relocation in china made our sample quite stable . migration with permanent resident permission still needed to be approved by authorities on a case - by - case basis in china . according to the 2000 china national population census , 2.68% of the rural population lived in provinces other than the provinces of their birthplaces ( 27 ) . our study sample was based on the residence registration system ; only subjects with permanent resident permission in local areas were involved in our study . therefore , we do not expect that intraprovince migration leading to measurement error in the coding of birth place is a major concern in our results ( 12 ) . second , subjects in our fetal - exposed cohort may have actually experienced severe famine during both the fetal period and the infancy period because the famine lasted approximately 3 years . it was therefore difficult to distinguish whether the fetal period or the infancy period was more important . however , the early childhood cohort also included subjects exposed to famine in infancy , which did not have a substantial influence on the risk of hyperglycemia . thus , our results indicate that the fetal period should be considered as the primary critical period . third , our subjects who experienced severe famine in the fetal period were in their early 40s in 2002 , and the cases of type 2 diabetes were few . the small numbers may partly explain why we did not observed significant associations with the risk of type 2 diabetes . we used the excess death rate as an indirect measure of famine exposure . with this method , we could not distinguish death due to famine from death due to unfavorable weather conditions or infections . we also did not have reliable information about individual food availability during the famine period . therefore , from our data , we can not conclude that the higher risk of hyperglycemia among subjects exposed to famine is exclusively due to malnutrition in early life . however , nutrition deficiency was highly prevalent during the chinese famine . china 's grain output declined by 15% in 1959 and in the following 2 years , and its food supply plunged further to 70% of its 1958 level ( 8) . as almost all foods were delivered through communal kitchens at that time , no social groups were spared from the effects of the famine ( 9 ) . however , since the famine effect on glucose intolerance did not depend on birth size in the dutch famine study ( 4 ) , we do not consider the lack of information about individual birth outcomes as a major limitation . in conclusion , we found that exposure to severe famine in fetal life increased the risk of hyperglycemia in adulthood . the mismatched nutrition postnatal environment represented by a western dietary pattern and improved economic status further increased susceptibility to hyperglycemia in those who experienced fetal exposure to famine . together with previous studies , our study emphasizes that early life environment is critical for the risk of hyperglycemia in adult life .

objectiveearly developmental adaptations in response to undernutrition may play an essential role in susceptibility to type 2 diabetes , particularly for those experiencing a mismatched rich nutritional environment in later life . we examined the associations of exposure to the chinese famine ( 19591961 ) during fetal life and childhood with the risk of hyperglycemia and type 2 diabetes in adulthood.research design and methodswe used the data for 7,874 rural adults born between 1954 and 1964 in selected communities from the cross - sectional 2002 china national nutrition and health survey . hyperglycemia was defined as fasting plasma glucose 6.1 mmol / l and/or 2-h plasma glucose 7.8 mmol / l and/or a previous clinical diagnosis of type 2 diabetes.resultsprevalences of hyperglycemia among adults in nonexposed , fetal exposed , early - childhood , mid - childhood , and late - childhood exposed cohorts were 2.4% , 5.7% , 3.9% , 3.4% , and 5.9% , respectively . in severely affected famine areas , fetal - exposed subjects had an increased risk of hyperglycemia compared with nonexposed subjects ( odds ratio = 3.92 ; 95% ci : 1.649.39 ; p = 0.002 ) ; this difference was not observed in less severely affected famine areas ( odds ratio = 0.57 ; 95% ci : 0.251.31 ; p = 0.185 ) . the odds ratios were significantly different between groups from the severe and less severe famine areas ( p for interaction = 0.001 ) . in severely affected famine areas , fetal - exposed subjects who followed an affluent / western dietary pattern ( odds ratios = 7.63 ; 95% ci : 2.4124.1 ; p = 0.0005 ) or who had a higher economic status in later life experienced a substantially elevated risk of hyperglycemia ( odds ratios = 6.20 ; 95% ci : 2.0818.5 ; p = 0.001).conclusionsfetal exposure to the severe chinese famine increases the risk of hyperglycemia in adulthood . this association appears to be exacerbated by a nutritionally rich environment in later life .

calcific aortic valve stenosis has become the most common acquired valve disorder with the highest prevalence in the 8th or 9th decade of life . comorbidities such as diabetes mellitus , stroke , coronary heart disease , peripheral artery disease , pulmonary disease , and renal impairment appear to markedly increase the risk of conventional valve replacement in the elderly and may limit the benefit of surgery in these patients . nevertheless , the outcome of untreated aortic stenosis is dismal once congestive heart failure , angina pectoris , or syncope occur . therefore alternative , less invasive treatment options are needed . since the first - in - man transcatheter aortic valve implantation ( tavi ) by cribier et al . in 2002 , more than 50000 procedures were performed worldwide and this intervention has become an accepted assumingly less invasive treatment alternative for high risk surgical patients . however , due the comorbidities in these patients , even tavi is associated with a number of complications that may lead to impaired outcome . their closer evaluation and the definition of risk factors as well as measures to reduce their occurrence are essential and require further research . acute kidney injury ( aki ) is a well - known complication of angiography with the use of iodinated contrast media that accounts for a significantly prolonged hospital stay and worse in - hospital outcome . furthermore aki has been shown to be an independent predictor of mortality [ 58 ] . the most important risk factor for aki in patients undergoing standard heart catheterization is preexisting chronic kidney disease [ 9 , 10 ] . other risk factors include volume depletion , hemodynamic instability , and the use of nephrotoxic drugs . tavi requires the administration of contrast media and preexisting kidney disease is frequent in the currently treated patient population . however , the incidence of aki , predictors of this complication , and its impact on outcome in patients undergoing tavi have so far been poorly defined . therefore , we sought to assess the incidence of aki , search for its predictors , and analyze its impact on 30-day as well as midterm outcome in a sizeable group of consecutive patients undergoing tavi . a total of 150 consecutive patients with symptomatic aortic stenosis who underwent tavi in our institution because they were either not suitable for conventional surgical valve replacement or were considered at high operative risk by a multidisciplinary team including cardiologists and cardiac surgeons were included in this study . ten out of 150 patients had been enrolled in a chronic dialysis program and were therefore excluded from analysis concerning aki . before intervention , all patients received left and right heart catheterization . according to the institution policy , written informed consent blood samples for hematology and serum chemistry were drawn one day prior to intervention and daily up to 72 hours after treatment . patients with a previously impaired kidney function ( estimated glomerular filtration rate , egfr < 60 ml / min/1,73 m ) received an intravenous prehydration protocol consisting of saline 0.9% with 1200 mg of n - acetyl - cysteine both 12 hours before and after the procedure . the vascular access site was evaluated by color - coded doppler sonography and ct - angiography . valve replacement was performed under general anaesthesia , except in one case which was done under local anaesthesia . all procedures were performed in the catheter laboratory using fluoroscopic guidance and nonionic isoosmolar contrast media iopromide ( ultravist 370 ( tm ) , schering ag , berlin , germany ) and transoesophageal echocardiography . the patients received either a 23 or 26 mm edwards - sapien valve prothesis . renal function at baseline and after 48 hours was determined from serum creatinine determined by the method of jaff . since creatinine is known to be an insufficient marker of renal function but estimated glomerular filtration rate ( egfr ) is considered most suitable we used the modification of diet in renal disease ( mdrd ) formula for calculation : ( 1)egfr ( ml / min/1,73 m2 ) = 186(creatinine , mg / dl)1,154(age , years)0,203 ( 0,742 in women ) . acute kidney injury is divided into three stages by the acute kidney injury network : stage 1 is defined as a rise in serum creatinine 26.5 mol / l compared to baseline values or an increase in serum creatinine of more than or equal to 50% or a reduction in urine output as documented oliguria of less than 0.5 ml / kg per hour . in this study aki was defined as a 26.5 mol / l rise in serum creatinine 48 hours after procedure compared to baseline data drawn 24 hours before intervention . logistic euroscore was calculated by the web - based system ( http://www.euroscore.org/ ) in advance and results taken for clinical decision making . sts score was evaluated retrospectively for further analysis including the predicted risk of renal failure ( http://209.220.160.181/stswebriskcalc261/de.aspx ) . renal failure by sts score is defined as an increase of serum creatinine > 176.8 mol / l , a 50% or greater increase in serum creatinine over baseline preoperative value , or new requirement for dialysis . differences in basic clinical characteristics between groups were tested by chi - square test for categorical and the anova f - test for continuous variables . uni- and multivariate predictors of aki were assessed by logistic regression analysis and odds - ratios ( or ) are reported . univariate predictors of mortality during followup were analyzed by cox regression and calculation of hazard rate ratios ( hr ) with 95% confidence intervals ( 95% ci ) . multivariate analysis of mortality was performed by cox regression analyses with potential covariates ( adjusted hr ) . as covariates for adjustment , those parameters were chosen which were found to have a p value lower than 0.05 in univariate analyses of death . the mean age in the patient group was 81 7 years . in total , 96 patients received valve replacement via transfemoral ( tf ) and 54 patients via transapical ( ta ) approach . patients with transapical approach were more frequently male and had significantly more underlying comorbidities such as hypertension , pad , chd , previous cabg , previous stroke , and impaired kidney function based on baseline serum creatinine measurement ( see table 1 ) . ten patients ( 7% ) had already been enrolled in a chronic dialysis program before intervention ( 5% of tf versus and 9% of ta patients , p = 0.006 ) and were therefore excluded from analysis concerning aki . the average amount of contrast media used in all patients was 147 58 ml . patients with transfemoral approach received with 160 57 ml of contrast media a significantly greater amount than patients with transapical access who received 125 53 ml ( p < 0.0001 ) . the rate of aki after pre - tavi diagnostic right and left heart catheterization in our patient population was 9.2% ( n = 13 ) . only 4 patients ( 2.8% ) developed aki after both diagnostic coronary angiography and tavi procedure ( median 15 days between diagnostic catheterization and valve procedure ) . after exclusion of the ten patients who had already been enrolled in a chronic dialysis program before tavi , 140 patients were left for the analysis concerning the occurrence of acute kidney injury . two patients without aki needed short - term dialysis ( one patient was hemofiltrated due to low cardiac output and consecutive renal impairment , another patient acquired septic shock with renal failure ) . there was no significant difference regarding weight , height , baseline creatinine , and hemoglobin values in pts . who developed aki after intervention compared to those who did not ( see table 2 ) . patients with aki were significantly younger ( 79 9 yrs versus 82 6 yrs , p = 0.008 ) and had more frequently comorbidities such as hypertension and previous cabg whereas differences in peripheral arterial disease , cerebrovascular disease , chd , and hypercholesterolemia did not reach statistical significance . this difference did not also reach statistical significance which could have been due to the sample size . the amount of contrast media used during the procedure was also very similar between groups ( 147 71 ml versus 148 56 ml , p = 0.93 ) . with aki than in those without aki ( 20 12 days versus 15 10 days , p = 0.03 ) . both , 30-day - mortality ( 29% versus 7% , p < 0.0001 ) and cumulative mortality after a median followup of 309 days were significantly higher in aki patients ( 43% versus 18% , p < 0.0001 ) . aki was associated with significantly worse survival ( hrr 2.7 , ci 1.345.41 , p = 0.006 , figure 1(a ) ) . mortality in aki pts . was even higher ( hrr 3.8 , ci 1.3710.37 , p = 0.01 , figure 1(b ) ) after adjusting for risk factors ( age , diabetes , pad , hypertension , previous myocardial infarction and cabg , left ventricular dysfunction , amount of contrast dye , baseline creatinine , and hemoglobin ) . predictors of aki occurrence in univariate and multivariate regression analysis are shown in tables 3 and 4 . of all included variables ( age , diabetes , hypertension , pad , previous cabg , myocardial infarction , left ventricular function , baseline creatinine , and hemoglobin and amount of contrast dye ) only age was found to be significantly associated with aki in univariate analysis and was detected as an independent predictor of aki in multivariate analysis ( or 0.93 , ci 0.870.99 , p = 0.03 ) . including vascular access site in the model , transapical approach was also a significant predictor of aki ( or 1.8 , ci 1.8518.4 , p = 0.003 ) . neither euroscore ( 27 19% versus 23 13 , p = 0.18 ) nor sts score ( 6.0 3.5% versus 6.0 3.4% , p = 0.97 ) predicted the marked difference in mortality rates between aki and non - aki pts ( figure 2 ) . when applying the sts score renal failure definition to our patient population , 19 pts . however , the predicted rate of renal failure by sts score was only 7.3% ( p = 0.023 ) for all pts . the predicted risk for renal failure based on the sts score did not significantly differ between the ta and tf treatment groups ( 6.8 3.5% versus 8.3 5.1% , p = 0.054 ) . the observed rate of aki was however significantly higher in the ta group ( 31% versus 11% , p = 0.001 ) and exceeded thereby markedly the predicted rate of renal failure 1.6-fold in the tf and 3.7-fold in the ta pts . acute kidney injury after the use of iodinated contrast media in angiography is known to account for a number of adverse effects such as prolonged hospital stay and to be an independent risk factor of mortality [ 710 ] . several investigators have shown that aki is a relatively frequent complication after tavi and that it is associated with an increased mortality [ 1417 ] . however it remains unclear which of the underlying comorbidities contribute most to the adverse outcome following aki . moreover , reliable predictors of aki in this patient population still need to be defined . in particular , the value of risk scores developed for patients who undergo open heart surgery remains so far unknown . in the present study , . found very similar rates with 19% for aki and 2% for temporary dialysis . while bagur et al . reported a lower rate of aki ( 11% ) and need of dialysis after tavi ( 1.4% ) , aregger et al . and kong et al . found markedly higher rates of 28% and 7.4% , and 28.8% and 6% , respectively . one explanation for this wide variation of aki rates could be that the average amount of contrast media used in these studies differed markedly , too . while it was 148 ml in the present study , bagur et al . with the lowest rate of aki reported < 100 ml . in contrast , aregger et al . with the higher rate of aki used 242 ml on average . the amount of contrast media used during angiography is indeed considered one major risk factor for the development of aki . nevertheless , in the present study no significant difference in contrast media use could be found between aki und non - aki patients . the observation that aki was not related to the amount of contrast used has also been reported by other groups [ 15 , 16 ] suggesting that other factors may be more important for the development of renal impairment in the population currently undergoing tavi . prehydration in addition to intravenous n - acetyl - cysteine application prior to contrast media exposure is a well - known measure to reduce aki rates in patients with renal impairment . in our study patients with an egfr < 60 ml / min/1,73 m were treated with 1000 ml of saline 0.9% and 1200 mg of n - acetyl - cysteine which may have prevented more patients from experiencing aki than without these protective measures . nevertheless , this prevention strategy in general and even more in this specific patient population is not effective enough to thoroughly avoid occurrence of aki . therefore hydration therapy may have contributed to the nonsignificant association between baseline creatinine and acute kidney injury risk . comparing aki rates and patients after diagnostic and tavi catheterization no correlation between aki after diagnostic and valve procedure could be seen implying the lack of a patient - inherent predisposition for aki occurrence after exposure to contrast media . this finding complies with the results of van linden et al . who stated that early contrast media exposure ( 17 days ) by cardiac catheter or ct - scan did not increase the risk of aki or rrt . in this context it should be kept in mind that after cardiac surgery without any use of contrast media , the rate of aki can also reach up to 30% with 1% requiring dialysis treatment [ 21 , 22 ] . bagur et al . reported a 25% incidence of aki after surgical aortic valve replacement in patients with preprocedural chronic kidney disease compared to 12% in patients undergoing tavi . the markedly adverse effect of the occurrence of aki on the outcome of tavi underlines the importance of identifying predictors of this complication as well as appropriate measures for its prevention . in the present study , 30-day mortality and midterm mortality were as high as 29% and 43% in patients with aki compared to only 7% and 18% in those who did not develop this complication . the difference in survival was even more pronounced when adjusting for differences in baseline characteristics . similar findings have been reported by other investigators [ 1417 , 23 ] . despite these rather consistent findings with regard to incidence of aki after tavi and its adverse impact on outcome , the data with regard to risk prediction and options for prevention of aki remain controversial . it appears obvious that preprocedural chronic kidney disease should be a major risk factor for the development of postprocedural aki . . indeed showed impaired renal function with moderately elevated serum creatinine values before intervention and aki occurrence unrelated to the amount of contrast media to be the strongest predictors of 1-year mortality among tavi patients . although baseline creatinine in the present study was slightly higher in the aki group ( 126.4 59.2 this may be due to small sample size and thus lack of statistical power . as a matter of fact , preinterventional serum creatinine was found to be a significant predictor of aki only in the study by elhmidi et al . whereas several other studies could not confirm this observation [ 14 , 15 , 17 , 19 ] . paradoxically , younger age turned out to be the only independent preprocedural risk factor for the development of aki in the present study . this observation must be seen with caution . to qualify for tavi instead of conventional surgery , younger patients must assumingly have been markedly sicker than older patients . hypertension was also found to be a predictor of aki in other studies [ 14 , 19 ] . without consistency , peripheral artery disease [ 15 , 16 ] , previous myocardial infarction , chronic obstructive pulmonary disease , systemic inflammatory response [ 16 , 17 ] , residual aortic regurgitation , and periprocedural red blood cell transfusion [ 14 , 15 , 19 ] have been reported to predict aki after tavi . in accordance with kong et al . , transapical tavi was found to be associated with a higher risk of aki in the present study . although this could be partially due to the worse baseline characteristics of these patients , transapical access remained a significant predictor after consideration of such differences . whether the more invasive nature of this approach , higher bleeding rates and requirement for blood cell transfusion account for this difference remains to be shown . in addition , similar to previous reports , the observed 30-day mortality was markedly lower than predicted by the logistic euroscore ( 11% versus 24% ) whereas the sts score was indeed lower ( 6% ) . this is in agreement with the observation of piazza et al . who found lower estimates of operative mortality by the sts score stating that this scoring system has suboptimal discriminatory power and calibration for tavi patients . sts score as a surgical risk algorithm obviously omits several risk factors in the tavi population leading to different patient selection and thus mortality rates . the present study also demonstrates that sts score for prediction of renal failure has little value for prediction of renal failure after tavi . this underlines once more the importance of developing appropriate scores for the risk of death as well as of the risk of renal failure and other complications in patient populations currently treated with tavi . in addition to preexisting factors , hemodynamic instability with consecutive extreme hypotension caused by rapid pacing , balloon valvuloplasty , and prosthesis deployment during tavi may account for a significantly higher risk of aki in patients undergoing tavi compared to simple angiography or pci . this must be considered when developing measures to reduce the occurrence of aki after tavi . although the data were collected prospectively in consecutive patients undergoing tavi , the analysis with regard to incidence and predictors of aki was performed retrospectively . potentially relevant factors such as red blood cell transfusion , after procedure thrombocytopenia and hemoglobin drop , procedure time , hemodynamic complication or the use of angiotensin converting enzyme inhibitors , and/or angiotensin receptor blockers could not be evaluated . although the study comprised a sizeable number of tavi patients , it reflects a single - center experience only and a much larger population is required to perform extensive multivariate analyses in order to better identify risk factors for the development of aki with relevant impact on the decision making in clinical practice . its occurrence does not appear to be primarily related to the amount of contrast dye used . the occurrence of aki markedly increases hospital stay as well as 30-day and midterm mortality even after consideration of the baseline risk profile . thus , improvements in predicting the risk of aki after tavi as well as effective measures to reduce the rate of this complication would be essential .

background . transcatheter aortic valve implantation ( tavi ) is widely used in high risk patients ( pts ) with aortic stenosis . underlying chronic kidney disease implicates a high risk of postprocedural acute kidney injury ( aki ) . we analyzed its occurrence , impact on hospital stay , and mortality . methods . 150 consecutive pts underwent tavi in our institution ( mean age 81 7 years ; logistic euroscore 24 15% ) . aki definition was a creatinine rise of 26.5 mol / l or more within 48 hours postprocedural . ten patients on chronic hemodialysis were excluded . results . aki occurred in 28 pts ( 20% ) . baseline creatinine was higher in aki pts ( 126.4 59.2 mol / l versus 108.7 45.1 mol / l , p = 0.09 ) . contrast media use was distributed evenly . both , 30-day mortality ( 29% versus 7% , p < 0.0001 ) and long - term mortality ( 43% versus 18% , p < 0.0001 ) were higher ; hospital stay was longer in aki pts ( 20 12 versus 15 10 days , p = 0.03 ) . predicted renal failure calculated sts score was similar ( 8.0 5.0% [ aki ] versus 7.1 4.0% [ non - aki ] , p = 0.32 ) and estimated lower renal failure rates than observed . conclusion . aki remains a frequent complication with increased mortality in tavi pts . careful identification of risk factors and development of more suitable risk scores are essential .

platinum drugs are a mainstay of cancer therapy . approximately half of all cancer patients receiving chemotherapy cisplatin , carboplatin , and oxaliplatin ( chart 1 ) three platinum complexes approved by the us fda for the treatment of human cancer are commonly applied to treat bladder , testicular , head and neck , ovarian , colon , and small cell and nonsmall cell lung cancers . despite such widespread use , the cytotoxicity of these drugs is not limited to cancer cells , and off - target activity results in emesis , alopecia , nausea , kidney damage , myelosuppression , and peripheral neuropathy . moreover , many tumors are either inherently resistant to the currently employed platinum - based therapies or acquire resistance during treatment . in an attempt to find molecules with improved potency , fewer side effects , and a novel spectrum of activity , researchers have prepared thousands of platinum complexes and tested them for anticancer activity . one strategy used to address the foregoing issues is to devise complexes that depart from the neutral , square - planar , dna - cross - linking cis - dia(m)mine a current manifestation explores cationic monofunctional platinum(ii ) complexes , which bear only one labile ligand and form one bond to the dna nucleobases . the significant difference in the interaction of monofunctional complexes with dna compared to classical bifunctional cross - linking compounds very likely contributes to the unique response that phenanthriplatin , or cis-[pt(nh3)2(phenanthridine)cl ] ( chart 1 ) , elicits when used to treat cancer cells . studies with the monofunctional compound pyriplatin ( chart 1 ) , cis-[pt(nh3)2(pyridine)cl ] , reveal that little distortion of the dna double helix is induced upon platination , and a similar situation is likely to be obtained with phenanthriplatin . this result is very different from the significant dna bending at 1,2-intrastrand cross - links that occurs following treatment with bifunctional platinum agents such as cisplatin . anonleaving group ligands are colored blue , and leaving group ligands are red . nonleaving group ligands are colored blue , and leaving group ligands are red . crystallographic and biochemical studies have revealed the mechanism by which pyriplatin exerts its anticancer activity , most likely transcription inhibition followed by consequent apoptosis . structural studies suggest that the steric bulk of the pyridine ring is crucial for activity . this hypothesis provides an explanation for the contrast between the activity of pyriplatin and the inactivity previously observed for monofunctional compounds such as [ pt(nh3)3cl ] and [ pt(dien)cl ] . a systematic variation of the n - heterocyclic ring am in compounds of the form cis-[pt(nh3)2(am)cl ] resulted in the discovery of the far more potent analog phenanthriplatin . in a preliminary screen of cultured human cancer cells , phenanthriplatin displayed significantly greater cytotoxicity than cisplatin and showed a pattern of activity distinct from that of either cisplatin / carboplatin or oxaliplatin . a more detailed understanding of the spectrum of activity was gained by analyzing the cytotoxicity of phenanthriplatin in the nci60 panel of cancer cells . the pattern of cell killing was uncorrelated with that of any other platinum agent in the nci database . unlike pyriplatin , the asymmetry of the phenanthridine ring renders phenanthriplatin chiral . that phenanthriplatin can exist as two distinct enantiomers is of potential importance because the two enantiomers may display different pharmaceutical activity . initial experiments revealed that phenanthriplatin binds dna and that it does so in a covalent , rather than intercalative , manner . moreover , studies with e. coli , analogous to those initially performed to investigate the mechanism of action of cisplatin , corroborate the hypothesis that the interaction of phenanthriplatin with dna is responsible for its anticancer effects . to gain more insight into the nature of the interaction of phenanthriplatin with dna , we prepared small molecule complexes that model its reactions with guanosine residues ( chart 2 ) . the n7 position of guanine is the most nucleophilic among dna bases and , as a result , it is the primary binding site for platinum agents . the model complexes cis-[pt(nh3)2(r - gua)(am)](otf)2 , where r - gua is a 9-alkylguanine , am is phenanthridine , and otf is trifluoromethanesulfonate or triflate , were therefore prepared using the triflate salt of phenanthriplatin as a synthetic precursor . the chirality of phenanthriplatin combined with coordination to r - gua creates diastereomers , the nature of which was investigated by x - ray crystallography and nmr spectroscopy . here we report the results of an investigation of these diastereomeric analogs of phenanthriplatin ( chart 2 ) , which were prepared to investigate whether the two phenanthriplatin enantiomers can be resolved on the physiological time scale . consideration of its dynamics is crucial for interpreting the conformational isomerism observed with the 9-alkylguanine model complexes . evidence in both the solid state and solution phase indicates that , when phenanthriplatin reacts with guanine , diastereomeric selection occurs among the possible conformational isomers that can form . the origin of this selection has been identified , as described in this article . the synthesis and characterization of the compounds under discussion are presented in the supporting information along with crystallographic details and specifications of the instruments used for physical measurements . h nmr spectra were acquired over a temperature range for 24 to investigate potential fluxional behavior exhibited by these compounds . the rate of exchange at the temperature at which two peaks coalesce was estimated using eq 1 , in which k is the rate of exchange at the coalescence temperature and is the difference in hz between the two signals in the low temperature ( slow exchange ) limit . this estimate was used to inform the initial guess in a full line shape analysis . simulated spectra were fit to experimental spectra at the different temperatures , and the corresponding rate constants were extracted . these rate constants were used to construct eyring plots and determine activation parameters.1 deuterated dmso solutions of 6 and 8 , both of which are 9-methylguanine complexes , were sparged with n2 for 2 min and then sealed in an nmr tube under a blanket of nitrogen . briefly , for each sample , a h nmr spectrum was acquired following preirradiation at the frequency of the methylguanine h8 ( h8 g ) resonance . the preirradiation power was chosen so as to just saturate the h8 g signal and eliminate it from the spectrum . a second spectrum was then acquired with identical parameters , but with the saturation frequency set to a region downfield and devoid of signals . no cross peaks between h8 g and any of the phenanthridine protons were observed in a noesy experiment . rotating - frame overhauser effect ( roesy ) spectra were collected on samples prepared in deoxygenated acetone - d6 . a mixing time of 200 ms was employed with a 90 pulse of 11 s . the complex cation of 6 was constructed in gaussview , and the planes of the phenanthridine and 9-ethylguanine ligands were set perpendicular to the coordination plane . two conformers were investigated , one in which the h6 proton of the phenanthridine ligand ( h6p ) and h8 g were on the same side of the platinum coordination plane and the other in which they were on opposite sides . the zero order relativistic approximation ( zora ) , along with the attendant tzv - zora basis set , was applied to treat relativistic effects . the resolution of the identity approximation and the appropriate auxiliary basis set were used to accelerate computations . the stationary nature of the structures obtained from geometry optimizations was confirmed using numerical frequency calculations . optimizations were conducted in either the gas phase or in solution by using an implicit conductor - like screening model ( cosmo ) . to model aqueous solvation , the dielectric constant of the polarizable continuum was set to 80.400 and the refractive index to 1.3300 . monofunctional complexes having the formula cis-[pt(nh3)2(am)cl ] , where am is an n - heterocyclic ligand , have previously been obtained as nitrate salts by treating cisplatin , cis-[pt(nh3)2cl2 ] , with 1 equiv of silver nitrate followed by an equivalent of am . alternatively , cis-[pt(nh3)2(am)cl]cl can be obtained by heating cisplatin with am to displace one of the chloride ligands . a major problem with these methods , however , is that neither silver - mediated halide abstraction nor direct ligand substitution proceeds selectively at just one coordination site . as a result , in addition to the desired cis-[pt(nh3)2(am)cl ] complex , appreciable amounts of cis-[pt(nh3)2(am)2 ] form together with unreacted cisplatin . here we use silver triflate to prepare 7 and 1 , triflate salts of pyriplatin and phenanthriplatin , respectively , to provide a much wider range and degree of solubility in organic solvents . as a result , addition of acetone to the residue that remains following removal of dmf from the synthesis mixtures of 1 and 7 dissolves the triflate salts of cis-[pt(nh3)2(am)cl ] and cis-[pt(nh3)2(am)2 ] . unreacted cisplatin , on the other hand , does not dissolve in acetone and can be removed by filtration . when ether is layered onto the acetone filtrate , crystals of cis-[pt(nh3)2(am)cl]otf deposit over the course of a few days . these crystals can be harvested before the more soluble cis-[pt(nh3)2(am)2]otf2 precipitates , providing access to analytically pure material . spectroscopic characterization of these complexes was consistent with that which had been previously reported for the nitrate salts of these cations . the assignment of the peaks in the h nmr spectrum of 1 was carried out using a combination of cosy and noesy spectra ( figure s4 ) . the nmr spectra for these compounds ( figure s19s22 ) show a multitude of peaks arising from different rotamers that interconvert slowly on the nmr time scale at room temperature . the syntheses of 5 , 6 , and 8 were achieved by treating either 1 or 7 with an additional equivalent of silver triflate and adding the appropriate 9-alkylguanine . as described above , the h nmr spectra of 24 exhibit peak multiplicity as a result of slow to intermediate exchange between conformational isomers . upon heating , these signals broaden , coalesce , and finally sharpen as the rate of exchange increases . although some regions of the spectra have complex overlapping features , other regions show well - resolved peaks and coalescence events . the line shapes of portions of the h nmr spectra of 24 that showed well - defined , baseline - resolved coalescing signals were simulated . the simulated spectra were fit to the experimental data by varying the rate constant . a two - site model was used for 2 and 4 , and a four - site model was used for 3 . an example of the simulated and experimental data for 4 is shown in figure 1 . data for all compounds can be found in the supporting information ( figures s23s25 ) . experimental and simulated line shapes of portions of the h nmr spectrum of 4 . the temperature at which the experimental data were collected and the rate constant used to generate the simulation are shown next to each set of data . the variation in rate constant with temperature can be used to determine the activation parameters for the interconversion . the enthalpy and entropy of activation , h and s respectively , are obtained from the first order rate constants using an eyring analysis ( figure 2 ) . eyring plots for all the coalescence events simulated are presented in the supporting information . the gibbs free energy of activation at a given temperature t , gt , can be obtained using eq 2.23the values of g298.15 for compounds 24 are collected in table 1 . using eq 3 , where kb is boltzmann s constant , h is planck s constant , and r is the universal gas constant , the rate constant at a given temperature , the lifetime of the molecule in a given conformational state at temperature t , t , for the process can be obtained by taking the inverse of the rate constant . the errors for g298.15 are obtained by standard propagation of the errors of h and s obtained from the least - squares linear regression eyring plots . the errors should be taken only as estimates of the true errors and are presented to indicate the relative precision with which the different determinations were made . the approximate nature of the error estimate arises from the fact that this analysis treats data across a logarithmic scale on equal footing . moreover , it equally weights the rate constants from near the coalescence point , which are more accurate , and rate constants obtained far from coalescence , which are less accurate . the error propagation was not carried out for the lifetimes because only modification by physical constants is involved . eyring plot of the conformational isomerism of 4 using the data from figure 1 . pertinent crystallographic data for 1 , 5 , 7 , and 8 are summarized in table s1 . the crystal structure of 1 ( figure 3 ) shows the cis-[pt(nh3)2(phenanthridine)cl ] cation to be similar in structure to that present in the structure of the nitrate salt , with bond lengths and angles falling within expected ranges . the geometry of the primary coordination sphere in both structures is essentially identical with an rmsd of 0.023 . the most significant differences are found in the orientation of the phenanthridine rings as shown in figure s30 . in the nitrate salt , the phenanthridine plane does not contain the line connecting the platinum atom and the nitrogen atoms of the phenanthridine and the trans ammine . this line instead forms an angle of 18 with the phenanthridine plane . in the triflate salt , this angle is only 10. an important aspect of the structure of the complex is that the asymmetry of the phenanthridine ligand about the platinum coordination plane produces a chiral molecule . molecular diagrams of the platinum complexes from the crystal structures of 1 , 5 , 7 , and 8 with thermal ellipsoids drawn at the 50% probability level . color code : n blue , o red , c gray , cl green , pt magenta , h open circles . the chirality originates about the bond between the platinum center and the phenanthridine nitrogen atom ( pt np ) and can be classified according to the conventions of axial chirality . np vector , the platinum coordination plane lies in front of the perpendicular plane of the phenanthridine ring . the direction in which the front ring needs to be rotated so as to have the priority substituent of the front plane coincide with the priority substituent of the back plane dictates the stereochemistry . clockwise rotation is denoted p and counterclockwise rotation m. the different enantiomers of phenanthriplatin are shown in figure 4 . ( a ) the enantiomers of phenanthriplatin and ( b ) the convention used to classify them . in part b , the complex is viewed along the ammine platinum phenanthridine vector . the coordination plane is shown as a darkened line , and the phenanthridine plane as a dashed line . the structure of 7 ( figure 3 ) , the triflate salt of pyriplatin , also displays expected bond lengths and angles . in this complex , unlike 1 , the line connecting the platinum atom and the nitrogen atoms of the pyridine and the trans ammine is essentially contained by the plane of the n - heterocycle . the ring deviates significantly , however , from a perpendicular orientation with respect to the platinum coordination plane . the dihedral angle of 60 between the pyridine and the coordination plane is consistent with the angle of 56 observed for trans-[pt(pyridine)2cl2 ] . details of the determination of the structure of a nonmerohedral trilling of this latter compound are presented in the supporting information . given the lack of steric or electronic factors to enforce strict perpendicularity between the pyridine ring and the coordination plane , the angle in both cases is most likely dictated by crystal packing interactions . the solution and refinement of the structure of 8 ( figure 3 ) proceeded smoothly , except for the presence of a void about the 0 , 0 , 1/4 special position containing disordered electron density . the density could not be successfully modeled as either a molecule of dmf , diethyl ether , or a 1:1 disorder of the two , any of which would be consistent with the 41 e within the void . one dmf molecule disordered across each of the 4 voids of 228 within the unit cell ( z = 8) would be consistent with the combustion analysis results obtained from this material . the pyridine and 9-methylgunanine rings are both canted in the same direction by 19 and 23 , respectively . the structure of 5 , the product of the reaction of activated 1 with 9-ethylguanine , was also solved ( figure 3 ) . the salt crystallized along with one water molecule , located on the 2-fold proper rotation axis , and one disordered acetone molecule . the presence of 0.5 equiv of water is consistent with the elemental analysis of this compound . the acetone present in the structure is not observed during combustion analysis and is probably removed during the vacuum drying of the substance . the phenanthridine ligands lie parallel to the ac plane , and the 9-ethylguanine ligands and the platinum coordination plane lie perpendicular to this crystallographic plane . the phenanthridine and 9-ethylguanine ligands of the square - planar complex cation the former is oriented essentially perpendicular to the coordination plane but the latter is canted by 23 , such that the guanine carbonyl oxygen approaches the ammine coordinated cis to it . preirradiation at the frequency of the h8 g signal in solutions of 6 and 8 induced perturbations in the signals of those protons that interact with h8 g in a through - space manner . in the difference spectrum of 8 , obtained as described above , negative peaks were seen arising from the ch3 protons of the 9-methyl group as well as the ortho hydrogen atoms of the pyridine ring ( figure s26 ) . in the difference spectrum of 6 , a negative peak was again seen for the ch3 protons of the 9-methyl group . negative peaks were also observed for h6 and h7 of the phenanthridine ring ( figure s27 ) owing to their close proximity to h8 g . the roesy spectrum of 6 also confirms the presence of the through - space interaction between h8 g and h6 of the phenanthridine ring ( figure s28 ) . geometry optimizations were performed on two conformational isomers of 5 that are related by a 180 rotation about the pt np bond . in calculations in which the starting geometry had both n - heterocyclic ligands perpendicular to the coordination plane , optimization did not significantly alter the geometries of either of the conformers , which exhibited a negligible difference in strain energy . an overlay of the optimized structures of both conformers is shown in figure 5 . overlay of the molecular mechanics optimized geometries obtained by setting the aromatic ligands perpendicular to the platinum coordination plane and rotating 180 about the platinum more rigorous geometry optimization using dft methods reproduced the canting of the alkylguanine ligand that was observed in the crystal structure of 5 ( figure 6 ) . in the gas phase , the distance of 2.72 between the carbonyl oxygen and the ammine nitrogen is sufficiently small to propose the presence of an intramolecular hydrogen bond . the calculations were also carried out with implicit aqueous solvation . as expected , the interaction between the carbonyl oxygen atom and the ammine nitrogen atom is attenuated , but the resulting on distance of 2.86 suggests that the interaction persists even in the presence of highly polar solvents . the canting of the guanine ring in ( a ) the crystal structure and ( b ) the dft - optimized structure of 5 . color code : n blue , o red , c gray , pt magenta , h atoms omitted for clarity . ( c ) schematic representation of the dihedral angle formed between the platinum coordination plane ( black ) and the phenanthridine ring of 5 in the mm calculations ( red ) , the crystal structure ( blue ) , the dft calculations with implicit water solvation ( purple ) , and the dft calculations in the gas phase ( green ) . studies with pyriplatin showed that this compound has a spectrum of activity that differs significantly from those of the clinically employed platinum anticancer drugs . the low potency of pyriplatin prompted a search for molecules that maintain this distinct spectrum of activity , but display higher activity . phenanthriplatin was developed as the result of a systematic variation of the n - heterocyclic amine ligand , am , of cis-[pt(nh3)2(am)cl ] . it was found to be 740-fold more potent that cisplatin across a variety of cell lines , and the distinct spectrum of activity was maintained . in an effort to better understand the mechanism of anticancer action of phenanthriplatin , we sought to investigate the structures of the adducts formed by phenanthriplatin and analogs of guanosine . for these studies , the triflate salt of the cis-[pt(nh3)2(am)cl ] cation was prepared to facilitate subsequent synthetic steps . in the process of analyzing the solid state structure of the compound , however , we realized an aspect of the structure of this complex that had previously gone unnoticed : in the solid state , the complex cation is chiral . the centrosymmetric space group of the structure requires that both enantiomers be present in the crystal in equal portions . the chirality arises from the asymmetry of the phenanthridine ring about the coordination plane to which it is perpendicular . different enantiomers of pharmaceutics typically display different activity because these molecules interact with biological systems that are inherently chiral . one example of direct relevance to the field of platinum anticancer research is oxaliplatin , which contains only one enantiomer in the form that is marketed for clinical use . the r , r isomer of trans - diaminocyclohexaneoxalatoplatinum(ii ) has greater activity than that of the enantiomeric s , s form . if phenanthriplatin is indeed a racemic mixture of two stable enantiomers , then one might have significantly different activity than the other . it is crucial to realize , however , that the two enantiomers can interconvert via rotation about pt np . if rotation about this bond is rapid at ambient or physiological temperatures , then the complex is effectively achiral . as will be discussed below , in addition to implications for the enantiomeric resolution of the compound , asymmetry of the phenanthridine ligand about the platinum coordination plane also has implications for its interaction with dna . investigation of the phenanthridine rotation is similar in nature to studies that have been carried out on models and retro - models of 1,2-d(gpg ) intrastrand cross - links formed by bifunctional platinum compounds . this similarity arises from the fact , as will become important below , that both the phenanthridine ligand and guanine derivatives coordinate in a manner that is asymmetric about the platinum coordination plane . the chirality present in models of the intrastrand [ pt(nh3)2{d(gpg ) } ] adduct was first recognized by cramer et al . the phenomenon has subsequently been investigated in great detail by marzilli , natile , and co - workers , among others . dynamic nmr spectroscopy is a method ideally suited to investigate whether rotation about a chemical bond is hindered . np in phenanthriplatin interconverts two enantiomers with identical nmr properties , however , and so this dynamic process will induce no change in the line shape of the spectrum , regardless of the rate at which it occurs . accordingly , the room temperature h nmr spectrum of 1 shows a single set of well - defined resonances . the signals in the aromatic region are particularly sharp , and the breadth of the signals arising from the two ammines is due to a combination of quadrupolar relaxation from n and coupling to csa - broadened pt . to investigate rotation about the pt np bond , the rotamers must be rendered diastereomeric . this goal was accomplished by replacing the two ammine ligands with the enantiomerically pure r , r - diaminocyclohexane ( dach ) chelate . the c2 symmetry of this ligand ensures that the coordination sites trans to the two coordinated nitrogen atoms are chemically equivalent . in the room temperature h nmr spectrum of 2 , the signals arising from the protons on the aromatic ring are all cleanly doubled ( figures 1 and s19 ) . moreover , raising the temperature of the sample induces broadening , coalescence , and subsequent sharpening of the signals ( figure s19 ) . this behavior is consistent with the presence of two rotamers that interconvert rapidly on the nmr time scale at elevated temperature . the conformations of these two rotamers are equivalent to those of the enantiomers of phenanthriplatin , i.e. m and p , giving rise to ( r , r)m and ( r , r)p diastereomers . detailed information about the energetics of the rotamer interconversion can be obtained by simulating the experimental line shapes obtained at different temperatures . the fit of the simulation to the experimental data can be optimized by varying k , the rate constant for interconversion . these k values obtained at different temperatures can be used to construct an eyring plot from which the activation parameters for the interconversion can be extracted . the gibbs free energy of activation for the interconversion of the ( r , r)m and ( r , r)p diastereomers of 2 at room temperature , g298.15 , was 70 kj mol , and the corresponding rate constant at this temperature was 3.2 s. the inverse of this rate constant reveals that 63% of a diastereomerically pure sample of 2 would racemize within about 300 ms of dissolution at ambient conditions . the validity of this conclusion , however , rests on the accuracy with which 2 models the structure of 1 . the most significant difference of relevance to the discussion at hand is in the n n angle formed by either the ammines of 1 or the dach of 2 . the former , obtained from the crystal structure of 1 , is 89.1 , and the latter , from the crystal structure of oxaliplatin , is 83.5. the smaller angle enforced by the chelator relieves the steric interactions that provide a barrier to rotation of the phenanthridine ring . the barrier obtained with 2 , therefore , provides an upper estimate to the rate of rotation in phenanthriplatin . np in phenanthriplatin will be sufficiently slower that enantiomeric resolution may be possible . to obtain a diastereomeric analog of 1 in which the n pt n angle formed by the ammines is left unperturbed , 4 was prepared . the addition of a second pt np bond as a center of chirality creates ( p , m ) , ( m , p ) , ( p , p ) , and ( m , m ) conformational isomers . the ( p , p ) and ( m , m ) designations describe a meso compound , and the ( p , m ) and ( m , p ) rotamers are enantiomers . hindered rotation about the pt np bonds would , therefore , give rise to two sets of signals . using a dynamic nmr analysis analogous to that described above for 2 , activation parameters can be extracted ( table 1 , figure s25 ) . in addition to adding a second chiral center , however , the second phenanthridine ligand may introduce steric bulk that inhibits pt np rotation to a degree greater than in 1 . to assess the influence of the cis disposition of two phenanthridine rings on pt np rotation , 3 was prepared . the set of conformers listed for 3 is also present for 4 , but the presence of the r , r - dach ligand renders all four rotamers diastereomeric : ( r , r)(p , m ) , ( r , r)(m , p ) , ( r , r)(p , p ) , and ( r , r)(m , m ) . dynamic nmr spectroscopy again reveals the activation parameters for interconversion ( table 1 ) through a simulation of the line shapes of the four distinct sets of h nmr signals that coalesce on heating ( figure s24 ) . a comparison of the results obtained from the dynamic nmr experiments on 24 is shown in scheme 1 . upon transitioning from 2 to 3 , the addition of the extra phenanthridine ligand raised the barrier to rotation , but the lifetime only changed by a factor of 1.7 . exchanging the chelating dach present in 3 for the two ammine ligands in 4 increased the lifetime by an order of magnitude . even so , the lifetime of a given diastereomer of 4 is about 5 s. importantly , unlike 2 , 4 provides an upper estimate of the barrier to rotation in phenanthriplatin . the results with 3 and 2 indicate that transitioning from 4 to 1 will lower the barrier to phenanthridine rotation . np bond in phenanthriplatin is rapid on the pharmacological time scale and that enhanced activity can not be obtained by isolating and administering one of the isolated enantiomers . the rotation about this bond does , however , play a significant role in the interaction of 1 with dna . the interaction of phenanthriplatin with dna can be modeled by preparing complexes with the formula cis-[pt(nh3)2(phenanthridine)(9-alkylguanine ) ] . compounds 6 and 5 ( chart 2 ) were prepared using 9-methyl- and 9-ethylguanine , respectively . this mode of coordination is corroborated by the shift in the h8gh nmr signal observed on binding to the platinum . as a result of this coordination geometry , the bond between the platinum center and the n7 of the guanine ( pt ng ) acts as a center of chirality in the same manner as pt np . multiplicity in the h signals was expected to arise due to the slow interconversion of ( pp , mg ) , ( mp , pg ) , ( pp , pg ) , and ( mp , mg ) , where the subscripts indicate the coordinate bond to either phenanthridine ( p ) or 9-alkylguanine ( g ) . surprisingly , however , only a single set of signals was observed ( figure s8 ) . cooling an nmr sample to 60 c did not induce any decoalescence or broadening ( figure s22 ) . in these molecules , therefore , either intramolecular rotation is occurring significantly more rapidly than in complexes 14 or one particular set of conformers is preferentially formed . note that , for instance , ( pp , pg ) and ( mp , mg ) are enantiomers and would produce identical nmr spectra . through - space dipolar interactions between nmr active nuclei provide an ideal means with which to probe three - dimensional molecular structure in solution . before investigating the more complex phenanthridine - containing compounds , however , a simpler pyriplatin analog was prepared and studied . in 8 , the pyridine ring is symmetric about the platinum coordination plane and so the platinum pyridine bond is not a center of chirality . the chirality about pt ng produces enantiomers but not diastereomers . in the crystal structure of 8 , there are 2 sets of protons that are close enough to suggest that a nuclear overhauser effect ( noe ) will occur involving h8 g : the ch3 of the 9-methyl group ( 2.52 ) and the ortho protons from the pyridine ring ( 3.56 ) . the methyl group provides a particularly convenient internal standard because the rigidity of the guanine ring ensures that the methyl protons will stay in close proximity to h8 g regardless of the relative orientation of the pyridine and guanine rings . a h saturation transfer nmr experiment was carried out with preirradiation at the frequency of the h8 g signal and revealed a through - space interaction between h8 g and the 9-methyl protons as well as the ortho protons of the pyridine ring ( figure s26 ) . in the ( mp , pg ) isomer of 6 , as well as the enantiomeric ( pp , mg ) , h8 g and h6p are on the same side of the coordination plane . in ( pp , pg ) and ( mp , mg ) , which are diastereomers of the first pair , h8 g and h4p are on the same side of the platinum coordination plane . only those protons that are on the same side of the platinum coordination plane as h8 g are expected to undergo through - space interactions with this nucleus . when a saturation transfer experiment analogous to that described for 8 was conducted with 6 , an interaction was observed between h8 g and the 9-methyl protons , as expected , and with the h6p proton . moreover , an interaction was also seen with h7p , further confirming that the ( mp , pg)/(pp , mg ) enantiomeric pair is preferentially formed in solution over the ( pp , pg)/(mp , mg ) pair . a though - space interaction was also observed in the roesy spectrum ( figure s28 ) . a crystal structure of 5 was solved ( figure 3 ) , and it also showed only the presence of ( mp , pg ) and ( pp , mg ) . the centrosymmetry of the space group c2/c in which the complex crystallized requires the presence of both enantiomers . molecular mechanics calculations were initially performed to interrogate the origin of the diastereoselectivity exhibited by 5 and 6 . calculations in which the aromatic ligands were set perpendicular to the coordination plane ( figure 5 ) revealed little energetic difference between the two diastereomeric forms . inspection of the crystal structure of 5 , however , reveals that the guanine ligand is not perpendicular to the coordination plane . rather , it cants so as to direct the carbonyl oxygen toward the cis - coordinated ammine . the on distance of 3.19 is too long to constitute a formal intramolecular hydrogen bond in the solid state , but such an interaction may occur in solution . a similar interaction between the carbonyl of a pyriplatin - platinated guanosine and the cis ammine of the platinum complex was observed in the crystal structure of pyriplatin - platinated dodecamer duplex dna . dft geometry optimization was able to reproduce the canting of the 9-alkylguanine . in the optimized gas phase geometry , inclusion of implicit aqueous solvation weakens the interaction somewhat , but the on separation remains short at 2.86 . the origin of the diastereoselectivity appears to result from this canting of the guanine ligand , which relieves steric congestion over one face of the platinum complex . one side of the phenanthridine ligand has a greater degree of steric bulk in the vicinity of the platinum center than the other , as illustrated in figure 6 . in the favored diastereomer , this bulkier portion of the phenanthridine is directed toward the vacant space formed by the canting of the guanine . it remains to be seen whether the conformational preference is maintained in full length dna polymers or during complexation with proteins that recognize platinum lesions . as a final comment n bonds in phenanthriplatin were not rapid , then different diastereomeric forms of the platinum adduct could be kinetically trapped regardless of the energetic preference that might exist for one conformer . instead , the unhindered bond rotation established above allows the complex to assume the thermodynamically favorable conformation , regardless of the manner in which it initially binds to the 9-alkylguanine . the solid state structure of phenanthriplatin highlights the fact that asymmetry of the phenanthridine ligand about the platinum coordination plane results in chirality . the use of model compounds with diastereomeric rotamers provides evidence that rotation about this bond in phenanthriplatin is rapid , eliminating any need to isolate and administer an enantiomerically pure compound . rapid rotation about the this bond also prevents the kinetic resolution of diastereomers in complexes of the type cis-[pt(nh3)2(phenanthridine)(9-alkylguanine ) ] , which mimic the interaction of phenanthriplatin with dna . the ( mp , pg ) and ( pp , mg ) diastereomers observed in both solution and the solid state are those in which the h8 g and h6p are located on the same side of the platinum coordination plane . the experimental data described above indicate that interaction between the guanine carbonyl and cis - coordinated ammine determines the preferential diastereomer formation .

the monofunctional platinum complex cis-[pt(nh3)2cl(am)]+ , also known as phenanthriplatin , where am is the n - heterocyclic base phenanthridine , has promising anticancer activity . unlike bifunctional compounds such as cisplatin , phenanthriplatin can form only one covalent bond to dna . another distinguishing feature is that phenanthriplatin is chiral . rotation about the pt n bond of the phenanthridine ligand racemizes the complex , and the question arises as to whether this process is sufficiently slow under physiological conditions to impact its dna - binding properties . here we present the results of nmr spectroscopic , x - ray crystallographic , molecular dynamics , and density functional theoretical investigations of diastereomeric phenanthriplatin analogs in order to probe the internal dynamics of phenanthriplatin . these results reveal that phenanthriplatin rapidly racemizes under physiological conditions . the information also facilitated the interpretation of the nmr spectra of small molecule models of phenanthriplatin - platinated dna . these studies indicate , inter alia , that one diastereomeric form of the complexes cis-[pt(nh3)2(am)(r - gua)]2 + , where r - gua is 9-methyl- or 9-ethylguanine , is preferred over the other , the origin of which stems from an intramolecular interaction between the carbonyl oxygen of the platinated guanine base and a cis - coordinated ammine . the relevance of this finding to the dna - damaging properties of phenanthriplatin and its biological activity is discussed .

studies have demonstrated that different ethnic groups may have different predisposing risk factors , epidemiologic patterns , and outcomes of stroke . this has been shown in african americans , caucasians , hispanics , arabs , and asians [ 16 ] . these differences could be due to differences in demographic or socioeconomic factors or in lifestyle . although epidemiologic studies on stroke were carried out in different parts of the world , including some arab countries [ 711 ] , there are no published data about palestine . furthermore , knowledge of the prevalence of stroke - related risk factors can help health decision makers to direct efforts toward reducing stroke - related morbidity and mortality . in palestine the private sector plays a minor role due to its high cost compared to the average income in palestine . currently , the palestinian national authority is in charge of west - bank and gaza strip , with a total population of 4,151,668 inhabitants . nablus district is the second largest district with a population of 362,159 native palestinian inhabitants . this study was conducted to identify the risk factors , in - hospital mortality , and discharge medications for patients with ischemic stroke admitted to al - watani government hospital , nablus , palestine . this one - year , retrospective , hospital - based study was conducted between september 01 , 2006 and august 31 , 2007 . all patients admitted to the hospital with acute ischemic stroke were included in the study . each diagnosed patient had been followed up until either death at the hospital or discharge . the data were collected by retrospective review of medical charts included age , gender , medications , serum creatinine ( scr ) level , risk factors , and post - stroke medical complications . then following were considered risk factors : age , hypertension ( htn ) , diabetes mellitus ( dm ) , congestive heart failure ( chf ) , atrial fibrillation ( af ) , ischemic heart disease ( ihd ) , smoking , previous stroke , and obesity [ 1215 ] . hyperlipidemia is a risk factor for stroke , but we did not include it because it had not been done routinely at the hospital . obesity was defined as a body mass index ( bmi ) > 30 for both genders . creatinine clearance ( crcl ) was calculated by using cokcroft - gault equations , and values for females were obtained by multiplying the result by 0.85 . the post - stroke complications considered were the presence of one or more of the following : constipation , seizure , depression , infection , limb pain , and gastrointestinal upset . analysis of data was carried out using the " statistical package for social sciences " ( spss ) program for windows version 15.0 ( spss inc . , univariate analysis was carried out using pearson chi - square for categorical variables and student 's t test for continuous variables . multiple logistic regression analysis was used to find independent predictors of in - hospital mortality . variables included in the regression model were those that had a significant p value ( p < 0.05 ) in the univariate analysis . analysis of data was carried out using the " statistical package for social sciences " ( spss ) program for windows version 15.0 ( spss inc . , univariate analysis was carried out using pearson chi - square for categorical variables and student 's t test for continuous variables . multiple logistic regression analysis was used to find independent predictors of in - hospital mortality . variables included in the regression model were those that had a significant p value ( p < 0.05 ) in the univariate analysis . we found that 186 stroke patients were admitted to the hospital during the study period ; of these , we studied 153 who were diagnosed with ischemic stroke . 45.8% were males and 54.2% were females , giving a male : female ratio of 0.84:1 . there was no significant difference between females and males in age . among the patients , 39.9% had previous attack , and 60.1% had a first ever stroke ( fes ) . patients with fes were younger than those with recurrent stroke ( 67.94 11.66 versus 70.80 10.19 years , respectively ) ; the difference in age between the two groups was not significant ( p = 0.11 ) . the prevalence of risk factors in the study sample was investigated ( figure 1 ) . patients had an average of 3.4 1.2 risk factors ( range 1 7 ) before the attack . htn was the most prevalent risk factor ( 66% ) of the patients , followed by dm ( 45.8% ) . renal dysfunction was also common : 33.9% of the patients had an estimated crcl < 60 ml / min . smoking was practiced by 19.6% of the patients , most of whom ( 99% ) were males . no significant difference was found in the prevalence of risk factors between males and females , with the exception of smoking which was significantly associated with males . however , chf and dm were more common in females , while htn was more common in males . prevalence of various risk factors in the stroke patients admitted to the hospital during the study period . htn : hypertension , dm : diabetes mellitus , bmi : body mass index , af : atrial fibrillation , chf : congestive heart failure , ihd : ischemic heart disease twenty - six ( 17% ) of the patients died during hospitalization . of the 127 survivors , four variables were significantly associated with in - hospital mortality : history of previous stroke ( p= 0.004 ) , crcl at admission ( p = 0.004 ) , number of post - stroke complications ( p = 0.001 ) , and age ( p = 0.043 ) . multiple logistic regression analysis indicated that the number of post - stroke complications ( p= 0.001 ) and previous stroke ( p = 0.03 ) were significant independent predictors of in - hospital mortality . table 1 summarizes the demographic and clinical characteristics of patients who survived versus those who died . table 2 shows multiple logistic regression model using the enter method for predicting mortality after stroke . results of univariate analysis of 153 ischemic stroke patients according to vital status at discharge . htn : hypertension , dm : diabetes mellitus , af : atrial fibrillation , ihd : ischemic heart disease , chf : congestive heart failure . continuous variables were expressed as mean sd , while categorical variables were expressed as frequency and percentage . p value was calculated using independent t test for continuous variables and chi - square test for categorical variables . multiple logistic regression by the enter method for identifying predictors of mortality after ischemic stroke . an average of 4.33 medications ( sd = 1.5 , range : 19 ) were prescribed per stroke survivor . seventy - four ( 92.5% ) stroke survivors with a diagnosis of hypertension were discharged on antihypertensive medications . ace - i were the most ( n = 62 , 50% ) was the class of antihypertensive agents most frequently prescribed for stroke survivors . the main exceptions were anti - coagulants , antibiotics , and anti - inflammatory agents , all of which were used at admission but were discontinued at discharge . there was no difference in the average number of medications prescribed for males and females at discharge ( p= 0.36 ) . we studied the characteristics of patients with ischemic stroke attacks at al - watani hospital , palestine . the mean age was higher than that reported in hospital - based studies from some other arab countries but lower than that reported from developed countries [ 16 , 17 ] . an important observation is the very low percentage ( 1.6% ) of stroke patients who were less than 45 years old . the prevalence of stroke in young patients seems to vary between different ethnic groups of various geographical areas . for instance , only 35% of all strokes occurred in individuals under 45 years old in some countries , while in others they constituted as much as 1930% [ 1820 ] . the male : female ( m : f ) ratio of 0.84:1 in this study was not in agreement with previous studies on stroke from other countries . one study in a province of saudi arabia found a m : f ratio of 1.8:1 . a second study among the national guard community in saudi arabia found a m : f ratio of 2.2:1 . a third study in saudi arabia found a ratio of 3.4:1 [ 2123 ] . however , a hospital - based study in saudi arabia found a 1:1 male : female ratio in stroke patients in general . the unexpected gender ratio in our study might indicate the presence of undiagnosed or uncontrolled stroke - related risk factors in women in palestine . the high prevalence of risk factors among the stroke victims indicates that control of these factors is important for the prevention of stroke . thus , screening , modification , and better control of existing risk factors , such as htn , dm , and cardiac diseases , should be the primary strategy for prevention of stroke . prospective observational studies have established that the risk of primary stroke is strongly related to blood pressure ( bp ) . lowering diastolic bp by 5 mmhg or systolic bp by 10 mmhg reduces the risk of stroke by an estimated 38% . in our study , most patients with htn were given anti - hypertensive agents , particularly ace - i , at discharge . about one - third of the patients had an estimated crcl < 60 ml / min . a recent study indicated that mild degrees of renal dysfunction are associated with increased risk of incident ischemic stroke or transient ischemic attack . recent studies have also established that patients with end stage renal disease ( esrd ) have 510fold higher risk of stroke compared to patients with non - esrd [ 28 , 29 ] . results of in - hospital mortality among stroke patients in this study were also close to those reported in most arab countries . four variables were significant risk factors of in - hospital mortality in stroke patients , namely , history of previous stroke , number of post - stroke complications , creatinine clearance , and age . the influence of age on stroke outcome is still a matter of debate . while several studies showed a negative influence , other studies showed no influence [ 3038 ] ; our results resemble those that showed a negative influence of age on stroke outcome . it is difficult to establish whether age influences stroke outcome itself , or through other factors associated with age . our finding regarding crcl as an independent predictor of early mortality in stroke patients is endorsed by some studies . for example , a study by friedman in 1991 indicated that serum creatinine was an independent predictor of mortality among 492 stroke survivors followed up for a mean period of 18 months . indicated that patients with creatinine clearance calculated at admission of < 51 ml / min had a higher mortality rate . finally , a recent study by fabjan et al . indicated that in patients with ischemic stroke , decreased crcl was associated with higher in - hospital mortality . interestingly , we found that prior use of anti - platelets was not associated with decreased risk of mortality . a hospital - based study carried out in kuwait found that non - se of anti - platelets is significantly associated with deleterious 30-day outcome . one way to reduce early mortality in stroke patients is to develop a stroke unit supervised by a specialized neurologist . admission of acute stroke patients into specialized hospitals seems to reduce the risk of in - hospital mortality . moreover , academic medical centers with vascular neurologists had lower rates of in - hospital mortality for patients with ischemic stroke . it seems that mortality in patients with stroke does not depend only on patient - related factors but also on hospital characteristics . most of the patients in the study had risk factors commonly present in stroke patients . better control of these risk factors might decrease the future incidence rate of stroke in palestine . the number of post - stroke complications and previous stroke were significant independent predictors of in - hospital mortality . medications prescribed at discharge for stroke survivors are consistent with the type of risk factors , especially htn , present in patient 's medical files .

objective : stroke is a major health problem , yet no studies on stroke have been reported from palestine . this one - year , hospital - based study was conducted to determine the prevalence of risk factors and the in - hospital mortality rate in patients with ischemic stroke . method : all patients admitted to al - watani government hospital and diagnosed with ischemic stroke between september 2006 and august 2007 were included in the study . data were obtained by retrospective review of medical charts . pearson chi - square and independent t test were used in the univariate analysis . multiple logistic regression analysis was used to determine the independent predictors of in - hospital mortality rates among the patients . statistical testing and graphics were carried out using spss 15 . results : we identified 153 ischemic stroke patients ( 83 females and 70 males ) of whom 92 were having a first - ever stroke ( fes ) . patients had several prevalent modifiable risk factors such as hypertension ( htn ) ( 66% ) , diabetes mellitus ( dm ) ( 45.8% ) , and renal reduced renal function ( crcl < 60 ml/ min ) ( 33.9% ) . twenty - six ( 17% ) of the patients died during hospitalization . four variables were significantly associated with in - hospital mortality : history of previous stroke ( p= 0.004 ) , crcl at admission ( p = 0.004 ) , number of post - stroke complications ( p = 0.001 ) , and age ( p = 0.043 ) . multiple logistic regression analysis indicated that the number of post - stroke complications ( p= 0.001 ) and previous stroke ( p = 0.03 ) were significant independent predictors of in - hospital mortality . conclusion : screening and better control of risk factors , especially htn , dm and renal dysfunction , are required to decrease the incidence and in - hospital mortality among patients with ischemic stroke .

cigarette smoking is the most important preventable cause of mortality all over the world . also , it is responsible for many non - communicable diseases such as cancers and cardiovascular diseases . moreover , cigarette smoking is the cause of half of the death of those who smoke for a long time . in the year 2000 , about 5 million adults died as a result of cigarette consumption ( about 12% of total deaths in the year 2000 ) . it is estimated that this rate reaches 8.3 million per year by the year 2030 , which 70% of these deaths will occur in developing countries . according to the who estimation , there are about 1.3 billion smokers worldwide that comprise one third of the world population over the age of 15 years . if this pattern of smoking remains unchanged , this rate will reach 2 billion by the year 2030 . meysamie a and colleagues reported that prevalence of cigarette smoking is 23.4% and 1.4% in iranian males and females , respectively . teachers and other groups like clergymen who can be role models , play an important role in persuasion or prevention of cigarette smoking among the youth . also , as students will enter the society and play key roles such as physicians , engineers , and teachers and so on ; studying their smoking behaviors is very important . therefore , students are also role model for the younger , as well as being a representative of youth in the population . the rate and tendency toward smoking among students has increased as shown by several studies . for example , a national survey in the us demonstrated that during a 5-year period , the rate of smoking among students increased from 22% to 29% . also , a study in iran found similar results showing that 5% of the female students in medical schools who were non - smokers in their first year of study became cigarette users in their seventh year of education . these rates among male students were 2% in the first and dramatically increased to 34% in the seventh year . heydari et al . also showed that prevalence of smoking was significantly higher in students in their last year of study compared with whom in the first year . in addition , several studies have demonstrated that prevalence of smoking was higher among students who had smoker teachers compared to those who had non - smoker . these imply the importance of study of prevalence of smoking in these groups and also their knowledge , attitude and practice on the matter . since the clergymen in any religion are one of the most influential groups in the society , study of their smoking habits has been of interest and several studies looked at the prevalence of smoking in this group . for example , a study on buddhist monks demonstrated that prevalence of daily smoking was 12% , which was lower than general population . this study also showed that buddhist monks with no history of smoking had a better knowledge and attitude towards the hazards of smoking . however , there is no study about smoking status of clergymen in islamic countries including iran . the aim of this study is to estimate and compare the prevalence of smoking in 3 groups of male teachers , clergymen and university students and also their knowledge , attitude and prediction of future smoking . this is the first study in iran , which compares not only the prevalence of smoking but also evaluates knowledge , attitude and their future prediction of smoking and investigates inter - relationship in these 3 groups . university students , clergymen and teachers were studied in tehran , iran during 2009 . in this cross sectional study , the knowledge , attitude towards tobacco consumption and their prediction of smoking in the next 5 years of participants were asked by questionnaire . since the understudy clergymen were all males , only males first , tehran islamic religious school and shahid beheshti university were randomly selected from the corresponding lists . then , four medical and randomly four non medical faculties from the shahid beheshti university were selected . one class per grade ( from grade 1 to 4 ) was also randomly selected in these faculties . besides , in tehran islamic religious school one class per grade ( from grade 1 to 5 ) was also selected randomly . the students and clergymen were all 18 - 25 years old . in each class , after explaining the aim of the study and also the confidentiality , all the students were invited to participate in the study . male teachers ( 20 - 29 years ) who thought boys in middle schools in tehran were also randomly selected . first , from the list of districts provided by the ministry of education and training 5 districts randomly selected . third , in each school , on average , about 10 teachers were randomly selected . for calculation of the required number of subjects in each of 3 groups , the formula for sample size calculation for proportion was used . considering p = 50% , = 0.05 and precision of 0.05 , a minimum sample size of 385 was computed for each group . a total of 1,271 university students were enrolled ( 765 medical and 506 non - medical ) . also , 549 clergymen and 551 teachers were randomly studied . therefore , in all 3 groups , we examined a larger population than the calculated required number , which generally can increase the power and precision of the analysis . to examine knowledge and smoking status of the study subjects , a self - report questionnaire was adapted from the standard questionnaire of the global adult tobacco survey . this questionnaire evaluates age of smoking initiation , place of birth , history of smoking ( one with consumption of at least 100 cigarettes was defined as a smoker person ) , smoking status at present , also knowledge about tobacco consumption ( including 4 general knowledge multiple choice questions with 4 options ) , attitude towards tobacco consumption ( 4 multiple choice questions with 5 options ) and contains one question about the probability of tobacco consumption in the next 5 years , which is presented in details by heydari et al . considering the subjects total scores of knowledge and attitude , the answers were categorized into 2 groups of poor / inappropriate ( no correct answer ) and moderate or good / appropriate ( at least one correct answer ) . the answer to the question about probability of smoking in the next 5 years was divided into 2 groups of yes or no . data was entered and analyzed using spss ( 11.5 ) and stata ( 11.0 ) . chi - squared test and logistic regression were used for comparison of smoking status , knowledge , attitude , and probability of smoking in the next 5 years among the 3 groups . in this study , 1,271 students , 549 clergymen , and 551 teachers were interviewed in tehran during 2009 . as table 1 shows , 395 ( 31.1% ) of students , ( 95% ci : 28.5%-33.6% ) had a history of smoking more than 100 cigarettes . this rate was 21.9% ( 95% ci : 18.3 - 25.3% ) among clergymen and 27.2% ( 95% ci : 23.4%-30.9% ) among teachers . as presented in this table , the highest prevalence was seen among students and the lowest among clergymen ( p < 0.0001 ) . logistic regression showed that prevalence of smoking was significantly higher among students and teachers than clergymen ( p < 0.0001 and p = 0.04 ) . however , the 3.9% difference in prevalence of smoking between students and teachers was not found significant ( p = 0.09 ) . also , among the 3 groups , there was borderline difference in age of smoking initiation ( p = 0.06 ) . neither , such difference was detected in terms of successful quit attempts , occasional smoking and daily cigarette consumption ( p = 0.99 ) . no significant difference was found in the amount of daily smoking ( less than 10 cigarettes , 11 - 20 cigarettes and more than 20 cigarettes ) between the 3 groups ( p = 0.64 ) . smoking status of male students , teachers and clergymen in tehran there was a significant difference between the understudy groups in terms of their knowledge , attitude and probability of smoking in the next 5 years ( p < 0.0001 ) as presented in table 2 . also , table 2 shows , 61.9% ( 787 ) of students had poor knowledge ; whereas , this rate was 38.8% ( 213 ) among clergymen and 42.1% ( 232 ) among teachers . inappropriate attitude ( tendency ) towards smoking was observed in 23.1% ( 294 ) of students , 10.2% ( 56 ) of clergymen and 12.7% ( 70 ) of teachers . in addition , 11.9% ( 151 ) of students , 5.8% ( 32 ) of clergymen and 7.3% ( 40 ) of teachers predicted that they will smoke cigarette in the next 5 years . it worth mentioning that the knowledge , attitude , and probability of smoking in the next 5 years of two groups of medical and non - medical students were not statistically significant ( p = 0.29 ; p = 0.28 ; p = 0.30 ; data not shown ) , respectively . this was the reason that we combined these two groups of the students for comparisons between 3 groups . knowledge , attitude and prediction of smoking in the next 5 years among male students , teachers and clergymen in tehran table 3 shows the odds ratio of smoking initiation in the understudy groups based on their level of knowledge , and attitude . odds ratio of smoking cigarette was not significant in students with poor knowledge ( or = 1.2 ; 95% ci : 0.9 - 1.6 ) . whereas , odds ratio of smoking cigarette in clergymen ( or = 3.1 ; 95% ci : 2.0 - 4.6 ) and teachers ( or = 2.7 ; 95% ci : 1.9 - 4.0 ) with poor knowledge was significantly higher than those with moderate or good level of knowledge . also , in all 3 groups , the odds ratio of smoking cigarette was higher among those with inappropriate attitude compared to those with appropriate attitude towards smoking . this chance was significantly more in clergymen than teachers and was the lowest amongst students ( p = 0.01 ; table 3 ) . likelihood of smoking in the next 5 years of male students , teachers and clergymen based on their knowledge and attitude in tehran in addition , when examining the effect of current smoking status on the likelihood of smoking cigarette in the next 5 years , the corresponding odds ratios for students , clergymen and teachers were 1.4 , ( 95% ci : 0.98 - 2.0 ) , 4.0 ( 95% ci : 1.9 - 8.2 ) and 2.9 ( 95% ci : 1.5 - 5.6 ) respectively , which were significantly different ( p = 0.0001 ) . the results of study of association between knowledge , attitude and prediction of smoking in the next 5 in three groups are presented in table 4 . as shown in this table , 25.9% ( 204 ) of students with poor level of knowledge had also inappropriate attitude towards smoking cigarette ; whereas , only 15% ( 32 ) of clergymen and 17.2% ( 40 ) of teachers with poor knowledge had this attitude ( p < 0.0001 ) . also , 7.8% ( 61 ) of students with poor knowledge predicted that they may smoke cigarette in the next 5 years , where these rates were 3.8% ( 8) and 4.3% ( 10 ) among clergymen and teachers , respectively ( p in addition , it was found that 20.4% ( 60 ) of students with inappropriate attitude predicted that they may smoke cigarette in the next 5 years however these were 28.6% ( 16 ) and 28.6% ( 20 ) in clergymen and teachers ( p = 0.008 ) . these finding revealed that although frequency of inappropriate attitude was higher among students , the chance of smoking in the next 5 years in this group was lower than clergymen and teachers . as it is stated the age rage of the students and clergymen were 18 to 25 years and of the teachers 20 to 29 years . when significant test was carried on grade ( as a representative of age ) of students and clergymen it was not significant ( p = 0.37 ) . also , given the rage of age ( 20 to 29 years ) , the mean age of teachers is not generally far from the other 2 groups to alter the findings . the results of this study showed that prevalence of smoking was higher among students ( 31.1% ) and teachers ( 27.2% ) in comparison with clergymen ( 21.0% ) and other males in general population ( 23.4% ) . although , the lowest consumption was seen among clergymen , it was not significantly lower than general population ( p = 0.40 ) . also , level of knowledge , attitude and prediction of smoking cigarette in the next 5 years were more favorable in teachers and clergymen . although , whenever the range of age extended or the females are enrolled the findings could be different . in general , limited studies have compared smoking status in different groups in a community , although there are many researches focusing on a specific group of people . they evaluated 280 medical students in isfahan university of medical sciences and found that prevalence of smoking was 34% among male students who were in last year of their training . another study conducted in saudi arabia showed a relatively similar prevalence of smoking among students of different majors . in this study , which was conducted on 202 medical and 300 non - medical students , they demonstrated that the rate of smoking was 27.8% and 39.5% among medical and non - medical students , respectively . frisch et al . in malaysia examined the pattern of smoking , level of knowledge and attitude of 146 medical and nursing students towards smoking and found that only 11% of male students and none of the female students smoked which is much lower than ours . the level of knowledge and attitude were significantly lower in students compared with other groups in our study . however , other studies conducted in different countries demonstrated that level of knowledge and attitude of medical students towards smoking were more appropriate . a study conducted in the united kingdom on 181 dental students showed that more than 90% of dental students had moderate or good knowledge and more than 80% had an appropriate attitude towards smoking pizzo et al . in a study aiming to examine prevalence of smoking among dental students and their knowledge and attitude towards quitting showed that of 220 students 65% of students had appropriate knowledge and 87% had appropriate attitude towards smoking cessation activities . although in their study , the level of knowledge and attitude of dental students were higher than our students , no significant difference was observed between prevalence of smoking . this indicates that appropriate knowledge and attitude alone can not result in a proper behavior and other confounding factors like socioeconomic and family issues should also be taken into account . , in the netherlands examined knowledge , attitude and rate of smoking in 3 groups of medical students ( 725 subjects ) , residents ( 126 subjects ) and psychology counselors ( 236 subjects ) and found that prevalence of smoking among medical students and residents was lower than the general population ; whereas , prevalence of smoking among counselors was not different from general population . they also found that counselors had poorer knowledge and more inappropriate attitude towards smoking compared to the other 2 groups . the results of glantz study on prevalence of smoking in comparison with general population were in contrast with ours , however he found similar association between knowledge , attitude and prevalence of smoking cigarette with our study . limited studies have been conducted on the smoking status among clergymen of other religions and those available have been mostly performed on buddhist monks . a study conducted on 318 buddhist monks in cambodia showed that 44% were smokers ; whereas , prevalence of smoking in cambodian general population was 65% . in addition , most monks had a poor knowledge about hazards of smoking but as the result of social stigma , prevalence of smoking among them was lower than the general population . another study conducted on buddhist monks in laos showed that prevalence of smoking among them was about 12% , which is much lower than the neighboring countries like cambodia . this study also demonstrated that lao monks had a good knowledge about hazards of smoking . the results of these 2 studies were in accord with those of ours demonstrating that islamic clergymen and buddhist monks both had a lower prevalence of smoking than general population and also had an appropriate level of knowledge in this respect . in general , the majority of studies performed on teachers only studied the prevalence of smoking . our study findings regarding high prevalence of smoking among teachers were in agreement with the findings of talay et al . , in turkey , and sorensen et al . in india . on the contrary , a study in bahrain on 1,140 teachers demonstrated that only 8.7% of bahraini teachers smoked , which was lower than their general population . they also reported that these teachers had acceptable level of knowledge about hazards of smoking . the results of this study revealed that clergymen and teachers with poor knowledge had lower chance for becoming a smoker . in all 3 groups , the odds ratio of smoking in those with inappropriate attitude was significantly different from those with appropriate attitude . however , this chance was not significantly different in clergymen than teachers . also , it was found that smoking status had no significant effect on the probability of smoking in the next 5 years among students . however , for clergymen and teachers , likelihood of smoking in the next 5 years among current smokers was significantly different from non - smokers . in a study on 5,112 teachers in malaysia it also showed that teachers attitude affected their smoking status , which is in concord with our study findings . another study conducted in bosnia on 273 physicians and nurses found a significant difference in their knowledge and attitude towards smoking . while , in each group , prevalence of smoking . also found a significant relationship between prevalence of smoking and level of knowledge and attitude in each group of medical students , psychologists and medical residents . preventing the initiation of smoking in the adolescents and decreasing the prevalence of smoking in adults are the most important methods for prevention of cancer and various diseases . these programs may include increased price of cigarettes , ban of smoking in public places , limiting cigarette advertisements , restricting tobacco advertising , and establishment of counseling and treatment centers for nicotine dependence . however , for implementation of such programs at the national level , a correct estimate of the prevalence of smoking in different social and occupational groups seems necessary . cigarette consumption has increased among the youth of various social levels in the recent years . this study also showed that prevalence of smoking was higher among male students ( which represents the youth in the community ) than general population . considering this increase , new strategies the strength of this study was looking at these 3 groups for the first time , having standard questionnaire , trained research staff and acceptable executive process . the weakness of the study was because the clergyman were male the other 2 groups were selected from males . also , limiting the information on grades of the students and clergymen and age rage of teachers and not recording exact age of participants was of the limitations of the study . this study showed that prevalence of smoking among male students and teachers was higher than general population and clergymen , who smoked equally . also , level of knowledge and attitude and prediction of future smoking in students were worse than teachers and clergymen , which is alarming .

background : students , clergymen and teachers as role models can be very important in encouragement or prevention of cigarette smoking in young people . the aim of this study was to compare prevalence of smoking in 3 male groups of teachers , clergymen and university students . also , study their knowledge and attitude towards it and the prediction of their future consumption.methods:in a cross sectional study in 2009 in tehran , iran , 1,271 male students , 549 clergymen and 551 teachers were randomly enrolled . each participant completed the global adult tobacco survey questionnaire . knowledge , attitude and prediction of smoking for the next 5 years were questioned in these 3 groups . chi - squared test and logistic regression were used for analysis . p < 0.05 was considered significant.results:prevalence of cigarette smoking was 31.1% , 21.9% and 27.2% among students , clergymen and teachers , respectively . smoking in students was not associated with poor knowledge but were in teachers and clergymen . the odds ratio of smoking in students , clergymen and teachers was higher among those with having inappropriate attitude towards it ( or = 1.6 , 6.1 and 4.5 ) . those with poor knowledge had an inappropriate attitude and predicted higher chance of cigarette consumption in the next 5 years ( p < 0.0001 ) . inappropriate attitude in all 3 groups resulted in higher prediction of future smoking ( p = 0.008).conclusions : this study revealed that the prevalence of smoking among male students and teachers was higher than general population and clergymen who equally smoked . also , level of knowledge and attitude of students were lower than teachers and clergymen .

toothbrushes play an essential role in oral hygiene and are commonly found in both community and hospital settings . toothbrushes may play a significant role in disease transmission and increase the risk of infection since they can serve as a reservoir for microorganisms in healthy , oral - diseased and medically ill adults . contamination is the retention and survival of infectious organisms that occur on animate or inanimate objects . in healthy adults , contamination of toothbrushes occurs early after initial use and increases with repeated use [ 2 , 3 ] . toothbrushes can become contaminated from the oral cavity , environment , hands , aerosol contamination , and storage containers . bacteria which attach to , accumulate , and survive on toothbrushes may be transmitted to the individual causing disease [ 4 , 5 ] . in the hospital there is a need for standardized nursing guidelines to prevent toothbrush contamination , which may increase the risk of infections from potentially pathogenic microorganisms and is clinically relevant for assessing the risks and benefits of oral care and informing nursing practice . this review of peer - reviewed literature was conducted to evaluate the cumulative state of knowledge related to toothbrush contamination , its possible role in disease transmission , and in preparation for a research study related to toothbrush contamination in critically ill adults . articles published from 1977 to 2011 , on human subjects and using the english language were obtained . the review included studies that evaluated toothbrush contamination in healthy and oral - diseased adults , guidelines for toothbrush and oral care in both healthy and medically ill persons , hospitalized and nonhospitalized patients , and interventions for reducing contamination of toothbrushes . the following databases were searched : pub med ( clinical inquiries and mesh ) , cinahl , cochrane library , national guidelines clearinghouse , web of science , and google scholar . key search terms used in the review were toothbrush , tooth brushing , colonization , bacterial contamination , contamination , oral hygiene , oral health , nursing practice , microbial contamination , and adults . a total of three separate searches were conducted in a systematic fashion using the inclusion and exclusion criteria and search terms . the first search ( search 1 ) identified articles in the selected databases and complete copies of articles that were considered to have met the inclusion criteria were obtained for further review ( table 1 ) . articles were excluded if they did not meet the inclusion criteria listed above , were conducted on a pediatric population , were duplicates from other databases , or only explored antibacterial methods . the second search ( search two ) included articles identified through cited articles and were reviewed following the same criteria . there were a total of 23 new articles identified through the second search . a third search ( search three ) was conducted one year after the first search in order to capture any recently published articles . after a review of the abstracts for the articles obtained through the three searches , a total of 88 relevant articles were identified for further evaluation . after inclusion criteria were applied , 38 articles were selected ; after exclusion criteria were applied , ten articles were retrieved to be read in their entirety and included in this review ( figure 1 ) . the selected studies are grouped by setting in vivo , in vitro , and studies that combined both types of settings . the sample sizes ranged from 3 to 103 with the majority of studies having a sample size under 30 . overall , the studies evaluated several perspectives related to toothbrush contamination to include : contamination , methods for decontamination , storage , design , and environmental factors . all of the studies examined toothbrush contamination and found significant bacterial retention and survival on toothbrushes after use [ 6 , 7 ] . glass found that toothbrushes from both healthy patients and patients with oral disease contained potentially pathogenic bacteria and viruses such as staphylococcus aureus , e. coli , pseudomonas , and herpes simplex virus . glass also found toothbrushes contaminated with herpes simplex virus 1 in numbers sufficient to cause an infection in the patient . bunetel et al . found that toothbrushes used by patients with existing oral disease quickly became contaminated . this study also found a significant relationship between repeated use and bacterial retention on toothbrushes and that the oral cavity can be inoculated from a contaminated toothbrush . several of the studies found that toothbrushes were contaminated before use [ 5 , 9 ] . mehta et al . found that 70% of the toothbrushes in their study became heavily contaminated with pathogenic microorganisms after use . studies by both taji and rogers and glass found extensive toothbrush contamination after use except in cases where an oral antiseptic , such as mouthwash , was used immediately prior to brushing . verran and leahy - gilmartin found that toothbrushes supported many different bacteria and the amount of growth was varied . bunetel et al . found that toothpaste , mouthwash , and oral antiseptics all decrease microbial load on toothbrushes . their study found that the toothbrushes became heavily contaminated after use . soaking the toothbrush in listerine for 20 minutes prior to and after brushing decreased the microbial load . the use of antimicrobial coated toothbrushes in adults with oral disease was explored by efstratiou et al . as a means to prevent toothbrush contamination . this study , however , found that coating the bristles with triclosan did not change bacterial growth but the use of toothpaste did . glass and jensen explored ultraviolet light as a means of decontamination and found this method to be effective at reducing the bacterial load on toothbrushes . the use of coated tufts and toothpaste was investigated in adult patients with oral disease . found that coated tuffs did not inhibit contamination but use of toothpaste did reduce contamination . mehta et al . found that an overnight immersion in chlorhexidine gluconate was highly effective in decreasing toothbrush contamination and chlorhexidine was more effective than listerine in reducing the microbial load of bacteria . sato et al . found that rinsing toothbrushes with tap water resulted in continued high levels of contamination and biofilm . . found that the use of regular and triclosan - containing toothpaste resulted in lower toothbrush contamination than no toothpaste use . toothbrushes can become contaminated through contact with the environment , and bacterial survival is affected by toothbrush storage containers . found that toothbrushes placed in closed containers and exposure to contaminated surfaces yielded higher bacterial counts than those left open to air . mehta et al . found that the use of a cap for toothbrush storage increased bacteria survival . in addition , glass found that bacteria survived more than 24 hours when moisture is present . toothbrush bristles range from soft to hard with different cluster patterns and plastic shapes while toothbrush handles included different plastic shapes and decorative moldings . bunetel et al . found that bacteria become trapped inside the bristles of the toothbrush and bacterial survival is dependent upon the bacteria ( aerobic versus anaerobic ) and toothbrush design . in addition , the researchers found that solid handles had less bacteria retention and that as the surface area increased , so did the microbial load . toothbrushes with bristles that are frayed and arranged closely together trapped and retained more bacteria . this finding was also echoed in a study by glass that explored the level of bacterial retention based on toothbrush brand , color and bristle pattern . contamination was the lowest in soft and round , clear , two bristle row toothbrushes . mehta et al . found that the retention of moisture and oral debris in the bristles increased bacterial survival . due to the limited number of publications specifically related to toothbrush contamination , it was necessary to conduct a preliminary evaluation of the majority of identified articles for this review . for example , several of the articles combined an in vivo examination of bacterial survival on actual patient 's toothbrushes and then conducted an in vitro autoinoculation experiment to examine decontamination methods on sterile toothbrushes in the laboratory . the selected studies all found that toothbrushes of healthy and oral diseased adults become contaminated with potentially pathogenic bacteria from the dental plaque , design , environment , or a combination of factors . the trend identified in the literature is to evaluate methods to reduce toothbrush contamination or toothbrush design rather than evaluating the process related to how the toothbrush initially becomes contaminated , is stored , or is disinfected . in a vulnerable population such as critically ill adults , pathogenic contamination may increase the risk of infection and mortality . although some interventions such as chlorhexidine , toothpaste , mouthwash , and ultraviolet sanitizers reduce bacterial survival , oral hygiene practices in the hospital setting by nurses vary . currently , there are no nursing guidelines related to toothbrush frequency of use , storage , and decontamination . in the hospital setting , the environment as a source of pathogenic bacteria is now a hot topic and the focus of many current infectious disease research studies . surfaces in close contact with the patient such as bed frames , countertops , sinks , bedside tables , linens , and mattresses may act as fomites . toothbrushes may come into contact with these surfaces prior to or after use thus increasing risk . while there is significant literature available on environmental contamination and risk for infection , no studies have specifically examined the toothbrush on more vulnerable hospital populations such as critically ill adults . toothbrush storage is inconsistent in both community and hospital environments and may increase exposure to pathogenic organisms . the storage conditions of toothbrushes play an important role in bacterial survival : toothbrushes stored in aerated conditions had a lower number of bacteria than those stored in plastic and bacterial growth on the toothbrush increased 70% in a moist , covered environment . in clinical practice , the author has observed that there is no standardized nursing protocol for the storage or replacement of toothbrushes and that some commonly observed nursing practices include storing the toothbrush in the bath basin with other bathing / personal supplies and linens , in a paper towel , in a plastic wrapper , on the bedside table , next to the sink , and in an oral rinse cup at the bedside . these practices may impact the contamination of toothbrushes . in this review , the majority of studies identified had small sample sizes . importantly , despite multiple studies supporting toothbrush contamination and the likely relationship between contamination and disease transmission , there are no studies that specifically examine toothbrush contamination and the role of environmental factors , toothbrush contamination and vulnerable populations in the hospital setting ( e.g. , critically ill adults ) , and toothbrush use in nursing clinical practice . additional descriptive studies to evaluate these relationships would be beneficial and informative for future research . the relationship between environmental factors , toothbrush contamination , and patient oral colonization would inform development of nursing oral care guidelines for adults that minimize risks related to toothbrush contamination .

toothbrushes are commonly used in hospital settings and may harbor potentially harmful microorganisms . a peer - reviewed literature review was conducted to evaluate the cumulative state of knowledge related to toothbrush contamination and its possible role in disease transmission . a systematic review was conducted on adult human subjects through three distinct searches . the review resulted in seven experimental and three descriptive studies which identified multiple concepts related to toothbrush contamination to include contamination , methods for decontamination , storage , design , and environmental factors . the selected studies found that toothbrushes of healthy and oral diseased adults become contaminated with pathogenic bacteria from the dental plaque , design , environment , or a combination of factors . there are no studies that specifically examine toothbrush contamination and the role of environmental factors , toothbrush contamination , and vulnerable populations in the hospital setting ( e.g. , critically ill adults ) and toothbrush use in nursing clinical practice .

the general public is exposed to multiple sources of information on medicine , either through traditional mass media ( e.g. , television , journals , and radio ) or through the internet ( e.g. , blogs , twitter , facebook , and the many features of web 2.0 ) . the media 's potential to reach large audiences gives it an important role in providing health - related information , shaping public health - related beliefs , and influencing health behaviors . however , medical information published in newspapers can often be based on scant evidence because of a lack of specialized knowledge of medical journalists , criticisms of medical information in newspapers , and a lack of available time to secure strong evidence prior to copy deadlines . in addition , information from newspapers , which should be understood by the general population , is sometimes an exaggeration or overstatement . erectile dysfunction is defined as a consistent or recurrent inability of a man to obtain and/or maintain a penile erection sufficient for sexual activity . a duration of symptoms of at least 3 months is considered acceptable to establish a diagnosis of erectile dysfunction , except in some instances of trauma or surgery . erectile dysfunction affected about 150 million men worldwide in 2000 , and this number is expected to double by 2025 as a result of improved life expectancy and the age - related nature of erectile dysfunction . this disorder is also associated with lower overall life satisfaction scores , mental health quality of life ( qol ) scores , and vitality qol scores . many patients look for information on erectile dysfunction from various media outlets , and commercials on treatment for erectile dysfunction have been developed . recently , sugita and miyakawa reported that the size of the japanese market for counterfeit phosphodiesterase-5 inhibitors was estimated to be about 2.5 times larger than that for genuine phosphodiesterase-5 inhibitors . the aim of the present study was to evaluate the accuracy of news information on erectile dysfunction from 10 nationwide newspapers . our present study will add to the growing body of research documenting the nature and influence of news information on erectile dysfunction in korea . articles on erectile dysfunction from 10 nationwide daily newspapers in korea from january 2011 through december 2011 were evaluated in this study . the newspapers included were the kyunghyang shinmun , the kukminilbo , the dong - a ilbo , the munhwa ilbo , the seoul shinmun , the segye ilbo , the hankyoreh , the hankok ilbo , the chosun ilbo , and the korea joongang daily . newspaper articles or columns that reported results of clinical or experimental studies were included ; however , articles that discussed disease occurrence , accidents , crime , policies , social phenomena related to medicine , questionnaire surveys , educational information , esoteric medical information from unreliable sources , and advertisements were excluded . news articles that summarized scientific congresses or journal articles that did not include direct quotations were also excluded . because a public set of data was used that did not include personal data , the present study was exempted from institutional review board review . the websites of both the chosun ilbo and korea joongang daily ( http://www.chosun.com and http://joongang.joinsmsn.com ) newspapers were used to search for relevant articles , whereas the website of the korean press foundation ( http://www.kinds.or.kr ) was used to search for articles from the other newspapers identified above . , we identified 20 articles in a pilot test to confirm the evaluation process for interpretation . all newspaper articles were separately extracted by two researchers ( y.s.h . and j.y.l . ) . differences in the results of the extraction and interpretation were discussed , and agreement was sought from a senior physician specializing in andrology ( k.s.c . ) . the articles were first categorized into articles with accurate statements and articles with inaccurate or misleading statements . on the basis of a previous study , articles with inaccurate or misleading statements were subclassified into four groups : 1 ) using inappropriate surrogate outcomes as clinical endpoints , 2 ) extrapolating nonhuman results to humans , 3 ) exaggerating the significance of results , and 4 ) using incorrect words . surrogate outcomes are defined as physiological or anatomical results ; however , clinical endpoints are defined as patient - related or functional outcomes , such as symptomatic improvement , recovery of normal activity , and survival . traditionally , surrogate outcomes have been interpreted as clinical endpoints , although the two should not be considered equivalent . for instance , an article on post - prostatectomy - induced erectile dysfunction that could be cured by mesenchymal stem cells demonstrated a typical example of confusing surrogate outcomes with end outcomes . results from animal experimental research or the use of cell lines may be inappropriately interpreted as equivalent to human data . although experimental , physiological , and animal studies are valuable and useful , they do not directly influence clinical decisions . finally , the use of incorrect words suggests that subjective and apocryphal terminologies may have been used . data extraction , sharing , and cooperation with our researchers were performed by using google drive ( http://drive.google.com ) and google docs ( google inc . , mountain view , ca , usa ) . a pearson chi - squared test and fisher exact test were used to compare distributions of categorical values , and statistical analyses were performed by using r ( r ver . 2.15.3 , r foundation for statistical computing , vienna , austria ; http://www.r-project.org ) . articles on erectile dysfunction from 10 nationwide daily newspapers in korea from january 2011 through december 2011 were evaluated in this study . the newspapers included were the kyunghyang shinmun , the kukminilbo , the dong - a ilbo , the munhwa ilbo , the seoul shinmun , the segye ilbo , the hankyoreh , the hankok ilbo , the chosun ilbo , and the korea joongang daily . newspaper articles or columns that reported results of clinical or experimental studies were included ; however , articles that discussed disease occurrence , accidents , crime , policies , social phenomena related to medicine , questionnaire surveys , educational information , esoteric medical information from unreliable sources , and advertisements were excluded . news articles that summarized scientific congresses or journal articles that did not include direct quotations were also excluded . because a public set of data was used that did not include personal data , the present study was exempted from institutional review board review . the websites of both the chosun ilbo and korea joongang daily ( http://www.chosun.com and http://joongang.joinsmsn.com ) newspapers were used to search for relevant articles , whereas the website of the korean press foundation ( http://www.kinds.or.kr ) was used to search for articles from the other newspapers identified above . searching was not performed by using the real newspaper . however , the korean press foundation supplied the nearest article in the printed version . , we identified 20 articles in a pilot test to confirm the evaluation process for interpretation . all newspaper articles were separately extracted by two researchers ( y.s.h . and j.y.l . ) . differences in the results of the extraction and interpretation were discussed , and agreement was sought from a senior physician specializing in andrology ( k.s.c . ) . the articles were first categorized into articles with accurate statements and articles with inaccurate or misleading statements . on the basis of a previous study , articles with inaccurate or misleading statements were subclassified into four groups : 1 ) using inappropriate surrogate outcomes as clinical endpoints , 2 ) extrapolating nonhuman results to humans , 3 ) exaggerating the significance of results , and 4 ) using incorrect words . surrogate outcomes are defined as physiological or anatomical results ; however , clinical endpoints are defined as patient - related or functional outcomes , such as symptomatic improvement , recovery of normal activity , and survival . traditionally , surrogate outcomes have been interpreted as clinical endpoints , although the two should not be considered equivalent . for instance , an article on post - prostatectomy - induced erectile dysfunction that could be cured by mesenchymal stem cells demonstrated a typical example of confusing surrogate outcomes with end outcomes . results from animal experimental research or the use of cell lines may be inappropriately interpreted as equivalent to human data . although experimental , physiological , and animal studies are valuable and useful , they do not directly influence clinical decisions . finally , the use of incorrect words suggests that subjective and apocryphal terminologies may have been used . data extraction , sharing , and cooperation with our researchers were performed by using google drive ( http://drive.google.com ) and google docs ( google inc . , mountain view , ca , usa ) . a pearson chi - squared test and fisher exact test were used to compare distributions of categorical values , and statistical analyses were performed by using r ( r ver . 2.15.3 , r foundation for statistical computing , vienna , austria ; http://www.r-project.org ) . after website searches , a total of 188 news articles on erectile dysfunction from 10 newspapers in 2011 were reviewed . 1 ) ; excluded articles were those discussing disease occurrence ( 16 ) , accidents ( 11 ) , crime and policy ( 7 ) , social phenomena related to medicine ( 5 ) , questionnaire surveys ( 4 ) , educational information ( 4 ) , esoteric medical information from unreliable sources ( 63 ) , and advertisements ( 57 ) . in addition , 21 articles that summarized scientific congresses or journal articles were also excluded . our exclusion criteria were based on the concept that simple reporting of an article without discussion of the outcomes of the research was not new health information . of the 47 articles that met our inclusion criteria , 27 ( 57.4% ) contained inaccurate or misleading statements on the basis of an evidence - based medicine evaluation . these included using inappropriate surrogate outcomes as clinical endpoints ( three cases , 6.4% ) , extrapolating nonhuman results to humans ( two cases , 4.3% ) , exaggerating the significance of results ( eight cases , 17.0% ) , and using incorrect words ( 14 cases , 29.8% ; fig . the rate of error was higher in information from korean sources than in information from international sources ( 22 cases vs. 5 cases ) . however , there were no significant differences between korean and foreign sources in any category ( table 1 ) . there were also no differences between articles written by general journalists and those written by medical journalists or specialists ( 13 cases by general journalists vs. 14 cases by medical journalists ) . after website searches , a total of 188 news articles on erectile dysfunction from 10 newspapers in 2011 were reviewed . 1 ) ; excluded articles were those discussing disease occurrence ( 16 ) , accidents ( 11 ) , crime and policy ( 7 ) , social phenomena related to medicine ( 5 ) , questionnaire surveys ( 4 ) , educational information ( 4 ) , esoteric medical information from unreliable sources ( 63 ) , and advertisements ( 57 ) . in addition , 21 articles that summarized scientific congresses or journal articles were also excluded . our exclusion criteria were based on the concept that simple reporting of an article without discussion of the outcomes of the research was not new health information . of the 47 articles that met our inclusion criteria , 27 ( 57.4% ) contained inaccurate or misleading statements on the basis of an evidence - based medicine evaluation . these included using inappropriate surrogate outcomes as clinical endpoints ( three cases , 6.4% ) , extrapolating nonhuman results to humans ( two cases , 4.3% ) , exaggerating the significance of results ( eight cases , 17.0% ) , and using incorrect words ( 14 cases , 29.8% ; fig . the rate of error was higher in information from korean sources than in information from international sources ( 22 cases vs. 5 cases ) . however , there were no significant differences between korean and foreign sources in any category ( table 1 ) . there were also no differences between articles written by general journalists and those written by medical journalists or specialists ( 13 cases by general journalists vs. 14 cases by medical journalists ) . good health is a prime global concern , and various factors including economic development play a significant role in shaping and defining a population 's perception of health . in particular , men 's health , including erectile dysfunction and andropause , are important matters in developing and developed countries . in asia , the men 's attitudes to life events and sexuality ( males ) study , which studies the prevalence and treatment of erectile dysfunction in china , japan , korea , malaysia , and taiwan , reported that 6.4% of 10,934 men between 20 and 75 years of age self - reported erectile dysfunction , and 83.5% found suitable treatments in the media . tannenbaum , in a survey on men 's health of 2,325 canadians between 55 and 97 years of age , reported that 35.7% of men surveyed , especially those of a younger age , showed an interest in erectile dysfunction . in an epidemiological study on erectile dysfunction in korean men , the rate of self - reported erectile dysfunction was 13.4% in 1,570 men aged 40 - 75 years , and the percentage of men scoring fewer than 17 points on the international index of erectile function scale was 32.4% , demonstrating a high prevalence of erectile dysfunction among korean men . thus , the prevalence of erectile dysfunction is high , particularly in developed countries like korea . it might be difficult for men with erectile dysfunction who are keen to receive treatment to find a urologist , schedule an interview , and finally receive medical treatment . reported on the numbers of prescriptions written for phosphodiesterase-5 inhibitors from each department within three medical institutions in korea . in their study , the mean rate of prescriptions for phosphodiesterase-5 inhibitors was approximately 46.4% from the urology departments , compared with 33.0% from endocrinology , 5.1% from neurology , 4.4% from cardiology , and 3.9% from family medicine , which indirectly suggests that the medical diagnosis and treatment of erectile dysfunction was relatively low in the urology departments of the three hospitals . mass media such as newspapers and television broadcasts are alternative sources of medical and health information . with the recent introduction of medical journalists , many medical news articles have appeared in newspapers and television broadcasts and are subsequently redistributed via the internet . unfortunately , as shown in our present study , the accuracy of these medicine - related articles can be unreliable . hwang et al . performed an evidence - based medical evaluation of health information from the television news . in that study , over an 8-month period , there were 85 such reports on the evening news , and 34 of them ( 40.0% ) were found to be inaccurate or misleading . in particular , using inappropriate surrogate outcomes as clinical endpoints was the most frequent error . this could be attributed to the misinterpretation of results owing to inappropriate study designs , or medical researchers assuming that anatomical or physiological endpoints in their studies could be translated directly to represent clinical outcomes in patients , and the ignorance of news reporters on the medical issues they were reporting . the reason for this might be that the researchers published exaggerated results , and the news media chose to focus on this misinformation of popular interest . the frequency of errors was higher in articles from korean sources than in articles from foreign sources . news articles from the foreign presses were re - reported by the korean media mainly because they were easily accessible to the journalists . on the contrary , domestic sources were often more interested in publicizing study researchers and their associated hospitals , often exaggerating claims although the level of korean media has been increasing , it may report biased content not based on exact evidence . because an increasing number of medical commentaries initially reported on television news and newspapers are now available online in media such as twitter , facebook , and youtube , the accuracy of primary sources ( i.e. , television news and newspapers ) has become more crucial . presently , the korean urological association and the korean society for sexual medicine and andrology do not use twitter or facebook aggressively , so as to reduce " retweeting " or " liking " misleading information and comments made by the television news or newspapers . to curtail these errors , evidence - based medical methodology must be agreed upon and practiced by all medical journalists and the news media . doctors must first embrace evidence - based medicine during the course of their training and clinical work and publish their research findings in relevant peer - reviewed journals before making their results public . additionally , medical journalists should spend more time studying and digesting medical - related information and receive training in evidence - based medicine . last but not least , both the korean urological association and the korean society for sexual medicine and andrology should constantly monitor misleading medical articles and request revisions whenever necessary . it is only through these measures that the general public can be spared the negative effects of medical misinformation . approximately 57% of all articles on erectile dysfunction from 10 nationwide daily newspapers were found to be inaccurate on the basis of an evidence - based medicine evaluation . to this effect , urologists , the korean urological association , and the korean society for sexual medicine and andrology play a paramount role in identifying articles containing inaccurate information . in addition , medical journalists should be trained to write articles based on evidence rather than sensation .

purposea rapid growth in the socioeconomic status of koreans has triggered an unprecedented explosion of health information for the general population . despite its obvious benefits , this increase in information could also result in potentially harmful effects for both consumers and professionals who do not use it appropriately . thus , this study was conducted to evaluate the quality and accuracy of health information on erectile dysfunction from 10 nationwide daily newspapers.materials and methodsthis study analyzed health information from 10 nationwide daily newspapers in korea from january 2011 through december 2011 . we reviewed the health information for quality by using evidence - based medicine tools and evaluated the accuracy of the information provided . articles that simply summarized scientific congresses or journal articles and that did not include direct quotations were excluded , as were advertisements.resultsa total of 47 articles were gathered . among them , 27 ( 57.4% ) contained inaccurate or misleading statements on the basis of an evidence - based medicine evaluation . these statements included using inappropriate surrogate outcomes as clinical endpoints ( three cases , 6.4% ) , extrapolating nonhuman results to humans ( two cases , 4.3% ) , exaggerating the significance of results ( eight cases , 17.0% ) , and using incorrect words ( 14 cases , 29.8% ) . the rate of error was higher in the information from korean sources than in that from international sources ( 22 cases vs. 5 cases).conclusionsapproximately 57% of all articles on erectile dysfunction from 10 nationwide daily newspapers were found to contain inaccuracies .

interleukin ( il)-6 is a cytokine featuring redundancy and pleiotropic activity . it was successfully cloned in 1996 as a b - cell differentiation factor , which promotes b - cell differentiation into antibody - producing cells . subsequent in vitro studies and analysis of il-6 transgenic mice have shown that il-6 acts not only on b cells but also on t cells , hepatocytes , hematopoietic progenitor cells , and various other cells [ 24 ] . one of the important functions of il-6 is the differentiation of cd4 nave t cells into effector cells . il-6 in the presence of tgf- promotes nave t - cell differentiation into th17 cells , while il-6 inhibits tgf--induced regulatory t - cell ( treg ) differentiation , causing imbalance between th17 and treg , which is a primary pathogenic factor in several autoimmune diseases . il-6 transmits its signal through its binding to transmembrane receptors or the soluble il-6 receptor ( il-6r ) [ 7 , 8 ] . after binding of il-6 to il-6r , the resultant il-6/il-6r complex associates with gp130 and induces homodimerization of gp130 , which triggers signal transduction system . the pathological significance of il-6 for diseases was first demonstrated in a case of cardiac myxoma . the culture fluid obtained from the myxoma tissues of a patient who presented with fever , arthritis with positivity for antinuclear factor , increased c - reactive protein ( crp ) levels and hypergammaglobulinemia and was diagnosed with undifferentiated connective tissue disease , contained a large quantity of il-6 , which suggested that il-6 might contribute pathologically to chronic inflammation and autoimmunity . subsequent studies have shown that dysregulation of il-6 production is implicated in the pathogenesis of castleman 's disease , rheumatoid arthritis ( ra ) , and various other autoimmune , inflammatory , and malignant diseases [ 24 ] . because of the biological activities of il-6 and its pathological role in diseases , it was anticipated that il-6 blockage would constitute a novel treatment strategy for autoimmune and inflammatory diseases [ 4 , 1315 ] . to this end , tocilizumab was developed , which is a humanized anti - il-6r monoclonal antibody ( ab ) of the igg1 class that was generated by grafting the complementarity determining regions of a mouse anti - human il-6r ab onto human igg1 . ra is a chronic , progressive inflammatory disease of the joints and surrounding tissues accompanied by intense pain , irreversible joint destruction , and systemic complications such as fatigue , anemia , and fever . at the local level , inflammatory cells invade the otherwise relatively acellular synovium leading to neovascularization , synoviocyte hyperplasia , and formation of pannus tissue , which in turn causes destruction of cartilage , erosion of the adjacent bone , and , ultimately , loss of function of the affected joint . the biological activities of il-6 such as proinflammatory activity , augmentation of synovial fibroblast proliferation , osteoclast differentiation , matrix metalloproteinase ( mmp ) , and vascular endothelial growth factor ( vegf ) production , as well as lymphocyte differentiation and its elevation in both serum and synovial fluids of patients with ra [ 1722 ] indicate that il-6 is one of the key cytokines involved in the development of ra . it has been demonstrated in animal model of ra , that are type ii collagen - induced arthritis ( cia ) , and antigen - induced arthritis , il-6 performs a major role in the development and progression of joint destruction , while il-6 deficiency generated by gene knockout or il-6 blockade by means of anti - il-6r ab reduces the incidence and severity of arthritis in these models [ 2328 ] . in the cia model , immunization with type ii collagen predominantly increased the frequency of th17 cells and treatment of mice with anti - il-6r ab during priming markedly suppressed the induction of th17 cells and arthritis development , while treatment with anti - il-6r ab on day 14 failed to suppress both th17 differentiation and arthritis . similarly , in a glucose-6-phosphate - isomerase- ( gpi-)induced arthritis model , administration of anti - il-6r ab on day 0 or 3 suppressed th17 differentiation and protected against arthritis induction , while injection of anti - il-6r ab on day 14 , at the peak of arthritis , did not bring about any improvement in arthritis . arthritis of anti - type ii collagen antibody - induced arthritis ( caia ) is another arthritis model , but , in this model , the priming phase of t cell dependent antibody generation is skipped . although il-6 is also elevated in this model , caia was profoundly suppressed in tnf mice but not in il-6 mice , indicating that tnf may play a more significant role in the development of caia than il-6 . these observations suggest that in the priming phase il-6 is a required factor for the activation of t cell response and production of antibodies specific for joint components and that in the effector phase tnf is the main generator of arthritis . we found that tocilizumab was not effective for clinical improvement in the condition of two patients with psoriatic arthritis , for whose development immune activation does not appear to be required . the clinical antiarthritic effect of tocilizumab is slower than that of tnf inhibitors , which may be due to the different pathological roles of il-6 and tnf in the development of ra ( figure 1 ) . as shown in table 1 , seven phase iii clinical trials of tocilizumab subsequent to phase i and ii studies demonstrated its efficacy either as monotherapy or in combination with disease - modifying antirheumatic drugs ( dmards ) for adult patients with moderate to severe ra [ 3440 ] . a cochrane database systematic review concluded that tocilizumab - treated patients taking concomitant methotrexate were four times more likely to achieve american college of rheumatology ( acr ) 50 improvement ( absolute % , 38.8% versus 9.6% ) and 11 times more likely to achieve disease activity score ( das ) remission ( 30.5% versus 2.7% ) than patients taking a placebo . furthermore , the samurai and lithe studies proved that radiological damage of joints was significantly inhibited by the treatment . the findings of the radiate trial showed that , among ra patients who had previously discontinued tnf inhibitors 50% achieved acr20 , 28.8% acr50 , and 12.4% acr70 responses . the acr improvement and das remission criteria include an acute - phase reactant component , so that there was concern that the effect of tocilizumab evaluated with these criteria might be overestimated . however , it was found that , even when criteria such as the simplified disease activity index ( sdai ) and clinical disease activity index ( cdai ) were used , remission rates for patients treated with tocilizumab were in the same range as those for patients treated with tnf inhibitors [ 42 , 43 ] . on the basis of the excellent results obtained for the efficacy of tocilizumab , it was approved in april 2008 for the treatment of ra in japan . the recommended posology of tocilizumab ( proprietary name , actemra ) is 8 mg / kg , every 4 weeks . subsequently , the european medicines agency approved tocilizumab ( proprietary name , roactemra ) for ra in january 2009 at a recommended dose of 8 mg / kg . in the united states , it was approved for ra in january 2010 , but the recommended starting dose is 4 mg / kg administered once every 4 weeks followed by an increase to 8 mg / kg depending on clinical response . while the dosage differs among countries , tocilizumab has now been approved for the treatment of ra in more than 90 countries worldwide . in addition to clinical trials , the efficacy of tocilizumab was reconfirmed in actual medical practice . the finding by the three recent studies , the german phase iiib real - life study ( tamara study ) [ 44 , 45 ] , the danish nationwide cohorts of ra patients ( danbio registry ) study , and the multicenter retrospective real - life study ( reaction study ) [ 47 , 48 ] are shown in table 2 . in the tamara study , 286 patients were registered for an analysis of the effectiveness and safety [ 44 , 45 ] , 41.6% of whom had previously been treated with tnf inhibitors . acr50 and acr70 response rates at week 24 were 50.7% and 33.9% , respectively , while 47.6% of the patients achieved das remission and 54.9% the european league against rheumatism ( eular ) good response . remission rates with the new acr / eular boolean - based criteria for clinical studies were 15.0% after 12 weeks and 20.3% after 24 weeks , and cdai and sdai remission rates were 24.1% and 25.2% , respectively . for the danbio registry in denmark , the disease activity decreased at all - time points , with remission rates for tocilizumab treatment of 39% after 24 weeks and 58% after 48 weeks . these response rates were comparable to those found for patients switching to their second tnf inhibitors and to the response rates previously observed in phase iii clinical trials . in japan , 229 patients were registered in the reaction study for an analysis of the effectiveness of tocilizumab [ 47 , 48 ] . clinical remission at week 52 was observed in 43.7% of the patients , radiographic non - progression in 62.8% , and functional remission in 26.4% . the retention rates at 24 and 52 weeks were 79.5% and 71.1% , respectively , and were the same for those with or without previous anti - tnf treatment . these results indeed show the efficacy of tocilizumab for the treatment of ra in actual medical practice . the safety and tolerability profiles of tocilizumab monotherapy for japanese ra patients obtained from six initial trials and five long - term extensions have been published . for these studies , 601 patients with a total exposure to tocilizumab of 2,188 patient - years ( pt - yr ) were enrolled . the incidence of adverse events ( aes ) , including abnormal laboratory test findings , was calculated as 465/100 pt - yr , with infections being the most common serious aes ( 6.2/100 pt - yr ) . of the patients treated more than 5 years , 59.7% met the das28 remission criteria at 5 years , which demonstrates the excellent tolerability and high efficacy of tocilizumab . in addition , a systemic literature review to assess the risk of aes for ra patients treated with tocilizumab reported that pooled odds ratios ( ors ) indicated statistical significance for an increased risk of aes for patients treated with 8 mg / kg of tocilizumab plus methotrexate compared with controls ( or = 1.53 ; 95%ci = 1.261.86 ) , as well as a heightened risk of infection ( or = 1.30 ; 95%ci = 1.071.58 ) . the results of an interim analysis of a postmarketing surveillance of all patients treated with tocilizumab in japan were recently reported . this analysis comprised 3,881 patients who received 8 mg / kg of tocilizumab every 4 weeks , and was observed for 28 weeks . occurrence of a total of 3,004 aes in 1,641 patients ( 167/100 pt - yr ) and 490 serious aes in 361 patients ( 27/100 pt - yr ) was reported . the most frequent ae and serious ae were infection at 31/100 pt - yr and 9/100 pt - yr , respectively , with the majority of infections being pneumonia and cellulitis . . abnormalities in laboratory test findings , such as increases in lipid and liver function parameters were common , and total and serious aes associated with laboratory test abnormalities were 35/100 pt - yr and 2/100 pt - yr , respectively . the increased lipid level resulting from tocilizumab administration is perhaps mediated by its effecting on lipoprotein receptor expression , since it was recently shown that overproduction of il-6 reduces blood lipid levels via upregulation of very - low - density lipoprotein receptors . in contrast , we and others observed that hba1c levels and insulin sensitivity improved as a result of tocilizumab treatment [ 53 , 54 ] . while white blood cell and neutrophil counts usually decreased just after tocilizumab injection , this was not related to the incidence of infection . twenty - five patients died for a standardized mortality ratio of 1.66 , which was similar to the results reported for a japanese cohort study of ra . the results of this analysis thus demonstrated that tocilizumab is acceptable in the actual clinical setting . seven cases of gastrointestinal ( gi ) perforation in six patients were reported in this postmarketing surveillance . in the worldwide roche clinical trials , 26 ( 0.65% ) cases of gi perforation were found among patients with ra treated with tocilizumab for a rate of 1.9/1,000 pt - yr and most cases appeared to be complications of diverticulitis . this rate is intermediate between the rates of gi perforations of 3.9/1,000 pt - yr for corticosteroids and 1.3/1,000 pt - yr for anti - tnf agents reported in the united health care database . the reactivation of tuberculosis is a major concern during anti - tnf treatment , but there is no medical consensus regarding the effect of il-6 blockade on tuberculosis . examined the effects of il-6 and tnf blockade on the development of tuberculosis infection in mice and observed that there was less tuberculosis infection for anti - il-6r ab than for anti - tnf ab . in addition , we showed that tuberculosis antigens - induced interferon ( ifn)- production was suppressed by the addition of tnf inhibitors ( infliximab and etanercept ) but not of tocilizumab . although it seems likely that the incidence of reactivation of tuberculosis is lower during tocilizumab treatment than that during anti - tnf treatment , further detailed studies will be needed to clarify this point . these include anti - tnf blockers ( infliximab , etanercept , adalimumab , golimumab , and certolizumab ) , an il-1 antagonist ( anakinra ) , a b - cell depletor ( rituximab ) , an il-6 receptor inhibitor ( tocilizumab ) , and a t - cell activation blocker ( abatacept ) . these biological modifiers target different molecules and b cells , leading to different clinical effects and causing different adverse effects . since no head - to - head comparative studies have been made of the efficacy of these various agents , it has not yet been determined which of these biologics should be selected for a given patient . currently , one of the anti - tnf drugs is chosen as a first - line biologic , but between 14 and 38% of patients show no or little response to anti - tnf treatment , with as many as 40% of patients discontinuing these drugs within a year and 50% within 2 years . the findings of the radiate trial showed that ra patients who had previously discontinued tnf inhibitors , mainly due to their inefficacy , achieved acr20/50/70 responses of 50% , 28.8% , and 12.4% , respectively , when tocilizumab was administered at 8 mg / kg every four weeks . at present , tocilizumab is likely to be prescribed as a second - line biologic therapy but will have to overcome significant competition from established anti - tnf therapies . it is anticipated that tocilizumab will be selected as a first - line biologic for moderately to severely active ra patients with certain complications . aa amyloidosis is a serious complication of ra , and amyloid fibril deposition causes progressive deterioration in various organs [ 59 , 60 ] . since the gene activation of serum amyloid a , a precursor protein of amyloid a fibril , depends primarily on il-6 [ 61 , 62 ] , tocilizumab administration was found to promptly reduce serum concentrations of saa , just as in the case of crp . three case reports showed the clinical ameliorative effect of tocilizumab on gastrointestinal symptoms due to intestinal amyloidosis [ 6365 ] , and amyloid a fibril deposits were found to have disappeared in two cases after three injections of tocilizumab [ 63 , 65 ] . this suggests that tocilizumab may be suitable as a first - line drug for ra patients who are complicated with or are at high risk of developing aa amyloidosis . remission induction is the current goal for ra , and with the development of biological modifiers , a growing number of ra patients has been able to achieve this goal . the long - term efficacy after cessation of tocilizumab followed by das28 remission was demonstrated in the dream ( drug - free remission after cessation of actemra monotherapy ) study . the continuous rate of tocilizumab - free efficacy was 35.1% at 24 weeks and 13.4% at 52 weeks . serum levels of il-6 and mmp-3 are useful markers for identifying patients who may be able to discontinue tocilizumab without risk of recurrence . in addition , the restore study ( retreatment efficacy and safety to tocilizumab in patients with rheumatoid arthritis at recurrence ) demonstrated that retreatment of all relapsed patients with tocilizumab resulted in re - remission . systemic lupus erythematosus ( sle ) is a multisystem autoimmune disorder with a broad spectrum of clinical presentations of unknown etiology that mainly affects young women . the pathogenesis of sle remains unclear , but the concept of apoptosis goes some way towards explaining how the immune system may recognize mainly intracellular antigens . defects in the clearance of apoptotic cells have been recognized in sle patients , leading to aberrant uptake by macrophages , which then present intracellular antigens to t and b cells , thus driving the autoimmune process . cytokine dysregulation is pervasive , and its expression profiles may serve as a marker of disease activity and severity . recent findings have highlighted type i interferon pathway or th17 cell activation in the pathogenesis of sle . levels of crp have been shown to rise in acute illness but not in sle flares , indicating that il-6 , a major regulator of crp production , has a minor role in sle development . however , recent findings suggest that crp dysregulation also plays a part in the pathogenesis of sle and sle may well be a potential target for il-6 blockade . urinary excretion and renal expression of il-6 was elevated in sle patients with active proliferating lupus nephritis [ 76 , 7881 ] , as were il-6 levels in the cerebrospinal fluid of sle patients with central nervous system involvement . compared to healthy controls , sle patients had significantly more il-6 secreting peripheral blood mononuclear cells [ 83 , 84 ] . lymphoblastoid cells isolated from sle patients produced higher levels of il-6 and blocking of il-6 inhibited anti - double - stranded dna ( dsdna ) ab production in vitro [ 85 , 86 ] , indicating that il-6 is involved in autoantibody production . in murine sle models , age - associated increases in serum il-6 , soluble il-6r , and abnormal expression of il-6r have been detected in mrl / lpr mice [ 8789 ] . in old nzb / w mice , anti - il-6 ab reduced and exogenous il-6 increased production of igg dsdna ab by b cells [ 90 , 91 ] . furthermore , il-6 administration exacerbated glomerulonephritis [ 92 , 93 ] , while il-6 blockade by means of anti - il-6r or anti - il-6 ab prevented the onset and progression of the disease [ 94 , 95 ] . mice with epidermal loss of junb reportedly developed an sle phenotype linked to increased epidermal il-6 secretion , and facial skin biopsies of sle patients displayed low levels of junb protein expression , high il-6 , and activated stat3 levels within lupus lesions . these findings led to an open - label phase i dosage - escalation study of tocilizumab ( 2 mg / kg , 4 mg / kg or 8 mg / kg , every 2 weeks for 12 weeks ) with an enrollment of 16 sle patients with mild - to - moderate disease activity . significant improvement in the modified safety of estrogens in lupus erythematosus national assessment version of the systemic lupus erythematosus disease activity index score was observed in 8 of the 15 evaluable patients , accompanied by a median reduction in anti - dsdna ab levels of 47% . the percentage of cd38cd19igd plasma cells in the peripheral blood , which was higher for sle patients than for normal controls ( mean 5.3% versus 1.2% ) , was significantly reduced to 3.1% at 6 weeks . systemic sclerosis ( ssc ) is a connective tissue disease , characterized by fibrosis of the skin and internal organs , vasculopathy , and immune abnormalities . il-6 in the serum of ssc patients was reportedly elevated and the level correlated with the skin severity score [ 100104 ] . moreover , the culture supernatants of peripheral blood mononuclear cells and skin tissues from ssc patients contained higher concentrations of il-6 than those from controls [ 105109 ] . in vitro studies demonstrated that il-6 may contribute to fibrosis by inducing collagen production and induce -smooth muscle actin ( -sma ) expression by dermal fibroblasts , leading to their differentiation into myofibroblasts . on the other hand , anti - il-6 ab suppressed procollagen type 1 production in fibroblasts derived from ssc patients in vitro . ssc serum mediated largely by il-6 was found to induce endothelial cell activation and apoptosis in endothelial cell - neutrophil cocultures . il-6 is also associated with humoral and cellular immunological abnormalities in ssc [ 98 , 99 ] . il-6 is thus thought to play a significant role in producing the characteristics of ssc . moreover , in a ssc model mouse , induced by immunization with topoisomerase i and complete freund 's adjuvant , loss of il-6 expression could ameliorate skin and lung fibrosis . we also examined the clinical effect of tocilizumab on two diffuse ssc patients who had been resistant to conventional treatment regimens . six months after the treatment , both patients showed softening of the skin with reductions of 50.7% and 55.7% for the total z - score determined with the vesmeter , a novel device for measuring the physical properties of the skin , and of 51.9% and 23% for the modified rodnan total skin score . histological examination showed thinning of the collagen fiber bundles and reduction of the number of -sma positive cells in the dermis . since there are few therapeutic drugs for ssc at the present time , these improvements suggest that tocilizumab appears to be a promising biologic for the treatment of ssc . the inflammatory myopathies encompass a group of heterogenous muscle diseases which share the common clinical features of slowly progressive symmetrical muscle weakness , decreased muscle endurance , and fatigue . they include polymyositis ( pm ) , dermatomyositis , and inclusion body myositis , but are generally considered to be distinct diseases with different pathophysiological mechanisms . muscles produce il-6 , and il-6 has been also shown to play a regulatory role in muscle wasting . among these inflammatory myopathies , pm appears to be another suitable target disease for tocilizumab . excessive il-6 expression has been found in the sera and infiltrating mononuclear cells in the muscles of pm patients [ 121123 ] . infiltrating cytotoxic t cells are thought to be involved in muscle fiber damage , and il-6 functions as a helper factor in the induction of cytotoxic t cells . moreover , in a model of myosin - induced experimental myositis it was shown that control mice developed clinically manifest muscle damage , whereas il-6-deficient mice showed no clinical or histological signs of muscle damage . in another model of pm , known as c - protein - induced myositis , intraperitoneal administration of anti - il-6r ab suppressed the severity of myositis preventatively as well as therapeutically . we tested the efficacy of tocilizumab in two pm patients who had been refractory to corticosteroids and immunosuppressive drugs . creatine phosphokinase levels of both patients normalized and mr images showed the disappearance of high - intensity zones in the thigh muscles . these findings suggest that tocilizumab may also be effective as a novel drug for refractory pm . dermatomyositis is a complement - mediated microangiopathy associated with destruction of capillaries , hypoperfusion , and inflammatory stress on the perifascicular regions , so that the pathology is different from that of pm . production of il-6 and type i interferon signature genes was recently proposed as a biomarker for disease activity in childhood dermatomyositis , which thus may be another disorder suitable for tocilizumab targeting . the pathological consequence of such inflammation is destruction of the vessel wall , which is histologically detected as fibrinoid necrosis . takayasu 's arteritis ( ta ) and giant cell arteritis ( gca ) belong to an entity designated vasculitis syndrome , and involve both large and medium - sized arteries [ 129 , 130 ] . the pathogenesis of ta and gca remains unclear , but it is clear that il-6 is involved in their development [ 129133 ] . tocilizumab treatment for a 20-year - old woman with refractory active ta improved the clinical manifestations and abnormal laboratory findings , and subsequent studies reported that tocilizumab treatment induced a rapid remission in 2 patients with ta and 5 patients with gca . surprisingly , two of the patients with gca went into remission without concomitant use of corticosteroids . moreover , tocilizumab was also shown to be effective as rescue treatment for three gca patients for whom the prednisone dose could not be tapered to less than 30 mg / day . positron emission tomography / ct scans revealed that in two patients generalized large - vessel vasculitis was detected during the active phase , which completely resolved upon a 6-month course of tocilizumab therapy . these reports strongly imply that il-6 inhibition may serve as an innovative strategy for the treatment of both ta and gca . however , several studies have suggested that gca patients with a lesser inflammatory response without an increase in il-6 expression were at a higher risk of developing ischemic manifestations than were other patients , since the angiogenic activity of il-6 offers protection against ischemia in such gca patients . these findings indicate that further clinical studies are required to evaluate the efficacy and safety of tocilizumab for gca and ta . it is worthy of note that il-6 has been also implicated in the development of other types of vasculitis syndrome such as polyarteritis nodosa ( pan ) and antineutrophil - cytoplasmic - antibody- ( anca ) associated vasculitis [ 139142 ] . however , so far there have been no reports about off - label use of tocilizumab for pan or anca - associated vasculitis . on the basis of excellent results of the efficacy of tocilizumab for castleman 's disease [ 143 , 144 ] and systemic juvenile idiopathic arthritis [ 145147 ] , it has been approved and used as the first - line biologic in japan . pilot studies and case reports with off - label use of tocilizumab also indicate the potential indications of this biologic for various other organ - specific autoimmune and chronic inflammatory diseases . these include relapsing polychondritis , acquired hemophilia a , autoimmune hemolytic anemia , adult - onset still 's disease [ 151165 ] , crohn 's disease , bechet 's disease with posterior uveitis , polymyalgia rheumatica [ 135 , 168 ] , remitting seronegative , symmetrical synovitis with pitting edema , spondyloarthritides [ 170175 ] , graft - versus - host disease [ 176 , 177 ] , tnf - receptor - associated periodic syndrome , and pulmonary arterial hypertension complicated with castleman 's disease or mixed connective tissue disease [ 179181 ] . further clinical trials are essential , however , to evaluate the efficacy and safety of tocilizumab for these diseases . acute il-6 synthesis provides a warning signal and protects the host from environmental stress , while its prolonged production causes the onset and progression of various autoimmune diseases . several clinical trials have verified the efficacy and safety of tocilizumab for ra , systemic juvenile idiopathic arthritis and castleman 's disease , resulting in approval of this innovative biologic for the treatment of these diseases . case reports of off - label use or pilot studies have also raised the possibility that tocilizumab could become the biological drug of choice for other systemic autoimmune diseases including sle , systemic sclerosis , polymyositis and large vessel vasculitis . at present , the mechanisms through which tocilizumab exerts its clinical ameliorative effects on phenotypically different autoimmune diseases are not completely understood . il-6 blockade may suppress autoantibody production or correct the imbalance of autoantigen - specific th17 and/or th1 versus treg . thus , clarification of the mechanisms as well as further clinical trials to evaluate the efficacy and safety of tocilizumab for these diseases are important issues .

interleukin ( il)-6 is a cytokine featuring redundancy and pleiotropic activity . while il-6 , when transiently produced , contributes to host defense against acute environmental stress , continuous dysregulated il-6 production plays a significant pathological role in several systemic autoimmune diseases . in response to the expectation that il-6 blockade would constitute a novel therapeutic strategy for the treatment of these diseases , tocilizumab , a humanized anti - il-6 receptor antibody , was developed . clinical trials have verified the efficacy and the safety of tocilizumab for patients with rheumatoid arthritis , resulting in approval of this innovative biologic for the treatment of rheumatoid arthritis in more than 90 countries worldwide . pathological analyses of the effect of il-6 on the development of autoimmune diseases and a considerable number of case reports and pilot studies have also indicated the beneficial effects of this antibody on other systemic autoimmune diseases , including systemic lupus erythematosus , systemic sclerosis , polymyositis , and large - vessel vasculitis .

this cross - sectional study was conducted on 182 epileptic patients who applied to the neurology polyclinics of the faculty of medicine at erciyes university , kayseri , turkey between november 2011 and november 2012 . inclusion criteria for the study were established for the participants as follows : to be literate , have no sign of intellectual disability or dementia , have no other disease than epilepsy , be between 18 - 75 years old , have been followed up for epilepsy for at least one year , and to be receiving antiepileptic treatment . the ethics committee of erciyes university , faculty of medicine , approved this study , and informed consent was obtained from the participants . data collection tools were employed through face - to - face interview with the participants after examinations in a specially allocated area in the neurology polyclinics . as data collection tools , we used the multidimensional scale of perceived social support systems , the beck depression inventory ( bdi ) and the assessment scale for coping attitudes ( cope ) . this form consisted of 16 questions concerning participants socio - demographical characteristics such as their age , gender , marital status , educational level , occupation , family type , income , and residence . it also covered introductory data regarding their disease such as the duration of the disease , seizure type , the frequency of seizures during the last one year ( monthly ) , duration of disease , the antiepileptic medications taken , how much the disease affected their family and academic life , and how the people around them perceived the disease . the scale was developed by zimmet et al14 in 1988 , and the study on its reliability and validity in turkey was conducted by eker et al.15 it is a 7-point likert type scale varying from absolutely no to absolutely yes . it has 3 subscales that are composed of 4 items to assess the sufficiency of support from 3 sources : family , friends , and other important or special individuals . the minimum score to be made on the subscales is 4 , while the maximum is 28 . the total scale score is obtained by summing up the scores from the subscales ; the minimum score is 4 and the maximum score is 84 . a high score from the scale indicates that the perceived social support is high.4,16 the cronbach - alpha internal consistency coefficient is 0.88 , and the reliability coefficient for subscales is 0.90 for perceived social support from friends , 0.83 for perceived social support from family and 0.92 for perceived social support from someone special . the inventory developed by beck et al17 is employed to determine the physical , emotional , and cognitive symptoms of depression . this is a multidimensional coping scale that was developed to assess the responses to stress in different ways.19 it is a self - rating scale that consists of 60 questions and 15 subscales . each subscale is composed of 4 questions and the possible scores from each subscale range from 4 to 16 . high scores from the scales allow us to interpret which coping attitudes are employed most by the individual . in our study , the cronbach - alpha internal consistency coefficient was found to be 0.79 . the study data was assessed using the ibm spss statistics for windows , version 20.0 . in the study , parametric and non - parametric analyses were conducted after checking the appropriateness of the variables for normal distribution in order to compare the continuous variables . to compare the average scores of 2 groups , we employed the student s t - test and/or mann - whitney u test , while we employed the kruskal - wallis test ( kw ) or one - way variance analysis to compare the averages of more than one group . dunn s test , one of the multiple comparison tests ( post - hoc ) , was employed to determine from which group the difference originated . . multiple linear regression analysis ( selection model : stepwise ) was conducted to find the independent variables that have an effect using the subtitles of the coping with stress strategies scale . as data collection tools , we used the multidimensional scale of perceived social support systems , the beck depression inventory ( bdi ) and the assessment scale for coping attitudes ( cope ) . this form consisted of 16 questions concerning participants socio - demographical characteristics such as their age , gender , marital status , educational level , occupation , family type , income , and residence . it also covered introductory data regarding their disease such as the duration of the disease , seizure type , the frequency of seizures during the last one year ( monthly ) , duration of disease , the antiepileptic medications taken , how much the disease affected their family and academic life , and how the people around them perceived the disease . the scale was developed by zimmet et al14 in 1988 , and the study on its reliability and validity in turkey was conducted by eker et al.15 it is a 7-point likert type scale varying from absolutely no to absolutely yes . it has 3 subscales that are composed of 4 items to assess the sufficiency of support from 3 sources : family , friends , and other important or special individuals . the minimum score to be made on the subscales is 4 , while the maximum is 28 . the total scale score is obtained by summing up the scores from the subscales ; the minimum score is 4 and the maximum score is 84 . a high score from the scale indicates that the perceived social support is high.4,16 the cronbach - alpha internal consistency coefficient is 0.88 , and the reliability coefficient for subscales is 0.90 for perceived social support from friends , 0.83 for perceived social support from family and 0.92 for perceived social support from someone special . the inventory developed by beck et al17 is employed to determine the physical , emotional , and cognitive symptoms of depression . high total scores indicate a high level and severity of depression . the cronbach - alpha internal consistency of bdi is calculated to be 0.950 . this is a multidimensional coping scale that was developed to assess the responses to stress in different ways.19 it is a self - rating scale that consists of 60 questions and 15 subscales . each subscale is composed of 4 questions and the possible scores from each subscale range from 4 to 16 . high scores from the scales allow us to interpret which coping attitudes are employed most by the individual . in our study , the cronbach - alpha internal consistency coefficient was found to be 0.79 . the study data was assessed using the ibm spss statistics for windows , version 20.0 . ( ibm corp . , armonk , ny , usa ) . in the study , parametric and non - parametric analyses were conducted after checking the appropriateness of the variables for normal distribution in order to compare the continuous variables . to compare the average scores of 2 groups , we employed the student s t - test and/or mann - whitney u test , while we employed the kruskal - wallis test ( kw ) or one - way variance analysis to compare the averages of more than one group . dunn s test , one of the multiple comparison tests ( post - hoc ) , was employed to determine from which group the difference originated . . multiple linear regression analysis ( selection model : stepwise ) was conducted to find the independent variables that have an effect using the subtitles of the coping with stress strategies scale . the median age of the 182 participants in the research population was 32 ( 18 - 75 ) , and 57.7% were women , 47.3% were single , 44% were primary school graduates , 82.45% had a nuclear family , 39% were housewives , 93.4% lived in a city , and 64.8% smoked . it was found that the participants employed emotion - oriented coping strategies most frequently . among the emotion - oriented coping strategies , religious coping ranked the first , positive reinterpretation and growth came second , and use of an instrumental social support method , which is one of the problem - oriented coping strategies , came third . the mean scores of the male participants were significantly high for positive reinterpretation and growth and use of instrumental social support ( p<0.05 ) . when the cope subtitles were compared according to the educational levels of participants , there was a statistically significant difference between the groups for behavioral disengagement and restraint . this difference originated from participants with preschool education and university education for behavioral disengagement , and from participants with university and other educational levels for restraint . participants with a traditional family structure had significantly lower mean scores for mental disengagement , denial , and religious coping than participants with a nuclear family structure . when the cope subtitles were compared according to occupations , there was a statistically significant difference between the groups for humor , restraint , substance use , and suppression of competing activities . this difference originated from the housewife and other occupations group for humor , and from worker , freelance and other occupations for restraint . participants who smoked had significantly lower scores than non - smoker participants for humor and behavioral disengagement ( p<0.05 ) ( table 2 ) . the seizures of 75.3% of the participants were generalized , and 41.8% had had no seizures during the last month . seventy - two percent of the participants stated that the response of those around them to their disease was negative , and 73.6% stated that their disease had no effect upon their communication ability . when the cope subtitles were compared according to seizure type , there was a statistically significant difference between the groups for positive reinterpretation and growth , focus on and venting of emotions , active coping , denial , humor , use of emotional social support , acceptance , and planning . this difference originated from the participants who had generalized or partial seizures . when the subtitles of the coping with stress strategies scale were compared according to the frequency of seizures in the last month , the difference was statistically significant for the groups for positive reinterpretation and growth , mental disengagement , denial , religious coping , and use of emotional social support . this difference originated from the participants who had one and more than one seizure a month for positive reinterpretation and growth and use of emotional social support , while for mental disengagement , denial , and religious coping it originated from participants who had more than one seizure a month and those who had no seizures . the mean scores of focus on and venting of emotions were significantly lower for participants who stated that the disease had an effect on their communication ability ( p<0.05 ) ( table 2 ) . our study found a positively significant correlation among the scores of denial , humor , and the duration of the disease there was a positively significant correlation between the scores of planning ( table 3 ) . a negatively significant correlation was found among the scores of suppression of competing activities , substance use , use of emotional social support , restraint , religious coping , use of instrumental social support , and the number of friends . while there was a negatively significant correlation between the scores of behavioral disengagement there was a positively significant correlation between the scores of use of instrumental social support and income . there was a positively significant correlation among the scores of humor , behavioral disengagement , and age ( p<0.05 ) ( table 3 ) . the correlation between the cope subscale scores of turkish epileptic patients according to different variables . there was a negatively significant correlation among the scores of planning , suppression of competing activities , acceptance , use of emotional social support , religious coping , active coping , use of instrumental social support , focus on and venting of emotions , mental disengagement , positive reinterpretation and growth , and depression ( p<0.05 ) ( table 3 ) . we found a negatively significant correlation among the scores of perceived social support from someone special , and in total and positive reinterpretation and growth , focus on and venting of emotions , use of instrumental social support , active coping , restraint , use of emotional social support , acceptance , suppression of competing activities , and planning . there was a negatively significant correlation with the scores of denial and humor ( p<0.05 ) ( table 3 ) . as a result of the multiple linear regression analysis , we found that the independent variables affecting the positive reinterpretation and growth subscale of the coping with stress scale were the type of seizures and interference of the disease in communication . the variables affecting the focus on and venting of emotions were the type of seizures and interference of the disease in communication . the variables affecting denial were family structure , smoking , and the type of seizures . the variable affecting the use of emotional social support , acceptance , and planning subscales was the type of seizures ( table 4 ) . multiple linear regression analysis of the factors affecting cope subscales of the turkish epileptic patients . in this study , we found that the participants most frequently employed emotion - oriented coping strategies . of the emotional coping methods , religious coping ranked first , while positive reinterpretation and growth ranked second . in a study on epileptic patients in iran , it was found that emotion - oriented coping strategies were employed more than problem - oriented coping strategies.21 in their studies , bautista et al22 stated that the most frequently employed basic coping strategies were acceptance , religious tendency , and searching for emotional support , while the less frequently used ones were substance use , denial , and humor . as in numerous studies,21,23,24 our study also suggests that religious belief and intuition are used as a major coping strategy to deal with this chronic disease ( epilepsy ) . based on these conclusions , the argument related to coping religiously is effective in coping because the spiritual dimension evokes a feeling to accept , to trust in god , and not to rebel by considering the disease as a difficulty originating from allah . however , to come to a final judgment , theist , and atheist groups should be compared . in our study , we found a positively significant correlation among the scores of behavioral disengagement , humor subscales , and age . bourgault - fagnou and hadjistavropoulos25 reported that the elderly were less sensitive and less anxious regarding their health than young people . this can be explained by the fact that as people age they know their disease better , and they , thus , have fewer negative perceptions about the disease ; also their coping capacity and rationalizing ability improve with age . the mean scores of the male participants from positive reinterpretation and growth and use of instrumental social support subscales were significantly higher . it is reported that coping strategies vary according to a variety of factors like age , gender , culture , and disease , and they have distinct characteristics for each individual.20 it is understood from our study that males are more successful in using instrumental social support , which is one of the problem - oriented coping strategies . we believe that this may be connected with attributed power perception , seeking a remedy , and self - expression , to the culture as well . the participants with higher levels of education had significantly lower scores for behavioral disengagement and restraint . cano et al26 stated that individuals with higher educational levels are more successful and experienced in using the coping strategies against the complex perceptions of feelings of pain . it is not surprising that as the educational level of individuals gets higher , they not only have a better capacity to apprehend the meaning and importance of the disease and its consequences , but also they do not have other tendencies ( for example , disengagement and restraint ) since they employ solution - oriented coping methods more successfully and consciously . the mean scores of the participants with a traditional family structure were significantly higher than participants with a nuclear family structure for mental disengagement , denial , and religious coping . it has been stated that spirituality has an influence on the coping abilities of participants with their disease.27 religious tendency , and the approaches observed in traditional family structures may lead them to be more effective since they are transferred in a hierarchical order ( from generation to generation ) . we found a positively significant correlation between income and the use of the instrumental social support subscale of cope and a negatively significant correlation with behavioral disengagement . while material income is an advantage in that it provides positive helpful support , since there are more opportunities to look for a remedy , it is understandable that it is also accompanied with a decrease in undermining behavior . as a result of multiple linear regression analysis , seizure type was the independent coefficient that affected positive reinterpretation and growth , focus on and venting of emotions , denial , religious coping , humor , use of emotional social support , acceptance , and planning subscales . we believe that those with generalized seizures are less successful in using coping strategies than those with partial seizures in many parameters , not only due to the severity of the disease but also to perception of the disease as well as to stigmatization , and since they lead to more severe biological outcomes . when the subtitles of the cope scale were compared according to the frequency of seizures during the last month , there was a statistically significant difference between the groups for positive reinterpretation and growth , mental disengagement , denial , religious coping , and use of emotional social support groups . the outcomes produced by epilepsy are reported to make coping with the disease more difficult than seizures do.28 having more seizures adversely affects the coping strategies due to their social - behavioral results , besides the fact that it creates distrust of the treatment provided , a more sick - person mode , and it develops a lack of self - confidence . we found a negatively significant correlation among the number of friends and the scores of using instrumental social support , religious coping , restraint , use of emotional social support , substance use , and suppression of competing activities subscales , while there was a negatively significant correlation with the scores of planning . participants employ not only positive strategies such as support from family and friends , using distracters , inclining to religion and spirituality , and using meditation techniques , but also negative strategies such as denial , self - accusation , or taking alcohol or drugs.29 people diagnosed with epilepsy experience many psychosocial problems , including the fear of having seizures.30 a life dependent on medications and other people adversely affects self - confidence . social support and acceptance of the disease by the family play an important role in surmounting such difficulties and social contacts and , within this context , family relations , and social contacts increase a patient s ability to cope with the disease.31 we found a positively significant correlation among the duration of disease and the scores of denial and religious coping subscales of the cope scale . having a strong faith in religion and an increase in the duration of the disease can explain their having a more submissive attitude in line with their faith and greater acceptance of the reality of the disease . the mean scores of participants who stated that their disease had an influence on their communication ability were significantly lower for the focus on and venting of emotions . the stigma and seizures that accompany epilepsy themselves adversely affect social communication.6 the presence of seizures and their unpredictable times in epileptics may result in social and communicative limitations , loss of functions , and loss of self - confidence . we found a negatively significant correlation among the depression scores , and the scores of positive reinterpretation and growth , mental disengagement , focus on and venting of emotions , use of instrumental social support , active coping , religious coping , use of emotional social support , acceptance , suppression of competing activities , and planning subscales , but we found a positively significant correlation with denial , humor , and substance use . these comorbidity rates are seen in correlation with endogenous and exogenous ( iatrogenic conditions ) factors and the severity and chronicity of epilepsy.32 depression is obviously common in epilepsy ; however , epilepsy is more common in depression . depression itself is a risk factor for epilepsy.33 lack of family support is a risk factor for depression in people with medical diseases.34 feelings of demoralization , helplessness and hopelessness , confusion , and subjective insufficiency are important for depression , and are determiners of which psychotherapy method should be preferred.6 in a study which investigated the coping strategies developed by patients with epilepsy and depression,22 it was observed that more than one third of the participants with major depression preferred coping through denial . lower life quality , higher inability , paroxysm of laughter , severe seizures , inability to work and inability to drive was also associated with depression . in general terms , being depressed decreases a person s capacity to cope in many aspects of life , and may lead to a tendency to use drugs because of depression . we found a positively significant correlation among the scores of perceived social support from a friend or someone special , and in total and positive reinterpretation and growth , focus on and venting of emotions , use of instrumental social support , active coping , restraint , use of emotional social support , acceptance , suppression of competing activities , and planning subscales , but we found a negatively significant correlation with denial and humor scores . people with epilepsy are frequently in fear of having a seizure and this affects their social relations , self - confidence , and academic success , and thus leads to making fewer friends , having a shorter married life , and more often developing anti - social behaviour.13 foladi et al3 stressed the greatest need of participants was for emotional support from their family and friends while taleghani et al35 stressed the significance of the presence of a supportive environment in which the problems , experiences , and anxieties of participants are discussed openly . those with epilepsy are in need of psychological and social support to cope with epilepsy.13 it is obvious from the positive correlation found among the perceived social support and problem - oriented and emotion - oriented coping strategies that social support offers the opportunity to use more conclusive methods and that participants employ more dysfunctional coping methods such as denial when such conditions are not available . a limitation of this study was the use of the bdi as the only assessment tool to assess the presence and severity of depressive symptoms . in conclusion , this study found that people with epilepsy employed emotion - oriented coping strategies most frequently . the most important variables affecting coping strategies with the disease among epileptics were age , gender , educational level , family structure , seizure type , and interference of the disease in the patient s communication ability . it is crucial for epileptics to keep losses related to their disease to a minimum by not only controlling the frequency and severity of seizures through optimal effective medication , but also by developing social support systems , taking the necessary precautions to protect their mental health and treating existing mental problems , if there are any .

objective : to investigate the perceived social support systems , and depression s effects on attitudes regarding coping strategies for the disease in patients with epilepsy.methods:this cross - sectional study was conducted on 182 epileptic patients who applied to the neurology polyclinics of the faculty of medicine at erciyes university , kayseri , turkey between november 2011 and november 2012 . as data collection tools , we used the multidimensional scale of perceived social support systems , beck depression inventory , and the assessment scale for coping attitudes.results:we found that epileptic patients most frequently employed emotion - oriented coping strategies . among the emotion - oriented coping strategies , religious coping ranked first , positive reinterpretation and growth came second , while using instrumental social support , which was one of the problem - oriented coping strategies , ranked third . the most frequently used non - functional coping methods were focus on and venting of emotions . the most influential variables on coping strategies of epileptic patients were age , gender , educational level , family structure , type of seizures , and the interference of the disease in communication . we found a negatively significant correlation among the scores of depression and emotion - oriented coping strategies , dysfunctional coping strategies , and problem - based coping strategies , while there was a positive correlation found between dysfunctional coping strategies and emotion - oriented coping strategies.conclusions:the most influential variables on the coping strategies of epileptic patients were age , gender , educational level , family structure , type of seizures , and the interference of the disease in communication .

the antimetabolite 5-fluorouracil ( fu ) is widely used in the treatment of solid tumors , including gastrointestinal , breast , head , and neck cancers . cardiotoxicity , a rare adverse effect of 5-fu , has a reported incidence of 1.2% to 18% . the patient presented with typical angina and electrocardiographic changes suggestive of an ischemic coronary event during the continuous infusion of 5-fu . the ischemia recurred when the infusion was stopped , and was relieved by administration of nitroglycerin followed by a sublingual calcium channel blocker . an 83-year - old man who had been diagnosed with stage iiic ( t4n2m0 ) adenocarcinoma of the ascending colon and who had undergone a right hemicolectomy was admitted to inje university seoul paik hospital to begin adjuvant chemotherapy , using the folfox4 regimen ( 400 mg / m 5-fu bolus infusion followed by the continuous infusion of 600 mg / m 5-fu for 22 hours on days 1 and 2 ; 200 mg / m leucovorin as a continuous infusion for 2 hours before 5-fu infusion on days 1 and 2 ; and infusion of 85 mg / m oxaliplatin on day 1 ) . baseline echocardiography performed 1 month before admission showed normal left ventricular systolic function and no regional wall motion abnormality . he was treated with 1,500 mg high - dose 5-fu ( 1,000 mg / m ) per day . on the morning of the 3rd day , he developed a severe , substernal , crushing chest pain during the continuous intravenous infusion of 5-fu ( cumulative dose 1,679 mg / m ) , which was partially relieved by administering sublingual nitroglycerin . the electrocardiogram ( ecg ) showed st segment elevation with a tall t wave in leads i , avl , and v4 - 6 , and reciprocal st segment depression in leads v1 - 2 ( fig . the troponin - i and ck - mb levels were 0.010 ng / ml ( reference range , 0.1 ) and 3.73 ng / ml ( reference range , 4.94 ) , respectively . severe hypokinesia of the lateral wall of the left ventricle was noted on a portable bedside echocardiogram . the 5-fu infusion was stopped , and the chest pain and electrocardiographic changes resolved after intravenous infusion of nitroglycerin at 30 g / min . emergency coronary angiography was then performed , which revealed significant stenosis in the proximal left circumf lex coronary artery ( lcx ) . intracoronary nitroglycerin ( 200 g ) was injected to exclude coronary vasospasm , but no change occurred ( fig . 2 ) . intravenous ultrasound ( ivus ) showed severe luminal narrowing with a heavy concentric plaque in the proximal lcx . percutaneous coronary intervention of the proximal lcx lesion was performed successfully with the implantation of a drug - eluting stent ( 3.5 16 mm ; taxus , national medical center , seoul , korea ) ( fig . the patient was transferred to the coronary - care unit , where 8 hours later , he reported a recurrence of the anterior chest pain . the ecg also showed st segment elevation and reciprocal st changes similar to those seen in the previous ischemic events ( fig . the chest pain and ecg changes persisted despite a 100 g / min nitroglycerin infusion . to rule out acute stent thrombosis , the chest pain and ecg changes were relieved after sublingual administration of 10 mg nifedipine . repeated coronary angiography showed a widely patent stent ( fig . 4 ) . the postprocedural troponin - i and ck - mb levels were 0.010 and 4.62 ng / ml , respectively . echocardiography performed the next day also showed the absence of the regional wall motion abnormality and normal left ventricular systolic function . we present here a case of severe cardiotoxicity mimicking acute anterolateral myocardial infarction occurring in a patient receiving 5-fu chemotherapy for adenocarcinoma of the colon . the cessation of 5-fu administration and the subsequent initiation of treatment with a sublingual calcium channel blocker and nitrate resulted in a successful outcome . although the mechanism by which 5-fu exerts its cardiotoxic effects is unknown , the resolution of the patient 's chest pain and the normalization of his ecg changes with a vasodilator strongly support the vasospastic hypothesis of 5-fu cardiotoxicity . one study postulated that 5-fu - associated cardiotoxicity is due to the uncoupling of the electromechanical mechanisms that underlie normal myocardial function , which might be mediated at the level of the cell membrane . recently , kuzel et al . suggested that 5-fu promotes a hypercoagulable state ( e.g. , coronary artery thrombosis ) and observed a significant increase in fibrinopeptide a and a decrease in protein c activity during 5-fu administration . the incidence of clinically apparent 5-fu cardiotoxicity is less than 10% in patients receiving the drug . patients with a history of coronary artery disease ( cad ) have a significantly increased risk of 5-fu - induced cardiotoxicity . although our patient did not have a history of cad , a large atheromatous plaque was found on coronary angiography and ivus . therefore , during 5-fu infusion , close , careful monitoring of patients , especially those with pre - existing cad or cad risk factors , is mandatory . prophylactic calcium channel blockers or nitrates should be administered to patients with cad during 5-fu administration , to prevent vasospasm . one study proposed that impaired renal function is also a risk factor for 5-fu cardiotoxicity . although it is not clear whether the cardiotoxic metabolites undergo renal excretion , the pathophysiological effect of 5-fu on the myocardium is likely to increase with decreased renal function . thus it is necessary to clarify which patients may benefit from optimum anti - angina prophylaxis and careful , close monitoring . the incidence of 5-fu - related cardiotoxicity appears to be dependent on the dosage and delivery system . infusion of 5-fu , which is now being used more frequently and at higher doses instead of bolus therapy , may be a significant factor in the development of 5-fu cardiotoxicity . in one study , nine patients treated with a higher - dose ( > 800 mg / m ) continuous infusion of 5-fu died suddenly . interestingly , despite stopping the 5-fu , the chest pain and ecg changes recurred in our case . one series reported that 19% of the patients developed reversible angina pectoris during treatment , which lasted for up to 12 hours after cessation of the infusion . the possible mechanisms of delayed angina are the late release of potent vasoactive 5-fu metabolites , which accumulate over time due to degradation of 5-fu . therefore , a calcium channel blocker or nitrates should be administered after stopping the 5-fu when 5-fu - induced cardiotoxicity occurs . the long - term outcome of patients with 5-fu - related cardiotoxicity has not been investigated extensively . as with our case , patel et al . recently reported interval improvements in the left ventricular wall motion abnormalities in echocardiography performed 8 to 15 days following the initial study . when cardiotoxicity occurs , 5-fu treatment is usually discontinued due to its very high recurrence rate ( 90% ) . the re - administration of 5-fu is not recommended , and a different chemotherapy regimen should be considered . interestingly , meydan et al . continued 5-fu chemotherapy in one group who experienced 5-fu cardiotoxicity due to the absence of an alternative drug and found that subsequent serious , hemodynamic consequences were easily controlled with nitrate treatment . for the remaining patients , either 5-fu was removed from the combination regimen or an alternative drug was started and no cardiotoxicity developed subsequently . in summary , although 5-fu - associated cardiac toxicity is rare , it may cause angina , myocardial infarction , and even sudden death . physicians should be aware of this potentially lethal side effect and should start the proper treatment when 5-fu cardiotoxicity develops . this case supports the vasospastic hypothesis of 5-fu cardiac toxicity , indicating that a calcium channel blocker may be effective for prevention or treatment of 5-fu cardiotoxicity .

cardiotoxicity associated with 5-fluorouracil ( fu ) is an uncommon , but potentially lethal , condition . the case of an 83-year - old man with colon cancer who developed chest pain during 5-fu infusion is presented . the electrocardiogram ( ecg ) showed pronounced st elevation in the lateral leads , and the chest pain was resolved after infusion of nitroglycerin . a coronary angiogram ( cag ) revealed that the patient had significant atherosclerosis in the proximal left circumflex artery . coronary artery spasm with fixed stenosis was considered , and a drug - eluting stent was implanted . after 8 hours , the patient complained of recurring chest pain , paralleled by st elevation on the ecg . the chest pain subsided after administration of intravenous nitroglycerin followed by sublingual nifedipine . repeated cag showed patency of the previous stent . this case supports the vasospastic hypothesis of 5-fu cardiac toxicity , indicating that a calcium channel blocker may be effective in the prevention or treatment of 5-fu cardiotoxicity .

lipoma is a common benign soft tissue neoplasm that sometimes may have mixed tissue components . ossification of a lipoma was first described in 1959 , and it is rarely reported7,16 ) . several names have been used to describe ossification of lipomas , including secondary calcification , ossified lipoma , ossifying lipoma , or osteolipoma , and some authors have used these terms interchangeably . as with classic lipomas , lipomas with ossifications may be found in any part of the body , but are usually found in the head , neck , oral cavity , and extremities , adjacent to bone . only two cases of osteolipoma arising in connection with vertebrae have been described . here , we present a case of an intramuscular osteolipoma on the posterior neck , independent of vertebrae , and review the clinicopathologic features of osteolipoma previously published in the literature . a 51-year - old female presented with a 5-year history of a painless , progressively enlarging mass on the posterior neck . physical examination revealed a soft , non - tender , uniform mass without surface abnormalities . computed tomography ( ct ) and magnetic resonance imaging ( mri ) showed a circumscribed mass involving the posterior neck muscles in the midline between the c2 and c6 spinous processes with a large calcified component ( fig . the ovoid mass was measured 4 cm in width , 6 cm in length , and 3 cm in height , without infiltration of the cervical spine . subsequently , the patient underwent a whole body bone scan , which revealed an amorphous calcification of the posterior neck without metastasis ( fig . the mass had a well - demarcated margin and the outer wall was very firm without adhesion to ligaments or paravertebral muscles . gross examination revealed a well - circumscribed mass composed of adipose and bony tissue , with red bone marrow visible on the cut surface ( fig . 3 ) . histological study showed a lipomatous lesion with large foci of osseous metaplasia at the periphery of the mass ; the bony portion was surrounded by mature adipose tissue ( fig . microscopic examination of the red bone marrow showed a meshwork of bone trabeculae and hematopoietic marrow elements ( fig . the patient was diagnosed with an intramuscular osteolipoma of the posterior neck and no recurrence was observed at the 6-month follow - up . a lipoma is a common soft tissue neoplasm , but lipomas with distinct osseous components are rare2 - 4,8,10 - 12,14,15 ) . as noted by heffernan et al.11 ) , in allen 's review of 635 cases , less than 1% of lipomas were ossified . further , as reported by de castro , et al.5 ) , when furlong analyzed histological findings from a group of 125 lipomas of the oral and maxillofacial region , there was no variant that presented as osseous metaplasia . lipomas presenting with osseous components have been reported as secondary calcification of lipoma or ossified lipoma4,12,14 ) . secondary calcification of lipoma involves calcification that progresses from the periphery inwards as a result of necrosis after impaired blood supply or repeated microtrauma5,14 ) . an ossified lipoma is defined as a histologic variant of lipoma that has undergone osseous metaplasia , and can be classified into 2 groups according to the predominance of the osseous component : ossifying lipoma and osteolipoma . if the adipose component of the lipoma is more dominant than the osseous component , it is an ossifying lipoma , whereas an osteolipoma presents with a dominant osseous component14 ) . osteolipomas have been reported in middle aged or elderly patients presenting with large painless masses with a long duration , even years , and they may be found incidentally2,7 ) . they have been reported at various sites located adjacent to bone or periosteum , including upper and lower extremity , oral mucosa , soft tissue of neck , and intracranial regions , including tuber cinereum , hypothalamus , suprasellar cistern , and the interhemispheric area1 - 5,7,10 - 15,17,18 ) . one patient was an 8-year - old female who presented with a progressively enlarging swelling over the lumbar region with a dermal sinus tract12 ) , and other was 20-year - old female with a cervical osteolipoma contained within the spinal canal14 ) . presented here , the osseous lipoma was located in the deep neck musculature , independent of bony vertebral structures . there are several hypotheses regarding the pathogenetic mechanisms that influence the osseous metaplasia of lipomas , but the details are still not clear . first , these tumours may originate from multipotent mesenchymal cells that can differentiate into lipoblasts , chondroblasts , fibroblasts , angioblasts , and osteoblasts . this could account for the several lipomatous variations of classic lipomas , i.e. lipoblastoma , lipomatosis , fibrolipoma , angiolipoma , myolipoma , myelolipoma , hibernoma , and atypical lipoma , and mesenchymoma is defined as a rare soft tissue lesion that is composed of fibrous tissue associated with two or more types of well differentiated mesenchymal cells that would not normally be found in the same area 2,7 ) . second , ossification may also have been induced by poor nutritional supply in the centre of a large lipoma after repetitive trauma , metabolic changes , or ischemia leading to transformation of fibroblasts into osteoblasts2,8 ) . fritchie et al.8 ) reported cytogenetic analyses of three osteolipomas and reported that the translocations in all cases were consistent with the karyotypic features of lipoma . other benign tumors that may contain bone including teratomas or dermoids , masses with secondary ossification due to trauma , liposarcomas with metaplastic changes , or congenital malformations , should be considered in the differential diagnosis . in addition , tumour calcinosis , calcification in a bursa , and other conditions such as ossifying fibromas , myositis ossificans , and osteosarcomas should be taken into consideration . the use of ct scanning provides excellent visualization of the calcified or ossified components of a lipoma and confirmation of proximity to adjacent bone , and mr imaging can also provide detailed information that is useful for further evaluation2,9 ) . definitive diagnosis of osteolipoma can be made with histopathologic findings after surgical excision , which is usually the recommended treatment . a histopathologic appearance of diffuse , mature ossification within adipose tissue and gross features demonstrating a dominant osseous component confirm the diagnosis . histologically confirmed osteolipomas are benign neoplasms , as with classic lipomas , and do not recur2,4 - 6,11 ) . to the best of our knowledge , it is the first case report of an intramuscular osteolipoma within the posterior neck independent of adjacent bone . we suggest that although osteolipoma is a rare variant , it is important to keep it in mind when a soft tissue mass of the posterior neck mixed with osseous component is encountered .

ossified lipoma or osteolipoma are rarely reported . it is defined as a histologic variant of lipoma that has undergone osseous metaplasia . osteolipoma presents with a dominant osseous component within a lipoma . we report a case of a histologically confirmed osteolipoma on the nuchal ligament independent of bone . the patient was a 51-year - old female who presented with a 5-year history of a painless , progressively enlarging mass on the posterior neck . computed tomography and magnetic resonance imaging showed a circumscribed mass compatible with fat between the c2 and c6 spinous processes with a large calcified irregular component . the mass with dual components was totally removed under general anesthesia and no recurrence was observed after 6 months of follow - up . we also reviewed the clinicopathologic features of previously reported osteolipomas in the literature and suggest that although osteolipoma is a rare variant of lipoma , it should be considered in the differential diagnosis when a lipoma of the posterior neck mixed with a bony component is encountered .

mycobacterium tuberculosis isolates from all culture - positive tb patients in seattle and king county , washington , during 20032004 were genotyped by spacer oligonucleotide typing and mycobacterial interspersed repetitive unit methods . we included patients who had an isolate that matched the outbreak strain or who had a social link to an already included patient . patient medical records were reviewed , and infectious periods were calculated . for sputum smear positive patients , the infectious period extended from 3 months before symptom onset or the first positive smear ( whichever was earlier ) until 2 weeks after the start of appropriate tb treatment or until the patient was placed into isolation or produced consecutively negative smears . for sputum smear negative patients , the infectious period extended from 1 month before symptom onset , the start of appropriate tb treatment , or the date that the patient was isolated ( whichever was earlier ) , until 2 weeks after the start of appropriate tb treatment or until patient isolation ( 7 ) . we interviewed patients to learn their contacts , activities , and locations frequented while they were contagious . additional contacts were found by outreach workers and a disease intervention specialist from the east - african community who was hired to work in the neighborhoods frequented by the patients . while in these neighborhoods , outreach workers and the disease intervention specialist recruited persons seen with patients or their contacts to be evaluated for tb and latent tb infection . contact activities , specifically those related to illicit drugs , were observed or self - reported . friends were defined as contacts of patients who spent time within a close - knit network of young men who exhibited similar marijuana - using behavior . other contacts were defined as the families and relatives of patients and those who were named but were not closely associated with this network . contacts received a tb evaluation including a tuberculin skin test ( tst ) to detect infection . patient 1 was first evaluated in december 2003 , when a chest radiograph suggested pulmonary tb ( i.e. , upper lobe cavitary infiltrate ) . however , only clarithromycin was prescribed , and the patient was lost to follow - up . he was again seen in an emergency room in april 2004 after the infection evolved into bilateral extensive pulmonary tb . eight patients were born in east africa ; a median of 13 years ( range 622 ) had passed since their arrival in the united states . patient 5 was a white woman who received illicit drugs from patient 1 . * unknown for 1 patient . patients were symptomatic and had findings indicating infectiousness : all had pulmonary tb , 7 had cavitary disease , and 8 had sputum that tested smear - positive for acid - fast bacilli . consecutive chest radiographs indicated progression to cavitary disease in < 75 days weeks in 3 patients and < 121 days in another patient . table 2 shows the dates of clear chest radiographs interpreted as normal and the first chest radiographs showing disease . while contagious , patients stayed in various locations , including cars , for most of the day . a single - bedroom apartment occupied by at least 1 patient while he was contagious was regularly visited by 2 other patients . numerous members of the friend network slept there on any given night , and many others would regularly visit during a 10-week period beginning in april 2004 ( figure ) . the occupants nailed boards over the apartment windows to conceal activities , primarily marijuana use , from outsiders . all reported frequent " hotboxing , " the practice of smoking marijuana with others in a vehicle with the windows closed so that exhaled smoke is repeatedly inhaled . considerable overlap in infectious periods was noted , which highlights the potential for simultaneous contact with multiple contagious patients . at least 31 ( 57% ) friend contacts spent time at the 1-bedroom apartment . after those with a past positive tst result were removed , 14 ( 64% ) of 22 screened friends and 6 ( 23% ) of 26 other contacts had a positive tst result . the risk for a positive tst result was 2.8 greater among friends than among other contacts ( 95% confidence interval = 1.36.0 ) . twenty - nine ( 54% ) friend contacts self - reported or were observed hotboxing . among the friends who reported or were observed hotboxing , 11 ( 79% ) of 14 who received a tst had a positive result . risk factors for tb include birth in a country with high tb prevalence ( 2 ) and incarceration ( 8) . although most patients in this outbreak were foreign - born and had histories of incarceration , genotyping results and epidemiologic findings suggest that tb was transmitted recently in the community rather than before immigration or during incarceration . frequent marijuana use has been reported among tb outbreak patients ( 9 ) and was the behavior linking these patients together . creative sharing of marijuana has been described recently as a factor for m. tuberculosis transmission . in australia , sharing a water pipe ( i.e. , " bong " ) was linked to transmission ( 10 ) . " shotgunning " refers to inhaling smoke from illicit drugs then exhaling it directly into another 's mouth ( 11 ) and was associated with m. tuberculosis transmission among a group of exotic dancers and their contacts ( 12 ) . this investigation noted that a similar activity , hotboxing , might have contributed to transmission . one patient with smear - positive cavitary disease reported daily hotboxing with friends , often for most of the day . in addition , marijuana smoking might induce cough , creating an ideal environment for transmission . many friends stayed and used marijuana at the single - bedroom apartment during the height of the outbreak . furthermore , by nailing boards over the windows , ventilation was limited , creating an environment similar to that of hotboxing . although progressive primary tb by nature is thought to be due to recent transmission , progressive primary tb with cavitation is uncommon ( 13 ) . however , frequent marijuana use and the setting of intense exposure may have played a role . in addition , poor nutrition and unhealthy lifestyles might have predisposed these young men to more rapid progression of disease . while no laboratory investigation to assess genetic susceptibility or strain virulence was conducted , these factors might have also contributed to the development of cases . this outbreak resembles an outbreak reported among regular patrons of a neighborhood bar ( 14 ) . both were fueled by a highly infectious source patient who spent extended amounts of time indoors with 1 group of persons who regularly used substances ( i.e. , alcohol or marijuana ) . the result in both situations was a higher than expected incidence of tb disease and latent tb infection . in the outbreak reported in this article , however , the substance of choice was illicit and further complicated the control of this outbreak . patients ' illicit drug activities promoted a reluctance to name contacts at risk and locations frequented . efforts had to revolve around meeting these young patients at times and locations convenient to the group . then after gaining the groups ' trust , outreach workers successfully found and screened contacts . many successful screenings took place on street corners and in parking spaces throughout the community . often outreach workers were successful only after spending hours driving throughout the community searching for patients and contacts . alternative strategies to name - based contact investigations may become increasingly critical to tb control as tb recedes further from the general population , yet persists within smaller guarded groups ( 15 ) .

matching mycobacterium tuberculosis isolates were noted among 11 young tuberculosis patients socially linked through illicit drug related activities . a large proportion of their friends , 14 ( 64% ) of 22 , had positive tuberculin skin - test results . the behavior of " hotboxing " ( smoking marijuana inside a closed car with friends to repeatedly inhale exhaled smoke ) fueled transmission .

dr . coyne - beasley receives research funding from merck & co. none of these funds were used in the conduction or completion of the research contained in this manuscript . this article was supported by grants from the national institutes of health 1r01ai113305 - 01 ( cates pi ) , and by the north carolina translational and clinical sciences institute , through support from the national center for advancing translational sciences ( ncats ) , national institutes of health , grant award number 1ul1tr001111 . the content is solely the responsibility of the authors and does not necessarily represent the official views of the nih .

a significant barrier to the delivery of hpv vaccine is reluctance by both healthcare providers and parents to vaccinate at age 11 or 12 , which may be considered a young age . this barrier has been called vaccine hesitancy in recent research . in this commentary , we suggest using social marketing strategies to promote hpv vaccination at the recommended preteen ages . we emphasize a critical public health message of a sexually transmitted infection ( sti ) as preventable and vaccination against hpv as a way to protect against its consequences . the message tackles the issue of vaccine hesitancy head on , by saying that most people are at risk for hpv and there is a way to prevent hpv 's serious consequences of cancer . our approach to this conversation in the clinical setting is also to engage the preteen in a dialog with the parent and provider . we expect our emphasis on the risk of sti infection will not only lead to increased hpv vaccination at preteen ages but also lay important groundwork for clinical adoption of other sti vaccines in development ( hiv , hsv , chlamydia , and gonorrhea ) as well as begin conversations to promote sexual health .

annually , 80,000 anterior cruciate ligament ( acl ) injuries occur in the united states with an estimated cost of almost a billion dollars . the most common mechanisms of acl injury are noncontact in nature , characterized by sudden deceleration prior to a landing motion or change of direction [ 2 , 3 ] . these noncontact injuries may be due to coordination failure involving a complete and momentary loss of normal protective muscle support . people at high risk of acl injury frequently demonstrate high dynamic knee valgus ( i.e. , knee abduction moment ) during landing from jumping , which may be due to decreased neuromuscular control [ 3 , 5 ] . current literature has proposed several laboratory - based tools to identify risk factors for acl injury . however , these screening tools require expensive 3d motion capture equipment , highly trained staff , and significant amount of time to administer and analyze rendering these tools inefficient and impractical for a clinical setting . several jumping and landing tests are used in the clinical setting , including the landing error scoring system ( less ) , the drop jump video screening test , and the tuck jump assessment ( tja ) [ 69 ] . for example , the tja protocol , unlike the other two tests , starts and stops from ground level instead of jumping from a box ; this better represents techniques encountered in normal jumping activities [ 6 , 8 ] . the tja protocol also requires participants to jump for 10 seconds , while the less and drop jump video screening test require only 1 to 2 jumps [ 69 ] . therefore , the tja evaluates a measure of performance endurance , introducing a potential fatigue effect that might highlight landing flaws not observable in 1 to 2 jumps [ 69 ] . plyometric assessment , identifying jumping and landing technique flaws pertaining to risk of acl injury [ 8 , 10 ] . although there are no published reports of the tja being widely used , the availability of test protocol and minimal equipment required may make this a favored tool to use in varied clinical settings with diverse personnel . the tja includes 10 technique flaws related to jumping and landing that are scored qualitatively as either having the flaw or not [ 6 , 8 ] . empirical evidence suggests a participant who demonstrates greater than or equal to 6 out of 10 technique flaws during the tja should be targeted for intervention to address flaws , such as correcting lower extremity valgus at landing [ 6 , 8 , 10 ] . the tja requires minimal equipment ( e.g. , video cameras and tape markers ) and takes only several minutes to administer . scoring follows standard criteria for each technique flaw and can be completed relatively quickly by watching video playback . these features make the tja a practical screening tool for injury risk assessment in a clinical setting for people of different educational backgrounds and levels of experience , as it is currently being used . previous literature reported the interrater reliability of the tja as high with percentage exact agreement ( pea ) of two testers across all scoring criteria of 93% ( range 80%100% ) when scoring 10 participants . the same study also reported intrarater reliability to be high ( pea of 96%100% ) . although reliability was reported high , several limitations in study design necessitate further study of reliability of the tja . specific information concerning training and background of tja scoring for the raters was not included , limiting generalizability to clinicians unfamiliar with jumping assessment . additionally , the small sample size may have allowed raters to remember previous scores , introducing bias , resulting in higher intrarater reliability . reliability has not been tested with raters of different educational backgrounds or levels of experience . demonstrating that scoring is consistent between raters of different educational and experiential backgrounds would allow implementation of the tja in the clinical and performance settings with improved understanding of accuracy . the purpose of this study was to investigate intrarater and interrater reliability of the tja with raters of different educational backgrounds and levels of clinical experience with healthy injury - free men and women . the hypothesis for this study was that the raters would demonstrate good intra- and interrater reliability for the tja and that there would be no difference in tja total score between raters of different educational and/or experiential backgrounds . a sample of 108 , both undergraduate and graduate , recreationally active students who were not currently involved in college athletics , were recruited for participation . all participants were healthy , injury - free men and women , 18 to 24 years old , without prior tuck jump training . from this cohort , the videos of 40 participants ( n = 13 men and n = 27 women ) were randomly selected for this reliability study . participants received a full explanation of the nature , purpose , and risks of the study and were given the opportunity to ask questions . the physical activity readiness questionnaire ( par - q ) was administered to screen for contraindications for testing . all participants signed an informed consent document approved by the institutional review board at northern arizona university before participating in the study . prior to participants completing the tja , height and weight were measured with a wall - mounted stadiometer and digital scale ( cardinal scale , webb city , mo , usa ) . the tja was performed using instructions from a previously published tja study by the developers of the test . initial set up for the tja required 2 two - dimensional video cameras ( sony handycam , sony corporation , san diego , ca and jvc camcorder jvc americas corporation , wayne nj ) on tripods to provide sagittal and frontal views of the participants . two pieces of masking tape were placed on the ground , parallel to each other 8 inches apart . participants were instructed to stand with one foot on each tape strip to ensure proper positioning for the cameras ( figure 1 ) . the participants were instructed in purpose and protocol of tja test which included : jumping repeatedly for 10 seconds with high effort level , bringing knees up as high as possible so both thighs were parallel with the ground , landing softly in the same footprint ( 2 pieces of tape ) with each jump , and then immediately begin the next jump . no feedback was given to participants while performing the assessment . technique flaws included : ( 1 ) lower extremity valgus at landing ( figure 2 ) , ( 2 ) thighs do not reach parallel ( peak of jump ) , ( 3 ) thighs not equal side - to - side ( during flight ) , ( 4 ) foot placement not shoulder width apart , ( 5 ) foot placement not parallel ( front to back ) , ( 6 ) foot contact timing not equal , ( 7 ) excessive landing contact noise , ( 8) pause between jumps , ( 9 ) technique declines prior to 10 seconds , and ( 10 ) does not land in same footprint ( excessive in - flight motion ) . additional figures depicting these technique flaws can be found in previously published tja studies [ 6 , 8 , 10 ] . the participants were rated as either demonstrating a technique flaw or not . per previously published literature , the flaws were then summed for the tja total score [ 6 , 8 ] . five raters of varying educational backgrounds and clinical experiences were chosen to analyze video and score the tja of the 40 participants . raters included a physical therapist with a doctor of physical therapy degree and 4 years clinical experience ( rater 1 ) ; the head strength and conditioning coach at a division 1 university with a masters of science in exercise science and strength and conditioning specialist certification and 7 years of clinical experience ( rater 2 ) ; the head athletic trainer as a certified athletic trainer at a division 1 university with 17 years of clinical experience ( rater 3 ) ; a third - year doctor of physical therapy student ( rater 4 ) ; and a first - year doctor of physical therapy student ( rater 5 ) . all raters were given identical flash drives with instructions , a microsoft access database to input scores , a copy of myer et al . that described the tja and scoring in detail , 80 video files for 40 participants ( frontal and sagittal plane view for each participant ) , and 2 video files for 1 example participant ( frontal and sagittal plane views ) previously scored independently by 3 of the authors of this paper ( l. a. dudley , c. a. smith , and m. warren ) . the sample video was chosen from the larger sample of 100 to assist with consistency training of the raters and was not included in the 40 participants evaluated by raters . raters were instructed to read an excerpt from myer et al . to create consistency between raters regarding the scoring procedures of the tja . this included looking at the tja scoring tool created with pictures of the first 6 technique flaws to ensure consistency with previously established tja scoring procedures . raters were then instructed to watch and score the example video , which had the purpose of ensuring consistency in procedures between all raters and establishing the same volume setting to accurately analyze technique flaw number 7 ( excessive contact landing noise ) . once raters accurately scored the example video , participants ' videos were scored independently , with no discussion amongst raters . raters were instructed to use frontal plane views to score technique flaws ( 1 ) , ( 3 ) , ( 4 ) , ( 6 ) , and ( 7 ) ; sagittal plane views for ( 2 ) and ( 5 ) ; and frontal and sagittal views for ( 8) , ( 9 ) , and ( 10 ) . raters were given instructions on how to change playback speed on the computer program to allow viewing of the video in slow motion . raters were encouraged to watch the videos as many times as necessary to give an accurate score . approximately 1 month later , 3 of the 5 raters scored the videos again to determine intrarater reliability . descriptive statistics were calculated as means with standard deviation for interval data and percents for categorical data . analysis of variance with tja total score as the dependent variable and rater as the independent variable was completed to assess differences in mean values between raters . intraclass correlation coefficients ( icc , 2,1 ) and associated 95% confidence intervals ( ci ) were calculated to determine intrarater ( 3 different raters at 2 timepoints ) and interrater ( 5 raters for first session and 3 raters for second session ) reliability of the total tja score . the clinical significance was defined as poor for an icc below 0.50 , moderate for 0.500.75 , and good for 0.75 or higher . icc was calculated as ( 1)icc=2b2b+2w , where b is the between person variance and w in the within person variance . maximum likelihood estimates of b and w were obtained from linear mixed models with rater as a fixed effect for interrater reliability . least square means were calculated for the mean tja scores for each rater , with p values calculated to determine significant differences in tja scores . all analyses were completed using sas , version 9.2 ( sas institute , inc . , cary , nc ) and an a priori alpha level of 0.05 was used to denote statistical significance . participants in this study were 40 university students , 18 to 24 years of age ( mean age standard deviation ( sd ) 21.0 1.6 years ) . there were 13 males and 27 females with an average height and weight of 170.8 8.9 cm and 67.4 14.7 kg , respectively . the average number of flaws identified on the tja ( tja total score ) were 6.30 1.76 ; and technique flaw ( 2 ) ( thighs do not reach parallel ) was the most frequently identified ( 87.5% of participants ) , using scoring from rater 5 who was randomly chosen . additionally , technique flaw ( 2 ) was the most consistently agreed upon by all raters . technique flaws ( 5 ) ( foot placement not parallel front to back ) and ( 9 ) ( technique declines prior to 10 seconds ) were the least agreed upon by all raters . analysis of variance showed differences between raters for mean tja total score ( f = 11.82 ; p < 0.001 ) . post hoc comparisons showed no consistent pattern in scoring by educational and/or clinical experience ( table 1 ) . interrater reliability between the 5 raters was poor ( icc = 0.47 ; 95% ci : 0.330.62 ) . however , interrater reliability between raters 1 , 2 , and 5 ( raters used for intrarater reliability ) improved in the second scoring session ( icc = 0.52 , 95% ci : 0.350.68 versus icc = 0.69 , 95% ci : 0.550.81 ) . intrarater reliability testing was completed by raters 1 , 2 , and 5 who scored the tja videos at two different timepoints . the purpose of this study was to investigate intrarater and interrater reliability of the tja with raters of different educational backgrounds and levels of clinical experience with healthy injury - free men and women . this study showed intrarater reliability to be poor to moderate for the 3 raters scoring videos of 40 participants . it is noteworthy that those with more education and experience were not more consistent in scoring the tja than those raters with minimal education and experience . rater 5 had highest intrarater reliability of the 3 raters and also had the most experience administering the tja . these results suggest the tja may not be used reliably , following published protocol , by a single clinician regardless of level of education or experience [ 6 , 8 , 11 ] . this study also showed poor interrater reliability between 5 raters when assessing tja performance of the same 40 participants . this suggests the tja may not be used reliably , following published protocol , when being scored by different raters [ 6 , 8 , 11 ] . however , interrater reliability improved the second session between the 3 raters that scored the videos in 2 separate sessions . additional training beyond what was done in the study , as well as practice scoring to achieve consensus between raters , may be required . these results are in contrast to herrington et al . who reported the tja had good intrarater and interrater reliability ( greater than 0.75 ) when 2 raters scored videos of 10 recreationally active university students at 2 different scoring sessions . did not specify raters ' educational background , level of experience , or previous training in the tja . however , as this study was written by a developer of the tja , the two raters likely had more experience in scoring the tja and were more familiar with the flaws than the 5 raters of this study . the improved interrater reliability in the current study from session one to session two suggested a possible learning effect . further , the small sample size used by herrington et al . could promote bias due to raters remembering the scores of only 10 participants . the authors of the current study took measures to ensure continuity with previously established tja procedures and scoring criteria . however , current tja instructions do not specify whether a technique flaw should be scored only if it is seen consistently throughout the test or if the technique flaw may be scored if seen only once during the test . due to this exclusion in the published protocol , it is likely that raters may inherently interpret this differently , thus creating scoring inconsistencies between raters . for example , during a 10 second tja a participant may demonstrate lower extremity valgus at landing only once throughout several jumps . one rater may score this as a technique flaw because it was demonstrated in the assessment ; however , another rater may not score this as a technique flaw because the majority of jumps did not demonstrate lower extremity valgus at landing . more training and specific tja instruction prior to administration may correct this issue and promote consistency between raters . the current study includes the most diverse set of raters to date , representing 3 professions that may administer the tja . this allows for greater external validity in the clinical and performance setting than other published reliability studies to date . however , there are limitations of the current study . participants in the current study were not competitive athletes , though were recreationally active university students . the results of this study suggest the tja may not be consistently scored , following the previously published protocol , although modifications to instructions and rater training may correct this . since , further research is necessary to determine whether more specific scoring guidelines or training in tja administration would improve reliability . using a published protocol and training of raters , the tja has poor to moderate interrater and intrarater reliability . there may be a learned effect with the tja since interrater reliability improved with repetition . enhanced training or scoring instructions may be required to improve reliability of the tja among professionals of varying education backgrounds and experience .

objective . the tuck jump assessment ( tja ) , a clinical plyometric assessment , identifies 10 jumping and landing technique flaws . the study objective was to investigate tja interrater and intrarater reliability with raters of different educational and clinical backgrounds . methods . 40 participants were video recorded performing the tja using published protocol and instructions . five raters of varied educational and clinical backgrounds scored the tja . each score of the 10 technique flaws was summed for the total tja score . approximately one month later , 3 raters scored the videos again . intraclass correlation coefficients determined interrater ( 5 and 3 raters for first and second session , resp . ) and intrarater ( 3 raters ) reliability . results . interrater reliability with 5 raters was poor ( icc = 0.47 ; 95% confidence intervals ( ci ) 0.330.62 ) . interrater reliability between 3 raters who completed 2 scoring sessions improved from 0.52 ( 95% ci 0.350.68 ) for session one to 0.69 ( 95% ci 0.550.81 ) for session two . intrarater reliability was poor to moderate , ranging from 0.44 ( 95% ci 0.220.68 ) to 0.72 ( 95% ci 0.550.84 ) . conclusion . published protocol and training of raters were insufficient to allow consistent tja scoring . there may be a learned effect with the tja since interrater reliability improved with repetition . tja instructions and training should be modified and enhanced before clinical implementation .

however , cognitive function tends to decline with age , which can affect the ability to drive safely . the accident rate per mile driven among drivers over the age of 65 has been found to be higher than that among middle - aged drivers , and when injured in a collision , these older drivers are also more likely to die or to sustain serious injuries2 . cognitive psychologists have demonstrated that the anterior attention system plays a major role in higher - level executive control of attention during more complex cognitive tasks , such as problem - solving and decision - making , especially when multi - tasking3 . for example , stephens and tunney5 provided evidence that chewing gum increases scores in immediate , delayed recall , and working memory tests . similarly , johnson and miles6 confirmed that chewing gum during learning and/or recall improves subsequent memory . the cognitive processes improved by chewing have been systematically investigated and include memory , attention , and executive function5 . specifically , gum - chewing during cognitive testing has been found to improve verbal working memory and immediate episodic long - term memory , sustained attention and language - based attention . the results for gum - chewing s effects on word - association executive function are conflicting7 . on the basis of these previous reports thus , the purpose of this study was to determine the effects of chewing gum on driving performance . to assess this , the subjects comprised 26 young drivers ( 10 females ) who held valid driving licenses ( mean driving distance , 87,200 31,134 km / year ; possession of driver s license , 3.40 2.20 years ) . all subjects were informed of the purpose and methods of the study prior to participation and provided their informed consent , following the ethical principles of the declaration of helsinki . exclusion criteria were ( i ) inability to chew gum for a period of approximately 30 min , ( ii ) age younger than 18 or older than 40 years , ( iii ) consumption of more than 40 units of alcohol per week , ( iv ) smoking more than 10 cigarettes per day , ( v ) currently taking medication , ( vi ) currently experiencing medical problems ( including dental problems ) or serious medical conditions , and ( vii ) allergies to gum6 . a stisim drive m400 driving simulator ( system technology inc . , hawthorne , ca , usa ) was used to provide subjects with various driving environments and to estimate their driving performance . the simulator consists of a car unit with adjustable car seats with a dashboard , a steering wheel , turn signals , and brake and gas pedals , and provides a 135 field of view of a simulated environment . realistic roadway scenery is projected on a wide computer monitor , placed in front of the center of the steering wheel . speed and gear numbers are displayed on the screen . auditory feedback is provided through speakers and includes the sound of the engine , braking , speeding around curves , wheels , and driving off the road . before assessment in the simulator , subjects practiced for 5 min . if a participant demonstrated comfortable performance in the driving trial , he or she was allowed to participate in the test trials . a 3-day break was provided after each trial : not chewing gum and chewing spearmint sugar - free gum . the order of the conditions was randomized . in the chewing - gum condition , participants chewed gum at their normal speed ( 1 hz ) during driving . in the control condition , the ~30-min driving scenario consisted of a two - lane highway in each direction , with a lane width of 3.5 m. subjects were instructed to drive in a steady lateral position in the right ( slower ) traffic lane , following a lead vehicle , while maintaining a steady speed of 80 km / h . participants were required to maintain speed limits , comply with traffic rules , and negotiate surrounding traffic . mean distance driven above the speed limit , lane position , mean distance driven across the center line , and mean distance driven off the road were estimated by the simulator equipment and used as estimates of the driver s abilities of brake , accelerator , and steering control . differences in driving performance between the no - chewing condition and the chewing gum condition and between genders were assessed using the paired t - test . the kolmogorov - smirnov test confirmed the data had a normal distribution ; therefore , parametric statistics were used . a p value < 0.05 was considered to indicate statistical significance in all tests . while chewing gum , drivers drove significantly shorter distances at speeds exceeding the speed limit ( 26.61% shorter ) and off the road ( 31.99% shorter ) than those while not chewing ( p < 0.05 ) . however , lane position and mean distance driven across the center line did not differ significantly ( p > 0.05 ; table 1table 1.effect of gum - chewing on driving capacity ( n=26)chewing gum condition ( mean sd)no gumchewing gummean distance driven above speed limit ( m)609.9 190.9456.5 166.9lane position ( m)6.79 0.466.81 0.54mean distance driven across center line ( m)59.2 34.343.5 32.9mean distance driven off road ( m)127.0 77.586.4 47.9p < 0.05 ) , and there was no effect of gender in either condition . safe driving demands the capacity to concentrate and to divide attention over multiple sensory events , including visual and auditory modalities ; it also requires high - level cognitive decisions in a rapidly changing environment8 . in other words , driving skills require the simultaneous integration of many types of information while coordinating movements3 . simulated driving studies indicate that measures of driving behavior , such as speed , lane position , and time / distance across the center line and off the road edge , correlate with a driver s comfort with the given driving conditions . driving is a complex task requiring appropriate responses to hazardous situations on the road , and research has shown that attention and executive function correlate with driving status . the stisim driving simulator is widely used to examine the driving abilities of drivers with poor performance9 . this study assessed mean speed , mean distance driven above the speed limit , lane position , mean distance driven across the center line , and mean distance driven off the road edge as proxies for driving performance . chewing previous studies have indicated that chewing gum during specific tasks improves reaction times10 and has positive impacts on activities requiring cognitive function . gum chewing may improve cognitive function by promoting regional cerebral blood flow and glucose delivery and by increasing the efficiency of attention and concentration11 , 12 . poor performance in a task requiring divided attention is frequently observed in adults with dementia , and previous studies have shown that divided attention problems also exist in many individuals with mild cognitive impairment13 . cognitively impaired older drivers often do not intend to restrict or stop driving in the future , and do not consider their cognitive problems to be serious ; thus , gum - chewing may reduce the risk of accidents among these individuals while their impairments remain mild3 . our conclusion is that chewing gum enhances alertness and attention while driving , because it makes drivers feel more comfortable during driving activities . also , this study has provided further evidence that driving simulators can be used to assess responses to road hazards in a safe environment . driving simulator recordings have previously been used to assess age - related changes in driving performance and cognition14 , and to predict potential future crashes by older drivers15 . further research may clarify the mechanism by which chewing gum improves performance in a task , such as driving , that requires sustained attention and executive function .

[ purpose ] the purpose of this study was to determine the effects of chewing gum on driving performance in a driving simulator . [ subjects ] in total , 26 young licensed drivers participated . [ methods ] the driving scenario was typical of an urban environment : a single - carriageway , two - way road consisting of a mix of curved and straight sections , with considerable levels of traffic , pedestrians , and parked cars . mean distance driven above the speed limit , lane position , mean distance driven across the center line , and mean distance driven off the road were used as estimates of brake , accelerator , and steering control . the results were compared with those of a non - chewing gum control condition . [ results ] the driving performance while chewing gum was significantly better : the mean distance driven above the speed limit was 26.61% shorter , and the mean distance driven off the road was 31.99% shorter . lane position and mean distance driven across the center line did not differ significantly between the two conditions . [ conclusion ] chewing gum appears to enhance driving performance during a sustained attention driving task .

life of the human being is intertwined with many changes and transformation from the clotting of the semen to the death and is divided into different stages according to specific criteria . , adolescents expand their eligibility bases , and gradually the responsibility of health care would be relegated to them by the parents . furthermore , the peers individuals have a more important role for the adolescents compared with the childhood period . the peers group provides a strong support for the adolescent and creates a sense of belongingness and power in them . therefore , it has an intense effect on the behavior and performance of the adolescents . the occurrence of an acute disease in this period leads to the creation of disturbance in the natural perfection process in the adolescence period . among these acute diseases , asthma is the most common disease in childhood , which is considered as the forerunner of acute diseases and simply can cause disability among the children . this disease is an important reason for the children 's absence from the school due to admission into the hospitals . the prevalence of children 's asthma all over the world is increasing due to numerous stressful factors , despite noticeable progressions in the field of disease controlling and access to different medications for its treatment . it can be envisaged as an epidemic disease , which has major effects on the health and the economical conditions of the society . the prevalence of this disease during the last few decades in the united states has been 16% among the children under 5 years old and nearly 74% among the adults . the percentage of the students with asthma in iran has been estimated as 10% . in recent years , due to various life problems in iran and the increased stress rate , the disease of asthma has been increased in the country like in most other societies . considering the high prevalence rate of this disease , training is one of the important methods for promoting the hygiene and health of the students through conducting different training projects by relying on their participation in different hygienic activities . moreover , caring through holding the training programs for the peer adolescents with acute diseases is one of the effective measures in order to train , empower , and cultivate the willpower in adolescents and also can help to change the risk - taking behaviors and decrease the side effects . recent conducted studies have shown the effectiveness of training peers in decreasing the side effects of the acute diseases e.g. choudhury et al . in 2009 investigated the effect of training the peers in preventing side effects of the cardiovascular diseases resulting from type ii diabetes . in addition , another study , which was conducted by mahat et al . in 2008 in new jersey , titles the effect of training peer adolescents who are susceptible to risk - taking sexual behaviors and preventing aids ; the findings indicated an obvious increase in decreasing these types of behaviors among the adolescents . since it is the most common reason for the frequent hospitalizations and school absence due to lack of information about the intensity of the disease and due to treatment among the asthmatic students and the peers of the adolescents with this disease can assist them better at the time of asthma attack because of having the possibility for an easy availability and a better trainability from the peer group and consequently decrease the incidence of side effects ; therefore , this study aimed to assess the effect of conducting training programs for high school students on the performance of the peers with asthma . this study was of a quase - experimental kind , conducted before and after intervention . the instruments for gathering information the questionnaire contain 5 demographic items , 14 knowledge measurement items , and a performance measurement checklist with 14 questions . having obtained necessary letters of permission from isfahan education organization , district 3 , those high schools in which students with asthma were being taught were selected . then , two female high schools , one for test group and the other for control group were chosen after convincing high school principals and achieving letters of consent from those students willing to participate ; 40 second - grade high school students were easily included in the study in each high school . the knowledge questionnaire and performance checklist developed by the researcher were presented to some professors of and specialists on asthma and allergy , and statistics advisors to decide their validity and the necessary amendments were made after collecting ideas and suggestions . in order to calculate reliability , the researcher , during a pilot study , presented the knowledge measurement to 10 qualified people , who were not going to be included by the study to fill them out . furthermore , the researcher , through an oral interview , tried to completed performance checklist and measured knowledge questionnaires and performance checklists cronbach as 87% and 89% , respectively . having homogenized the two groups demographically , the pre - test was conducted , and knowledge questionnaire was answered by the two groups measure performance , and through simulating an asthma attack , the researcher completed related checklist by observing activate of peers during attack . having examined pre - test results and determined the matters required for education , the training intervention for test group was conducted by the researcher through lecturing , group discussion , collective methods , and activating accompanied by questioning . the members of test group were firstly divided into two equal subgroups , and training program ( four 60-minute training sessions held at 9 - 10 and 10 - 11 am ) was performed for both subgroups while high school personnel were acting as coordinators . during first session , different types of illness and its clinical symptoms were presented via lecture and questioning after acquainting students with asthma definition . during second session , the content of the first session was firstly gone through , and some questions about first session 's ideas were asked and students replied while discussing in 5-member circle . then , content of the second session , including asthma attack reasons and its preventive methods , were offended by giving lecture and questioning . as the third session started , questioning and group discussion were adopted , and it was followed by presenting this session 's content including athletic asthma and its preventing methods . training of required activities and assistance in case of attack was presented to students providing individual students with mdi devices simultaneously , showing the pictures through powerpoint , and imitating by the researcher herself . furthermore , the indirect training was offered during fourth session and through presenting researcher - developed booklets . after a one - month break following the last training session , knowledge questionnaires and performance checklists were again filled out by both the groups , and the data was analyzed by spss 16 software . the t - paired test , independent - t test , mann - whitney test , and dependent - t test were utilized for comparing examined variables before and one month after intervention in both test subgroups , the scores average between the two subgroups at one point in time educational level and people 's age average in both subgroups , respectively . statistically p was held significant at < 0.05 . the average of an age was 16 in both groups examined in this study , and there was no difference between two groups . 82.5% of fathers of students in both groups had under diploma education , 5% of them in test group and 5 - 7% of them in control group enjoyed academic education , and the two groups were not statistically different ( p = 0.95 ) . furthermore , 87.5% of test group 's mothers and 75% of control group 's mothers had under diploma studies ; besides 0% of test groups mothers and 5% of control groups therefore , there was no significant difference between the two groups ( p = 0.13 ) . the occupation of a large portion of fathers and mothers of the two group 's members was self - employed and housewife , respectively the comparison of peoples knowledge and performance scores average , before and one month after training for both groups , is presented in the following table , showing there is no significant difference between the scores before and one month after training in control group . however , the difference is significant in test group ( p < 0.001 ) . as observed in [ table 1 ] , the average of knowledge and performance scores of the two groups have no significant difference , but the given difference will grow significant after training ( p < 0.001 ) . the comparison of mean of examined variables scores of test and group before and after training the results indicated that the amount of knowledge of peers of those students suffering from asthma would grow significantly after performing training intervention . the results of a study by pit erg et al . , aiming at examining the effect of training of peers of students with asthma , indicated that the mean of student 's knowledge scores of test group increased significantly one month after training intervention . the peers of students with asthma could assist them during attacks regarding study 's results , helping the patient and family 's fear and stress device , and consequently prevent from creating difficulties facing learning process and incurring high expenses for treatment . in a study by smith et al . , on training the peers of students with asthma , the level of knowledge and attitude of students in test group increased after training intervention compared to control group . in addition , the attitude level of students in test group showed a significant difference compared to before intervention , confirming the present study 's findings and showing its ability to increase students knowledge on asthma . 's study on training risky behaviors to adolescents peers predisposed to aids has reported similar findings , indicating that the training peers of students with chronic illnesses could consequently preventing from heightening their conditions . the results of piggy et al . , study conducted in the u.s . , have reported similar findings in case of diabetes . have also reported that training of the peers of those students suffering from diabetes helped their athletic performance improve . in another study by nazar et al . , entitled an examination of training effect on student 's nutritional behaviors , it was found that the amount of knowledge and nutritional performance of students had improved significantly after training . in a study by frail et al . , on performance of students with obesity , a significant difference was found in students behaviors and selection level of foods and meals after performing training program compared to before it . , in a study aimed at examining the effect of training peers of adolescents , predisposed to sexual risky behaviors in zambia , found that the knowledge of test group students on using condom in prevention aids increased one month after training . moreover , test group students attitude on using preventive instruments in order to prevent aids increased while no difference was observed in control group . kirby et al . , in their systematic examination , have reported in effectiveness of schools curricula and peers training on prevention from hiv . in a study on assessment of peers training effect on adolescents regarding aids preventing behaviors and sexual illness in cameroon , found that there is a significant correlation between training by peers and knowledge enhancement in the case of new control aptiva methods and sexual illness symptoms . the results of mohammadzadeh et al . showed that the mean knowledge score of teachers was ( 12 2.3 ) . this means that the mean knowledge score was intermediate . because of adolescents tendency toward peers and creating popularity in group , its consequent social support produced by this group , training student , in particular by peers of students with chronic illnesses , could prevent from stress and depression among affected students and be some strategy to enhance their health . since the trainings were offered during student 's classes , there was some interruption between class hours and training time ; the researcher could decrease the limitation greatly in collaboration with high school principle . the average of an age was 16 in both groups examined in this study , and there was no difference between two groups . 82.5% of fathers of students in both groups had under diploma education , 5% of them in test group and 5 - 7% of them in control group enjoyed academic education , and the two groups were not statistically different ( p = 0.95 ) . furthermore , 87.5% of test group 's mothers and 75% of control group 's mothers had under diploma studies ; besides 0% of test groups mothers and 5% of control groups mothers had academic studies . therefore , there was no significant difference between the two groups ( p = 0.13 ) . the occupation of a large portion of fathers and mothers of the two group 's members was self - employed and housewife , respectively the comparison of peoples knowledge and performance scores average , before and one month after training for both groups , is presented in the following table , showing there is no significant difference between the scores before and one month after training in control group . however , the difference is significant in test group ( p < 0.001 ) . as observed in [ table 1 ] , the average of knowledge and performance scores of the two groups have no significant difference , but the given difference will grow significant after training ( p < 0.001 ) . the comparison of mean of examined variables scores of test and group before and after training the results indicated that the amount of knowledge of peers of those students suffering from asthma would grow significantly after performing training intervention . the results of a study by pit erg et al . , aiming at examining the effect of training of peers of students with asthma , indicated that the mean of student 's knowledge scores of test group increased significantly one month after training intervention . the peers of students with asthma could assist them during attacks regarding study 's results , helping the patient and family 's fear and stress device , and consequently prevent from creating difficulties facing learning process and incurring high expenses for treatment . in a study by smith et al . , on training the peers of students with asthma , the level of knowledge and attitude of students in test group increased after training intervention compared to control group . in addition , the attitude level of students in test group showed a significant difference compared to before intervention , confirming the present study 's findings and showing its ability to increase students knowledge on asthma . 's study on training risky behaviors to adolescents peers predisposed to aids has reported similar findings , indicating that the training peers of students with chronic illnesses could consequently preventing from heightening their conditions . the results of piggy et al . , study conducted in the u.s . , have reported similar findings in case of diabetes . have also reported that training of the peers of those students suffering from diabetes helped their athletic performance improve . in another study by nazar et al . , entitled an examination of training effect on student 's nutritional behaviors , it was found that the amount of knowledge and nutritional performance of students had improved significantly after training . in a study by frail et al . , on performance of students with obesity , a significant difference was found in students behaviors and selection level of foods and meals after performing training program compared to before it . , in a study aimed at examining the effect of training peers of adolescents , predisposed to sexual risky behaviors in zambia , found that the knowledge of test group students on using condom in prevention aids increased one month after training . moreover , test group students attitude on using preventive instruments in order to prevent aids increased while no difference was observed in control group . kirby et al . , in their systematic examination , have reported in effectiveness of schools curricula and peers training on prevention from hiv . in a study on assessment of peers training effect on adolescents regarding aids preventing behaviors and sexual illness in cameroon , found that there is a significant correlation between training by peers and knowledge enhancement in the case of new control aptiva methods and sexual illness symptoms . the results of mohammadzadeh et al . showed that the mean knowledge score of teachers was ( 12 2.3 ) . this means that the mean knowledge score was intermediate . because of adolescents tendency toward peers and creating popularity in group , its consequent social support produced by this group , training student , in particular by peers of students with chronic illnesses , could prevent from stress and depression among affected students and be some strategy to enhance their health since the trainings were offered during student 's classes , there was some interruption between class hours and training time ; the researcher could decrease the limitation greatly in collaboration with high school principle . since training of peers of students with asthma has been effective , helping the knowledge increase and performance enhance , the performance of this kind of program is suggested in the case of other chronic illness .

background : asthma is the most prevalent disease during childhood , known as the most important reason for children 's disability adolescences truancies , thanks to their hospitalization and as a result intensification of disease symptom.material and methods : the present study is quasi - experimental kind , implemented with two groups . sampling method was straightforward . 80 student 's second - grade high school students constitute participants . both groups were homogenized considering age , gender , education , and parent 's vocation . first of all , a letter agreement was received from students . then , the knowledge and accomplishment of students were measured by means of relevant questionnaire and checklist in advance of educational intervention . in the next step , during 4 sessions , educational content specified beforehand was presented to students via interview , lecture , group discussion , and display methods . the knowledge and accomplishment of both groups students was measured through questionnaire and checklist . finally , the data was analyzed by spss 16 and statistical test of t - paired , independent - t , man - whitney , and anova.results:the finding demonstrated that group 's student accomplishment registered 91.8 1.3 while being 2.2 0.6 before holding session . in addition , their knowledge increased significantly , registering 99.6 1.2 while it had measured as 1.3 0.3 before sessions . the results of paired t - test indicated that the average of difference between knowledge and accomplishment grades of two groups was significant ( p<0.05).conclusions : regarding the finding of the present study and the fact that asthma is on the rise resulting in an increase in truancies as well as stressing the efficacy of training peers suffering from the disease , the implementation of the curriculum could be necessary .

the cytokine interferon- is critical for protection against viral and bacterial infections and tumor development . its biological activities require the phosphorylation of stat1 at a single tyrosine residue ( stark and darnell , 2012 ) . stat activation , as it transforms the stat1 dimers into dna binding transcription factors . stat1 inactivation , namely the enzymatic reversal of tyrosine phosphorylation , accordingly is equally important for physiological signaling ( liu et al . , 2011 ) . the tyrosine phosphatase tc45 is the major stat1-inactivating enzyme ( ten hoeve et al . , therefore is required for understanding the physiological regulation of ifn signaling as well as for the development of therapeutic stat1 modulators , e.g. , for viral and immune diseases ( borden et al . , 2007 ) . in the cell nucleus , stat1 inactivation is ultimately limited by the kinetics of dna binding , whereby stat1 is available for dephosphorylation only in its dna unbound state ( meyer et al . , 2003 ) . recent results indicate that dephosphorylation is a multistep process that requires stat1 dimers to undergo extensive spatial reorientation ( zhong et al . , 2005 ; mertens et al . , 2006 ) hydrodynamic modeling of analytical ultracentrifugation results obtained with purified stat1 indicated moreover that the reorientation of the recombinant stat1 dimers is considerably slower ( t1/2 2040 min ; wenta et al . , 2008 ) than the dephosphorylation of endogenous stat1 in living cells ( t1/2 < 15 min ; haspel et al . , 1996 ) . in fact , the acetylation of two particular lysine residues of stat1 was reported to enhance its dephosphorylation by facilitating recruitment of tyrosine phosphatase tc45 ( krmer et al . , 2009 ) , but this claim was subsequently invalidated ( antunes et al . , 2011 ) . another posttranslational modification , namely sumo conjugation , can enhance the dephosphorylation of stat1 by increasing its solubility , yet sumo does not itself partake in the actual dephosphorylation step ( droescher et al . , 2011 ) . the only stat1-interacting protein known to directly enhance the dephosphorylation reaction thus is -arrestin1 ( mo et al . , 2008 ) . the -arrestins are two ubiquitous proteins that are best known for their role as cytoplasmic adapters in the regulation of g protein - coupled receptors and other signaling molecules ( dewire et al . , 2007 ) . additional functions for -arrestins in the nucleus have also been described ( kang et al . , 2005 ) . propose a model whereby -arrestin1 , but not -arrestin2 , promotes the dephosphorylation of nuclear stat1 by acting as a scaffold to directly facilitate recruitment of phosphatase tc45 . this made -arrestin1 an interesting object for our studies of stat1 dimer reorientation and its effects on dephosphorylation . here we present the results of our experiments , which contrary to expectations provide evidence against the reported negative - regulatory role of -arrestin1 in stat1 signaling . at first we wanted to confirm that overexpression of -arrestin1 diminishes ifn-induced transcription of a stat1-dependent reporter gene in hela cells . we used c - terminally green fluorescent protein ( gfp)-tagged human -arrestin1 and n - terminally flag - tagged rat -arrestin1 , which in agreement with its evolutionary conservation can supplant functions and interactions of the human homolog ( scott et al . , 2006 ; both constructs were overexpressed in hela cells but did not diminish ifn-induced reporter gene activity ( figures 1a and 1b ) . we noted that increased -arrestin1 transfection led to an apparent rise in both constitutive and induced reporter gene activity , which overproportionally affected the former . consequently , when depicted as fold induction ( [ induced transcription]/[constitutive transcription ] ) , as done by mo et al . ( 2008 ) , the transcriptional activity indeed appeared to drop with increased -arrestin1 levels ( figures 1a and 1b ) . we identified reduced expression of the -galactosidase control gene as a cause ( figure 1c ) , probably due to titration of limiting coactivators by the cotransfected -arrestin expression vectors . we therefore varied the reporter gene to control gene ratio between 1.5:1 and 25:1 to test whether a different transfection protocol could reveal the reported inhibitory effect of -arrestin1 on stat1-mediated gene induction ( figure 1d ) . however , interferon - inducible reporter gene activity was the same for all reporter - to - control - gene ratios used , and an inhibitory effect of -arrestin1 was not recognizable ( figure 1e ) . fold induction was likely due to an error associated with the cotransfection of -arrestin1 expression vectors . it was hence not possible for us to rule out a similar error for their published data . based on the published work , we expected that overexpression of -arrestin1 in hela cells would diminish tyrosine phosphorylation of the endogenous stat1 . however , this was not the case , since stat1 phosphorylation kinetics was identical under all conditions examined regardless of the presence of increased -arrestin1 ( figure 2a ) . to avoid the limitations of transfection assays , we obtained mouse embryonic fibroblasts ( mefs ) deficient in -arrestin1 and control fibroblasts ( kohout et al . , 2001 ) . first , we used a commercially available antibody to confirm -arrestin1 expression in wild - type but not the -arrestin1-deficient mef ( figure 2b ) . we then studied the tyrosine dephosphorylation of stat1 in the wild - type cells and the -arrestin1-deficient cells ( figure 2c ) . this was done by treating the cells for 60 min with ifn before adding the protein kinase inhibitor staurosporine for up to 60 min to terminate abruptly the ongoing protein phosphorylation . as expected , after 1 hr in the presence of staurosporine the concentration of tyrosine - phosphorylated stat1 contrary to the previous report , however , stat1 dephosphorylation was not diminished in the cells lacking -arrestin1 ( figure 2c ) . thus , stat1 activation kinetics was unchanged regardless of whether -arrestin1 was overexpressed or deleted . we then used -arrestin1-deficient mefs in conjunction with the primer sequences and qpcr conditions of mo et al . ( 2008 ) to determine ifn-induced expression of gbp1 , cxcl10 , and isg15 genes . according to their work interferon - induced transcription of these genes is 5- to 25-fold higher for -arrestin1-deficient mefs than for wild - type cells . in our hands , in contrast , lack of -arrestin1 did not result in increased transcription . in fact , transcription was impaired in the -arrestin1-deficient cells , an observation we also made for the five ifn-inducible genes we tested in addition ( figure 3 ) . it remains to be determined if -arrestin1 has a stat1-independent stimulatory role specifically in interferon signaling , or whether the observed effects on gene transcription rather are a reflection of -arrestin1 s involvement in a multitude of cellular functions . these cells are transiently transfected to reconstitute -arrestin1 expression ( mo et al . , 2008 ) . we question the appropriateness of this experimental setup to discern the biological activities of individual -arrestins . for example , the authors presume comparable -arrestin1 expression in wild - type cells and their reconstituted mefs based on western blotting results ( see their figure 4c ) . yet this experiment does not fully clarify the issue in the absence of information about transfection efficiencies and hence cellular expression levels . if , however , the authors presumption is indeed correct , the problem arises that gene expression is upregulated 5- to 25-fold in the arrestin1/2 double - deficient mefs , whereas reconstitution with -arrestin1 merely halves gene expression according to mo et al . this discrepancy indicates that -arrestin double knockout mefs do not revert to wild - type characteristics with regard to stat1 signaling upon transfection of -arrestin1 . as this limits the value of -arrestion1/2 doubly deficient cells for studying the effects specifically of -arrestin1 on stat1 , we have instead used cells deficient solely in -arrestin1 . in the final set of experiments flag - tagged stat1 and gfp - tagged -arrestin1 were expressed in hek293 t cells either alone or together . the interaction of -arrestin1 and stat1 , which was reported to be maximal in ifn-treated cells , was then analyzed by anti - flag immunoprecipitation . alternatively , stat1-flag was coexpressed with gfp - tagged stat1 as a positive control since stat1 forms equally stable homodimers before and after stimulation with ifn ( wenta et al . , as shown in figure 4a , top panel , probing with anti - flag antibody of extracts from cells cotransfected with flag - tagged stat1 and gfp - tagged--arrestin1 ( lanes 1 and 2 ) or gfp - tagged stat1 ( lanes 5 and 6 ) , respectively , showed an 98 kda band expected for flag - tagged stat1 . reprobing with anti - gfp antibody ( middle panel ) revealed a band of 80 kda band in lanes 1 and 2 expected for gfp - tagged -arrestin1 ; and a band of 110 kda in lanes 5 and 6 expected for gfp - tagged stat1 . probing with anti--arrestin1 antibody ( bottom panel ) labeled an 80 kda band in lanes 1 and 2 , but not 5 and 6 , confirming -arrestin1 overexpression . these results demonstrated that both stat1 and -arrestin1 were well - expressed in the cotransfected cells . when the material that coprecipitated with flag - tagged stat1 was probed with anti - gfp to detect coprecipitating -arrestin1-gfp , shown in the middle panel , lanes 3 and 4 , a band that comigrated with gfp - tagged--arrestin1 reprobing with anti--arrestin1 antibody nonetheless confirmed this band to contain -arrestin1 ( bottom panel ) . since the same antibody was used for detection of -arrestin1 and stat1 , namely anti - gfp , it was possible to provide also a quantitative assessment for the efficiency with which these two proteins coprecipitated with the flag - tagged stat1 . the quantitative comparison , which was done after normalization of the signals for precipitated -arrestin1 and stat1 using their respective inputs , demonstrated that the interaction of stat1 with itself was three to six times stronger than with -arrestin1 . in our experiments the signal intensities for coprecipitating -arrestin1 were not only weak but moreover did not increase when extracts from ifn-treated cells were used ( middle and bottom panels , compare lanes 3 and 4 ) . as an additional control for the specificity of the observed interaction presumably between -arrestin1 and stat1 , we performed immunoprecipitations using anti - flag - tag antibody in the absence and presence of its antigen , namely flag - tagged stat1 ( figure 4b ) . as seen in the previous experiments , anti - flag - tag immunoprecipitations with extracts that contained both -arrestin1 and the flag - tagged stat1 gave a weak -arrestin1 signal ( middle and bottom panels , lane 7 ) . this signal was expectedly missing when -arrestin1 was missing in the extracts ( middle and bottom panels , compare lane 7 to lane 6 ) . however , the converse experiment using extracts devoid of flag - tagged stat1 did not result in absent or at least diminished precipitation of -arrestin1 ( middle and bottom panels , compare lanes 7 and 8) . we therefore concluded that the observed precipitation of -arrestin1 was not due to interactions specifically with stat1 . stat1 activation is indispensable for the execution of interferon- functions such as antimicrobial protection . interacting proteins that can reduce the activity of stat1 are therefore of pharmaceutical interest and potentially of clinical relevance , as was suggested for -arrestin1 by mo et al . however , our experiments did not identify -arrestin1 as a stat1-interacting protein , and they showed that -arrestin1 did not reduce the activation of stat1 . we accordingly found that the transcription of interferon- target genes was not inhibited in the presence of -arrestin1 . in summary , our data do not support a negative - regulatory role for -arrestin1 in interferon- signaling . immortalized mefs deficient in -arrestin1 and wild - type control cells were provided by dr . t , hela , and mef cells were kept in growth medium , dmem supplemented with 10% fbs and 1% penicillin / streptomycin , in a humidified incubator with 5% co2 at 37c . cells grown to 70%90% confluence were transfected using lipofectamine according to the manufacturer s recommendations ( invitrogen ) , followed by subsequent experimentation after 24 hr . plasmids encoding human -arrestin1-gfp ( arrb1-gfp ) and rat flag--arrestin1 ( flag - arrb1 ) were provided by dr . the interferon--dependent luciferase reporter gene 3xly6e gas - luc containing a triple stat1 binding site was described ( wen et al . , 1995 ) . human ( # 407306 ) and mouse ifn ( # 407303 ) were obtained from merck millipore . hela cells were transfected on 24-well plates with 0.25 g / well plasmid encoding -galactosidase , 0.25 g / well 3xly6e gas - luc reporter gene , and the indicated amounts of plasmid encoding -arrestin1 tagged with either gfp or flag . the amount of transfected dna was kept constant by adding plasmid pbluescript ( pbs ) . after 24 hr , the cells were left untreated or treated for 6 hr with 50 u / ml human ifn , followed by cell lysis in buffer containing 25 mm glycylglycine , 15 mm mgso4 , 4 mm egta , 1% triton x-100 , 1 mm dtt ( ph 7.8 ) . luciferase activity was determined with luciferase - assay - system ( promega ) according to the manufacturer s instructions . -galactosidase activity was photometrically measured at room temperature at a wavelength of 405 nm after incubation ( 3060 min ) of the extracts ( 5 l ) in 280 l buffer containing 0.75 mm nah2po4/na2hpo ( ph 7.4 ) , 1 mm mgcl2 , 1 mg / ml o - nitrophenyl--d - galactoside . control experiments ( figure 1d ) with varying ratios of luciferase and -galactosidase expression plasmids were done with hela cells under the conditions described before . the [ luciferase:gal ] ratios stated were obtained by using the following combinations of plasmid 3xly6e gas - luc and psv-gal : [ 1.5:1 ] 0.3 and 0.2 g / well , [ 5:1 ] 0.42 and 0.08 g / well , [ 10:1 ] 0.495 and 0.05 g / well , and [ 25:1 ] 0.5 and 0.02 g / well . in addition , a fixed amount ( 0.25 g / well ) of plasmid encoding -arrestin1 ( arrb1-gfp ) or pbs was included as indicated . the control experiments of figure 1c were done with hela cells on 12-well plates cotransfected with 0.2 g / well of plasmid psv-gal and the indicated amounts per well of flag - arrb1 . stat1 phosphorylation as shown in figure 2a was determined using hela cells on 6-well plates transfected with 0.8 g / well of the indicated vectors expressing -arrestin1 or -galactosidase as the control . twenty - four hours later the cells were passaged ( dilution 1:2.5 ) , and after a further 24 hr the cells were left untreated or treated for 1 hr with 50 u / ml human ifn , followed by a medium change and incubation with ifn - free growth medium for the indicated time periods . stat1 dephosphorylation ( figure 2c ) was detected using wild - type and -arrestin1-deficient mefs left untreated or treated with 50 u / ml mouse ifn for 1 hr . subsequently , 0.5 m tyrosine kinase inhibitor staurosporine ( # 569397 , calbiochem ) was added to the cells for the indicated lengths of time , followed by cell lysis in boiling sds sample buffer . cells were extracted on ice for 30 min in buffer containing 2 mm egta , 0.2 mm edta , 1 mm na - vanadate , 50 mm naf , 280 mm nacl , 0.5% ( v / v ) np-40 , 10% ( v / v ) glycerol , 1 mm dtt , protease inhibitors pmsf ( 0.54 mm ) and complete ( roche ) , and 50 mm tris / hcl ( ph 7.4 ) . soluble whole - cell extracts were resolved by sds - page and were analyzed by quantitative western blotting using a li - cor odyssey system as described ( droescher et al . , 2011 ) . anti - mouse stat1 ( sc-591 ) , anti - human stat1 ( sc-345 ) , anti - gfp ( sc-8334 ) , and anti--arrestin1 ( sc-9182 ) were purchased from santa cruz biotechnology . anti--actin ( a5441 ) and anti - flag m2 ( f3165 ) were from sigma . anti - phospho - tyr701 stat1 ( # 9171 ) was from cell signaling technology ; anti--galactosidase ( a11132 ) was from molecular probes . irdye800cw - conjugated anti - mouse ( # 926 - 32212 ) and anti - rabbit ( # 926 - 32213 ) igg secondary antibodies were purchased from li - cor bioscience . t cells grown on 10 cm plates were transfected with 2.5 g each of plasmids encoding stat1-flag and -arrestin - gfp or stat1-gfp . twenty - four hours later cell cultures were passaged ( dilution 1:2 ) , and after a further 24 hr the cells were left untreated or treated for 1 hr with 50 u / ml ifn. soluble whole - cell extracts , containing 500 g protein as determined by bradford assay ( biorad ) , were rotated at 4c with 5 l anti - flag m2 for 90 min before adding 12 l ( settled volume ) protein a / g plus agarose ( sc-2003 , santa cruz ) and rotating for another 2 hr . after centrifugation ( 1,000 g ) at 4c for 4 min , the precipitate was washed twice with whole - cell extraction buffer and eluted in sds sample buffer for western blotting . mef cells were grown overnight in serum - depleted growth medium ( 1% ( v / v ) fbs ) , followed by incubation in growth medium with or without 100 u / ml mouse ifn. subsequently , total rna was extracted , followed by reverse transcription , and expression of selected genes was measured by qpcr using sybr green . the primers used for gbp1 , cxcl10 , and isg15 were as described by mo et al . expression of psmb9 was determined with primer pair 5-cgggggtgtcgtggtgggctctg and 5-cgccggcactcctcaggggtcat ( reverse ) , and that of irf9 with 5-gcctttgccccatccccatctc and 5-cccctggccctggaagtactgg ( reverse ) . for further experimental details and the primers for cxcl9 , irf1 , and icam1 ,

summarysignal transducer and activator of transcription 1 ( stat1 ) is activated by tyrosine phosphorylation upon interferon- ( ifn ) stimulation , which results in the expression of genes with antiproliferative and immunomodulatory functions . the inactivation of stat1 occurs through tyrosine dephosphorylation by the tyrosine phosphatase tc45 . it was proposed that recruitment of tc45 required the direct interaction of stat1 with the scaffold protein -arrestin1 , making -arrestin1 an essential negative regulator of stat1 and ifn signaling ( mo et al . , 2008 ) . we tested the relevance of -arrestin1 for stat1 activity . our results do not confirm -arrestin1 as a stat1-interacting protein . the stat1 phosphorylation / dephosphorylation cycle was found to be unaffected by both the overexpression and the genetic deletion of -arrestin1 . accordingly , -arrestin1 did not inhibit stat1 transcriptional activity or the induction of ifn target genes in response to ifn. our data indicate that -arrestin1 is dispensable for stat1 dephosphorylation and the termination of ifn signaling .

a 53-year - old caucasian female with complaints of dry eyes and a burning sensation in the eyes presented to our ophthalmology clinic 5 years ago . her medical history revealed that the symptoms had persisted for 10 years , with some relief with lubricating eye drops used every 0.51 h. prior serology studies for antinuclear antibodies ( ana ) , ro and la were all negative . she denied any symptoms of dryness of her mouth , and she had no other comorbidities . clinical examination showed dry eyes , with slit lamp examination revealing thickening and hyperemia of the eyelids . she was put on genteal gel alternating with artificial tears ( restasis eye drops ) four times / day . during the course of the next 5 years , she underwent multiple surgeries for persistent eye dryness , including three procedures of silicone punctual plug placement in each eyelid , permanent thermal punctual occlusion , and later , resection of the canaliculus due to repeat reopening of the punctum despite three permanent thermal occlusions and continued patient discomfort secondary to refractory dryness of the eyes . additional evaluation to determine the etiology of dry eyes was done . it revealed the presence of antibodies to salivary gland protein 1 ( sp1 ) and parotid secretory protein ( psp ) , leading to the diagnosis of sjogren 's syndrome ( ss ) . a 68-year - old caucasian female with a known history of rheumatoid arthritis ( ra ) presented to our ophthalmology clinic with complaints of persistent dry eyes and irritation for the last 25 years . she had tried artificial tears , restasis eye drops and genteal gel with some relief . her prior workup by her rheumatologist included antibodies to ro and la , which were both negative . on physical evaluation , she was noted to have dry eyes . slit lamp examination revealed hyperemia and thickening of the eyelids . her laboratory evaluation in our clinic included antibodies to ro and la that were negative and antibodies to ana and rheumatoid factor , which were both positive . evaluation of additional autoantibodies revealed the presence of antibodies to sp1 and psp , leading to the diagnosis of ss . antibody testing for sp1 and psp in both patients was done at immco diagnostic laboratory , buffalo , n.y . a 53-year - old caucasian female with complaints of dry eyes and a burning sensation in the eyes presented to our ophthalmology clinic 5 years ago . her medical history revealed that the symptoms had persisted for 10 years , with some relief with lubricating eye drops used every 0.51 h. prior serology studies for antinuclear antibodies ( ana ) , ro and la were all negative . she denied any symptoms of dryness of her mouth , and she had no other comorbidities . clinical examination showed dry eyes , with slit lamp examination revealing thickening and hyperemia of the eyelids . she was put on genteal gel alternating with artificial tears ( restasis eye drops ) four times / day . during the course of the next 5 years , she underwent multiple surgeries for persistent eye dryness , including three procedures of silicone punctual plug placement in each eyelid , permanent thermal punctual occlusion , and later , resection of the canaliculus due to repeat reopening of the punctum despite three permanent thermal occlusions and continued patient discomfort secondary to refractory dryness of the eyes . additional evaluation to determine the etiology of dry eyes was done . it revealed the presence of antibodies to salivary gland protein 1 ( sp1 ) and parotid secretory protein ( psp ) , leading to the diagnosis of sjogren 's syndrome ( ss ) . a 68-year - old caucasian female with a known history of rheumatoid arthritis ( ra ) presented to our ophthalmology clinic with complaints of persistent dry eyes and irritation for the last 25 years . she had tried artificial tears , restasis eye drops and genteal gel with some relief . her prior workup by her rheumatologist included antibodies to ro and la , which were both negative . her medications included methotrexate for her ra for the last 30 years . on physical evaluation , her laboratory evaluation in our clinic included antibodies to ro and la that were negative and antibodies to ana and rheumatoid factor , which were both positive . evaluation of additional autoantibodies revealed the presence of antibodies to sp1 and psp , leading to the diagnosis of ss . antibody testing for sp1 and psp in both patients was done at immco diagnostic laboratory , buffalo , n.y . ss is an autoimmune disease starting in the lacrimal and salivary glands but with eventual systemic involvement of multiple other organs . ss can also occur secondary to other autoimmune diseases such as lupus and ra , known as secondary ss . patients with ss typically present with a dry , gritty sensation in the eyes and a dry mouth . at this stage typically , involvement of the lachrymal and submandibular glands occurs before involvement of the parotid glands . due to this , the presentation of dry eyes may occur much earlier in the disease process and precede the presence of a dry mouth . about 5% of the patients with ss develop b cell lymphoma , most commonly occurring in the salivary glands and gastrointestinal tract [ 1 , 2 , 3 ] . the diagnostic criteria for ss include clinical criteria : a schirmer test with a result of < 5 mm of wetting/5 min or a rose bengal score of > 4 ( van bijsterveld ) as well as ocular and oral symptoms . daily persistent dry eyes for more than 3 months , use of lubricants more than 3 times / day , a recurrent feeling of sand or gravel in the eyes as well as a dry mouth for more than 3 months with a glass of water required to eat dry foods further support a diagnosis of ss . in our clinics , the limit for schirmer 's test is set at 3 mm to reduce positive results in the normal population to < 15% . histological criteria for ss include focal sialoadenitis , and serological criteria include ana , rf and antibodies to ro and/or la . the diagnosis of ss is excluded if there is a history of head and neck radiation treatment , hepatitis c , aids , lymphoma , sarcoidosis , graft versus host disease or anticholinergic drugs . the 2 patients described in this case report met the clinical criteria for ss based on dry eyes and a positive schirmer test but lacked antibodies to ro or la . the autoantibodies that are most commonly believed to be associated with ss are ro and/or la antibodies . ro ( ss - a ) is an extractable nuclear antigen that is composed of proteins 52 kd and 60 kd combined with cytoplasmic rna species [ 1 , 6 ] . antibodies to ro are found in patients with autoimmune mediated systemic connective disorders including ss . antibodies to ro are detected in 4060% of the patients [ 1 , 6 , 7 ] . la ( ss - b ) is a 48-kd protein and an extractable nuclear antigen . antibodies to la are detected in < 20% of the patients but they are highly specific to ss [ 6 , 7 ] . however , there are multiple other antibodies being studied , including antibodies to muscarinic receptor 3 , tissue kallikrein , alpha - fodrin , carbonic anhydrase ii and vi , psp and sp1 . the significance of these autoantibodies has not been fully appreciated [ 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 ] . one of the mouse models used to study ss is the interleukin 14 alpha transgenic ( il14tg ) mouse . it reproduces all events noted to occur in ss patients in the same relative time frame [ 16 , 17 , 18 ] . the use of this animal model led to the discovery of the novel autoantibodies in ss . these include antibodies to sp1 , carbonic anhydrase 6 ( ca6 ) and psp . the presence of these autoantibodies in the sera of patients with ss has been confirmed . as per these studies , only 25% of the il14tg mice developed ro and/or la antibodies . interestingly , the time course in the development of the autoantibodies showed that the sp1 and ca6 antibodies were present very late in the disease . our first patient had symptoms of dry eyes requiring multiple procedures to help control them . our second patient had dry eyes for many years along with the comorbidity of ra . however , a diagnosis of ss was not made in either of our patients due to the lack of ro and/or la antibodies . the detection of sp1 and psp antibodies in our patients led to the diagnosis of primary ss in the first and secondary ss in the second patient . these cases stress the importance of using autoantibodies besides ro and la in the diagnosis of ss . further studies are in progress to define whether antibodies to sp1 and psp not only define early stages of ss but also particular forms of ss . md , and l.s . , phd , participated in the discovery of the autoantibodies to sp1 and psp . the suny at buffalo school of medicine holds a patent on these autoantibodies that is licensed to immco and nicox .

purposethe purpose of this report is to describe 2 patients with persistent severe dry eyes , positive schirmer tests for sjogren 's syndrome ( ss ) but lacking antibodies to either ro or la . these patients were diagnosed to have ss by detecting antibodies to salivary gland protein 1 ( sp1 ) and parotid secretory protein ( psp ) . this report emphasizes the existence of patients with ss who lack antibodies to either ro or la and may therefore be misdiagnosed . detection of novel autoantibodies , including antibodies to sp1 and psp , are helpful in identifying these patients . initial presentation may simply be dry eyes.methodstwo patients who presented to our ophthalmology clinic are described . one of the patients underwent multiple procedures over a period of 10 years for severe xerophthalmia . the other patient had rheumatoid arthritis and xerophthalmia . however , in both patients , chronic xerophthalmia had been considered to be idiopathic because antibodies ro and la were negative . further serologic testing revealed antibodies to sp1 and psp.resultstwo patients who lacked antibodies to ro and la but not to sp1 and psp were diagnosed as having ss.conclusionpatients presenting with unexplained dry eyes may not always show the serology markers in the current criteria for ss , anti - ro and anti - la . in these cases , investigation for novel , early antibodies to sp1 and psp is of importance in the diagnosis of ss .

rectal polypectomy causes thinning ( or even perforation ) of the rectal wall in addition to thermic injury at the polypectomy site . we present a rare case of spontaneous rectal perforation after uncomplicated nerve sparing endoscopic extraperitoneal radical prostatectomy in a patient with a previous history of rectal polypectomy at the perforation site . this conservative therapy for such rectal perforations is indicated if the patient 's general condition remains stable without any signs of infection . adequate time interval should be given to allow healing and avoid adding further thermal wall damage which may obscure healing leading to complications like fistula . conservative therapy for small missed rectal perforations constitutes an attractive , feasible and non invasive treatment entity . following this principle coloscopy is a routine examination for men over 50 years in germany . from the surgical point of view the other one is the perforation of the gut which is a severe and possibly lethal complication with a reported incidence of 0.1% to 0.9% [ 1 - 4 ] . most rectal injuries during nerve sparing endoscopic extraperitoneal radical prostatectomy ( nseerpe ) occur while transecting rectourethralis muscle cutting further into rectal wall . we present a rare case of spontaneous rectal perforation after uncomplicated nseerpe in a patient with a previous history of rectal polypectomy at the perforation site , which was successfully treated conservatively . in november 2005 , an otherwise healthy 71-year - old man with localised prostate cancer underwent nseerpe in our hospital . he gave history of polypectomy 10 cm from anus 4 weeks before , otherwise no clinical or laboratory abnormalities . coloscopy revealed bleeding from intraluminal wall laceration over a haematoma in polypectomy site ( figure 1a ) which was sealed with fibrin glue . ct revealed pelvic haematoma without detectable connections between rectum , bladder or abdomen ( figure 1b ) . ct at 16day showed established free connection between rectum and haematoma with some air without abdominal connections ( figure 2a , b ) . later , there was complete resorption of haematoma and healing of fistula as shown by abdominal ct ( figure 2c ) and coloscopy at 3month . psa is still undetectable . ( a ) rectoscopy view showing bleeding from wand haematoma without any fistula ( 2postoperative day ) . ( b ) ct examination showing pelvic haematoma without any signs of rectal perforation ( 3postoperative day ) . ( a. and b ) coronal and sagittal abdominal ct sections showing free connection between rectum and prostate bed with haematoma , contrast material and some air ( 16postoperative day ) . in this case rectal perforation could be either due to laceration because of previous polypectomy or missed iatrogenic perforation . owing to the intimate anatomical relation to prostate , it is our believe that this rectal wall thinning ( or perforations ) together with thermic injury at site of polypectomy should be given enough time to heal with adequate scare , otherwise it well predispose to laceration followed by perforation / necrosis in this site during or after operative manipulations . however care should be taken to exclude merely secondary haemorrhage due to clot dissolution in these cases which may manifest itself days to weeks after coloscopy ( 0.3 - 6.1% ) . although practiced in the beginning of open retropubic prostatectomy , most laparoscopic groups , including ours , leave the urethra intact during most of the dissection . instead , the plane between the seminal vesicles , prostate , and rectum is developed , progressing from the base of the prostate as close as possible to the apex . most of these injuries are visually identified intraoperatively and commonly repaired in a 2-layer suture with or without interposition of omentum / fat between the rectum and the vesicourethral anastomosis [ 5 - 7 ] . in case of missed perforations , signs and symptoms will be related to the size and site of the perforation , adequacy of bowel preparation , amount of peritoneal soilage , underlying bowel pathology ( e.g. thin walled colon from colitis or ischemia may result in a larger perforation than a healthy colon ) and finally , overall clinical condition of the patient . radiology often establishes the diagnosis however a localized perforation may demonstrate lack of pneumo peritoneum and necessitates other diagnostic procedures . management remains a controversial issue in that it can be effectively done by operative and nonoperative measures . generally , nonsurgical management is indicated if the patient 's general condition remains stable , the pneumoperitoneum does not increase in size , there are no signs of peritonitis and if the patient 's condition improves in response to conservative treatment . surgery is most definitely indicated in the presence of a large perforation , in the setting of generalized peritonitis or ongoing sepsis , the presence of concomitant pathology , unremitting colitis or perforation proximal to an obstructing distal lesion . finally , in the patient who deteriorates with conservative management . however , the best treatment for rectal injury during nseerpe remains prevention . although the primary reason is certainly anatomical since the plane of dissection is close to the rectum , another reason could be weakness of the rectal wall e.g. following polypectomy . in our opinion the later case needs 2 months in order to heal adequately and withstand operative manipulations . also to avoid adding further thermal wall damage to this site which may later obscure healing leading to more serious complications like fistula . according to this principle we have not faced this complication in following similar cases . nevertheless , the use of an intrarectal device / air should be emphasized in difficult cases due to surgeon inexperience , inflamed prostate , large volume gland , narrow and/or deep pelvis or previous rectal operations . these simple manoeuvres help in identifying the site of perforation and/or lacerations , if exist . about 2 months interval should be given to allow adequate healing before the operation and avoid adding further thermal wall damage which may obscure healing leading to complications like fistula . conservative therapy for these small missed rectal perforations constitutes a feasible and non invasive treatment entity . however in the presence of a large perforation , generalized peritonitis , ongoing sepsis or if the patient condition deteriorates surgery is indicated . intra - operative use of an intrarectal device / air should be emphasized in difficult cases . the authors disclose any commercial association that might pose a conflict in connection with the submitted article .

introductionrectal polypectomy causes thinning ( or even perforation ) of the rectal wall in addition to thermic injury at the polypectomy site.case reportwe present a rare case of spontaneous rectal perforation after uncomplicated nerve sparing endoscopic extraperitoneal radical prostatectomy in a patient with a previous history of rectal polypectomy at the perforation site . the patient could be treated conservatively . there was complete healing of the fistula without any effect on functional results . this conservative therapy for such rectal perforations is indicated if the patient 's general condition remains stable without any signs of infection.conclusionspolypectomy is an important risk factor for rectal perforation during nseerpe . adequate time interval should be given to allow healing and avoid adding further thermal wall damage which may obscure healing leading to complications like fistula . conservative therapy for small missed rectal perforations constitutes an attractive , feasible and non invasive treatment entity . following this principle we have not faced this complication in following similar cases .

falls of the elderly always lead to serious health issues as the decline of their physical fitness . fracture is the most common injury in fall of an elderly and there is also a certain possibility to get coma , brain trauma , and paralysis . at most fall situations , the fall process is the main source of injury because of the high impact . but sometimes the late medical salvage may worsen the situation . that means the faster the salvage comes , the less risk the elderly will face . mems ( microelectro mechanical systems ) sensors have simplified the design and implementation of sensor system . location based service ( lbs ) makes it more convenient to locate the elderly in health monitoring . beside these cameras are distributed at limited space to offer pictures or videos of human activities to implement fall detection algorithm . external supports such as motion sensors could be used to enhance computer vision based fall detection method , and a data fusion algorithm can operate the validation and correlation among the two subsystems to raise robust performance of fall detection . these computer based methods work effectively in indoor environment , but they are hard to realize in outdoor environment as the deployment of cameras is always limited . there are several kinds of detection methods which differ in constitution of motion sensors and detection algorithms . a single triaxial accelerometer can provide object 's accelerations in three directions which include the influence of gravity . the influence of gravity or dynamic acceleration is available by using a low pass filter or a high pass filter . some kinds of angular movement information can also be calculated based on the relationship between acceleration components and their vector sum . a triaxial magnetometer can detect magnetic strength in three directions , and it can also provide angular motion information in the horizontal plane . but the environment magnetic field may disturb the geomagnetic field which reduces the reliability of the magnetometer outputs , for instance , in some steel structure architecture or near some objects with strong electromagnetism . as angular information can also be extracted from accelerometer measurements , a state space filter such as the kalman filter is a commonly used technique to combine angular motion information . beside these , sensors such as barometer can also assist pure motion sensors at human gait recognition . but , in fact , using more sensors means more power consumption , and it is a challenge to design a proper algorithm to fuse different kinds of sensors . a single triaxial accelerometer is quite enough for human fall detection as sufficient information could be extracted from its measurements . besides this , the accelerometer coordinate does not have to be fixed if only the magnitude of sum vector is needed , and that is quite convenient for wearable application . in this paper , a fall detection system based on a wearable device is developed . the hardware and software realization of the device is mainly based on a single triaxial accelerometer and gps / gsm module . the device uses an efficient fall detection algorithm with less resource and power consumption , which means that it is a proper design for outdoor application . then it will get the elderly 's geographic position and send fall alarm short message to caregivers . so the elderly who has fallen can get timely help to minimize the negative influence . information like linear movements ( e.g. , displacement , velocity , and acceleration ) and angular movements ( e.g. , angle , angular velocity , and angular acceleration ) could be obtained directly or indirectly . beside these , frequency domain parameters could be extracted from basic sensor measurements by techniques such as fft and wavelet [ 11 , 12 ] . for single triaxial accelerometer application , accelerations and derived angular parameters could be used as recognition features . fall detection algorithm design according to the recognition feature , fall detection algorithms are classified as threshold based and machine learning based . for threshold based method , threshold of recognition feature is set by the designer before application which makes the algorithm have rapid response and less resource consumption . but the choice of threshold needs both rigorous schemes and adequate experiments . for machine learning based design , the classification of fall and normal activities is available with the assistance of technologies such as support vector machine ( svm ) and neural network [ 14 , 15 ] . machine learning assistance may enhance system robustness to some extent , but its algorithm design is always high computing resource consumed which limits its application in wearable device . as the compact wearable device requires low power consumption and a single triaxial accelerometer could provide effective information , threshold based fall detection algorithm will be used in this system . algorithm used in this fall alarm system when a real fall happens , collision between human 's body and ground will produce obvious peak value at the sum acceleration a which has magnitude as ( 1)a = ax2+ay2+az2 , where ax , ay , and az present accelerometer measurements of three axes . the system uses the sum acceleration as the first step to distinguish high intensity movements from others . but normal motions such as jumping or sitting also produce peak values , which mean that additional detection features are required . as human 's motion has low acceleration , it is feasible to get gravity component in each axis by using a low pass filter . if gravity components could be separated before and after human 's fall , then it is possible to calculate the rotation angle of accelerometer coordinate in 3d space , which is also equivalent to the rotation angle of gravity vector relative to fixed coordinate . quaternion is an effective tool to describe rotation movement in human 's gait change which also includes falling . a quaternion could be described as ( 2)q = q0+q1i+q2j+q3k which has magnitude as ( 3)q = q02+q12+q22+q32 . unit quaternion which has magnitude q = 1 can be described as ( 4)q = cos2+sin2qxi+sin2qyj+sin2qzk. as shown in figure 3 , rotation angle of q equals . the rotation axis is orthogonal to the rotation plane and its direction is in accordance with right hand screw rule . qx , qy , and qz are three components of the unit vector which describes the orientation of the quaternion at the fixed coordinate . besides rotation movement , quaternion can also describe a vector in 3d space , such as the gravity vector g which could be described as a quaternion ( 5)g=0+gxi+gyj+gzk.gx , gy , and gz are three components of g which has quaternion magnitude as ( 6)g = gx2+gy2+gz2=g . a unit quaternion q is used to describe human 's falling movement , which can also be divided into three rotation quaternions q1 , q2 , and q3 to simplify the calculation as shown in figure 4 . with the help of gravity vector information before and after human 's falling movement as shown in figure 3 , these three separated quaternions are all available . q 1 could be expressed as ( 7)q1=cos12+sin12sini+sin12cosj which has rotation angle and rotation axis information as ( 8)1=arctangbefore , zgbefore , x2+gbefore , y2,sin=gbefore , ygbefore , x2+gbefore , y2,cos=gbefore , xgbefore , x2+gbefore , y2 . quaternion q2 can be calculated as ( 9)q2=cos22+sin22k which has rotation angle as ( 10)2=arctan2gafter , ygafter , xarctan2gbefore , ygbefore , x . quaternion q3 is ( 11)q3=cos32+sin32sini+sin32cosj with rotation angle and rotation axis information as ( 12)3=arctangafter , zgafter , x2+gafter , y2,sin=gafter , ygafter , x2+gafter , y2,cos=gafter , xgafter , x2+gafter , y2 . then the rotation of the fall movement can be expressed by quaternion multiplication as ( 13)gafter = qgbeforeq=q3q2q1gbeforeq1q2q3 , where q=q0-q1i -- q2j -- q3k- is the conjugate quaternion of q. quaternion algebra is normally implemented based on basic matrix algebra . quaternion multiplication used above could be realized by matrix multiplication as ( 14)qgbefore = mq0gbefore , xgbefore , ygbefore , z = q0q1q2q3q1q0q3q2q2q3q0q1q3q2q1q00gbefore , xgbefore , ygbefore , z . the whole rotation quaternion can also be decomposed as ( 15)q = q3q2q1=mq3mq2q1.q1 , q2 , and q3 are all available based on gravity information before and after the falling movement . at last , it is possible to get four elements of quaternion q by the equation above and the rotation angle could be calculated as ( 16)=2arctanq12+q22+q32q0 . when an object is falling , magnitude of will approach 90 which is also a character different from most normal activities . the wearable device will be mounted on human 's waist at first to reflect the motion of human body closely , and the device will record gbefore while the elderly is standing still . there is no special requirement of the device orientation but only keep stationary during the wear . athreshold means the threshold of sum acceleration magnitude and tthreshold means the threshold of oscillation time duration after the break of athreshold . when measurements in three different axes have been acquired , the sum acceleration |a| will be calculated . when a real fall happens , sum acceleration will reach peak value of |a| athreshold . in a real falling , the fluctuation of acceleration will stop in time duration tthreshold and then the sum acceleration is |a| g as the elderly will lie on the ground . then the acceleration a is recorded as gafter . , the system will consider it as a fall if the rotation angle || 90. ti 's 16 bits mcu msp430f1611 is used to control the whole system and imply the detection algorithm . the measurement range of accelerometer could be set at 2 g , 4 g , 8 g , or 16 g , and the maximal sampling rate is 3.2 khz . as human 's activities are normally at low frequency bands , 100 hz is a proper sampling rate for human fall detection . there is an inner digital filter in adxl345 which could weaken noise and reduce the burden of digital signal processing in mcu to some extent . the measurements will be sent through iic ( interintegrated circuit ) bus communication between the sensor and the mcu . sim908 can offer gps and gsm service on serial port communication with mcu , and it can also work in low power mode . each hardware component of the wearable device is working under low voltage and the detection algorithm does not need complex calculation resource , so the power consumption of the whole device is quite low . a 1200 mah , 3.7 v polymer lithium battery is quite enough to provide the need of the wearable device for a couple of days . hardware structure of the detection device is shown in figure 6 , and the pcb board prototype is shown in figure 7 . one of them is the software design in wearable device , and the other is in the caregiver 's handset . after initialization of the system , gbefore would be extracted from acceleration measurements by using a low pass filter when the elderly is standing . if a fall has been detected , the wearable device will locate the user and send alarm short message to caregivers immediately . if the user withdraws the alarm by pressing a button manually , the device will get back to fall detection state and a short message will be sent to inform the caregivers . there is a url ( universal resource locator ) which links to a web map in the alarm short message . the fall location information will be highlighted on a web map when the caregiver opens this link . system test of the fall detection system has been conducted based on the system design described above . the sampling rate of accelerometer is set at 100 hz and the measurement range is 16 g with a maximum precision of 4 mg . mcu will read raw measurements from sensor 's inner fifo and apply the detection algorithm . the test objects are three different volunteers at the ages of 23 , 42 , and 60 , respectively . based on analysis of these volunteers ' experiment data , athreshold and tthreshold are set as 2 g and 2 s , respectively . in order to get g when standing and lying after the fall , sum acceleration a which has magnitude between ( 1 0.3 ) g and ( 1 + 0.3 ) g will be considered as gbefore and gafter . considering that the tilt of the ground or the lying posture of the elderly may affect the rotation angle , rotation angle between ( 90 30) and ( 90 + 30) will infer that the elderly has fallen . system test contains five kinds of activities of daily living ( i.e. , walking , jumping , squatting , sitting , and resting ) and four kinds of fallings ( i.e. , forward , backward , leftward , and rightward ) . each kind of motion has been repeated 20 times on each volunteer , and the detection results of the proposed algorithm and an acceleration threshold based algorithm are listed in table 1 . the sensitivity and specificity [ 21 , 22 ] of the proposed system can be got from the test data in table 1 . test results of acceleration threshold based algorithm show lower sensitivity and specificity at 91.6% and 88.7% , respectively . figure 9 shows the accelerations in different kinds of fallings which all trigger the fall alarm correctly and the corresponding rotation angles are 91.9 , 100.5 , 104.7 , and 114.1. figure 10 shows acceleration during activity of lying down and resting which has rotation angle of 117.0 and similar waveform to falling , but the peak acceleration is much lower . they all do not have rotation about 90 ; even some of the peak values are quite high , so the fall alarm has not been triggered . an alarm sms ( short message service ) containing a map url has been received by the handset as shown in figure 12 when a fall has been detected . clicking the url will open a map in web browser on which the fall location will be displayed accurately as shown in figure 13 . this paper developed a fall detection system based on a single triaxial accelerometer based wearable device . there is no special requirement of the device 's mounting orientation because the algorithm does not claim the axes of accelerometer to be fixed strictly . the system has low power consumed hardware design and highly efficient algorithm which could extend the service time of the wearable device . as normal activity of resting also has similar rotation as falling , it may trigger fall alarm when the body hits ground heavily . so the choice of athreshold is quite important to distinguish falling from heavily lying activity . sufficient sample number collected from subjects with different age and gender will improve the reliability and robustness of the threshold . beside these , technologies such as svm and neural network are considerable to seek out a proper classification method based on the features used in this system .

fall detection is a major challenge in the public healthcare domain , especially for the elderly as the decline of their physical fitness , and timely and reliable surveillance is necessary to mitigate the negative effects of falls . this paper develops a novel fall detection system based on a wearable device . the system monitors the movements of human body , recognizes a fall from normal daily activities by an effective quaternion algorithm , and automatically sends request for help to the caregivers with the patient 's location .

the basic helix - loop - helix ( bhlh ) family of transcriptional regulators are key players in a wide array of developmental processes in metazoans , including neurogenesis , myogenesis , hematopoiesis , sex determination and gut development ( reviewed in ) . the bhlh domain is approximately 60 amino acids long and comprises a dna - binding basic region ( b ) followed by two helices separated by a variable loop region ( hlh ) ( reviewed in ) . the hlh domain promotes dimerization , allowing the formation of homodimeric or heterodimeric complexes between different family members . over 400 bhlh proteins have been identified to date in organisms ranging from the yeast saccharomyces cerevisiae to humans ( see , for example ) . in previous work , we took advantage of the complete sequencing of the nematode and fly genomes to extract a large , and possibly complete , set of bhlh genes from these two organisms . a phylogenetic analysis of the amino acid sequences of these bhlhs , together with a large number ( > 350 ) of bhlh from other sources , in particular from mouse , led us to define 44 orthologous families ( that is , groups of orthologous sequences that derive from the duplication of a common ancestor ) , among which 36 include bhlh from metazoans only , and 2 have representatives in both yeasts and metazoans ( table 1 ) . we also identified two bhlh motifs present only in yeast , and four that are present only in plants . the 44 families of animal bhlh defined by our phylogenetic analyses families have been named according to the name ( or its common abbreviation ) of the first discovered or best - known member of the family . the number of members per family in worm , fly and human ( complete genomes ) as well as in mouse , sea squirt , and pufferfish ( uncompleted genomes ) is reported . each family has been tentatively assigned to a high - order group using the classification of atchley and fitch and ledent and vervoort . genes that can not be assigned to any families are categorized as ' orphan ' genes . beta3 and oligo are closely related families , one c. elegans gene ( f38c2.2 ) belongs to the beta3 family while another ( dy3.3 ) is equally related to both beta3 and oligo families . mad and mnt are closely related families , one ciona gene ( not7 ) belongs to the mad family while another ( lqw20007 ) is equally related to both mad and mnt families . tf4 and mlx are closely related families , one c. elegans gene ( t20b12.6 ) is equally related to both families . the hif , sim , and trh families form a strongly supported monophyletic group ( bootstrap value , 95% ) . a few genes that are included in this group can not be clearly related to one of the three families ( see additional data for details ) . the hey , hairy and enhancer of split families genes form a well - supported monophyletic group ( group e ; see figure 1 ) . two clear families ( hairy and hey families ) with high bootstrap support emerge from this group . all the remaining sequences have been grouped in a single family ( named enhancer of split ) , which has no real phylogenetic support . in addition , we defined higher - order groups which include several evolutionarily related families that share structural and biochemical properties . the different groups were named a , b , c , d , e and f , in agreement with the nomenclature of atchley and fitch . figure 1 shows the phylogenetic relationships between animal families and their tentative inclusion into the different higher - order groups . a neighbor - joining ( nj ) tree showing the evolutionary relationships of the 44 animal bhlh families listed in table 1 is shown . we used one gene ( usually from mouse ) per family to construct this tree . although there are strong theoretical reasons for preferring the unrooted tree , we show a rooted tree because it is easier to display compactly and more clearly represents the relationships at the tip of the branches . this tree is just a representation of an unrooted tree with rooting that should be considered arbitrary . , we show a tree in which branch lengths are not proportional to distances between sequences . some of these groups ( a and e ) are monophyletic groups , others ( d and f ) correspond to only one family , and yet others ( b and c ) are paraphyletic ( the last common ancestor of the different families that constitute the group is also that of bhlhs that do not belong to that group ) . a subgroup of group a families ( the atonal ' superfamily ' ) is also highlighted and is displayed in more detail in figure 2 . some examples of phylogenetic relationships among human and mouse bhlh . rooted nj trees are shown . numbers above branches indicate per cent support in bootstrap analyses ( 1,000 replicates ) . as in figure 1 , the rooting should be considered arbitrary . mouse genes are shown in red , human genes in blue , and other species in black . species abreviations are as followed : br , brachydanio rerio ; ce , caenorhabditis elegans ; ci , ciona intestinalis ; dm , drosophila melanogaster ; gg , gallus gallus ; tr , takifugu rubripes ; xl , xenopus laevis . ( a ) evolutionary relationships among atonal ' superfamily ' members ( see figure 1 ) . the different constituting families are pointed out . for sake of simplicity , only mouse , this tree is rooted using the closely related twist gene from mouse ( see figure 1 ) as outgroup . in all cases , a human and a mouse sequence cluster together with high bootstrap values , indicating orthology relationships . this tree is rooted using the closely related math1 gene from mouse ( see figure 1 ) as outgroup . whereas one human and one mouse bhlh ( n015926 and pmeso1 , respectively ) are clearly orthologs , there is no one - to - one relationship between two mouse bhlh ( mesp1 and mesp2 ) and three human bhlh ( n010356a , b , c ) , although these bhlh cluster together with a high bootstrap value . this tree is rooted using the closely related mitf gene from mouse ( see figure 1 ) as outgroup . two human genes have clear mouse orthologs but two others ( q9hap2 and n005106 ) have no such orthologs . this tree is rooted using the closely related hen1 gene ( nscl family ) from mouse ( see figure 1 ) as outgroup . the lyl1 and lyl2 mouse genes are collectively orthologs to one human gene ( p12980 ) , indicating a probable gene duplication specific to mouse . in brief , groups a and b include bhlh proteins that bind core dna sequences referred to as e boxes ( canntg ) , respectively cacctg or cagctg ( group a ) and cacgtg or catgttg ( group b ) . group c corresponds to the family of bhlh proteins known as bhlh - pas , as they contain a pas domain in addition to the bhlh . group d corresponds to hlh proteins that lack a basic domain and are hence unable to bind dna . group e includes proteins related to the drosophila hairy and enhancer of split bhlh ( her proteins ) . these proteins bind preferentially to sequences referred to as n boxes ( cacgcg or cacgag ) . they also contain two characteristic domains in addition to the bhlh , the ' orange ' domain and a wrpw peptide in their carboxy - terminal part . group f corresponds to the coe family , which is characterized by the presence of an additional domain involved both in dimerization and in dna binding , the coe domain . the completion of the human genome sequencing project now allows us to derive the complete set of bhlh present in a vertebrate genome . tblastn searches on the human genome draft sequence enabled us to identify 125 different human bhlhs . after exhaustive searches with blastp in protein databases and the use of the smart database ( simple modular architecture research tool ) we also identified additional fly , worm and mouse bhlh sequences ( total number : 58 in fly , 39 in worm , and 102 in mouse ) . in addition , we made tblastx searches on the incompletely sequenced genomes of the pufferfish takifugu rubripes and the sea squirt ciona intestinalis and retrieved 84 and 18 different bhlhs , respectively . we also retrieved , through blastp searches , eight different bhlh genes from the completely sequenced yeast genome . phylogenetic analysis of all these sequences allowed us to define 44 orthologous families of bhlh proteins in metazoans ( the 38 families defined in our previous report plus 6 additional ones , arising out of the additional sequences used in this analysis ) . our work now enables comparison of the putative complete repertoires of bhlhs in metazoans belonging to the two main subdivisions of bilaterian animals ( the bilateria ; see for a recent overview of the classification of metazoans ) - the deuterostomes ( human ) and the protostomes ( fly and worm ) . this comparison gives us the opportunity to analyze evolution of the diversity of the bhlhs on a metazoan - wide scale , thus giving useful insights into the evolution of multigenic families . in addition , our results allow us to reconstruct the minimum complement of bhlh genes that were present in the bilaterian common ancestor . to isolate human bhlh genes , we made tblastn searches on the human genome draft sequence , as described in materials and methods . we eventually got 125 different human bhlh sequences , which are listed in table 2 . all retrieved sequences were used to make blastp searches against protein databases in order to detect those sequences that were already identified . we found that 80 sequences were already present in protein databases ; 45 of the retrieved sequences from the human genome correspond to previously uncharacterized genes . we similarly retrieved , by tblastn , 84 and 18 different bhlh sequences from the incompletely sequenced genomes of the pufferfish t. rubripes and the sea squirt c. intestinalis , respectively ( see additional data files ) . in addition , we retrieved the complete set of bhlh genes present in the fly ( total 58 ) , worm ( 39 ) , and yeast ( 8) genomes , as well as all the cloned mouse bhlh genes to date ( 102 ) , as described in materials and methods . these sequences with their accession numbers and some information ( genomic localization and orthology relationships ) the complete list of bhlh genes from homo sapiens human sequences are identified using their accession number from swissprot , trembl , smart or ncbi genome project sequences . in the latest case , this accession number nt_xxxxxx.y ( xxxxxx identifies the contig and y the version of the draft ) has been abbreviated as nxxxxxxx . gene names are those reported in protein databases or have been assigned by us on the basis of the orthology relationships with mouse genes ( these names are marked by an asterisk ) . the identification of the contig in which each of the bhlh gene is included is also given . in a few cases ( marked with a question mark ) , we were unable to retrieve , in the genome sequence , previously cloned genes . this may be due to the fact that these genes lie in still unsequenced regions of the genome , or to some limitations of the current version of blast ( see text for details ) . chromosomal localizations are given as reported in the ncbi human genome sequence database ( locuslink and/or omim ) . the complete list of bhlh genes from drosophila melanogaster gene names ( with commonly used synonyms in some cases ) and their usual abbreviation are as reported in flybase , except those marked by an asterisk . in these cases , we propose names based on the orthology relationships with well - characterized vertebrate genes . sequences are listed with the family in which there are included ( or stated as orphan genes ) , their chromosomal localization ( position on the polytene chromosomes map as found in flybase ) , and their accession number . the ' orphan ' gene delilah clearly belongs to the high - order group a and is most probably a highly divergent neurod family gene ( see for discussion ) . the complete list of bhlh genes from caenorhabditis elegans gene identifications are those of the c. elegans genome project . the localization of the genes referred to the worm recombination genetic map as found in wormbase . sequences marked with an asterisk form a well supported monophyletic group and encode proteins with two bhlh ( see text for details ) . the complete list of bhlh genes from mus musculus mouse genes are listed with the family in which they are included , the identification of their human ortholog(s ) ( ? indicates that no clear ortholog was found , see text for details ) , and their accession number . in most cases , we report here only one name ; synonyms can be found in the protein databases using the reported accession numbers . the complete list of bhlh genes from saccharomyces cerevisiae yeast genes are listed with the family in which they are included and their accession number . to carry out evolutionary analyses of multigene families requires one to distinguish orthologs , which have evolved by vertical descent from a common ancestor , from paralogs , which arise by duplication and domain shuffling within a genome . failure to do so can result in functional misclassification and inaccurate molecular evolutionary reconstructions . the overall similarity ( as determined by the blast e - value ) is often used as a criterion to determine orthology relationships within large data sets such as complete genomes , but there is evidence that more rigorous phylogenetic reconstructions are required to confidently determine orthologies . we therefore constructed phylogenetic trees to define groups of orthologous sequences , as we did previously ( see materials and methods ) . we determined 44 orthologous families that contain most of the metazoan bhlh families ( table 1 and additional data ) . the criterion we used to define orthologous families was as in ; that is , orthologous families are monophyletic groups found in the gene trees constructed by different phylogenetic methods and whose monophyly is supported by bootstrap values larger than 50% . we named each family according to its first discovered member or , in a few cases , its best - characterized member . this analysis gave similar results to that described in , except that the additional sequences included in the present phylogenetic analyses led us to define six additional families of bhlhs from metazoans , compared with our previous report . we found a total of 125 and 102 different bhlh sequences in human and mouse , respectively ( tables 2 and 5 ) . these sequences were used to make phylogenetic reconstructions as described above and in materials and methods . this allows us to infer orthology relationships between mouse and human sequences . two sequences were considered as orthologs if they are more closely related to each other than to any other mouse or human sequences . this can be easily detected in the phylogenetic trees , as the two sequences will form an exclusive monophyletic group ( figure 2a ) . among the 125 human sequences , 94 can be accurately related to 1 ( or in a few cases 2 , see below ) mouse genes ( table 2 ) and , conversely , human orthologs can be confidently assigned to 93 of the 102 mouse genes ( table 5 ) . among the 31 human genes and 9 mouse genes that do not show clear orthology relationships to any mouse or human genes , respectively , 8 human genes and 6 mouse genes are members of families in which phylogenetic relationships are uncertain - the mesp , e12 and coe families ( figure 2b and additional data ) . the mesp family contains four human genes and three mouse genes , the e12 family seven human and four mouse genes , and the coe family four human and four mouse genes . some of these genes can not be clearly linked to each other ( see figure 2b for an example ) . it is , however , conceivable that such relationships do exist but that phylogenetic reconstruction methods fail to detect them . we therefore consider that , in the mesp family for example ( figure 2b ) , three of the four human genes correspond to the three mouse genes , and so , to date , one human gene lacks an ortholog among the cloned mouse genes . applying the same reasoning to the e12 and coe families leads us to conclude that at least 26 human genes ( 20% of the total ) do not have orthologs among the mouse bhlh genes cloned to date and only 3 mouse bhlhs ( 3% ) have no orthologs in the bhlh set we derived from the human genome sequence draft . figure 2c shows a typical phylogenetic tree of a family containing human genes that lack mouse orthologs . the fact that only three mouse genes lack human orthologs strongly argues that , although our analysis was made on a draft version of the human genome sequence , the set of bhlh we retrieved is likely to be almost complete , and hence gives a highly accurate view of the bhlh repertoire of a human being . additional blast searches for human orthologs of the three mouse bhlhs that lacked orthologs ( scleraxis , dermo-1 and s - myc ) were unsuccessful , suggesting that these orthologs either do not exist in humans or are not in the draft sequence . we were recently made aware that there is some incompatibility between the current version of blast and the human genome sequence ( probably due to the large number of ns ( unassigned nucleotides ) in the sequence ) , which makes blast unable to locate some of the best or even exact matches of small query sequences ( j.a.m . this may explain why we missed the four genes cited above , and also why , in a few cases , we were unable to find known cloned human genes in the genome sequence ( see table 2 ) . we also found eight cases in which two human genes group together ( with high statistical support ) to the exclusion of any other genes and are often orthologs of a single mouse gene ( figure 2b and additional data ) . conversely , we found two cases in which two mouse genes are , collectively , orthologs of a single human gene ( figure 2d ) . this may reveal relatively recent duplications specific to the human or mouse lineage . in agreement with this , in all cases amino - acid identity between the two duplicates is high and is not confined to the bhlh . in addition , we found that in two cases ( human sequences q9uh92/n005106 and q02363/n005999 ) , one of the two duplicates lacks introns . this strongly suggests that the duplications have occurred by retrotransposition , a type of event that appears to be rather frequent in humans . in both cases , the copy lacking introns has stop codons in the bhlh , suggesting that it is a pseudogene . among the 39 bhlh from the worm , 6 can not be assigned to any family ( orphan genes ; see tables 1 and 4 ) . five of these have an unusual architecture in they contain two bhlh domains ( see also ) . phylogenetic analysis of these proteins indicates that they result from the duplication of an ancestral gene that already contained two bhlhs ( figure 3 ) . both bhlh domains are loosely related ( on the basis of overall similarity ) to her proteins ( group e ; figure 1 ) , but their inclusion in group e is not supported by phylogenetic reconstruction ( figure 3 ) . in addition , they lack the orange domain , which is characteristic of most her proteins and provides them with functional specificity . they also lack the wrpw motif found in the carboxy - terminal region of almost all her proteins and which allows interaction with the groucho represser protein . moreover , they lack a conserved proline in the basic domain that confers dna - binding specificity on the her proteins . a rooted nj tree is shown that depicts the phylogenetic relationships of the five worm proteins with two bhlh domains . mouse genes representative of some of the animal families have been included in this analysis . numbers above branches indicate per cent support in bootstrap analyses ( 1,000 replicates ) . as in figure 1 the sequences of the first bhlh of each worm proteins are shown in blue , the second in red . both form monophyletic groups with high bootstrap values , indicating that these proteins originate from an ancestral protein that already had two bhlh domains . there is , furthermore , a weaker support ( 40% bootstraps ) for an association of the two bhlh domains into a monophyletic group ( not shown in the figure , as only nodes with 50% or more support are shown ) , suggesting that the ancestral protein may have acquired its two bhlh domains through tandem duplication rather than by association of unrelated bhlh domains . no other protein with two bhlhs has been reported in other metazoans and we were unable to find such proteins in the fly and human genomes . a protein with two bhlh domains is found in rice ( oryza sativa ; protein p0498b01.20 ; accession number bab61947 ) but its sequence is completely unrelated to that of the worm protein . several bhlh proteins do contain other dna - binding and/or dimerization domains in addition to their bhlh , such as the pas domain , leucine zippers or the coe domain . it is conceivable that these domains may cooperate and thereby confer particular functions on the proteins containing them . similarly , the presence of two bhlhs might modify the specificity of the proteins containing them . bhlh genes are found in all major subdivisions of the eukaryotes : metazoans , fungi and plants . it seems , therefore , that the bhlh motif was established in early eukaryote evolution . we have found eight different bhlh genes in the unicellular eukaryote , the yeast s. cerevisiae . most of these genes were already cloned and have been functionally characterized ( reviewed in ) . these genes often regulate biochemical pathways ( such as phosphate utilization , phospholipid and amino - acid biosynthesis , glycolysis ) through the transcriptional activation of more - or - less large sets of genes involved in these pathways . orthologs of these genes are found in other distantly related yeasts such as schizosaccharomyces pombe and kluyveromyces lactis ( our unpublished observations ) , indicating an ancient origin for the different bhlh genes among yeasts . the relatively small number of bhlh genes found in the unicellular yeast contrasts with the large number found in multicellular eukaryotes such as animals and plants . we report here the existence of 39 different bhlh genes in c. elegans , 58 in d. melanogaster , and 125 in humans . preliminary analysis of plant genomes , in particular of arabidopsis thaliana and o. sativa , similarly indicates a large number of bhlh genes ( more than 100 in the completely sequenced genome of a. thaliana , our unpublished observations ) . this important diversification of the bhlh repertoire in animals and plants has occurred independently , as plant and animal bhlh genes are never found in a same family . the current view of eukaryote phylogeny suggests that fungi and animals are more closely related to each other than to the plants . nevertheless , we found that only two families contain both yeast and animal genes ( see table 1 ) , suggesting that the common ancestor of fungi and animals may have possessed even fewer bhlh genes than the present - day yeasts . in the near future , the genome projects currently underway on various ' basal ' eukaryotes ( see ) may give important insights into the very early evolutionary history of the bhlh family . we suggest that the diversification of bhlh genes is directly linked to the acquisition of multicellularity and hence to the recruitment of genes involved in cell functions such as metabolism into the developmental processes required to build multicellularity . indeed , in animals , bhlh genes are generally involved in development and in tissue - specific gene regulation ( reviewed in ) a similar situation may exist in plants , although very few bhlh genes have been functionally characterized . in addition , in both animals and plants , the diversification of bhlh genes seems to have occurred early in the evolution of these lineages . indeed , our phylogenetic analysis of animal bhlh genes shows that most belong to 44 different orthologous families . of these families , 43 contain representatives from both protostomes and deuterostomes , and must therefore be represented in their common ancestor ( often called urbilateria ) , which lived in pre - cambrian times ( 600 million years ago ) . in addition , the few bhlh genes that have been cloned from cnidarians , which are not bilaterians , are clearly included in families ( see the twist , myod and asc families in additional data ) , suggesting that the establishment of at least some families predates the divergence of bilaterians and non - bilaterians . further analyses of bhlh genes in cnidarians , sponges and slime molds will help to resolve the issue of the early evolution of bhlh genes in animals . our preliminary analyses of plant bhlh genes are consistent with an early diversification in plants , as in animals . indeed , many a. thaliana bhlh genes have clear orthologs in a distantly related plant , o. sativa , whose genome has been partially sequenced ( our unpublished observations ) . arabidopsis is a eudicotyledon and oryza a member of the liliopsida ( a monocotyledon ) , and given the phylogenetic relationships of these clades this suggests that the possession of numerous bhlh genes might be ancestral to angiosperms . further analysis of the evolution of bhlh in plants will require the completion of the genome projects currently underway on rice and tomato ( a eudicotyledon of a different lineage from arabidopsis ) , as well as the isolation of bhlh in a broader spectrum of plant species , in particular in basal angiosperms and non - angiosperms . comparison of the bhlh repertoires found in the protostomes and the deuterostomes gives important insights in to the evolution of the bhlh family in metazoans . the conclusions that can be drawn are completely consistent with those presented in our previous work but the inclusion of the probable complete set of bhlh from a vertebrate strengthens these conclusions . most families ( 43/44 ) contain genes from protostomes ( fly and/or nematode ) and deuterostomes , indicating that these families were already present in the last common ancestor of both protostomes and deuterostomes , that is , of all bilaterians . the fact that most families contain both protostome and deuterostome genes also suggests that there was no addition of new bhlh types in the corresponding lineages , and therefore no important diversification of the ancestral repertoire . this may represent the emergence of new bhlh types in the vertebrate lineage , or alternatively a loss of ancestral types in both fly and nematode . the analysis of bhlh genes from molluscs or annelids might help to settle this question . it is now widely believed that the bilateria ( the triploblastic metazoans ) are composed of three main lineages : deuterostomes ( which include vertebrates and echinoderms ) and protostomes , which themselves include two large groups , the ecdysozoans ( for example , arthropods and nematodes ) and the lophotrochozoans ( for example , annelids , molluscs and flatworms ) ( reviewed in ) . therefore , the finding of ortholog genes in vertebrates and lophotrochozoans but not in fly and nematode would strongly suggest that gene loss(es ) has ( have ) occurred in the ecdysozoan lineage . similarly , the case of families that contain vertebrate and either worm or fly genes is best explained by gene losses that occurred , inside the ecdysozoan clade , in either lineage after the arthropod / nematode divergence . this occurred in the fly lineage for very few families ( 4/44 ) , suggesting the existence of a strong pressure to maintain the entire bhlh repertoire . the much larger number of families ( 13/44 ) that have vertebrate and fly members but no nematode representative suggests that extensive bhlh gene losses have occurred in the worm lineage . strikingly , the worm lacks the important cellular and developmental regulator myc . a similar absence of important developmental regulators , such as hedgehog , toll / il-1 and jak / stat pathway elements has also been reported in the nematode . in addition , a large number of nematode genes ( 6/39 ) can not be clearly assigned to specific families ( orphan genes ) . this is probably due to the high divergence rate reported for nematode genes in general and which we found within our specific data set ( and data not shown ) . interestingly , however , some nematode sequences have diverged very little from their fly or mouse counterparts . these include the few functionally characterized c. elegans bhlh genes that show overall functional conservation with their vertebrate and/or fly orthologs ; for example , the c. elegans orthologs of twist and myod are involved in muscle formation , and the orthologs of atonal and neurod ( lin-32 and cnd-1 ) have a role in nervous - system development . the genetic control of developmental processes such as neurogenesis and myogenesis relies on small sets of interacting genes ( syntagms ) . the function of syntagms crucially relies on specific molecular interactions among their members , hence imposing strong structural constraints on them and preventing structural diversification ( for discussion on syntagms and evolution , see ) . this may explain why such networks are strongly conserved throughout metazoan evolution and why nematode genes involved in such networks have been subject to special constraints . an extensive increase of bhlh family complexity has occurred in vertebrates : the most frequent number of different bhlh genes per family is one in fly ( 30/44 ) and worm ( 27/44 ) , and two in human ( 14/44 ; but 20/44 human families do in fact contain more than two genes ) . most bhlh families ( 32/44 ) , as with other gene families , have more members in vertebrates than in other phyla ( table 1 ) . of these families , 14 ( 32% ) contain four or more vertebrate genes ( table 1 ) and hence may reveal the occurrence of two whole - genome duplications ( the 2r hypothesis ) in early vertebrate evolution . in the most popular version , this is thought to have occurred by one duplication at the root of the vertebrates and a second in the gnathostomata lineage , after its divergence from agnatha ( reviewed in ) . several recent analyses , however , tend to refute ( at least , do not support ) this hypothesis ( reviewed in ) . for example , the current mammalian gene number estimations based on the human draft sequence , ests and comparisons with other vertebrates propose that the human genome would contain no more than 35,000 genes ; that is , about twice the number of fly and worm . this might , however , result from gene loss during or after the rounds of duplication . in addition , phylogenetic analyses of gene families that comprise four members cast doubt on the 2r hypothesis . as pointed out by hughes , the presence of four members in a vertebrate gene family by itself does not support the genome duplication hypothesis . support may only come from families whose phylogenetic tree shows a topology of the ( ab ) ( cd ) form , that is , two pairs of two closely related paralogs . hughes discussed the phylogenies of 13 protein families important in development , and found that only one of them shows an ( ab ) ( cd ) topology . similar results were recently obtained by martin and hughes et al . on several other families with much more rigorous phylogenetic tests . these results have led to the alternative hypothesis that the abundance of duplicated genes in vertebrates compared to invertebrates may be due to a high rate of local duplications , rather than entire genome duplications ( reviewed in ) . phylogenetic trees of the 14 bhlh families that contain four or more members do not clearly show such ( ab ) ( cd ) topologies ( see additional data ) . we have , however , to note that the phylogenies inside families often have only poor resolution and it is therefore difficult to draw firm conclusions from them . to isolate human bhlh genes , we made tblastn searches on the human genome draft sequence , as described in materials and methods . we eventually got 125 different human bhlh sequences , which are listed in table 2 . all retrieved sequences were used to make blastp searches against protein databases in order to detect those sequences that were already identified . we found that 80 sequences were already present in protein databases ; 45 of the retrieved sequences from the human genome correspond to previously uncharacterized genes . we similarly retrieved , by tblastn , 84 and 18 different bhlh sequences from the incompletely sequenced genomes of the pufferfish t. rubripes and the sea squirt c. intestinalis , respectively ( see additional data files ) . in addition , we retrieved the complete set of bhlh genes present in the fly ( total 58 ) , worm ( 39 ) , and yeast ( 8) genomes , as well as all the cloned mouse bhlh genes to date ( 102 ) , as described in materials and methods . these sequences with their accession numbers and some information ( genomic localization and orthology relationships ) the complete list of bhlh genes from homo sapiens human sequences are identified using their accession number from swissprot , trembl , smart or ncbi genome project sequences . in the latest case , this accession number nt_xxxxxx.y ( xxxxxx identifies the contig and y the version of the draft ) has been abbreviated as nxxxxxxx . gene names are those reported in protein databases or have been assigned by us on the basis of the orthology relationships with mouse genes ( these names are marked by an asterisk ) . the identification of the contig in which each of the bhlh gene is included is also given . in a few cases ( marked with a question mark ) , we were unable to retrieve , in the genome sequence , previously cloned genes . this may be due to the fact that these genes lie in still unsequenced regions of the genome , or to some limitations of the current version of blast ( see text for details ) . chromosomal localizations are given as reported in the ncbi human genome sequence database ( locuslink and/or omim ) . the complete list of bhlh genes from drosophila melanogaster gene names ( with commonly used synonyms in some cases ) and their usual abbreviation are as reported in flybase , except those marked by an asterisk . in these cases , we propose names based on the orthology relationships with well - characterized vertebrate genes . sequences are listed with the family in which there are included ( or stated as orphan genes ) , their chromosomal localization ( position on the polytene chromosomes map as found in flybase ) , and their accession number . the ' orphan ' gene delilah clearly belongs to the high - order group a and is most probably a highly divergent neurod family gene ( see for discussion ) . the complete list of bhlh genes from caenorhabditis elegans gene identifications are those of the c. elegans genome project . the localization of the genes referred to the worm recombination genetic map as found in wormbase . sequences marked with an asterisk form a well supported monophyletic group and encode proteins with two bhlh ( see text for details ) . the complete list of bhlh genes from mus musculus mouse genes are listed with the family in which they are included , the identification of their human ortholog(s ) ( ? indicates that no clear ortholog was found , see text for details ) , and their accession number . in most cases , we report here only one name ; synonyms can be found in the protein databases using the reported accession numbers . the complete list of bhlh genes from saccharomyces cerevisiae yeast genes are listed with the family in which they are included and their accession number . to carry out evolutionary analyses of multigene families requires one to distinguish orthologs , which have evolved by vertical descent from a common ancestor , from paralogs , which arise by duplication and domain shuffling within a genome . failure to do so can result in functional misclassification and inaccurate molecular evolutionary reconstructions . the overall similarity ( as determined by the blast e - value ) is often used as a criterion to determine orthology relationships within large data sets such as complete genomes , but there is evidence that more rigorous phylogenetic reconstructions are required to confidently determine orthologies . we therefore constructed phylogenetic trees to define groups of orthologous sequences , as we did previously ( see materials and methods ) . we determined 44 orthologous families that contain most of the metazoan bhlh families ( table 1 and additional data ) . the criterion we used to define orthologous families was as in ; that is , orthologous families are monophyletic groups found in the gene trees constructed by different phylogenetic methods and whose monophyly is supported by bootstrap values larger than 50% . we named each family according to its first discovered member or , in a few cases , its best - characterized member . this analysis gave similar results to that described in , except that the additional sequences included in the present phylogenetic analyses led us to define six additional families of bhlhs from metazoans , compared with our previous report . we found a total of 125 and 102 different bhlh sequences in human and mouse , respectively ( tables 2 and 5 ) . these sequences were used to make phylogenetic reconstructions as described above and in materials and methods . two sequences were considered as orthologs if they are more closely related to each other than to any other mouse or human sequences . this can be easily detected in the phylogenetic trees , as the two sequences will form an exclusive monophyletic group ( figure 2a ) . among the 125 human sequences , 94 can be accurately related to 1 ( or in a few cases 2 , see below ) mouse genes ( table 2 ) and , conversely , human orthologs can be confidently assigned to 93 of the 102 mouse genes ( table 5 ) . among the 31 human genes and 9 mouse genes that do not show clear orthology relationships to any mouse or human genes , respectively , 8 human genes and 6 mouse genes are members of families in which phylogenetic relationships are uncertain - the mesp , e12 and coe families ( figure 2b and additional data ) . the mesp family contains four human genes and three mouse genes , the e12 family seven human and four mouse genes , and the coe family four human and four mouse genes . some of these genes can not be clearly linked to each other ( see figure 2b for an example ) . it is , however , conceivable that such relationships do exist but that phylogenetic reconstruction methods fail to detect them . we therefore consider that , in the mesp family for example ( figure 2b ) , three of the four human genes correspond to the three mouse genes , and so , to date , one human gene lacks an ortholog among the cloned mouse genes . applying the same reasoning to the e12 and coe families leads us to conclude that at least 26 human genes ( 20% of the total ) do not have orthologs among the mouse bhlh genes cloned to date and only 3 mouse bhlhs ( 3% ) have no orthologs in the bhlh set we derived from the human genome sequence draft . figure 2c shows a typical phylogenetic tree of a family containing human genes that lack mouse orthologs . the fact that only three mouse genes lack human orthologs strongly argues that , although our analysis was made on a draft version of the human genome sequence , the set of bhlh we retrieved is likely to be almost complete , and hence gives a highly accurate view of the bhlh repertoire of a human being . additional blast searches for human orthologs of the three mouse bhlhs that lacked orthologs ( scleraxis , dermo-1 and s - myc ) were unsuccessful , suggesting that these orthologs either do not exist in humans or are not in the draft sequence . we were recently made aware that there is some incompatibility between the current version of blast and the human genome sequence ( probably due to the large number of ns ( unassigned nucleotides ) in the sequence ) , which makes blast unable to locate some of the best or even exact matches of small query sequences ( j.a.m . this may explain why we missed the four genes cited above , and also why , in a few cases , we were unable to find known cloned human genes in the genome sequence ( see table 2 ) . we also found eight cases in which two human genes group together ( with high statistical support ) to the exclusion of any other genes and are often orthologs of a single mouse gene ( figure 2b and additional data ) . conversely , we found two cases in which two mouse genes are , collectively , orthologs of a single human gene ( figure 2d ) . this may reveal relatively recent duplications specific to the human or mouse lineage . in agreement with this , in all cases amino - acid identity between the two duplicates is high and is not confined to the bhlh . in addition , we found that in two cases ( human sequences q9uh92/n005106 and q02363/n005999 ) , one of the two duplicates lacks introns . this strongly suggests that the duplications have occurred by retrotransposition , a type of event that appears to be rather frequent in humans . in both cases , the copy lacking introns has stop codons in the bhlh , suggesting that it is a pseudogene . among the 39 bhlh from the worm , 6 can not be assigned to any family ( orphan genes ; see tables 1 and 4 ) . five of these have an unusual architecture in they contain two bhlh domains ( see also ) . phylogenetic analysis of these proteins indicates that they result from the duplication of an ancestral gene that already contained two bhlhs ( figure 3 ) . both bhlh domains are loosely related ( on the basis of overall similarity ) to her proteins ( group e ; figure 1 ) , but their inclusion in group e is not supported by phylogenetic reconstruction ( figure 3 ) . in addition , they lack the orange domain , which is characteristic of most her proteins and provides them with functional specificity . they also lack the wrpw motif found in the carboxy - terminal region of almost all her proteins and which allows interaction with the groucho represser protein . moreover , they lack a conserved proline in the basic domain that confers dna - binding specificity on the her proteins . a rooted nj tree is shown that depicts the phylogenetic relationships of the five worm proteins with two bhlh domains . mouse genes representative of some of the animal families have been included in this analysis . numbers above branches indicate per cent support in bootstrap analyses ( 1,000 replicates ) . as in figure 1 the sequences of the first bhlh of each worm proteins are shown in blue , the second in red . both form monophyletic groups with high bootstrap values , indicating that these proteins originate from an ancestral protein that already had two bhlh domains . there is , furthermore , a weaker support ( 40% bootstraps ) for an association of the two bhlh domains into a monophyletic group ( not shown in the figure , as only nodes with 50% or more support are shown ) , suggesting that the ancestral protein may have acquired its two bhlh domains through tandem duplication rather than by association of unrelated bhlh domains . no other protein with two bhlhs has been reported in other metazoans and we were unable to find such proteins in the fly and human genomes . a protein with two bhlh domains is found in rice ( oryza sativa ; protein p0498b01.20 ; accession number bab61947 ) but its sequence is completely unrelated to that of the worm protein . several bhlh proteins do contain other dna - binding and/or dimerization domains in addition to their bhlh , such as the pas domain , leucine zippers or the coe domain . it is conceivable that these domains may cooperate and thereby confer particular functions on the proteins containing them . similarly , the presence of two bhlhs might modify the specificity of the proteins containing them . bhlh genes are found in all major subdivisions of the eukaryotes : metazoans , fungi and plants . it seems , therefore , that the bhlh motif was established in early eukaryote evolution . we have found eight different bhlh genes in the unicellular eukaryote , the yeast s. cerevisiae . most of these genes were already cloned and have been functionally characterized ( reviewed in ) . these genes often regulate biochemical pathways ( such as phosphate utilization , phospholipid and amino - acid biosynthesis , glycolysis ) through the transcriptional activation of more - or - less large sets of genes involved in these pathways . orthologs of these genes are found in other distantly related yeasts such as schizosaccharomyces pombe and kluyveromyces lactis ( our unpublished observations ) , indicating an ancient origin for the different bhlh genes among yeasts . the relatively small number of bhlh genes found in the unicellular yeast contrasts with the large number found in multicellular eukaryotes such as animals and plants . we report here the existence of 39 different bhlh genes in c. elegans , 58 in d. melanogaster , and 125 in humans . preliminary analysis of plant genomes , in particular of arabidopsis thaliana and o. sativa , similarly indicates a large number of bhlh genes ( more than 100 in the completely sequenced genome of a. thaliana , our unpublished observations ) . this important diversification of the bhlh repertoire in animals and plants has occurred independently , as plant and animal bhlh genes are never found in a same family . the current view of eukaryote phylogeny suggests that fungi and animals are more closely related to each other than to the plants . nevertheless , we found that only two families contain both yeast and animal genes ( see table 1 ) , suggesting that the common ancestor of fungi and animals may have possessed even fewer bhlh genes than the present - day yeasts . in the near future , the genome projects currently underway on various ' basal ' eukaryotes ( see ) may give important insights into the very early evolutionary history of the bhlh family . we suggest that the diversification of bhlh genes is directly linked to the acquisition of multicellularity and hence to the recruitment of genes involved in cell functions such as metabolism into the developmental processes required to build multicellularity . indeed , in animals , bhlh genes are generally involved in development and in tissue - specific gene regulation ( reviewed in ) . a similar situation may exist in plants , although very few bhlh genes have been functionally characterized . in addition , in both animals and plants , the diversification of bhlh genes seems to have occurred early in the evolution of these lineages . indeed , our phylogenetic analysis of animal bhlh genes shows that most belong to 44 different orthologous families . of these families , 43 contain representatives from both protostomes and deuterostomes , and must therefore be represented in their common ancestor ( often called urbilateria ) , which lived in pre - cambrian times ( 600 million years ago ) . in addition , the few bhlh genes that have been cloned from cnidarians , which are not bilaterians , are clearly included in families ( see the twist , myod and asc families in additional data ) , suggesting that the establishment of at least some families predates the divergence of bilaterians and non - bilaterians . further analyses of bhlh genes in cnidarians , sponges and slime molds will help to resolve the issue of the early evolution of bhlh genes in animals . our preliminary analyses of plant bhlh genes are consistent with an early diversification in plants , as in animals . indeed , many a. thaliana bhlh genes have clear orthologs in a distantly related plant , o. sativa , whose genome has been partially sequenced ( our unpublished observations ) . arabidopsis is a eudicotyledon and oryza a member of the liliopsida ( a monocotyledon ) , and given the phylogenetic relationships of these clades this suggests that the possession of numerous bhlh genes might be ancestral to angiosperms . further analysis of the evolution of bhlh in plants will require the completion of the genome projects currently underway on rice and tomato ( a eudicotyledon of a different lineage from arabidopsis ) , as well as the isolation of bhlh in a broader spectrum of plant species , in particular in basal angiosperms and non - angiosperms . comparison of the bhlh repertoires found in the protostomes and the deuterostomes gives important insights in to the evolution of the bhlh family in metazoans . the conclusions that can be drawn are completely consistent with those presented in our previous work but the inclusion of the probable complete set of bhlh from a vertebrate strengthens these conclusions . most families ( 43/44 ) contain genes from protostomes ( fly and/or nematode ) and deuterostomes , indicating that these families were already present in the last common ancestor of both protostomes and deuterostomes , that is , of all bilaterians . the fact that most families contain both protostome and deuterostome genes also suggests that there was no addition of new bhlh types in the corresponding lineages , and therefore no important diversification of the ancestral repertoire . this may represent the emergence of new bhlh types in the vertebrate lineage , or alternatively a loss of ancestral types in both fly and nematode . the analysis of bhlh genes from molluscs or annelids might help to settle this question . it is now widely believed that the bilateria ( the triploblastic metazoans ) are composed of three main lineages : deuterostomes ( which include vertebrates and echinoderms ) and protostomes , which themselves include two large groups , the ecdysozoans ( for example , arthropods and nematodes ) and the lophotrochozoans ( for example , annelids , molluscs and flatworms ) ( reviewed in ) . therefore , the finding of ortholog genes in vertebrates and lophotrochozoans but not in fly and nematode would strongly suggest that gene loss(es ) has ( have ) occurred in the ecdysozoan lineage . similarly , the case of families that contain vertebrate and either worm or fly genes is best explained by gene losses that occurred , inside the ecdysozoan clade , in either lineage after the arthropod / nematode divergence . this occurred in the fly lineage for very few families ( 4/44 ) , suggesting the existence of a strong pressure to maintain the entire bhlh repertoire . the much larger number of families ( 13/44 ) that have vertebrate and fly members but no nematode representative suggests that extensive bhlh gene losses have occurred in the worm lineage . strikingly , the worm lacks the important cellular and developmental regulator myc . a similar absence of important developmental regulators , such as hedgehog , toll / il-1 and jak / stat pathway elements has also been reported in the nematode . in addition , a large number of nematode genes ( 6/39 ) can not be clearly assigned to specific families ( orphan genes ) . this is probably due to the high divergence rate reported for nematode genes in general and which we found within our specific data set ( and data not shown ) . interestingly , however , some nematode sequences have diverged very little from their fly or mouse counterparts . these include the few functionally characterized c. elegans bhlh genes that show overall functional conservation with their vertebrate and/or fly orthologs ; for example , the c. elegans orthologs of twist and myod are involved in muscle formation , and the orthologs of atonal and neurod ( lin-32 and cnd-1 ) have a role in nervous - system development . the genetic control of developmental processes such as neurogenesis and myogenesis relies on small sets of interacting genes ( syntagms ) . the function of syntagms crucially relies on specific molecular interactions among their members , hence imposing strong structural constraints on them and preventing structural diversification ( for discussion on syntagms and evolution , see ) . this may explain why such networks are strongly conserved throughout metazoan evolution and why nematode genes involved in such networks have been subject to special constraints . an extensive increase of bhlh family complexity has occurred in vertebrates : the most frequent number of different bhlh genes per family is one in fly ( 30/44 ) and worm ( 27/44 ) , and two in human ( 14/44 ; but 20/44 human families do in fact contain more than two genes ) . most bhlh families ( 32/44 ) , as with other gene families , have more members in vertebrates than in other phyla ( table 1 ) . of these families , 14 ( 32% ) contain four or more vertebrate genes ( table 1 ) and hence may reveal the occurrence of two whole - genome duplications ( the 2r hypothesis ) in early vertebrate evolution . in the most popular version , this is thought to have occurred by one duplication at the root of the vertebrates and a second in the gnathostomata lineage , after its divergence from agnatha ( reviewed in ) . several recent analyses , however , tend to refute ( at least , do not support ) this hypothesis ( reviewed in ) . for example , the current mammalian gene number estimations based on the human draft sequence , ests and comparisons with other vertebrates propose that the human genome would contain no more than 35,000 genes ; that is , about twice the number of fly and worm . consistent with this , many gene families in vertebrates have fewer than four genes . this might , however , result from gene loss during or after the rounds of duplication . in addition , phylogenetic analyses of gene families that comprise four members cast doubt on the 2r hypothesis . as pointed out by hughes , the presence of four members in a vertebrate gene family by itself does not support the genome duplication hypothesis . support may only come from families whose phylogenetic tree shows a topology of the ( ab ) ( cd ) form , that is , two pairs of two closely related paralogs . hughes discussed the phylogenies of 13 protein families important in development , and found that only one of them shows an ( ab ) ( cd ) topology . similar results were recently obtained by martin and hughes et al . on several other families with much more rigorous phylogenetic tests . these results have led to the alternative hypothesis that the abundance of duplicated genes in vertebrates compared to invertebrates may be due to a high rate of local duplications , rather than entire genome duplications ( reviewed in ) . phylogenetic trees of the 14 bhlh families that contain four or more members do not clearly show such ( ab ) ( cd ) topologies ( see additional data ) . we have , however , to note that the phylogenies inside families often have only poor resolution and it is therefore difficult to draw firm conclusions from them . we identified the probable full complement of bhlh in three different metazoans that are representative of the two major subdivisions of the animal kingdom , the protostomes ( c. elegans and d. melanogaster ) and the deuterostomes ( humans ) . most of these families ( 43/44 ) have protostome and deuterostome members , and must therefore have been represented in their common ancestor before the cambrian radiation which saw the emergence of all present - day phyla , and many extinct ones . morphologically , these ancestors ( also called urbilateria ) were probably coelomates with antero - posterior and dorso - ventral polarity , rudimentary appendages , some form of metamerism , a heart , sense organs such as photoreceptors and a complex nervous system . genetically , they possessed numerous homeobox genes ( among which are at least seven hox genes ) , several intercellular signaling pathways ( tgf- , hedgehog , notch , egf ) , at least four pax genes , and 38 c2h2 zinc - finger proteins . the functional conservation that is often observed between protostome and deuterostome orthologs indicates that some of the developmental functions associated with the present - day genes were already established in urbilateria , further indicating the genomic and developmental complexity of these ancient ancestors . the full set of bhlh sequences in the fly , worm , and yeast were obtained mostly by blastp searches against the new releases of the complete genomic sequences of c. elegans , d. melanogaster , and s. cerevisiae . mouse bhlh genes were obtained by blastp searches against the more recent versions of the non - redundant database at ncbi and the sanger protein databases . in addition , we retrieved and analyzed all the bhlhs from these organisms that are listed in the smart database . the comparison with the lists of bhlhs found in the smart database and published by other groups strongly suggests that we retrieved the full set of bhlh genes present in the fly , yeast , and worm genomes , as well as all the cloned mouse bhlh genes to date . tblastn searches were done at the ncbi on the human genome and at the doe joint genomic institute ( university of california and the us department of energy ) for the pufferfish and sea squirt genomes . we used as query two different sequences ( usually one from mouse and one from fly or worm ) of each of the families we defined previously . searches were done at two stringencies , e < 1 and e < 0.01 , with all other parameters set to default . the blast searches detected some sequences that display only low overall similarity with the query , or similarities only to a part of the bhlh domain . we checked these sequences by hand and found that in all cases they did not correspond to bona fide bhlh domains . we hence did not include these sequences in our subsequent analyses . during the course of our work , the data presented in this paper come from the third version ( april 2001 ) . careful examination of the fourth version ( july 2001 ) did not give additional data . a final check has been done on the latest release ( version 6 ) in november 2001 with no significant changes , except that some contigs have been renamed and two sequences were no longer found . we do not include these two sequences ( which were closely related duplications of existing bhlh genes ) as they may represent artifacts of the genome sequence assembly process . we can not exclude the possibility , however , that they are bona fide bhlh genes that were no longer detected as a result of limitations of the current version of blast ( see results and discussion ) . protein alignments were made using clustalw with no adjustment of the default parameters and were subsequently edited and manually improved in genedoc multiple sequence alignment editor and shading utility ( version 2.6.001 ) . the evaluation of percentage conservation of residues in multiple sequence alignments was done using the blosum62 similarity scoring table . only the bhlh motif ( determined as in ) , plus a few flanking amino acids , was used in most of our analyses because the remaining parts of proteins from independent clades are either not homologous or have diverged so much that the alignments are meaningless . the facilities of the belgian embnet node were used for sequence analysis using genedoc software and for most of the protein alignments using clustalw . distance trees were constructed with the neighbor - joining ( nj ) algorithm using paup 4.0 based on a dayhoff pam 250 distance matrix . the resultant trees were bootstrapped ( 1,000 bootstrap replicates ) to provide information about their statistical reliability . bootstraps were made with paup 4.0 , parameters set to default values . given the large number of sequences ( > 300 ) , we were unable , because of computer calculation limitations , to perform maximum - parsimony ( mp ) and maximum - likelihood ( ml ) analyses on the multiple alignment that contains all sequences . we made several additional alignments that include only those bhlh sequences that belong to a particular high - order group ( figure 1 ) . nj , mp and ml trees were constructed from these alignments and were fully congruent with the nj trees constructed from the general alignments . the mp analysis was performed using paup 4.0 with the following settings : heuristic search over 100 bootstrap replicates , maxtrees set up to 1,000 due to computer limitations , other parameters set to default values . maximum likelihood ( ml ) was done using treepuzzle 4.0.2 . the ml was performed using the quartet - puzzling tree - search procedure with 25,000 puzzling steps , using the jones - taylor - thornton ( jtt ) model of substitution , the frequencies of amino acids being estimated from the data set , with an uniform rate of substitution . the trees were displayed with the tree view program ( version 1.5 ) , saved as pict files , converted into jpeg files using graphic converter , and then annotated using adobe photoshop and adobe illustrator . the full set of bhlh sequences in the fly , worm , and yeast were obtained mostly by blastp searches against the new releases of the complete genomic sequences of c. elegans , d. melanogaster , and s. cerevisiae . mouse bhlh genes were obtained by blastp searches against the more recent versions of the non - redundant database at ncbi and the sanger protein databases . in addition , we retrieved and analyzed all the bhlhs from these organisms that are listed in the smart database . the comparison with the lists of bhlhs found in the smart database and published by other groups strongly suggests that we retrieved the full set of bhlh genes present in the fly , yeast , and worm genomes , as well as all the cloned mouse bhlh genes to date . tblastn searches were done at the ncbi on the human genome and at the doe joint genomic institute ( university of california and the us department of energy ) for the pufferfish and sea squirt genomes . we used as query two different sequences ( usually one from mouse and one from fly or worm ) of each of the families we defined previously . searches were done at two stringencies , e < 1 and e < 0.01 , with all other parameters set to default . the blast searches detected some sequences that display only low overall similarity with the query , or similarities only to a part of the bhlh domain . we checked these sequences by hand and found that in all cases they did not correspond to bona fide bhlh domains . we hence did not include these sequences in our subsequent analyses . during the course of our work , the data presented in this paper come from the third version ( april 2001 ) . careful examination of the fourth version ( july 2001 ) did not give additional data . a final check has been done on the latest release ( version 6 ) in november 2001 with no significant changes , except that some contigs have been renamed and two sequences were no longer found . we do not include these two sequences ( which were closely related duplications of existing bhlh genes ) as they may represent artifacts of the genome sequence assembly process . we can not exclude the possibility , however , that they are bona fide bhlh genes that were no longer detected as a result of limitations of the current version of blast ( see results and discussion ) . protein alignments were made using clustalw with no adjustment of the default parameters and were subsequently edited and manually improved in genedoc multiple sequence alignment editor and shading utility ( version 2.6.001 ) . the evaluation of percentage conservation of residues in multiple sequence alignments was done using the blosum62 similarity scoring table . only the bhlh motif ( determined as in ) , plus a few flanking amino acids , was used in most of our analyses because the remaining parts of proteins from independent clades are either not homologous or have diverged so much that the alignments are meaningless . the facilities of the belgian embnet node were used for sequence analysis using genedoc software and for most of the protein alignments using clustalw . distance trees were constructed with the neighbor - joining ( nj ) algorithm using paup 4.0 based on a dayhoff pam 250 distance matrix . the resultant trees were bootstrapped ( 1,000 bootstrap replicates ) to provide information about their statistical reliability . bootstraps were made with paup 4.0 , parameters set to default values . given the large number of sequences ( > 300 ) , we were unable , because of computer calculation limitations , to perform maximum - parsimony ( mp ) and maximum - likelihood ( ml ) analyses on the multiple alignment that contains all sequences . we made several additional alignments that include only those bhlh sequences that belong to a particular high - order group ( figure 1 ) . nj , mp and ml trees were constructed from these alignments and were fully congruent with the nj trees constructed from the general alignments . the mp analysis was performed using paup 4.0 with the following settings : heuristic search over 100 bootstrap replicates , maxtrees set up to 1,000 due to computer limitations , other parameters set to default values . maximum likelihood ( ml ) was done using treepuzzle 4.0.2 . the ml was performed using the quartet - puzzling tree - search procedure with 25,000 puzzling steps , using the jones - taylor - thornton ( jtt ) model of substitution , the frequencies of amino acids being estimated from the data set , with an uniform rate of substitution . the trees were displayed with the tree view program ( version 1.5 ) , saved as pict files , converted into jpeg files using graphic converter , and then annotated using adobe photoshop and adobe illustrator . additional data files are available with the online version of this paper as follows : multiple alignments ( in rich text format ) on which the trees displayed in the figures are based . a list of all bhlh sequences from human in rich text format multiple alignments for each family of bhlh proteins : the achaete - scute a family , the achaete - scute b family , the ahr family , the ap4 family , the arnt family , the atonal family , the beta3 and oligo families , the bmal family , the clock family , the e12/e47 family , the extramacrochaete family , the enhancer of split family , the fig alpha family , the hairy family , the hand family , the hey family , the mad and mnt families , the max family , the mesp family , the mist family , the mitf family , the myc family , the myod family , the myor family , the net family , the neurod family , the neurogenin family , the nscl family , the paraxis family , the ptf a and b families , the scl family , the src family , the srebp family , the tf4 and mlx families , the trh , hif , and sim families , the twist family , and the usf family . representative phylogenetic trees ( nj trees bootstrapped 1,000 times to provide statistical support to the nodes , usually rooted with a sequence from a closely related family . in a few cases , closely related families are shown in the same phylogenetic tree ) of each family of bhlh proteins : the achaete - scute a family , the achaete - scute b family , the ahr family , the arnt family , the atonal family , the beta3 , mist , and oligo families , the bmal family , the clock family , the e12/e47 family , the extramacrochaete family , the enhancer of split family , the fig alpha family , the hairy family , the hand family , the hey family , the mad and mnt families , the max family , the mesp family , the mitf family , the myc family , the myod family , the myor family , the net family , the neurod family , the neurogenin family , the nscl family , the paraxis family , the ptf a and b families , the scl family , the src family , the srebp family , the tf4 and mlx families , the trh , hif , and sim families , the twist family , and the usf family . species name abbreviations are as in the figure legends and as below : av , asteris vulgaris ; avim , avian myelocytomatosis virus cmii ; bb , branchiostoma belcheri ; bf , branchiostoma floridae ; bm , bombyx mori ( domestic silkworm ) ; caebr , caenorhabditis briggsae ; cc , ceratitis capitata ; cp , cynops pyrrhogaster ; cs , cupiennius salei ; cyca , cyprinus carpio ; ds , drosophila simulans ; dv , drosophila virilis ; dy , drosophila yakuba ; hr , halocynthia roretzi ; hv , hydra vulgaris ( hydra attenuata ) ; ilo , ilyanassa obsoleta ; jc , juonia coenia ( precis coenia ) ( peacock butterfly ) ; kl , kluyveromyces lactis ; lv , lytechinus variegatus ( green urchin ) ; nv , notophtalmus viridens ; ol , oryzias latipes ( japanese medaka ) ; om , oncorhyncus mikis ; pc , podocorine carnea ; pv , patella vulgata ( common limpet ) ; rn , rattus norvegicus ; sb , spermophilus beecheyi ; sc , saccharomyces cerevisiae ( baker 's yeast ) ; sp , schizosaccharomyces pombe ; spu , strongylocentrotus purpuratus ( purple urchin ) ; st , silurana tropicalis ; tc , tribolium castaneum ; tricho , trichinella spiralis multiple alignment on which figure 1 is based click here for additional data file a list of all bhlh sequences from human click here for additional data file multiple alignment of the bhlh of achaete - scute a family proteins click here for additional data file multiple alignment of the bhlh of achaete - scute b family proteins click here for additional data file multiple alignment of the bhlh of ahr family proteins click here for additional data file multiple alignment of the bhlh of ap4 family proteins click here for additional data file multiple alignment of the bhlh of arnt family proteins click here for additional data file multiple alignment of the bhlh of atonal family proteins click here for additional data file multiple alignment of the bhlh of beta3 and oligo families proteins click here for additional data file multiple alignment of the bhlh of bmal family proteins click here for additional data file multiple alignment of the bhlh of clock family proteins click here for additional data file multiple alignment of the bhlh of e12/e47 family proteins click here for additional data file multiple alignment of the bhlh of extramacrochaete family proteins click here for additional data file multiple alignment of the bhlh of enhancer of split family proteins click here for additional data file multiple alignment of the bhlh of fig alpha family proteins click here for additional data file multiple alignment of the bhlh of hairy family proteins click here for additional data file multiple alignment of the bhlh of hand family proteins click here for additional data file multiple alignment of the bhlh of hey family proteins click here for additional data file multiple alignment of the bhlh of mad and mnt families proteins click here for additional data file multiple alignment of the bhlh of max family proteins click here for additional data file multiple alignment of the bhlh of masp family proteins click here for additional data file multiple alignment of the bhlh of mist family proteins click here for additional data file multiple alignment of the bhlh of mitf family proteins click here for additional data file multiple alignment of the bhlh of myc family proteins click here for additional data file multiple alignment of the bhlh of myod family proteins click here for additional data file multiple alignment of the bhlh of myor family proteins click here for additional data file multiple alignment of the bhlh of net family proteins click here for additional data file multiple alignment of the bhlh of neurod family proteins click here for additional data file multiple alignment of the bhlh of neurogenin family proteins click here for additional data file multiple alignment of the bhlh of nscl family proteins click here for additional data file multiple alignment of the bhlh of paraxis family proteins click here for additional data file multiple alignment of the bhlh of ptf a and b families proteins click here for additional data file multiple alignment of the bhlh of scl family proteins click here for additional data file multiple alignment of the bhlh of src family proteins click here for additional data file multiple alignment of the bhlh of srebp family proteins click here for additional data file multiple alignment of the bhlh of tf4 and mlx families proteins click here for additional data file multiple alignment of the bhlh of trh , hif , and sim families proteins click here for additional data file multiple alignment of the bhlh of twist family proteins click here for additional data file multiple alignment of the bhlh of usf family proteins click here for additional data file representative phylogenetic tree of the achaete - scute a family of bhlh proteins click here for additional data file representative phylogenetic tree of the achaete - scute b family of bhlh proteins click here for additional data file representative phylogenetic tree of the ahr family of bhlh proteins click here for additional data file representative phylogenetic tree of the arnt family of bhlh proteins click here for additional data file representative phylogenetic tree of the atonal family of bhlh proteins click here for additional data file representative phylogenetic tree of the beta3 , mist , and oligo families of bhlh proteins click here for additional data file representative phylogenetic tree of the bmal family of bhlh proteins click here for additional data file representative phylogenetic tree of the clock family of bhlh proteins click here for additional data file representative phylogenetic tree of the e12/e47 family of bhlh proteins click here for additional data file representative phylogenetic tree of the extramacrochaete family of bhlh proteins click here for additional data file representative phylogenetic tree of the enhancer of split family of bhlh proteins click here for additional data file representative phylogenetic tree of the fig alpha family of bhlh proteins click here for additional data file representative phylogenetic tree of the hairy family of bhlh proteins click here for additional data file representative phylogenetic tree of the hand family of bhlh proteins click here for additional data file representative phylogenetic tree of the hey family of bhlh proteins click here for additional data file representative phylogenetic tree of the mad and mnt families of bhlh proteins click here for additional data file representative phylogenetic tree of the max family of bhlh proteins click here for additional data file representative phylogenetic tree of the mesp family of bhlh proteins click here for additional data file representative phylogenetic tree of the mitf family of bhlh proteins click here for additional data file representative phylogenetic tree of the myc family of bhlh proteins click here for additional data file representative phylogenetic tree of the myod family of bhlh proteins click here for additional data file representative phylogenetic tree of the myor family of bhlh proteins click here for additional data file representative phylogenetic tree of the net family of bhlh proteins click here for additional data file representative phylogenetic tree of the neurod family of bhlh proteins click here for additional data file representative phylogenetic tree of the neurogenin family of bhlh proteins click here for additional data file representative phylogenetic tree of the nscl family of bhlh proteins click here for additional data file representative phylogenetic tree of the paraxis family of bhlh proteins click here for additional data file representative phylogenetic tree of the ptf a and b families of bhlh proteins click here for additional data file representative phylogenetic tree of the scl family of bhlh proteins click here for additional data file representative phylogenetic tree of the src family of bhlh proteins click here for additional data file representative phylogenetic tree of the srebp family of bhlh proteins click here for additional data file representative phylogenetic tree of the tf4 and mlx families of bhlh proteins click here for additional data file representative phylogenetic tree of the trh , hif , and sim families of bhlh proteins click here for additional data file representative phylogenetic tree of the twist family of bhlh proteins click here for additional data file representative phylogenetic tree of the usf family of bhlh proteins click here for additional data file we are grateful to lionel christiaen who made us aware of the takifugu and ciona genome projects , and to marc colet and robert herzog for comments on the manuscript . this work has been supported by the federal office for scientific , technical and cultural affairs ( v.l . ) and the centre national de la recherche scientifique , the institut franais de la biodiversit , and the universit paris - sud ( m.v . ) .

backgroundthe basic helix - loop - helix ( bhlh ) proteins are a large and complex multigene family of transcription factors with important roles in animal development , including that of fruitflies , nematodes and vertebrates . the identification of orthologous relationships among the bhlh genes from these widely divergent taxa allows reconstruction of the putative complement of bhlh genes present in the genome of their last common ancestor.resultswe identified 39 different bhlh genes in the worm caenorhabditis elegans , 58 in the fly drosophila melanogaster and 125 in human ( homo sapiens ) . we defined 44 orthologous families that include most of these bhlh genes . of these , 43 include both human and fly and/or worm genes , indicating that genes from these families were already present in the last common ancestor of worm , fly and human . only two families contain both yeast and animal genes , and no family contains both plant and animal bhlh genes . we suggest that the diversification of bhlh genes is directly linked to the acquisition of multicellularity , and that important diversification of the bhlh repertoire occurred independently in animals and plants.conclusionsas the last common ancestor of worm , fly and human is also that of all bilaterian animals , our analysis indicates that this ancient ancestor must have possessed at least 43 different types of bhlh , highlighting its genomic complexity .

microleakage between the root canal filling and root - canal walls may adversely affects the results of root - canal treatment.1 apical leakage is considered to be common cause of endodontic failure.2 hence , different endodontic filling materials , sealers and techniques have been introduced to the dental community in an attempt to improve apical seal.1 various materials have been used in root canal treatment in an attempt to achieve success . but , a combination of gutta - percha and a sealer are used most commonly . gutta - percha is considered an impermeable core material ; therefore , leakage through an obturated root canal is expected to take place at the interfaces between the sealer and dentin or the sealer and gutta - percha , or through voids within the sealer.3 apical sealing is desirable to prevent passage of bacteria and their endodotoxin apically . in vitro evaluation of apical dye penetration is used to estimate the sealing ability which is corresponding to in vivo amount of micro leakage with particular sealer.2 many techniques were used to evaluate the leakage of sealers such as ; colored dye penetration radio labeled tracer penetration dissolution of hard tissue clearing of teeth , spectrometry of radioisotopes electrochemical and gas chromatography . however , many studies showed no significant difference between these techniques.1 the aim of this in vitro study was to quantitatively evaluate the sealing properties of three different root - canal sealers ; tubliseal , sealapex and ah26 using a spectrophotometric method . thirty - six extracted sound mandibular molar natural teeth specimens with complete root and free from caries or cracks were collected , stored , disinfected and handled as per the recommendations and guidelines laid down by occupational safety and health administration and centers for disease control and prevention.4 the teeth were then placed in 0.9% physiologic saline solution for ten days prior to access cavity preparation . the samples were divided into three experimental groups ; group i : sealapex , group ii : tubliseal and group iii : ah26 with 12 samples in each group . the samples were then coated with nail varnish all over the root surface except 2 mm around the apical foramen . 2 ml of freshly prepared 2% methylene blue dye was taken in each vial and the apical third of the root was suspended in the dye for 72 h. samples were washed with distilled water , nail varnish removed and then placed in 20 ml of 35% nitric acid for 72 h. standard solutions of 1% , 0.5% , 0.2% , 0.05% , 0.02% and 0.01% of methylene blue in 35% nitric acid were prepared and stored for 72 h. the standard solutions and the nitric acid solutions were filtered and centrifuged for 1 min after 72 h. the supernatant was subjected to spectrophotometric analysis using a filter of 670 nm . the amount of leakage was extrapolated from a standard linear regression curve constructed from stock standard methylene blue dye solutions . obtained data were statistical analyzed with kruskal wallis and mann whitney u - tests using spss software version 20 ( ibm ) . the results of the quantitative evaluation of the sealing properties of the three root - canal sealers are shown in table 1 . , tubliseal showed a significant difference ( p > 0.005 ) . in comparison to sealapex comparisons made between tubliseal and ah26 showed no significant difference ( table 2 , chart 1 ) . mean and standard deviation values of volumetric dye penetration in groups with reference to transmission . three - dimensional obturation of the root canal system with a fluid impervious seal is an important factor for successful endodontic therapy . the root canal filling should seal the canal both apically and coronally to prevent the passage of microorganism to apex or vice versa.5 most reliable method is the use of gutta - percha cones with sealer cement . sealers based on zinc oxide - eugenol ( tubliseal ) , calcium hydroxide ( sealapex ) , epoxy resins ( ah26 ) were included in the present study . in present quantitative dye leakage study , tubliseal demonstrated least dye leakage in comparison with other experimental groups ( table 1 ) . our results were in contrary to study by masoud and saleh where they found more microleakage in tubliseal group than other groups.6 sealapex is a calcium hydroxide type sealer . calcium hydroxide used as root canal sealer since it stimulates periapical tissues in order to maintain health or promote healing and secondly for its antimicrobial effects.7 it has been observed in some studies that , calcium hydroxide sealers showed a significant volumetric expansion during setting because of water absorption , which increases its solubility . the present in vitro investigation indicated maximum leakage value with sealapex among the experimental groups . in a contradictory to our results , cobankara et al . ( 2006 ) observed sealapex with better apical sealing than the other sealers ( ah plus and rc sealer ) at 7 , 14 , and 21 days.3 ah26 is an epoxy resin based sealer that provides easy handling characteristics , good flow , good sealing to dentin and prominent antimicrobial activity.6 kumar et al . observed more micro leakage with zinc oxide - based sealer and least with resin based sealers , this is contradictory to our study.8 in our study , there was no statistical significant difference between tubliseal and ah26 in micro leakage . observed least amount of dye penetration for ah plus and endorez group.9 even though , the current study did not indicate any statistically significant difference between ah26 and sealapex , it is time to question the overall efficacy of calcium hydroxide sealers on the grounds that the reparative and the calcification capabilities attributed to calcium hydroxide are generally desirable before completing the obturation . ah26 , resin based sealer provided a better apical seal when compared with sealapex even though the results were statistically not significant . it mixes easily , flows well , and has ample working time , good radio - opacity , comparable solubility , good adhesion and good biocompatibility.3 in the present study , there was no significant difference between group ii and iii and group i and iii . did nt find any significant difference between the tested three groups ; sealer 26 , enfoflas and resin group . but they observed higher microleakage in sealer 26 group compared with control.11 joseph and sing evaluated the apical sealing with four root canal sealers ; ah26 , sealapex , endoflas fs and ah plus and observed no significant differences between all groups except between ah plus and endoflas.5 nagas et al . observed significantly lower overall leakage with ah plus group , whereas no difference was found between master cone points.12 kopper et al . observed significant dye penetration for ah plus , endofill and sealer 26.13 cobankara et al . observed better sealing values for roekoseal after 21 days when compared to ketac - endo and ah plus , and there was no statistically significant difference.1 dultra et al . found no statistical difference between groups for apical leakage ( endofill , ah plus , endorez and epiphany).14 in addition , before accepting a new material for routine clinical use further experiments should also be performed to evaluate the other aspects of the materials physical and biological properties such as biocompatibility , solubility , disintegration , radio - opacity and dimensional stability . however , these in vitro studies do provide comparative information of the relative performance of sealers tested under the same conditions in each particular study and clinicians can use this information to possibly choose a better sealer . in the present study , tubliseal sealer showed least microleage compared with sealapex and ah26 sealer . it is important to remember before declaring any root canal sealer as most acceptable that the results of the dye penetration studies indicate only the relative sealing ability of root canal fillings in vitro and they do not indicate their ability to prevent the penetration of bacteria into filled root canals in vivo .

background : the aim of this study was to quantitatively analyze the amount of dye leakage with ah26 , sealapex and tubliseal sealers in endodonticaly treated teeth.materials and methods : a total of 36 extracted mandibular molar specimens were divided into three groups ; group i : sealapex , group ii : tubliseal , group ii : ah26 with 12 samples in each group . standard access cavity and biomechanical preparation was done with step back flare technique . obturations were done using respective sealers in the three different groups . then , samples were subjected to spectro photometric analysis using a filter of 670 nm . spectrophotometric analysis was performed to quantitatively analyze the amount of dye leakage with all three sealers.results:tubliseal exhibited the least microleakage . in comparison to sealapex , tubliseal showed a significant difference . compared to sealapex , ah26 showed no significant difference . comparisons made between tubliseal and ah26 showed no significant difference.conclusion:in the present study , tubliseal sealer showed least microleage compared with sealapex and ah26 sealer .

hepatitis c virus ( hcv ) is a major public health problem all over the world ; it is currently estimated that about 85% of those infected with hepatitis c virus will become a chronic carrier and may develop severe end stage liver diseases including cirrhosis and hepatocellular carcinoma . currently , the most important risk factor for infecting by hepatitis c is intravenous drug use ( idu ) which is the most associated with the sharing of drug injection equipments especially needles , syringes and other paraphernalia . infection with hcv among prison inmates is usually higher than that among the general population mainly because of past history of intravenous drug use and possibly high risk addiction - related behaviors in prison . during the last few years , much attention has been given to the prevalence of blood born diseases including hepatitis c among prisoners . according to limited research studies in iran , prevalence of hcv infection among prisoners with a history of drug injecting varies between 31.5% to 47% in different parts of the country.[68 ] however , a study in a local prison in fars province revealed 78% prevalence rates of hcv infection among incarcerated drug users . to address the often hidden phenomenon of hcv infection in prisons , considering the limited data on the epidemiology of hcv infection and related risk behaviors in our region , in this study we have focused on this infection and transmission risk factors among prisoners with history of idu in isfahan that could potentially be incorporated into current and future harm reduction initiatives us in iran . in a cross - sectional study , according to self - report and confirm by head of prison 's healthcare , the prison inmates who had intravenous drug history or current idus entered into study at march 2009 . since the most of our enrollees had sclerosis in peripheral vessels , blood sampling was mostly taken from the femoral vein by the health personnel of the prison setting . blood samples were then sent to the laboratory of infectious diseases research center during 3 hours in cold box to be tested by eilsa ( diapro - italy ) for the presence of hcv antibody . incarcerated with idu histories were also asked about their demographic characteristics and hcv - related risk behaviors with an interviewer assisted questionnaire . face and content validity of the questionnaire were evaluated by specialists and its reliability was confirmed by chronbach alpha = 0.74 . the research protocol was approved by the ethical committee of isfahan university of medical sciences in iran . all available means were used to guarantee privacy during interviews and confidentiality . instead of a block for the patient 's name , each questionnaire and test tube had an anonymous identification code , which was used for reporting laboratory results , too . statistical analysis was performed using spss for windows ( version 16.0 , 2007 , spss inc , chicago , il , usa ) . univariate analysis was used to assess association between being hcv positive and related risk factors . the variables that were significant in the univariate analysis ( p - value < 0.05 ) were included in the multiple logistic regression to estimate adjusted odds ratio ( aor ) and 95% confidence intervals ( ci ) . a total of 943 incarcerated idus ( 938 male and 5 female ) participated in the study . the median age of participants was 32.6 years ( range : 18 - 67 ) . the majority of participants ( 98.6% ) were iranian and 523 ( 55.5% ) had ever been married . among the married persons 451 ( 92% ) had been married once , 35 ( 7% ) had been married twice and 5 ( 1% ) had been married more . 143 ( 15.1% ) cases mentioned a history of traveling to another countries . the socio - demographic characteristics of participants overall , the prevalence of hcv - ab seropositivity was 41.6% [ table 1 ] . the median frequency of iv injections was 30 times per month ( range 1500 ) , the median duration of addiction was 12 years ( range 0.5 - 57 ) , the median frequency of incarceration was 3 times ( range 1 - 37 ) , and the median of total duration of incarceration was 3.66 years ( range 0.08 - 35 ) . frequency of hcv transmission related factors within study population 71.76% of the participants had illegal sex ( contact with other than spouse ) . among whom , 43% of men had history of having sex with another man ( msm ) , 64.2% had an intercourse with commercial sex workers and 30.9% of them had idu sexual partner . according to self reports , only 31% of them had ever used a condom during sex . within all studied samples with idu history , 13.3% had a single sexual partner and the others had 2 or more , in their lifetime . table 3 shows the odds ratio for some risk characteristics of idus using the logistic regression model . the results of this study indicated that overall prevalence of hcv ab seropositivity among idu inmates in isfahan province is 41.6% . in similar studies in iran , similar results were observed . in zakizad study , the seroprevalence of hepatitis c infection in sari addict prisoners has been reported as 30.8% . in another study by khani et al . the prevalence of hcv was 47.7% among drug addict prisoners in zanjan . in a study which was done in 3 provinces in iran ( isfahan , chahamahal bakhtiary and lorestan ) in 2003 , 34.7% of male prisoners who had been imprisoned for various drug - related offenses including purchase or sale and consumption , were hcv ab positive . in mashhad , the seroprevalence of hcv in incarcerated idus had been reported as 59.4% . in mohammad alizadeh study , the hcv antibody positivity among drug abusers in the central prison of hamedan was 30% . in a study on drug abusers admitted to prison in guilan province , of 460 inmates , 45.4% were hcv antibody positive which in intravenous drug abusers the prevalence reached to 88.9% . the prominent aspect of our study in comparison with other similar studies in iran , is the high number of participants whom all of them had history of intravenous addiction . as a matter of fact , this is one of the first large prevalence studies of blood- born infections among incarcerated idus in iran . in this study , the history of shared drug injection inside prison was one of the main incarceration - related risk factors . this association , has also been reported in other studies in the world.[1315 ] it seems that , the lack of access new needle / syringe in prisons and consequently needle sharing practice is the main reason of the hepatitis c epidemic in prisoners . our data , highlight for comprehensive and integrated interventions for incarcerated idus to prevent hcv transmission among idu population and community . effectiveness of needle programs in reducing needle sharing among idus has been shown in many countries . in our country , an outreach program for blood born infections prevention was supported by the united nation and the ministry of health of iran in 2003 . multiple incarcerations was the another independent risk factor in our subjects which show that the high rates of arrests and incarcerations lead to drug - related offences or other confounding factors inside prison , such as violence . our findings suggest that preventive interventions are compelling now for idus to ensure their safe passage throughout incarceration . in our study , other important modes of transmission were frequency and length of drug injection . according to these results , harm reduction strategies need to be expanded to prevent new hcv infections , particularly among young injectors.[1920 ] in our study , history of marriage was a protective factor for hcv - infection in the subjects after controlling for all other variables in the logistic - regression model . sexual transmission could play a role in sporadic or community - acquired hcv infections . however , there is contradictory finding regarding the association between marriage and hcv infection in the world . vandelli and colleagues indicated that the risk of sexual transmission of hcv within heterosexual monogamous couples is extremely low or even null . it is shown that , those who have multiple sexual partners , including female sex workers , men having sex with men , and attendees of sexually - transmitted diseases ( stds ) clinics are the main risk groups for hepatitis c. in the acute hepatitis surveillance study in usa , 18% of newly infected individuals reported sexual contact with an hcv - infected person or multiple sexual partners as their only risk factor for hcv acquisition . for the individual with chronic hcv infection , the estimated risk of sexual transmission of virus was 0% to 0.6% per year for those in monogamous relationships , and 1% per year for those with multiple sexual partners . so , it will be useful to encourage young people to get married to prevent sexually transmitted diseases in our society . in conclusion , according to progressive reported prevalence rates of hcv in the world and the importance of the current and potential burden of hcv - related complications , it is important to primary prevention of hcv infection that will be addressed through implementation of safe injection practices . the mortality associated with hepatitis c is expected to double in the next 10 - 20 years . it is conservatively estimated that the direct costs related to hepatitis c will be $ 10.3 billion during the years 2010 - 2020 . the indirect costs of hepatitis c ( eg , the loss of productivity during that era ) are estimated to total another $ 54.3 billion due to premature death and $ 20.6 billion due to disability . in addition , the total cost of therapy for hepatitis c is estimated to be $ 10,000-$12,000 . so , primary prevention will be more cost - effective than secondary prevention of morbidity and mortality from hcv infection through provision of interferon - based therapy . it is good opportunities in prison to have large number of idus over longer period to do specific preventive activities such as needle / syringe exchange and counsel imprisoned idus .

objectives : hepatitis c virus ( hcv ) infection is a major public health problem worldwide with serious complications . according to the importance of intravenous drug use ( idu ) as the main risk factor for hcv infection and transmission and prison as the main source of risky behaviors , this study conducted to define hcv infection and related risk factors in prison inmates with history of idu in isfahan province , iran.methods:this is a cross sectional study which the prison inmates with idu history in voluntary basis were enrolled . a validated questionnaire was asked and blood sample was obtained from each subject for the presence of hcv antibody . odds ratio and logistic regression were used for data analysis and p - value < 0.05 considered significant.results:i943 inmates with history of idu participated in the study . the overall prevalence of hcv antibody was 41.6% . the main independent risk factors were number of injection in the month [ or : 1.006 ( 1.002- 1.011 ) ] , length of drug addiction [ or : 1.05 ( 1.004 - 1.098 ) ] , multiple incarceration [ or : 1.15 ( 1.05 - 1.23 ) ] and use of needle / syringe share inside prison [ or : 4.19 ( 2.22 - 7.9 ) ] . in our study , marriage was a protective factor for hcv infection [ or : 0.34 ( 0.18 - 0.64 ) ] as well.conclusions:according to relatively high prevalence of hcv infection and associated risk factors which observed in this study it is important to primary prevention in prisons through syringe / needle exchange and counsel with imprisoned idus .

lipomas represent uncommon neoplasms of the oral cavity ; only 1% to 5% of cases occur at this site . based on their histopathological features of conspicuous multiple components , lipomas can be divided into some subclasses [ 2 , 3 ] , and the most common is the fibrolipoma [ 1 , 3 , 4 ] . cases reporting on lipoma with osteo / chondroid differentiaion were only 16 cases as a result of retrieving literature from 1960 to 2008 . further , other names of this subclassification exist in literature ; osteo / chondrolipoma , ossifying / osseous lipoma , lipoma with chondro / osseous metaplasia , and lipoma with cartilaginous / osseous change . the obscure etiology of osseous / chondroid differentiaion led to confusion of the name . to clarify the etiology of osseous / chondroid differentiaion in lipoma , the authors report additional 2 cases of oral lipoma / fibrolipoma with osseous and/or chondroid differentiation and describe the clinical , histopathological , and immunohistological features of these . review of the literature and clinicopathological analysis from the files of the oral pathology department , nihon university school of dentistry at matsudo from 1995 to 2007 of these cases were performed and compared . two cases that had been diagnosed as lipoma with osteo / chondroid differentiation were retrieved from the files mentioned earlier . in these cases , sections of 2 cases were deparaffinized in xylene and dehydrated in tris - buffered saline ( ph 7.6 ) . primary antibodies against e29 ( epithelial membrane antigen ( ema ) ) , qbend 10 ( cd34 class ii ) , ki - s5 ( ki-67 antigen ) , 5.8a ( myo d1 ) were purchased from a commercial source ( dako , denmark ) , and sc-12488 ( runx-2 ) , sc-20095 ( sox-9 ) , sc-71992 ( s-100 chain ) , sc-71993 ( s-100 chain ) , sc-55520 ( fgf-1 ) , and sc-57494 ( vegf ) were purchased from a commercial source ( santa cruz biotechnology , inc , usa ) . for detection of the antigen , the dextran polymer method ( chem mate envision kit , dako , denmark ) was used . to improve detection , the deparaffinized sections were pretreated by microwave heating with citrate buffer ( ph 6 ) . the primary antibodies were generally used at a dilution of 1 : 50 , and the incubation time was 1 hour at room temperature . positive controls consisted of specimens of schwannoma for s-100 and ; squamous cell carcinoma for ema , cd34 and ki-67 ; ossifying fibroma for fgf-1 and runx-2 , normal lung tissue for sox-9 ; and inflammatory granulation tissue for myod-1 . as a negative control , mouse igg1 ( ki-67 , vegf , cd34 , s-100 ) , igg2a ( ema , s-100 ) and igg2b ( fgf-1 ) , goat igg ( runx-2 ) and rabbit igg ( sox-9 ) were used instead of the primary antibodies . at least 500 nuclei were counted in 5 high - power ( x400 ) fields and ki-67 labeling index was calculated . mean intratumor microvessel density was obtained by calculating the average counts of these 5 fields . the protocol was approved by the committee on studies involving human beings of nihon university school of dentistry at matsudo ( ec 05 - 002 ) . clinical data were retrieved from patient records , and all cases were reviewed microscopically and subclassified . the literature from 1960 to 2008 of lipoma with osseous / chondroid differentiation was reviewed . case 1a 28-year - old woman presented with a painless relatively hard mass on the dorsal surface in the midline of the tongue that had recently grew slightly . the patient 's mother first noticed a tiny nodule when the patient was 6 months old . examination showed a well - defined hard nodular mass , approximately 16 16 9 mm in size , which was sharply demarcated within the muscles of the tongue and freely mobile . a yellowish tinge was visible through the overlying mucous membrane and the lesion was firm on palpation . clinical diagnosis of lipoma with calculus was made and the tumor was completely excised from the tongue under general anesthesia . gross examination showed a yellowish soft to hard smooth mass measuring 16 15 12 mm in size . microscopically , the tumor consisted of a well - circumscribed mass of fatty tissue with a cellular chondroid component . chondromatous nodules within uniform adipose tissue were seen in large areas of mature fat cells supported by fibrous connective tissue ( figure 1 ) . pathological diagnosis was made as lipoma with chondroid differentiation . a 28-year - old woman presented with a painless relatively hard mass on the dorsal surface in the midline of the tongue that had recently grew slightly . the patient 's mother first noticed a tiny nodule when the patient was 6 months old . examination showed a well - defined hard nodular mass , approximately 16 16 9 mm in size , which was sharply demarcated within the muscles of the tongue and freely mobile . a yellowish tinge was visible through the overlying mucous membrane and the lesion was firm on palpation . clinical diagnosis of lipoma with calculus was made and the tumor was completely excised from the tongue under general anesthesia . gross examination showed a yellowish soft to hard smooth mass measuring 16 15 12 mm in size . microscopically , the tumor consisted of a well - circumscribed mass of fatty tissue with a cellular chondroid component . chondromatous nodules within uniform adipose tissue were seen in large areas of mature fat cells supported by fibrous connective tissue ( figure 1 ) . case 2a 59-year - old man presented with a relatively painless hard mass on the left side of the lower labial vestibule surface , which appeared the sense of incompatibility 2 months prior to consultation . examination showed a well- defined hard nodular mass , approximately 5 mm in diameter , and freely mobile , and covered with mucosa of normal aspect and color . there was no tenderness , no sign of inflammation . to retrieve more - detailed relativity with the surrounding tissue , ct was taken and showed a lesion in the left side of the lower lip that appeared to be a small mass with areas of little calcification within . gross examination showed a yellowish soft to hard smooth mass measuring 9 5 5 mm in size . microscopically , the tumor consisted of a well - circumscribed mass of mature fat cells supported by fibrous connective tissue septa and myxoid tissue characterized by spindle cells . a focal island of consecutive chondroid and woven bone component was surrounded by spindle / fusiform - shaped mesenchymal cells throughout the lesion ( figure 2 ) . pathological diagnosis was made as fibrolipoma with osseous / chondroid differentiation . a 59-year - old man presented with a relatively painless hard mass on the left side of the lower labial vestibule surface , which appeared the sense of incompatibility 2 months prior to consultation . examination showed a well- defined hard nodular mass , approximately 5 mm in diameter , and freely mobile , and covered with mucosa of normal aspect and color . there was no tenderness , no sign of inflammation . to retrieve more - detailed relativity with the surrounding tissue , ct was taken and showed a lesion in the left side of the lower lip that appeared to be a small mass with areas of little calcification within . gross examination showed a yellowish soft to hard smooth mass measuring 9 5 5 mm in size . microscopically , the tumor consisted of a well - circumscribed mass of mature fat cells supported by fibrous connective tissue septa and myxoid tissue characterized by spindle cells . a focal island of consecutive chondroid and woven bone component was surrounded by spindle / fusiform - shaped mesenchymal cells throughout the lesion ( figure 2 ) . the patients made an uneventful recovery , and after 15 years ( case 1 ) and 1-year ( case 2 ) follow - up , there was no sign of recurrence . all chondrocytes were stained for s-100 & , some for runx-2 ( figure 3(a ) ) and some of the outer layer for sox-9 . spindle cells were divided into 2 groups ; peripheral spindle cells around osteo / chondroid tissue and distant spindle cells inside the myxoid area . peripheral spindle cells around chondroid tissue stained diffusely for s-100 & and sox-9 ( figure 3(b ) ) , though peripheral spindle cells around osteoid tissue only stained for runx-2 ( figure 3(c ) ) . distant spindle cells inside the myxoid area stained diffusely for s-100 and focally for s-100 , myod , sox-9 , and cd34 . as for the number of vessels detected by cd34 , there were 5.0 vessels in this tumor , and there was no differences among the inside areas . none of the cases stained for either ema or fgf-1 or with vegf . between 1995 and 2007 , the files for all cases of oral lipoma at this department showed 909 cases of nonepithelial tumors in the oral soft tissue . of all the lipoma cases , 27 occurred in males and 19 in females ; their mean age was 53.8 years ( range : 2872 years ) . most lesions were located in the buccal mucosa ( n = 16 ) , margin of tongue ( n = 10 ) , lower lip ( n = 7 ) , gingiva ( n = 4 ) , and others . microscopically , 28 cases ( 61.0% ) were classified as lipoma and 18 ( 39.0% ) as fibrolipoma . the literature from 1960 to 2008 of lipoma with osseous / chondroid differentiation was reviewed . fourteen reports ( including 16 cases ) were described , and our cases are number 17 and 18 . of all cases , 10 occurred in males and 8 in females ; their mean age was 52.4 years ( range : 2181 years ) . most lesions were situated in the tongue ( n = 6 ) , lower lip ( n = 5 ) and other areas . microscopically , 6 cases ( 33.3% ) had an osseous component , 11 cases ( 61.1% ) had a chondroid component , and 1 case ( our case , 5.6% ) had an osseous / chondroid component . according to our review of the english - language literature , which included our cases , 6 cases were described as lipoma with osseous component [ 57 , 9 , 14 , 15 ] , 11 cases as chondroid component [ 4 , 813 , 16 , 17 ] and 1 case as fibrolipoma with osseous / chondroid components . our case 2 is namely the first report of fibrolipoma with osseous / chondroid differentiation . osteolipomas are less common than chondrolipomas in the whole body , and almost the same tendency was described in oral lesion . lipoma with osseous / chondroid differentiaion is extremely rare and occurs mainly in large long - standing lipomas [ 15 , 19 ] . case 1 also showed 28-year duration , and this might cause the osseous / chondroid to change into lipoma . as with many tumors , further indistinct etiology of osseous / chondroid change in lipoma has been discussed and most researchers mentioned that their origin is from different types of undifferentiated mesenchymal cells . the hypothesis is that the neoplastic transformation occurs in multipotential undifferentiated mesenchymal cells that later differentiate into lipoblasts , chondroblasts , or osteoblasts and fibroblasts . another hypothesis is that only the adipose cells have a neoplastic transformation , and that the cartilage and bone is produced by differentiation of undifferentiated mesenchymal cells of stroma in chondroblasts or osteoblasts . immunohistochemical results in this study indicated that there was no proliferative characteristic in mesenchymal ( spindle ) cells with small positive rates by ki-67 . chondrocytes and peripheral spindle cells around chondroid tissue with positive findings for s-100 , , and sox-9 had chondromatous characteristics . sox-9 belongs to the sox ( sry - related high - mobility group box ) family of transcription factors and is a key regulator of developmental processes including chondrogenesis . runx-2 is essential for skeletal mineralization when it stimulates osteoblast differentiation of mesenchymal stem cell and mature chondrocyte differentiation . positive findings for runx-2 in spindle cells around osteoid components and some of chondrocytes indicated osteoblast / chondrocyte characteristics of them . these characteristics are synthesized and thought to be differentiation on account of small positive rate for ki-67 and negative findings for vegf and fgf-1 in this study . osseous / chondroid formation occurred in the central part of these tumors in which a small amount of vessels existed . multipotential undifferentiated mesenchymal cells had undergone differentiation to osteoblast / chondroblast by topical modification , such as nutrition disorder or asphyxia . the bone formation adjacent to chondroid tissue should not be dystrophic calcification because of its positive findings for runx-2 in spindle cells surrounding chondrocytes . these immunohistochemical results supported the conclusion that endochondral ossification and/or perichondral ossification had occurred in these tumor . these results supported lipoma with osseous / chondroid differentiation . lipomas are benign mesenchymal neoplasms composed of mature adipocytes and are rare soft tissue tumors with a 2.2% incidence rate of the whole body . a 20% incidence rate of lipoma has been reported in the head and neck region . a 5% incidence rate in oral lesion of this study showed lower than in the head and neck region , but this rate is almost the same tendency compared to previous reports [ 1 , 20 , 21 ] . the average age of the patient of lipoma was 53.8 years old , and male - to - female ratio was 1.4 : 1.0 in this study . our study supports the current reports that lipomas are generally more common in males than in females ( soft tissue ) , and occurred in adult patients most often between the ages of 40 and 60 years [ 1 , 3 ] . the predominant locations were the buccal mucosa and the margin of the tongue and the lower lip , where there is plenty of adipose tissue . the patients ' characteristics and location distribution , cheek and tongue , were similar to previous reports [ 1 , 20 , 22 ] . microscopically , 28 cases ( 61.0% ) were classified as lipoma and 18 ( 39.0% ) as fibrolipoma . fujimara have reported , but our case 2 is the first report of fibrolipoma with osseous / chondroid differentiation . the two cases this reports contributed to pile up of rare lipoma / fibrolipoma with osseous / chondroid differentiation case , and , in addition , immunohistochemical result is contributed to the clarification of the etiology .

the purpose of this study was to determine the clinicopathological and immunohistochemical features of lipoma / fibrolipoma with rare occasions as osseous and/or chondroid differentiation in the oral cavity . two cases of the tumors , who presented with a painless , relatively hard mass on the oral mucosa , were studied . these were consisted of a well - circumscribed mass of fatty tissue with chondroid and significant fibrous component intermixed with the lobules of fat cells with chondroid and woven bone component , respectively . immunohistochemical study revealed that peripheral spindle cells around chondroid tissue stained diffusely for s-100 & and sox-9 , though peripheral spindle cells around osteoid tissue only stained for runx-2 . according to review of the literature , lipoma / fibrolipoma with osseous and/or chondroid differentiation was 18 cases . also fibrolipoma with osseous and chondroid differentiation is the first to be reported here . these results indicated that the cartilage / bone is produced by differentiation of undifferentiated mesenchymal cells of stroma .

staphylococcus ( s. ) intermedius and s. pseudintermedius are both staphylococcus intermedius group ( sig ) members . these microorganisms are opportunistic pathogens that cause skin and nosocomial infections in dogs and cats . s. pseudintermedius is the predominant sig species in korea and until recently was misclassified as s. intermedius . increased identification of methicillin - resistant sig ( mrsig ) isolates has emphasized the importance of public health precautions for veterinary staff and pet owners . many current studies have evaluated methicillin - resistant characteristics and the staphylococcal cassette chromosome mec ( sccmec ) of sig isolates , particularly s. pseudintermedius . although type i to type viii sccmec types have been identified , there is no significant studies have been conducted to examine sccmec types isolated from veterinary hospitals located nationwide in korea . sccmec types may produce staphylococcal exotoxins including staphylococcal enterotoxins ( ses ) , exfoliative toxins ( ets ) including s. intermedius exfoliative toxin ( siet ) , and toxic shock syndrome toxin 1 ( tsst 1 ) ; these toxins are associated with atopic dermatitis , mastitis , food poisoning , canine pyoderma , and chronic otits . siet ( encoded by the siet gene ) , first described by terauchi et al . , plays a potential role in the pathogenesis of canine pyoderma and chronic otitis . there may also be a greater chance of sig isolates to colonization and/or expand in veterinary staff , companion animals , and hospital environments . therefore , this study is focused on the genetic identification of staphylococcal exotoxins , sccmec types , and genetic relatedness by pulsed - field gel electrophoresis ( pfge ) of the sig isolates in korea . samples of s. pseudintermedius ( n = 167 ) and s. intermedius ( n = 11 ) were isolated ; their identity was confirmed by gram - staining and biochemical testing such as coagulase , dnase production and hydrolysis . polymerase chain reaction ( pcr ) was carried out with primers targeting s. intermedius nuclease and 16s rrna genes . to differentiate s. pseudintermedius from s. intermedius and s. delphini , pcr - restriction fragment length polymorphism analysis was performed using pta gene - specific primers ( pta_f1:5'-aaa gac aaa ctt tca ggt aa-3 ' , and pta_r1 : 5'-gca taa aca agc att gta ccg-3 ' ) , and the restriction enzyme mboi ( new england biolabs , usa ) . samples were collected from eight veterinary hospitals from four different regions in korea between november 2006 and january 2010 as previously described . pcr analysis to detect the methicillin - resistance gene ( meca ) pcr was also performed to amplify genes encoding sea ( sea ) , seb ( seb ) , sec ( sec ) , sed ( sed ) , see ( see ) , seg ( seg ) , seh ( seh ) , sei ( sei ) , tsst 1(tsst-1 ) and ets ( eta , etb , etd , and siet ) using primers and pcr conditions used in other previous studies . fri913 , fri 361 , fri 472 , fri 569 , mnhoch , and rn4220 strains were used as either positive or negative controls for superantigen genes . a superantigen gene obtained in this study was further analyzed by dna sequencing using the vector nti align x program ( invitrogen , usa ) . all mrsig isolates including 49 methicillin - resistant s. pseudintermedius ( mrsp ) and three methicillin - resistant s. intermedius ( mrsi ) were subsequently classified as sccmec type i to viii by either single or multiplex pcr assays using protocols in other previous studies including primers and pcr conditions . genomic dna plug kits ( bio - rad , usa ) with smai ( new england biolabs , usa ) digestion were used for the pfge analysis of 39 s. pseudintermedius isolates from a private referral veterinary hospital collected at different times ( eight isolates from november 2006 , 24 isolates from april 2008 , four isolates from june 2009 , and three isolates from october 2009 ) and six s. intermedius isolates ( all collected during april 2008 ) . pfge was performed according to protocol used in previous study and the manufacture 's instruction ( bio - rad , usa ) except for an initial pulse time of 5 sec and final time of 40 sec for 21 h . analysis of the sig isolates ( n = 178 ) for ses and tsst 1 genes showed that only a single s. pseudintermedius isolate from a veterinary staff member ( h1 - 23 ) harbored the enterotoxin c ( sec ) gene ( fig . 2 ) revealed that the amplified sec gene the canine type c se gene ( seccaine ) . a total of 166 out of 178 sig isolates ( 155 s. pseudintermedius and 11 s. intermedius ) isolated from veterinary staff members ( n = 38 , 95% ) , companion animals ( n = 107 , 93% ) , and veterinary hospitals ( n = 21 , 91.3% ) harbored siet originally detected in s. intermedius . however , other et genes such as eta , etb , and etd , known to originate from s. aureus , were not identified . sccmec typing of the 49 mrsp isolates identified 38 type v isolates , one type iv isolate , and 10 non - identifiable isolates . pfge analysis showed that sig isolates from a private referral animal hospital ( collected between 2006 and 2009 ) recovered from veterinary staff , companion animals , and the environment had the same band patterns ( fig . 3 ) . six s. pseudintermedius isolates ( two from companion animals collected in september 2006 , three from veterinary staff members , and one from companion animals collected in april 2008 ) and two s. pseudintermedius isolates ( one from a veterinary hospital environment isolated in april 2008 , and one from a companion animal isolated in june 2009 ) showed the same band patterns ( fig . since sig is closely related to s. aureus , studies have been performed to determine whether sig has adapted the eta , etb and etd toxins from s. aureus by pcr test with specific primers . however , 166 out of 178 ( 93.3% ) sig isolates harbored the siet toxin originating from s. intermedius . this result and those of other studies imply that the majority of sig isolates harbor the siet gene . although the siet gene was present in 93.3% of the sig isolates ( from 108 dogs and two cats ) in this study , only 14 dogs had a history of various skin disease including allergy and prolonged inflammation lesion in skin ( n = 13 ) or otitis ( n = 1 ) ( data not shown ) . therefore , other factors such as the general health of an animal and existence of other sig virulence factors may play an important role in outbreaks of various kinds of skin disease or otitis . the sec gene was detected in a single isolate in the present study , which was identified as seccanine by dna sequencing . however , this isolate was isolated from a veterinary staff member , and no additional seccanine isolates were identified in the veterinary hospital where this individual worked . this suggested that the isolate might be transmitted from an in- or outgoing companion animal with which the veterinary staff was in contact . the low incidence of toxins in this study could be secondary to the small number of isolates collected from companion animals that a history of skin disease or otitis . only 11.3% of s. pseudintermedius in a previous study had exotoxins although all samples were taken from patients diagnosed with pyoderma or chronic otitis and referred to a veterinary teaching hospital . although the majority of sccmec types were type v ( 78.9% ) , one isolate was type iv . in a previous study , 23 isolates ( 85.2% ) , three isolates ( 11.1% ) and one isolate ( 3.7% ) from veterinarians , staff , students , companion animals and environment in the veterinary hospitals were determined to be an mrsp hybrid sccmec type i~ii , type v , or non - identifiable , respectively . a previous european and north american study identified 75 hybrid sccmec type ii~iii isolates ( 72.8% ) , two type iii isolates ( 1.9% ) , six type iv isolates ( 5.8% ) , 14 type v isolates ( 13.6% ) , four type vii isolates ( 3.9% ) , and two non - identifiable isolates ( 1.9% ) from diseased and healthy dogs in veterinary diagnostic laboratories of different countries . this demonstrated that the majority of mrsig isolates in korea harbor the sccmec type v whereas the hybrid type ii~iii is the main sccmec type found in veterinary hospitals in japan , europe , and north america . in the present study , pfge analysis of the 39 s. pseudintermedius and six s. intermedius isolates from a private referral veterinary hospital ( collected during november 2006 , april 2008 , june 2009 , and october 2009 ) showed that 20 isolates ( lines 1~6 , lines 7~9 , lines 10~15 , lines 16~18 , and lines 19~20 ) had the same band patterns . moreover , some isolates obtained on different sampling dates showed the same band patterns ( six isolates from lines 12~17 , and two isolates from lines 21~22 ) . these results suggest potential contamination or expansion of s. pseudintermedius and s. intermedus isolates among veterinary staff , companion animals , and veterinary hospital environments , and colonization by these specific strains for more than 13 or 18 months in the same hospital . in conclusion , eta , etb , etd genes were not detected but siet toxin was found in 166 isolates in the current study . pfge analysis results of isolated from h animal hospital showed that s. pseudintermedius isolates collected in over a period of 13 and 18 months from veterinary staff , companion animals , and the hospital environment had the same band patterns . s. pseudintermedius infections in humans , spread of mrsp populations , and association of sig with canine pyoderma and chronic otitis has been previously reported . therefore , sig , especially mrsig , may have significant clinical implications for companion animals with skin infections or chronic otitis that is of concern for veterinary staff , companion animal owners , and healthy companion animals .

the staphylococcus ( s. ) intermedius group ( sig ) has been a main research subject in recent years . s. pseudintermedius causes pyoderma and otitis in companion animals as well as foodborne diseases . to prevent sig - associated infection and disease outbreaks , identification of both staphylococcal exotoxins and staphylococcal cassette chromosome mec ( sccmec ) types among sig isolates may be helpful . in this study , it was found that a single isolate ( one out of 178 sig isolates examined ) harbored the canine enterotoxin sec gene . however , the s. intermedius exfoliative toxin gene was found in 166 sig isolates although the s. aureus - derived exfoliative toxin genes , such as eta , etb and etd , were not detected . sccmec typing resulted in classifying one isolate as sccmec type iv , 41 isolates as type v ( including three s. intermedius isolates ) , and 10 isolates as non - classifiable . genetic relatedness of all s. pseudintermedius isolates recovered from veterinary staff , companion animals , and hospital environments was determined by pulsed - field gel electrophoresis . strains having the same band patterns were detected in s. pseudintermedius isolates collected at 13 and 18 months , suggesting possible colonization and/or expansion of a specific s. pseudintermedius strain in a veterinary hospital .

the brain is a highly complex organ that consists of various types of neurons and glia . how the complex brain architecture develops from neural stem cells ( nscs ) is one of the central questions of neuroscience . much interest has been paid to the mechanisms of nsc maintenance , proliferation , migration , and differentiation into various cell types . a second focus of interest in neurogenesis is the fact that neurogenesis continues throughout life in particular brain regions . the long - held belief that neurons are no longer regenerated once development of the central nervous system ( cns ) ceases was challenged about 50 years ago . progress in the several decades since has made adult neurogenesis in the hippocampus and olfactory system widely accepted . the mechanisms underlying adult neurogenesis and its functional significance have been pursued as an important focus of neurogenesis research . further , the finding that newly generated neurons and glia from nscs can be used in the treatment of degenerative disease and trauma of the cns has attracted the wide interest of neuroscientists and clinicians in stem cell biology . these therapeutic approaches are strongly facilitated by recent advances in es cells and ips cells . in addition , further interest has been shown in the correlation between impaired neuro / gliogenesis and psychiatric diseases , including mood disorders and chronic stress syndromes , along with recent progress in basic research into these diseased states . a closer understanding of the nature of nscs and neuro / gliogenesis is therefore crucial to various aspects of neuroscience , including the development , remodeling , and restoration of brain architecture and function . stem cell and neuro / gliogenesis biology is also strongly linked to the plastic potential of the cns . nscs show remarkable plasticity in differentiating into a variety of cell types that play a critical role in the formation of the complex brain architecture . after the brain develops to maturity , its plastic function is generally exhibited through the plasticity of preexisting neurons generated during the developmental and neonatal periods . it is notable , however , that nscs in the mature brain likely confer far stronger plastic potential . in addition , restoring appropriate neuro / gliogenesis in the diseased brain can be regarded as the process of adjusting the brain s plasticity for structural and functional restoration . thus , stem cell and neuro / gliogenesis biology highlights the remarkable plastic potential of the brain . this research field provides an opportunity to integrate structural plasticity and functional plasticity at the cellular , circuit and behavioral levels and has attracted wide interest from basic scientists , clinicians , and society . in 2015 , the japanese association of anatomists and the physiological society of japan held a joint annual meeting in kobe , japan . one symposium , neurogenesis from embryo to adult , attracted a large audience and generated vigorous discussion . in this review article , we summarize the five presentations we made in this symposium , with the goal of providing a comprehensive understanding of the current progress and future directions of this research field . first , itaru imayoshi introduced the role of transcription factors in the self - renewal and differentiation of nscs in the developing neuroepithelium . dr . imayoshi revealed the pivotal role of the expression dynamics of basic helix - loop - helix ( bhlh ) transcription factors , oscillatory or sustained , on the nsc self - renewal and fate decision . second , tatsunori seki raised the question of how nscs produce granule cells in the embryonic and early postnatal hippocampus and how the early neurogenesis is succeeded by adult neurogenesis . seki revealed that the first progenitors of granule cells arising in the dentate anlage in the developing hippocampus express glial fibrilar acidic protein ( gfap ) and that these gfap - expressing progenitors form a proliferative zone in the subpial and hilar zones during the perinatal stage and later in the subgranular zone , where neurogenesis continues throughout life . third , nobuaki tamamaki introduced the emerging view that adult neurogenesis may also occur in the neocortex . tamamaki revealed that neuronal progenitors can be induced in the leptomeninges of the neocortex by electrical stimulation and showed their migration and differentiation in the neocortex . fourth , masahiro yamaguchi introduced how adult - born olfactory neurons are selected for survival or death in the olfactory bulb . dr . yamaguchi revealed that the key mechanism of activity - dependent selection is the integration of sensory inputs from the periphery and top - down inputs from higher brain regions . lastly , yoshitaka hayashi , yoshitaka tatebayashi , and seiji hitoshi introduced the relation between major depressive disorder and oligodendrocyte genesis in the human prefrontal cortex . hayashi et al . established a method to quantify oligodendrocytes and their progenitors and revealed the dysfunction of oligodendrocyte development in the diseased prefrontal cortex . nscs are multipotent and self - renewable cells that give rise to neurons , astrocytes , and oligodendrocytes in the brain . nscs of the lateral ventricular wall in the forebrain undergo changes in morphology and produce different progeny as brain development proceeds . nscs begin as neuroepithelial cells , become radial glial cells , and then finally develop many astrocytic characteristics in the adult brain . during neural development , nscs ( neuroepithelial cells / radial glial cells ) initially undergo symmetric cell division : each nsc divides into two nscs . by repeating symmetric cell division , these cells then undergo asymmetric cell division : each nsc divides into two distinct cell types , one nsc and one immature neuron / neuronal precursor or a basal progenitor cell . immature neurons or neuronal precursor cells migrate outside the ventricular zone ( vz ) into the outer layers , where they become mature neurons , whereas basal progenitor cells migrate into the subventricular zone ( svz ) , proliferate further , and give rise to more neurons . by repeating asymmetric cell division , after the production of neurons , nscs finally differentiate into glial cells , but some of them are maintained as nscs in the postnatal and adult brain . bhlh transcription factors play pivotal roles in the self - renewal of nscs and fate determination of neurons , astrocytes , and oligodendrocytes [ 813 ] . repressor bhlh factors such as hes1 regulate the self - renewal of nscs as downstream effectors of notch signaling , whereas proneural bhlh factors such as ascl1 ( also called mash1 ) promote neuronal differentiation . however , in addition to hes1 , some bhlh fate determination factors , such as ascl1 and olig2 , have roles in nsc maintenance or proliferation . it is not completely understood how these various and sometimes opposing functions of each bhlh factor in the self - renewal and fate - choice events of nscs are achieved . showed that transcription of some bhlh genes is differentially regulated in nscs and differentiating cells , prompting imayoshi et al . to analyze the expression dynamics of bhlh factors in further detail . . indicated that bhlh factors are expressed by nscs in an oscillatory manner and that one of them becomes dominant during the fate choice event ( fig . they then proposed that the multipotent state of nscs correlates with the oscillatory expression of several bhlh factors , whereas the differentiated state correlates with the sustained expression of a single bhlh factor [ 8 , 9 , 15].fig . 1expression dynamics of bhlh factors in multipotency and cell fate choice . in multipotent nscs , levels of hes1 and ascl1 oscillate with periods of 23 h , while that of olig2 oscillates with a period of 58 h. by contrast , during cell fate choice , one of the bhlh factors is expressed in a sustained manner , while the others are repressed expression dynamics of bhlh factors in multipotency and cell fate choice . in multipotent nscs , levels of hes1 and ascl1 oscillate with periods of 23 h , while that of olig2 oscillates with a period of 58 h. by contrast , during cell fate choice , one of the bhlh factors is expressed in a sustained manner , while the others are repressed to examine the functional significance of these bhlh factor expression dynamics , a novel optogenetic method ( photo - activatable gal4/uas system ) was developed to artificially manipulate the expression patterns of bhlh factors with blue light illumination . this analysis demonstrated that sustained expression of ascl1 induces neuronal differentiation , whereas oscillatory expression of ascl1 activates cell proliferation of nscs . thus highlighted the importance of the expression dynamics of bhlh factors in the self - renewal , multipotency , and fate determination of nscs [ 8 , 9 , 15 ] ( fig . during the embryonic period , neurons of both the neocortex and hippocampus are initially generated from the vz of the pallium : the neocortex develops from the dorsal and lateral pallium and the hippocampus from the medial pallium . in the neocortex , neurogenesis occurs only during the embryonic and early postnatal stages and ceases by the early postnatal stage , as typically occurs in most brain regions . however , neurons are exceptionally produced in the adult hippocampus , where dentate granule cells arise from gfap - expressing astrocyte - like adult neural progenitor cells , which characteristically differ from embryonic neural progenitor cells [ 2 , 1618 ] . in an attempt to understand how neurogenesis persists in the hippocampus , seki et al . explored the neurogenic process of dentate granule cells from the embryonic to late postnatal stages with special reference to gfap - expressing progenitors , namely when these progenitors appear during the embryonic period and how they form the granule cell layer . analysis using gfap - gfp transgenic mice showed that a distinct gfap - gfp - expressing cell population first appears in the vz of the medial pallium around a ventricular indentation or in the dentate notch at the dorsal edge of the fimbria at e13.514.5 . these are radially oriented cells with apical and/or basal processes that seem to migrate from the vz to the marginal zone , a region below the pia surface . no gfap - gfp expression is found in the dorsal or lateral pallium , which develops into the neocortex . during the perinatal period , gfap - gfp cells increase in number and form a migratory stream in the suprafimbrial region , as well as in the subpial region between the fimbria and dentate gyrus , alternatively called the fimbrio - dentate junction or fimbrio - dentate juncture . three proliferative zones are formed by gfap - gfp - expressing cells in these regions : the primary dentate matrix in the vz , secondary dentate matrix in the suprafimbrial area and regions below the pia membrane and hippocampal fissure , and the tertiary dentate matrix in the core region of the dentate gyrus , the putative hilus . gfap - gfp cells in the migratory stream and the developing dentate gyrus are positive for sox2 and ki67 , suggesting that they contain proliferative progenitors . they also express neurogenin2 in the vz , tbr2 and neurod in the migratory stream and developing dentate gyrus , and prox1 in the developing dentate gyrus . it is therefore concluded that distinctive gfap - expressing progenitors arising around the dentate notch form germinal regions in the migratory stream and developing dentate gyrus and gradually differentiate into granule neurons . this also indicates that in the dentate gyrus , astrocyte - like neural progenitors continue to generate granule neurons from the beginning of development and throughout life . the site of origin , proliferative activity , and migratory pattern of the gfap - gfp - expressing cells agree well with those suggested by studies with h - thymidine autoradiography , and nestin - gfp . although previous reports showed the existence of gfap - expressing cells and fibers in the embryonic hippocampus [ 21 , 23 , 24 ] , these are thought to belong to the radial type of astrocytes that function as scaffolds for migrating neuroblasts . however , the existence of abundant gfap - gfp - expressing cells with neuronal marker proteins clearly demonstrates that principally gfap - expressing cells and fibers are not derived from radial astrocytes , but rather gfap - expressing migrating spindle - shaped neuronal progenitors and precursors . interestingly , the dentate gyrus shares some similarities with the cerebellum in terms of their morphogenesis . for example , the hippocampus and cerebellum are derived from neuroepithelium around the edge ( cortical hem and rhombic lip ) of the dorsal portions of the telencephalon and metencephalon , respectively , both of which are attached to the choroid plexus ( fig . 2 ) . both progenitors are generated in the ventricular zone ( primary proliferative zone ) and migrate to the subpial region , in which secondary proliferative zones ( secondary dentate matrix and external granular layer ) are formed . the progenitors in the secondary proliferative zone give rise to small interneurons or granule cells . production of both neurons in the subpial regions continues into the early postnatal period to form the cerebellar and dentate granule cell layers . these facts suggest that there may be a similar mechanism in early postnatal neurogenesis in the subpial regions of the dentate and cerebellum . furthermore , the dentate gyrus , unlike the cerebellum , establishes a tertiary proliferative zone ( tertiary dentate matrix ) in the hilus . during the late postnatal period , the progenitors in this tertiary dentate proliferative zone are gradually confined to the subgranular zone , where neurogenesis persists in adults.fig . 2 a progenitors of granule cells in the developing hippocampus . gfap - gfp - expressing progenitors ( green ) arise from the dentate notch ( arrowhead ) and migrate to the dentate gyrus . chp choroid plexus , cp cortical plate , egl external granular layer , gcl granule cell layer , sdm secondary dentate matrix , sgz subgranular zone , svz subventricular zone , tdm tertiary dentate matrix , vz ventricular zone ( modified from ) a progenitors of granule cells in the developing hippocampus . gfap - gfp - expressing progenitors ( green ) arise from the dentate notch ( arrowhead ) and migrate to the dentate gyrus . chp choroid plexus , cp cortical plate , egl external granular layer , gcl granule cell layer , sdm secondary dentate matrix , sgz subgranular zone , svz subventricular zone , tdm tertiary dentate matrix , vz ventricular zone ( modified from ) although adult neurogenesis is a common phenomenon in the non - mammalian neocortex , the scale of adult neurogenesis in the neocortex appears to decrease significantly as the phylogeny approaches the human being . in apparent contrast , the limbic area of the telencephalon appears to be a site of continuous generation of neurons . in particular , the medial edge of the mammalian telencephalon , the dentate gyrus , produces granule cells constantly throughout life in humans , while the svz surrounding the lateral ventricle continuously produces gabaergic granule cells for the olfactory bulb . granule cells produced in the dentate gyrus are integrated into the circuitry of the hippocampus and save new information , whereas granule cells produced in the svz of the lateral ventricle migrate rostrally and are integrated into the olfactory circuitry . in other words , these new neurons are not integrated into the neocortex . however , if a significant number of new neurons is produced in the neocortex and integrated into the neocortical circuit , we can not deny the possibility that these new neurons ruin the circuits for memory and thought in the human neocortex . macklis reported that the synchronous apoptotic degeneration of corticothalamic neurons may induce generation of the same number of neurons in the neocortex and may recreate the same circuit as was originally present . in this case , neurogenesis in the neocortex would not disturb the circuit in the neocortex and would be welcomed . our goal here is to stimulate the interest of many researchers to join the hunt for neuron progenitors in the mouse neocortex . as the first hunter , costa et al . sought the source of neuron progenitors using a neurosphere assay method and found a novel source of neuron progenitors outside the two major germinative zones ( vz and svz ) , namely the marginal zone of the neocortex . these neuronal progenitors in the marginal zone of the developing cerebral cortex were notably distinct from those in the vz and svz . as the second hunter of the neuron progenitors in the adult neocortex , ohira and colleagues sought nscs and neuronal progenitor cells ( npcs ) in the adult rat neocortex . the vast majority of neocortical neurons were eliminated and the remnant contained more nscs and npcs . they then observed npcs in neocortical layer 1 of adult rats . on injection of a simple retrovirus to visualize these npcs in layer i , they found that no npcs or related cells were neurons , but rather had a glia - like appearance . moreover , none of the npcs had axons . as the third hunter of the neuron progenitors in the adult neocortex , nakagomi and colleagues found that neural stem / progenitor cells ( nspcs ) reside in many regions of the cns , including the svz of the lateral ventricle , subgranular zone of the hippocampal dentate gyrus , cortex , striatum , and spinal cord . in addition , they demonstrated that the leptomeninges ( pia mater and arachnoid membrane ) also exhibit nspc activity in response to ischemia and reported that the nspcs express nspc markers , such as nestin , and form neurosphere - like cell clusters with self - renewal activity . when the mouse brain was stimulated by electrodes and nestin - immunoreactivity ( -ir ) in the leptomeninges was examined , neurogenic progenitors were clearly revealed ( fig . 3 ) . if the electrical stimulation of the brain was continued , nestin - ir was replaced with gad67-ir or neurod6-ir in granule cells . when the neocortex was continually stimulated for up to 1 or 2 weeks , neuron progenitors in the leptomeninges migrated into the neocortex and projected dendrites into the cortical plate and axons into the white matter . in this case , we consider that the newly generated neocortical neurons would not ruin the circuit in the neocortex.fig . nestin immunoreactive sites are in green piaprogenitors on the outer surface of the pia mater induced by kindling stress . adult - born olfactory neurons are generated in the svz and migrate rostrally to the olfactory bulb ( ob ) , the first relay in olfactory information processing . they then differentiate into granule cells ( gcs ) or periglomerular cells , which act as interneurons in the ob . in rodents , roughly one percent of total ob gcs are newly generated each day , and more than 60 % of gcs in the adult ob are estimated to be adult - born gcs [ 3 , 34 ] . under normal conditions only half of the new gcs succeed in living longer than 1 month after generation , while the other half are eliminated by apoptosis . initial excess neurogenesis and subsequent elimination commonly occur in both embryonic and adult neurogenesis . because proper neuronal selection between life and death is crucial to refining the neuronal circuitry throughout the life of the animals , it is important to understand the mechanism of how adult - born neurons are appropriately selected . first , the selection of adult - born new gcs depends on olfactory sensory experience . exposure to novel odors increases the survival rate of new gcs , while olfactory sensory deprivation remarkably increases the apoptosis of new gcs [ 3 , 35 ] . second , the selection of new gcs depends on brain state . in many cases brain plasticity is influenced by the brain state , most notably by the wake - rest / sleep cycle . in the hippocampus and neocortex , consolidation of spatial memory is considered to occur during the rest / sleep period that follows the spatial learning . on the supposition that the selection of new gcs may occur during the time course of olfactory sensory experience and rest / sleep , yokoyama et al . examined whether the fate decision of new gcs is associated with feeding behavior . under food restriction , mice initially showed extensive eating behavior following food availability and then showed grooming , resting , and sleeping , which are typical postprandial ( after - meal ) behaviors . interestingly , apoptosis of gcs increased approximately two - fold during the postprandial behaviors , whereas perturbation of these postprandial behaviors remarkably suppressed this increase in gc apoptotic elimination . further , electroencephalogram analysis showed that postprandial slow - wave sleep ( sws ) was well correlated with the gc elimination . so , what mechanisms promote gc elimination during postprandial behaviors , particularly during postprandial sws ? neurons in the olfactory cortex ( oc ) repeatedly generated synchronized spike discharges during sws . importantly , this synchronized firing of oc neurons generated synchronized top - down synaptic inputs to the ob , which were considered as the candidate signal that promoted gc elimination . in fact , electrical stimulation of the top - down inputs from the oc to the ob promoted gc elimination , and pharmacological blockade of the top - down inputs during the postprandial period suppressed gc elimination . these observations demonstrate that top - down inputs during the postprandial period crucially promote gc elimination . these findings raised the further question of whether enhanced gc elimination occurs only during the postprandial behaviors or during other behavioral states also . komano - inoue et al . examined whether fear responses to noxious stimuli induce gc elimination . mice delivered an electrical foot shock showed startle and fear responses during and immediately after shock delivery . interestingly , gc elimination was very rapidly enhanced in mice that showed these behavioral responses extensively . moreover , this enhanced gc elimination after shock delivery was inhibited by the suppression of neuronal activity in the oc . these results show that gc elimination can be promoted during various behaviors and that top - down inputs from the oc to the ob represent the common signal for inducing gc elimination . one intriguing possibility is that behaviors associated with important life events , such as food eating and experience of noxious stimulation , might promote reorganization of the ob neuronal circuit , including the enhanced gc elimination . the oc receives synaptic inputs not only from the ob but also from other nonolfactory brain regions , such as the amygdala and prefrontal cortex . important life events might recruit wide brain areas for activation and thereby potentiate the activity of the oc and top - down inputs from the oc to the ob to reorganize the ob neuronal circuitry . in conclusion , the life and death of new gcs in the ob is determined by the interplay between bottom and up olfactory sensory inputs from the periphery and top - down inputs from higher cortical regions ( fig . gcs are presumed to receive these inputs at different synapses in different layers of the ob [ 3 , 41 ] . it is important to understand the manner of integration of these synaptic inputs onto a given gc . it is also important to reveal what kind of information is represented by the top - down inputs . considering that the top - down inputs reflect the activity of higher cortical regions , they might represent the subjective meaning of the odor stimuli for the animal , including the valence of the odor and odor - evoked emotional and motivational responses . the fate of new gcs thus needs to be understood with regard to the activity of large neuronal networks.fig . 4activity - dependent selection between survival and death for new gcs in the ob . a model of how synaptic inputs integrate and determine the fate of new gcs . during olfactory experience , some newly generated gcs ( green ) receive odor input ( red arrows for right gc ) while others do not ( left gc ) . top - down input from the oc to the ob ( blue arrow ) , which typically occurs during the sleep state , eliminates new gcs that did not receive an odor input ( left gc ) and leave new gcs that did receive an odor input to survive ( right gc ) . thus , top - down input from the oc might reflect the activity of various brain regions . this figure was modified from activity - dependent selection between survival and death for new gcs in the ob . a model of how synaptic inputs integrate and determine the fate of new gcs . during olfactory experience , some newly generated gcs ( green ) receive odor input ( red arrows for right gc ) while others do not ( left gc ) . top - down input from the oc to the ob ( blue arrow ) , which typically occurs during the sleep state , eliminates new gcs that did not receive an odor input ( left gc ) and leave new gcs that did receive an odor input to survive ( right gc ) . thus , top - down input from the oc might reflect the activity of various brain regions . major depressive disorder ( mdd ) is a debilitating mental illness that causes a persistent feeling of sadness , loss of interest , deep despair , and death by suicide . brain imaging studies of patients with mdd have demonstrated abnormalities in gray matter volume , metabolism , and blood flow in the prefrontal cortex and hippocampus [ 4244 ] . neuropathological studies in these brain regions in mdd have long been of interest in psychiatry . a small number of histological assessments of the prefrontal cortex from patients with mdd revealed abnormalities in the size of neurons and the density of oligodendrocytes [ 4547 ] . however , the stereological techniques often used for the quantification of numbers of cells in brain sections pose a serious methodological challenge to neuropathologists . using flow cytometry , hayashi et al . established a novel cell - counting method to quantify the number of nuclei of total cells , neurons , and oligodendrocytes in brain regions of interest from postmortem brains ( fig . the nuclei were obtained by mechanical homogenization of unfixed frozen brain , because the homogenization process breaks the cell membrane and cytoplasm but leaves the nucleus intact . the nuclei in suspension were stained with 7-aad , neun , and olig2 as markers of dna ( total nuclei ) , neurons , and oligodendrocytes , respectively , making it possible to quantify the number , size , and fluorescence intensity of the cells with a flow cytometer . furthermore , the fluorescence intensity of olig2 immunostaining is stronger in oligodendrocyte precursor cells ( opcs ) than in mature oligodendrocytes . the validity and accuracy of this novel cell - counting method have been verified by estimating the number of total and neuronal nuclei from the whole cerebral cortex of rats . this method has been applied to the gray matter of the frontopolar ( brodmann area 10 ) and inferior temporal ( brodmann area 20 ) cortices of postmortem brains from mdd patients and healthy controls . in the present report , hayashi et al . found a reduction in olig2 oligodendrocyte - lineage cells in the gray matter of the frontopolar cortex from mdd patients compared to that from healthy controls ( fig . by measuring the fluorescence intensity , they were able to separate the olig2 cell population into olig2 mature oligodendrocytes and olig2 opcs ( fig . detailed olig2 population analysis showed a significant reduction in the densities of both olig2 and olig2 nuclei in the frontopolar cortex of mdd patients compared to healthy controls ( fig . these results suggest that the frontopolar cortex , which is considered to be closely associated with the pathogenesis of mdd , processes the oligodendrocyte abnormality and that patients with mdd have a dysfunction in oligodendrocyte development ( such as in proliferation and/or maturation ) in childhood and/or adolescence.fig . 5quantification of frontopolar cortex from mdd and healthy control . a negative controls of flow cytometry staining with igg . b a novel cell counting method can separate nuclei from postmortem brain into neurons ( neun ) and oligodendrocytes ( olig2 ) , astrocytes , and microglia ( neun / olig2 ) . c the olig2 nuclei were further divided into two populations of strongly and weakly olig2-positive nuclei , which are oligodendrocyte precursor cells and oligodendrocytes , respectively . the density of olig2 ( d ) , olig2 ( e ) , and olig2 ( f ) nuclei in 1 mg of gray matter from the frontopolar cortex ( 10 cells / mg ) . * p < 0.05 by unpaired t test . results of immunostaining with ki-67 , a marker of proliferating cells , in the hippocampus ( g ) and subependymal zone ( h ) of adult cynomolgus monkeys . a part of this figure was rearranged from quantification of frontopolar cortex from mdd and healthy control . a negative controls of flow cytometry staining with igg . b a novel cell counting method can separate nuclei from postmortem brain into neurons ( neun ) and oligodendrocytes ( olig2 ) , astrocytes , and microglia ( neun / olig2 ) . c the olig2 nuclei were further divided into two populations of strongly and weakly olig2-positive nuclei , which are oligodendrocyte precursor cells and oligodendrocytes , respectively . the density of olig2 ( d ) , olig2 ( e ) , and olig2 ( f ) nuclei in 1 mg of gray matter from the frontopolar cortex ( 10 cells / mg ) . * p < 0.05 by unpaired t test . results of immunostaining with ki-67 , a marker of proliferating cells , in the hippocampus ( g ) and subependymal zone ( h ) of adult cynomolgus monkeys . a part of this figure was rearranged from continuous neurogenesis in the adult brain is observed in the hippocampus and subependymal zone in rodents as well as cynomolgus monkeys ( macaca fascicularis ) ( fig . volumes of literature report that chronic stress suppresses neurogenesis in rodents and that antidepressants stimulate neurogenesis in the subgranular zone in the hippocampus and the subependymal zone in the lateral ventricles [ 50 , 51 ] . although newly generated oligodendrocytes were observed in the adult hippocampus of a non - human primate , oligodendroglial generation from nscs in the adult brain is controversial . recently , opcs that express ng2 chondroitin sulfate were recognized as generators of newly myelinating oligodendrocytes in the adult brain . the proportion of opcs was reported to constitute about 5 % of total cells in rodent brains , which appears comparable to the number of olig2 nuclei in the gray matter of the frontopolar cortex of the human brain ( fig . because myelination in humans continues from adolescence into the 5th or 6th decade of life ( see review ) , adult oligodendrogenesis in the prefrontal cortex and hippocampus may play a significant role in the pathology of mdd [ 57 , 58 ] . thus , it is an urgent issue to understand the molecular mechanisms underlying the generation of opcs from nscs , maintenance of opcs , and production and maturation of oligodendrocytes from opcs in the adult primate brain and to clarify the causal relationship between the dysfunction of adult opcs / oligodendrocytes and pathogenesis of mdd . these topics indicate that stem cell and neuro / gliogenesis biology is involved in various aspects of neuroscience . this biology encompasses a wide range of animal ages , from the embryo to adulthood ; wide brain regions , from the developing neuroepithelium to higher neocortical regions ; and various conditions of the brain , including development , maturation , and disease . collectively , this research field aims to reveal the structural and functional plasticity of the cns including its genesis , remodeling , and restoration , by incorporating multidisciplinary approaches at the molecular , cellular , circuit , and behavioral levels . the broad scope of stem cell and neuro / gliogenesis study raises questions about inherent similarities and differences in the properties of nscs at different animal ages and in different brain regions , as well as in the process of neuro / gliogenesis in the developing , mature , and diseased brain . it appears important to understand both the intrinsic and extrinsic mechanisms of the nsc fate decision , including cell - to - cell interaction between immature cells , between newly generated cells and preexisting cells , and between new healthy cells and old diseased cells . further , with regard to functional aspects , how are new neurons and glia incorporated into neural circuits to develop , improve , and restore brain function ? to understand this , we need to know more about the constitution of functional neural networks and the interactions of the network with new neurons and glia . we also need to determine how neural activity contributes to the appropriate incorporation of new neurons and glia into functional networks . recent advances in optogenetic and pharmacogenetic approaches will facilitate these studies . to obtain a comprehensive view , it is crucial to integrate knowledge under different situations and to try to reveal similarities and differences among these situations . the 2015 joint meeting was an ideal opportunity to facilitate the interchange of knowledge among researchers with different backgrounds . we hope that this review will further facilitate this interaction and raise the interest of those who are not directly engaged in this research field . we wish to emphasize that neuro / gliogenesis from nscs is a drastic process involving significant structural and functional changes in the cells and that integration of newly generated neurons and glia into neural networks is accompanied by significant structural and functional changes in the network . thus , stem cell and neuro / gliogenesis biology provides an ideal platform for the integration of structural and functional plasticity and will contribute to our understanding of the remarkable plastic potential of the cns . we also hope that progress in this field will facilitate the interaction of neurobiologists and clinicians and encourage many people by revealing the tremendous plasticity that is buried in our own brain .

neurons and glia in the central nervous system ( cns ) originate from neural stem cells ( nscs ) . knowledge of the mechanisms of neuro / gliogenesis from nscs is fundamental to our understanding of how complex brain architecture and function develop . nscs are present not only in the developing brain but also in the mature brain in adults . adult neurogenesis likely provides remarkable plasticity to the mature brain . in addition , recent progress in basic research in mental disorders suggests an etiological link with impaired neuro / gliogenesis in particular brain regions . here , we review the recent progress and discuss future directions in stem cell and neuro / gliogenesis biology by introducing several topics presented at a joint meeting of the japanese association of anatomists and the physiological society of japan in 2015 . collectively , these topics indicated that neuro / gliogenesis from nscs is a common event occurring in many brain regions at various ages in animals . given that significant structural and functional changes in cells and neural networks are accompanied by neuro / gliogenesis from nscs and the integration of newly generated cells into the network , stem cell and neuro / gliogenesis biology provides a good platform from which to develop an integrated understanding of the structural and functional plasticity that underlies the development of the cns , its remodeling in adulthood , and the recovery from diseases that affect it .

foot and mouth disease ( fmd ) is an economically important disease of cloven - hoofed livestock such as cattle , sheep , and other domestic animals in addition to several wild - life species . the causative agent of the fmd is the foot and mouth disease virus ( fmdv ) . the disease is considered one of the most important barriers to the worldwide trade of livestock and animal products . fmdv isolates sampled from the world have been categorized into seven different serotypes , a , o , c , asia1 , and south african territories 1 ( sat1 ) , sat2 , and sat3 , according to antigenic levels ; and a large number of variants have appeared within each serotype . out of the three serotypes ( o , a , and asia1 ) found to be prevalent in iran in recent years , serotype a has been found to be more antigenically and genetically diverse than the others . the fmdv possesses a single - stranded rna molecule consisting of about 8,200 nucleotides within an icosahedral capsid composed of structural proteins . the open reading frame encodes a single polyprotein which can be cleaved into four structural proteins ( vp4 , vp2 , vp3 , and vp1 ) and eight non - structural proteins ( l , 2a , 2b , 2c , 3a , 3b , 3c , and 3d polymerase [ 3d ] ) . in general , mutations or deletions in structural proteins may help the fmdv to evade immune responses of the host whereas mutations or deletions in non - structural proteins can inhibit viral replication and protein processing . the fmdv usually infects cells by binding to integrin receptors via a long flexible loop ( g - h loop ) of vp1 ( 1d ) . the sequence of this loop contains a conserved arginineglycine - aspartic acid ( rgd ) tripeptide motif which is characteristic of ligands that bind to integrin receptors . the vp1 protein is encoded by the vp1 coding region of viral rna ; this coding region is 627~639 bp long and produces a protein containing 209~213 amino acid residues depending on the serotype . this gene contains a taa stop codon and is responsible for encoding a protein containing 470 amino acid residues . similar to other picornaviruses , the fmdv 3d protein is a viral - encoded rna polymerase . among the different fmdv serotypes and subtypes , both the nucleotide and amino acid sequences of 3d uninterrupted co - circulation of the virus in the environment and the absence of viral polymerase proofreading activity during the replication process lead to the appearance of various genetic isolates . analysis of the viral genome sequence is a crucially important approach for monitoring field isolates in areas where the disease is endemic . previous studies have shown that iran has one of the highest reported rates of fmd cases per year . in iran , fmd is largely controlled by vaccinating cattle and sheep with vaccines prepared against isolates that are likely to be encountered in the region . spread of the disease is promoted by the presence of large populations of susceptible animals , low vaccination rates , prevalence of multiple serotypes ( including serotypes a , asia1 , and o ) , and unlimited movement of susceptible animals in the country . furthermore , there is endemic co - circulation of multiple genotypes of type a virus . the high incidence of fmd has allowed the identification of new variants of the virus over the last 7 years . the general objective of this study was to determine the nucleotide sequences of genes encoding vp1 and 3d proteins of a type a fmdv ( a / iran87 ) highly adapted to cell culturing , and to compare them to other corresponding sequences available in the genbank database . the razi vaccine and serum research institute ( iran ) generously provided samples of the fmdv used for our study . clinical specimens of fmdv which included samples in tongue epithelium tissue were collected in 1987 from infected calves displaying clinical symptoms of fmd in an iranian field located in tehran . the a / iran87 strain was initially detected and isolated nearly more than two decades ago . thus , the highly passaged virus investigated in this study was not virus isolated from the field . the tissue samples were used to infect baby hamster kidney 21 ( bhk-21 ) cells . the virulent isolate was passaged in ba ( cell line derived from pig kidney ) and bhk-21 cell monolayers . propagation steps included six passages in ba cells and then four passages in bhk-21 cells . thereafter , it has been used for the development of fmd vaccines during the past years as a vaccine seed . the infected cell culture supernatant from the ~150th passage was clarified and stored at -70 before use . total rna was extracted from fmdv - infected bhk-21 cells using an rnx plus kit ( cinnagen , iran ) according to the manufacturer 's instructions . briefly , the cell culture was centrifuged at 85,000 g at 4 for 2 h. the pellet was resuspended in 200 l of pbs , mixed well with 400 l of rnx reagent , and incubated at room temperature for 5 min . the pellet was washed with 75% ethanol and dissolved in 20 l of rnase - free water . rt - pcr was carried out in 50 l of reaction mixture containing 10 l of 5 reaction buffer , 4 l of mixed dntps ( 2.5 mm each ) , 1 l of amv enzyme ( titan one tube rt - pcr system kit ; roche diagnostic , germany ) , 1 l of each primer ( 10 pmol each ) , 4 l of rna template , 2.5 l dtt , 3 l 25 mm mgcl2 , and 23.5 l of h2o . the following conditions were used for amplification : 42 for 30 min , 94 for 3 min ; 30 cycles of 94 for 30 sec , 52 for 30 sec , 72 for 40 sec ; then followed by 72 for 5 min . the vp1 and 3d coding regions of fmdv ( 621 and 690 bp , respectively ) were amplified using standard methods with a one - step rt - pcr system and specific primer combinations . 5'-gacatgtcctggtgcatctg-3 ' for 3d , and forward : fmg13 5'-accaggatgatgattggcag-3 ' and reverse : fm15 5'-tttcactcctacggtgtcgc-3 ' for vp1 . amplified pcr products of the expected length were separated by electrophoresis in a 1% agarose gel , stained with ethidium bromide , and visualized under a uv transilluminator . after successful amplification of the target dna sequences , fragments were purified using a gel extraction kit ( roche , germany ) following the recommendations of the supplier . ligation was performed with plasmid vector ptz57r / t ( fermentas , germany ) in 0.165 g , 0.18 pmol ends and 0.54 pmol ends purified pcr fragment in 1 ligation buffer , polyethylene glycol ( peg 4000 ) , 5 units of t4 dna ligase , and up to 30 l of deionized water at 22 for 16 h. the ligated products were used to transform chemically competent cells ( xl1-blue cells ) and white colonies grown on lb plates were randomly selected . the cloned pcr products were purified using plasmid purification kit ( roche diagnostics , germany ) according to the manufacturer 's instructions and sequencing was carried out in both directions using a t7 promoter primer ( mwg biotech , germany ) . the published sequences of 66 fmdv type a isolates recovered from different parts of the world were included in this analysis and compared to the corresponding sequence of the a / iran87 isolate . reference fmdv sequences were obtained from the national center for biotechnology information ( ncbi , usa ) . the sequences used for each gene were first examined to exclude ambiguous sequences which were incomplete or frame - shifted . to determine the degree to which genetic diversity was observed in the vp1 and 3d proteins , multiple alignments and comparisons of the predicted amino acid sequences of isolates were carried out ( fig . nucleotide sequence homology / divergence was calculated using the megalign project of the dnastar software package ( ver 5.1 ; dnastar , usa ; data not shown ) . the histories of the type a fmdv field isolates , including year of isolation , accession number , and geographical distribution , are presented in table 1 . the nucleotide sequences of the vp1 and 3d coding regions of a / iran87 have been submitted to genbank ( accession no . total rna was extracted from fmdv - infected bhk-21 cells using an rnx plus kit ( cinnagen , iran ) according to the manufacturer 's instructions . briefly , the cell culture was centrifuged at 85,000 g at 4 for 2 h. the pellet was resuspended in 200 l of pbs , mixed well with 400 l of rnx reagent , and incubated at room temperature for 5 min . the pellet was washed with 75% ethanol and dissolved in 20 l of rnase - free water . rt - pcr was carried out in 50 l of reaction mixture containing 10 l of 5 reaction buffer , 4 l of mixed dntps ( 2.5 mm each ) , 1 l of amv enzyme ( titan one tube rt - pcr system kit ; roche diagnostic , germany ) , 1 l of each primer ( 10 pmol each ) , 4 l of rna template , 2.5 l dtt , 3 l 25 mm mgcl2 , and 23.5 l of h2o . the following conditions were used for amplification : 42 for 30 min , 94 for 3 min ; 30 cycles of 94 for 30 sec , 52 for 30 sec , 72 for 40 sec ; then followed by 72 for 5 min . the vp1 and 3d coding regions of fmdv ( 621 and 690 bp , respectively ) were amplified using standard methods with a one - step rt - pcr system and specific primer combinations . amplified pcr products of the expected length were separated by electrophoresis in a 1% agarose gel , stained with ethidium bromide , and visualized under a uv transilluminator . after successful amplification of the target dna sequences , fragments were purified using a gel extraction kit ( roche , germany ) following the recommendations of the supplier . ligation was performed with plasmid vector ptz57r / t ( fermentas , germany ) in 0.165 g , 0.18 pmol ends and 0.54 pmol ends purified pcr fragment in 1 ligation buffer , polyethylene glycol ( peg 4000 ) , 5 units of t4 dna ligase , and up to 30 l of deionized water at 22 for 16 h. the ligated products were used to transform chemically competent cells ( xl1-blue cells ) and white colonies grown on lb plates were randomly selected . the cloned pcr products were purified using plasmid purification kit ( roche diagnostics , germany ) according to the manufacturer 's instructions and sequencing was carried out in both directions using a t7 promoter primer ( mwg biotech , germany ) . the published sequences of 66 fmdv type a isolates recovered from different parts of the world were included in this analysis and compared to the corresponding sequence of the a / iran87 isolate . reference fmdv sequences were obtained from the national center for biotechnology information ( ncbi , usa ) . the sequences used for each gene were first examined to exclude ambiguous sequences which were incomplete or frame - shifted . to determine the degree to which genetic diversity was observed in the vp1 and 3d proteins , multiple alignments and comparisons of the predicted amino acid sequences of isolates were carried out ( fig . nucleotide sequence homology / divergence was calculated using the megalign project of the dnastar software package ( ver 5.1 ; dnastar , usa ; data not shown ) . the histories of the type a fmdv field isolates , including year of isolation , accession number , and geographical distribution , are presented in table 1 . the nucleotide sequences of the vp1 and 3d coding regions of a / iran87 have been submitted to genbank ( accession no . genetic comparison of the field isolates to the vaccine strain is of significant importance for testing the usefulness of the existing vaccine strain as well as selecting new vaccine strain(s ) . during virus circulation , amino acid substitutions accumulate at the different positions of the protein sequence . in the present study , a highly passaged field isolate of fmdv ( a / iran87 ) named mardabad or a87 isolate was selected for genetic analysis . sequences of the protein coding regions ( vp1 and 3d ) from the high - passage cell - adapted vaccine strain were determined by pcr amplification and sequencing . comparison of the amino acid sequences revealed amino acid changes in both structural and non - structural proteins . the number of sequence differences exhibited by each of the isolates showed that a / iran87 contains four amino acid substitutions at positions 17 , 26 , 50 , and 57 in the 3d coding region ( fig . 1 ) . there is clear evidence of novel amino acid substitutions ( alaser , aspglu , gluasn , and alagly ) in two domains of the 3d protein . comparison of the vp1 protein among different variants showed that a - iran - vaccinal contained a change at position 179 . 1a shows that amino acid changes among the vp1 genes of the field isolates consist of numerous nucleotide substitutions and deletions compared to the consensus sequence . comparison of the predicted amino acid sequences of vp1 region in the field isolates revealed amino acid deletions in three iranian vaccine isolates ( a - iran - vaccinal , a iran04 , and a / iran87 ) . as shown in fig . 1a , deletions occurred within the 13 amino acid positions ( 168 to 180 ) of the vp1 region . three - dimensional analysis of 3d protein showed that the aspglu substitution occurred in a beta sheet located in a small groove of the protein . the variations were not distributed uniformly along the genes ; there were areas of high and low incidence of nucleotide sequence variation ( data not shown ) . the region of the vp1 gene between amino acids 157 and 183 contained a high degree of variation and encoded an important immunogenic site on the viral surface . consequently , nucleotide changes in this region are most likely involved in the appearance of new antigenic variants . the vp1 and 3d nucleotide sequences of the selected fmdv - a subtypes isolated from outbreaks in iran and other countries were used to construct a fmdv - a vp1 and 3d - based sequence similarity tree . 2 shows a phylogenetic tree that was constructed based on the sequence alignment of 35 genomes of the vp1 region and 42 genomes in the 3d region which are distinctly divided into different lineages . as depicted in fig . 2a , the a / iran87 isolate clustered with a saudi arabian and five iranian isolates into a branch separate from other type a isolates . 2b demonstrates that a / iran87 and all isolates examined in the present study originated from different geographical areas and did not cluster in relatively similar lineages based on 3d sequences . all the field isolates shared comparatively lower homology ( 90~93% ) with the a / iran87 isolate in the 3d coding region ( data not shown ) . the topology of the phylogenetic tree indicated that there was no remarkable similarity between a / iran87 and all other isolates in the 3d coding region . space - filling and worm styles of the three - dimensional structures of the 3d and vp1 proteins from the a / iran87 isolate were observed . analysis of the 3d protein structure showed that the aspglu substitution occurred in a beta sheet located within a small groove of the protein . in order to examine the long amino acid deletion in the three - dimensional structure of the vp1 protein , homology - based modeling of the protein domains it indicated that , 13 amino acid deletions in the vp1 protein of a / iran87 isolate caused a change in three - dimensional structure of protein in the g - h loop region . detailed knowledge of the molecular characteristics of the major fmdv immunogenic components would be useful for monitoring various processes like evolution , genetic diversity , and virus origin , and for developing protective vaccines . among the fmdv serotypes , fmdv variants also appear during continuous infection of animals on farms or proliferation in cell cultures . furthermore , genetic diversification among fmdv subtypes can be caused by events occurring during recombination . along with above mechanisms , there is continuous co - circulation of multiple genotypes of fmdv type a which may lead to recombination ; this progresses gradually between closely related isolates when multiple viral genotypes exchange of different genomic regions between fmdvs by recombination is directly linked to fmdv diversification . recombination , particularly in the structural protein - coding region , may offer selective advantages to the virus . this can be great concern to areas where multiple fmdv genotypes co - circulate . the presence of extensive antigenic variation among different fmdv isolates can hinder vaccination against fmdv . genetic polymorphisms amongst different type a subtypes demonstrated that four nucleotides in the 3d coding region of isolate a / iran87 ( 49 , 78 , 148 , and 170 ) were changed from g to t , c to a , g to a , and c to g , respectively , resulting in amino acid substitutions ala17ser , asp26glu , glu50asn , and ala57gly , respectively . in contrast to the a / iran87 isolate , the remaining 41 isolates showed no corresponding changes at these positions . analysis of the sequence data revealed that the a / iran87 isolate also contained 30 nucleotide deletions in region 501~540 of the vp1 nucleotide sequence , which led to long amino acid deletions ( rgdlgslaarvaa ) in the g - h loop . it should be noted that the rgd sequence was deleted from the g - h loop . the three - dimensional and antigenic structures of many different serotypes of the fmdv have been examined . in spite of losing the rgd sequence and acquiring a asp26glu substitution in a beta sheet located in a small groove of the 3d protein , the virus grew in a bhk-21 suspension cell culture and exerted cytopathic effects after 16~18 h. since this strain is used as a vaccine strain , it may be inferred that the rgd does not have a key role in the virus binding to cells during infection . was found to be deleted , such a natural deletion in vp1 gene of fmdv is a novel phenomenon and has not been previously reported in compared isolates . the vp1 capsid protein contains a mobile loop between the g and h strands on the virus surface ; this protein not only contains the major immunodominant epitopes of the virion but also a highly conserved rgd amino acid sequence motif . several studies using different approaches have indicated that naturally developed fmdv isolates attach to cells via the highly conserved rgd motif . fmdvs have been shown to use multiple rgd - dependent integrins of the v subgroup to initiate infection ; these include v3 , v6 , v1 , and v8 . on the other hand , these viruses are capable of entering cells via non - integrin pathways . tissue culture - adapted fmdvs are able to utilize heparan sulfate ( a glycosaminoglycan found on the cell surface ) as a receptor for entering the cells . it has recently been found that fmdv can utilize unknown receptors except for integrin and heparan sulfate . this indicates the presence of other receptors and possible alternative mechanisms for viral particle entrance into the cells . this conclusion is also supported by the finding that destruction of the g-h loop rgd motif by site - directed mutagenesis created viruses which were able to replicate and assemble , but were not capable of initiating further infection cycles because of their inability to interact with cell receptors . previous findings from studies of synthetic peptides suggested that the carboxy - terminus of the vp1 protein situated in the vicinity of the rgd motif is required for rgd - mediated cell binding . the g - h loop of the vp1 protein on the viral particle surface is considered to be the dominant epitope for several fmdv types and the most variable part of the particle . antibody - based responses against the loop have the ability to neutralize fmdvs in vitro and are protective in vivo . for example , replacing the whole or partial g - h loop leads to the doubling of antibody titers against type a virus . new findings in this field are in complete agreement with a previous report documenting that the only mutation which did not increase heterologous responses was deletion of the rgd - motif ( 3a ) . in another study , the rgd sequence was deleted from a genetically engineered fmdv ( type a12 ) , thereby rendering the virus particles incapable of binding to cell surfaces . protective immunity in cattle has been obtained by utilizing particles from which the receptor binding site was deleted as a vaccine , revealing that this deletion has no significant effect on the antigenic properties of viruses with the rgd sequence . many reports have suggested that fmdv subtypes can be obtained in the field by immune selection during outbreaks of fmdv which have not been effectively neutralized in animal populations with incomplete immunization . thus , key changes in protein sequences that give rise to differences in immunogenicity and virulence can be seen among viruses isolated from partially immunized animals . in addition , it is possible that other significant changes take place when the virus moves from one host cell type to another . for instance , this may occur when fmdv spreads from one species to another ( from cattle to swine and then back to cattle ) or when fmdv cultured in bhk cells infects animal herds . for a number of years , it has been observed that fmdv easily adapts to many different tissue culture cell types . a recent report on a single passage of subtype a12 ( avr1 ) in bhk cells showed that some viral variants selected from this passage contained amino acid changes at residues 148 and 153 of the vp1 , including ones that have a limited capability for eliciting cross - neutralizing antibodies . the rapid generation of immunogenically distinguishable variants among fmdv subtype a viruses may affect the large - scale production of vaccines in bhk cells . adaptation for growth in tissue culture can be used to select viruses that have antigenic and cell attachment site(s ) different from those of the parental virus . amino acid residues 134~158 form a g-h loop structure which contains the main fmdv immunogenic epitope . in fmdv type c , mutation of residues 138~140 and 148~150 in the vp1 region has been shown to affect fmdv antigenicity . mutation frequency in the vp1 gene sequence of the fmdv has been estimated to be 1.6 10~6.4 10 substitutions for each nucleotide per year . g - h loop - specific antibody responses are known to play a major role in immunity induced by the current fmdv vaccines . considering the high sequence variability of this loop within and among the serotypes , it will be valuable to direct immune responses against other protective epitopes that are not as variable . residues 140~160 of vp1 have been previously shown to induce neutralizing antibodies against fmdv types o and a . due to a novel deletion of the 13 amino acid region including the rgd motif in the vpi capsid protein of a / iran87 , the effect of this motif in receptor binding and cell infection must be determined . this protein was initially called the fmdv infection - associated antigen since antibodies to this antigen are be detected in serum from recovery animals . the 3d position is located at the c - terminus of the polypeptide next to the 3c gene product . the 3d coding region produces a 470-amino acid protein with a molecular weight of ~55 kda . the pivotal role of 3d in the viral replication process is attributed to the fact that the 3d gene is highly conserved , particularly in the functional motifs . since rna polymerase is necessary for viral replication , the degree of sequence identity among the 3d genes is much greater than that of vp1 gene sequences . a wealth of three - dimensional structural information is currently available for a large number of rna - dependent rna polymerases from different families of positive- and double - stranded rna viruses , including three different members of the picornaviridae family . considering the fact that this strain can infect bhk-21 cells and has been used as a vaccine strain for nearly two decades , it may be concluded that deletion of rgd has no critical role in virus binding to cells during the initiation of infection . the findings of the current study demonstrated that the cell - adapted vaccine strain a / iran87 containing 13-amino acid deletions in the vp1 protein can be considered as a novel variant of fmdv type - a .

the nucleotide sequence of the vp1 ( 1d ) and partial 3d polymerase ( 3dpol ) coding regions of the foot and mouth disease virus ( fmdv ) vaccine strain a / iran87 , a highly passaged isolate ( ~150 passages ) , was determined and aligned with previously published fmdv serotype a sequences . overall analysis of the amino acid substitutions revealed that the partial 3dpol coding region contained four amino acid alterations . amino acid sequence comparison of the vp1 coding region of the field isolates revealed deletions in the highly passaged iranian isolate ( a / iran87 ) . the prominent g - h loop of the fmdv vp1 protein contains the conserved arginine - glycine - aspartic acid ( rgd ) tripeptide , which is a well - known ligand for a specific cell surface integrin . despite losing the rgd sequence of the vp1 protein and an asp26glu substitution in a beta sheet located within a small groove of the 3dpol protein , the virus grew in bhk 21 suspension cell cultures . since this strain has been used as a vaccine strain , it may be inferred that the rgd deletion has no critical role in virus attachment to the cell during the initiation of infection . it is probable that this fmdv subtype can utilize other pathways for cell attachment .

an age - group stratified ( 1824 , 2534 , 3544 , 4554 , 5564 , 6574 , and 7584 y ) geographically representative random sample of 2,604 swedish inhabitants with an equal number of women and men was selected by swedish national tax agency in 2007 . the respondents were divided into equally large summer and autumn groups to cover the possible seasonal variation in f&v consumption . a questionnaire enclosed with an invitation letter the respondents were also given a web - based questionnaire as an alternative measure ; this was done to encourage respondents who find a traditional pen - and - paper questionnaire too time - consuming to complete the survey . as compensation , the respondents could receive a short report of the main results of the study , if they wished . a few weeks after the first circular , a reminder letter was sent to the non - responders . to increase the response rate , a second reminder was sent to all non - respondents in january 2008 . for a detailed picture of the study design , the returned questionnaires were treated confidentially , the respondents gave their informed consents , and the swedish research council 's guidelines for good research practice ( 30 ) were followed in conducting the present study . detailed information regarding consumption of different kinds of f&v was collected using a 13-item self - administered 24-h recall and a 12-item food frequency questionnaire ( ffq ) . respondents were asked to report consumed amounts either in deciliters or number of portions in the self - administered 24-h recall ( e.g. did you eat apples yesterday and , if yes , how many? ) . the ffq included eight response categories for each item ( never, less than once / week , once / week, 24 times / week, 56 times / week, once / day, two times / day, and more than twice a day ) , indicating the average consumption frequencies for salad / raw and cooked vegetables as well as for fresh fruit and fruit juice ( e.g. how often do you usually eat fresh fruit? ) . both the ffq and the self - administered 24-h recall were based on a validated questionnaire where no differences were observed between the 24-h recall and one - day weighed food record of f&v ( 31 ) . in addition , the ffq showed a correlation coefficient of 0.73 for total f&v with a seven - day food record ( 31 ) . the present questionnaire was also pre - tested on 20 swedish adults , and one focus group interview ( n=5 ) was held in which respondents discussed f&v . mean daily consumption of different types of fruits , vegetables , as well as total consumption ( including berries and juice , excluding potatoes ) on a population - level was calculated from the self - administered 24-h recall . data on different sociodemographic variables ( gender , age , household size , country of birth , and level of education ) are presented in table 1 . level of pa was measured by asking how many hours per week the respondents engage in exercise that causes them to perspire or become breathless . respondent characteristics and fruit and vegetable consumption ( portions / day ) according to the self - administered 24-h recall note : groups with significantly ( p0.01 ) higher consumption with bolded figures . tukey hsd post test : 0.5 h vs. 1.12.5 h , p<0.001 ; 0.5 h vs. > 2.5 h , p<0.001 . the analyses were undertaken separately for women and men as well as for different demographic groups . means and 99% confidence intervals [ 99% cis ] were calculated for daily f&v consumption ( portions / day ) . respondents reporting unusually high consumption of > 15 f&v portions / day were excluded from the analyses ( five men and five women ) . two - sample t - tests and one - way anova were used to determine whether mean f&v consumption from the self - administered 24-h recall differed between subgroups . where needed based on normality tests and regarding the large sample size , the variable was used in its original form . mann whitney test was used to analyze consumption data from the ffq . due to the large number of significance tests conducted , a p - value < 0.01 ( two - sided ) was considered statistically significant throughout the study . detailed information regarding consumption of different kinds of f&v was collected using a 13-item self - administered 24-h recall and a 12-item food frequency questionnaire ( ffq ) . respondents were asked to report consumed amounts either in deciliters or number of portions in the self - administered 24-h recall ( e.g. did you eat apples yesterday and , if yes , how many? ) . the ffq included eight response categories for each item ( never, less than once / week , once / week, 24 times / week, 56 times / week, once / day, two times / day, and more than twice a day ) , indicating the average consumption frequencies for salad / raw and cooked vegetables as well as for fresh fruit and fruit juice ( e.g. how often do you usually eat fresh fruit? ) . both the ffq and the self - administered 24-h recall were based on a validated questionnaire where no differences were observed between the 24-h recall and one - day weighed food record of f&v ( 31 ) . in addition , the ffq showed a correlation coefficient of 0.73 for total f&v with a seven - day food record ( 31 ) . the present questionnaire was also pre - tested on 20 swedish adults , and one focus group interview ( n=5 ) was held in which respondents discussed f&v . mean daily consumption of different types of fruits , vegetables , as well as total consumption ( including berries and juice , excluding potatoes ) on a population - level was calculated from the self - administered 24-h recall . data on different sociodemographic variables ( gender , age , household size , country of birth , and level of education ) are presented in table 1 . level of pa was measured by asking how many hours per week the respondents engage in exercise that causes them to perspire or become breathless . respondent characteristics and fruit and vegetable consumption ( portions / day ) according to the self - administered 24-h recall note : groups with significantly ( p0.01 ) higher consumption with bolded figures . tukey hsd post test : 0.5 h vs. 1.12.5 h , p<0.001 ; 0.5 h vs. > 2.5 h , p<0.001 . the analyses were undertaken separately for women and men as well as for different demographic groups . means and 99% confidence intervals [ 99% cis ] were calculated for daily f&v consumption ( portions / day ) . respondents reporting unusually high consumption of > 15 f&v portions / day were excluded from the analyses ( five men and five women ) . two - sample t - tests and one - way anova were used to determine whether mean f&v consumption from the self - administered 24-h recall differed between subgroups . where needed based on normality tests and regarding the large sample size , the variable was used in its original form . whitney test was used to analyze consumption data from the ffq . due to the large number of significance tests conducted , a p - value < 0.01 ( two - sided ) was considered statistically significant throughout the study . a total of 1,304 questionnaires were returned and properly filled out , giving a response rate of 51% . approximately one - third of the respondents chose the web - based questionnaire . roughly one - third ( 37% ) had a university - level education , and 49% had been employed during the past six months . about 12% of the respondents were born outside sweden , compared to roughly 17% in the total swedish population ( 33 ) . sociodemographic characteristics of the respondents and total f&v consumption in each subgroup are presented in table 1 . according to the self - administered 24-h recall , total f&v consumption was 2.2 ( 99% ci 2.02.4 ) portions per day in the lowest quartile among women ( n=187 ) and 1.9 ( 1.72.1 ) daily portions among men ( n=201 ) . the highest quartile of women ( n=159 ) consumed 9.9 ( 9.310.4 ) portions on the examined day compared to 9.4 ( 8.410.5 ) portions among men ( n=143 ) . mean number of total f&v portions varied from 4.9 to 5.8 among women and 4.2 to 5.0 among men in different age groups , but consumption did not differ statistically across age groups . men born in sweden had lower total f&v consumption than did men born in another country ( p=0.006 ) . women with 0.5 h self - reported pa / week had lower f&v consumption than those reporting 1 h pa / week ( p<0.001 ) . also among men , those reporting 0.5 h pa / week had lower f&v consumption than those reporting 2.5 h pa / week . consumption of different types of f&v , based on the self - administered 24-h recall , by gender and age group is presented in table 2 . fresh fruits , as well as raw vegetables and salad , were consumed in the greatest amounts , and more by women than by men ( p<0.001 ) . comparisons between women and men within each age group showed that 55- to 74-year - old women consumed more fruits in total ( p0.001 ) , and more fresh fruits ( p0.002 ) , than men . women between 55 and 64 years also had higher total vegetable consumption than men in the same age group ( p=0.002 ) . overall consumption of berries and cooked vegetables was relatively low , and no gender differences were shown . mean consumption of different fruit and vegetable types ( n of self - reported portions / day , [ 99% confidence interval ] ) by gender and age group ( 24-h recall ) note : p - values for gender differences within age group . the ffq also showed that fresh f&v were consumed most often ( table 3 ) . more women than men in all age groups consumed fresh fruit every day ( 63.5% vs. 38.9% for all age groups combined , respectively ; p<0.001 ) ) . almost one half of women ( 48.3% ) consumed salad or raw vegetables every day compared to 28.8% among men ( p<0.001 ) . men reported a higher consumption frequency only for canned fruit ( p=0.002 ) , although the overall consumption frequency was very low . percentages of daily users of different fruit and vegetable types and gender differences in f&v consumption frequencies within age groups ( food frequency questionnaire ) note : bolded figures are statistically significant differences . once / day + two times / day + more than twice a day . mann whitney test for gender differences in consumption frequencies ( eight categories as presented in methods ) . comparisons between women and men using the web - based or pen - and - paper questionnaire showed no differences in total f&v consumption . age differences between these two groups could not be seen , whereas women choosing the web - based questionnaire had higher level of education . according to the self - administered 24-h recall , total f&v consumption was 2.2 ( 99% ci 2.02.4 ) portions per day in the lowest quartile among women ( n=187 ) and 1.9 ( 1.72.1 ) daily portions among men ( n=201 ) . the highest quartile of women ( n=159 ) consumed 9.9 ( 9.310.4 ) portions on the examined day compared to 9.4 ( 8.410.5 ) portions among men ( n=143 ) . mean number of total f&v portions varied from 4.9 to 5.8 among women and 4.2 to 5.0 among men in different age groups , but consumption did not differ statistically across age groups . men born in sweden had lower total f&v consumption than did men born in another country ( p=0.006 ) . women with 0.5 h self - reported pa / week had lower f&v consumption than those reporting 1 h pa / week ( p<0.001 ) . also among men , those reporting 0.5 h pa / week had lower f&v consumption than those reporting 2.5 h pa / week . consumption of different types of f&v , based on the self - administered 24-h recall , by gender and age group is presented in table 2 . fresh fruits , as well as raw vegetables and salad , were consumed in the greatest amounts , and more by women than by men ( p<0.001 ) . comparisons between women and men within each age group showed that 55- to 74-year - old women consumed more fruits in total ( p0.001 ) , and more fresh fruits ( p0.002 ) , than men . women between 55 and 64 years also had higher total vegetable consumption than men in the same age group ( p=0.002 ) . overall consumption of berries and cooked vegetables was relatively low , and no gender differences were shown . mean consumption of different fruit and vegetable types ( n of self - reported portions / day , [ 99% confidence interval ] ) by gender and age group ( 24-h recall ) note : p - values for gender differences within age group . the ffq also showed that fresh f&v were consumed most often ( table 3 ) . more women than men in all age groups consumed fresh fruit every day ( 63.5% vs. 38.9% for all age groups combined , respectively ; p<0.001 ) ) . almost one half of women ( 48.3% ) consumed salad or raw vegetables every day compared to 28.8% among men ( p<0.001 ) . men reported a higher consumption frequency only for canned fruit ( p=0.002 ) , although the overall consumption frequency was very low . percentages of daily users of different fruit and vegetable types and gender differences in f&v consumption frequencies within age groups ( food frequency questionnaire ) note : bolded figures are statistically significant differences . once / day + two times / day + more than twice a day . mann whitney test for gender differences in consumption frequencies ( eight categories as presented in methods ) . comparisons between women and men using the web - based or pen - and - paper questionnaire showed no differences in total f&v consumption . age differences between these two groups could not be seen , whereas women choosing the web - based questionnaire had higher level of education . on average , respondents in the present study reported slightly higher f&v consumption than expected based on earlier findings in sweden ( 13 , 14 ) . some other recent studies ( 25 ) have also reported changes in dietary habits during the past decade . however , the methodology used in the present study differed from the latest national food intake survey ( 13 ) , and the results are not directly comparable with each others . in line with our findings , previous studies have also shown that men tend to have lower f&v consumption than women ( 1518 ) . a study from finland showed that gender differences in vegetable consumption have actually increased ( 34 ) . both the self - administered 24-h recall and ffq showed higher f&v consumption in women when all age groups were combined , while the data from the ffq showed larger differences between women and men within different age groups . earlier studies have shown that men slightly underestimate their f&v consumption , whereas the opposite applies to women ( 33 ) . thus , the gender differences revealed here may turn out to be slightly less significant , but the similar findings from two measures indicate that these differences actually exist . the self - administered 24-h recall showed higher consumption of fresh fruit particularly for middle - aged and older women , which is in line with results from a recent finnish study ( 18 ) . on the other hand , the ffq showed that women in many age groups were more frequent consumers of not only fruit , but also salad and other raw vegetables . f&v it seems that swedes are not used to including f&v in mixed dishes , but rather eat them separately as they are . on the other hand , the authors are aware of the limitations of the study questionnaire , where only one open - ended item on mixed dishes was included . this may have led to some underreporting , indicating that actual consumption may be higher than reported . the present study further showed some interesting differences between women and men in factors associated with f&v consumption . men born outside sweden had higher f&v consumption than men born in sweden did , which may reflect the different habits associated with other food cultures . for instance , non - western immigrants in denmark have been reported to have higher odds for daily consumption of cooked and raw vegetables ( 35 ) , and iranians living in sweden also show higher consumption of f&v ( 36 ) . however , because the specific country of birth is not reported in the present study , no conclusions regarding specific food cultures can be drawn . pa was the only variable of those investigated that was associated with total f&v consumption in both genders . therefore , we may speculate that f&v consumption is an integrated part of a lifestyle in which pa also plays an important role . however , the limitations of only using a single question to measure level of pa are recognized ( 25 ) , and more detailed assessments are encouraged to gain better understanding on the associations between pa and consumption of f&v . from a methodological point of view , the ffq may enable better prediction of average consumption over a longer time period , whereas the self - administered 24-h recall was used to more accurately assess consumption over a specified period . also , there is a certain risk of over - reporting actual consumption in the ffq , but this was not regarded as a problem when ranking individuals and subgroups according to their consumption ( 37 ) . in addition , although consumption has not been reported in grams , consumption frequency as such is a good measure of consumption , because portion sizes have not been reported to differ between high and low consumers ( 12 ) . we are aware of the possibility of a type i error for false significant findings ( 38 ) due to the many significance tests conducted . however , because the main findings are significant at a level well below 1% , and both dietary assessment measures showed similar findings , this is not regarded as a problem . in the present study , all days of the week were equally represented in the data ( data not shown ) , which eliminates the possible bias caused by consumption differences across the weekdays . seasonal consumption variation can also be excluded as a confounding factor , because the data consist of roughly equally large groups of participants in different seasonal groups . the aim was to show an average consumption of the population over one year and not to compare seasonal variation as such . the present results have to be interpreted with caution owing to the relatively large proportion of non - respondents ( 49% ) , although the drop - out rate was nearly as high in the latest nationwide dietary survey used as a reference here ( 13 ) . men in general , and the youngest age group ( 1824 years ) , but also the oldest women ( 7584 years ) were somewhat underrepresented in the present survey . however , the relatively high total number of respondents should be representative of actual f&v consumption levels in sweden , although in most cases , respondent selection in postal surveys probably leads to overrepresentation of people who are more interested in the topic , or who already have healthier dietary habits . the cross - sectional design limits conclusions about the temporal or causal nature of the findings , but the study design can provide answers that address the study aims . from a data collection perspective , the present study gives valuable experience in using a web - based questionnaire as an alternative response measure . giving respondents the opportunity to choose between a traditional pen - and - paper and a web - based questionnaire was intended to increase the response rate and attract particularly younger respondents , but age differences could not be identified in the present study ( data not shown ) . in accordance with danish experiences ( 39 ) , the majority of respondents preferred the paper version to the web - based questionnaire . however , using the internet may still be worthwhile , as it saves both environmental and labor resources . the present study showed average f&v consumption close to the recommended level of five portions per day . fresh f&v showed the highest consumption , whereas berries and processed f&v products were less popular . university education was positively associated with higher f&v consumption for women , and men born outside sweden showed higher consumption than did men born in sweden . respondents who reported more hours per week engaged in doing pa had higher f&v consumption in both genders . although total consumption seems to have increased , the role of other underlying factors besides the sociodemographic factors and pa studied here remains unclear . in addition , other perspectives on f&v consumption , such as possible seasonal variation in availability of different f&v types , should be taken into consideration when implementing new dietary interventions or guidelines . the authors have not received any funding or benefits from industry to conduct this study .

backgroundfruit and vegetables ( f&v ) are strongly associated with health . the latest swedish national dietary survey from a decade ago showed that consumption of f&v was below recommended levels . however , current consumption in different subgroups is not well known.objectiveto investigate the consumption of various f&v types in swedish adults grouped according to sociodemographic factors and self - reported physical activity ( pa).designa cross - sectional survey using a quantitative pen - and - paper or web - based questionnaire in a population - based random sample of adults 1884 years ( final n=1,304 ; 51% ) . a self - administered 24-h recall and a food frequency questionnaire ( ffq ) were used to measure f&v consumption . data on gender , age , education level , country of birth , and pa ( hours / week ) were included as grouping variables . besides descriptive data , two - sample t - tests and non - parametric tests were performed . a p - value < 0.01 was regarded as significant.resultsmean f&v consumption based on the self - administered 24-h recall was close to the recommended five portions / day : 5.4 ( 99% ci 5.15.6 ) portions / day among women and 4.7 ( 4.45.0 ) portions / day among men ( p<0.001 ) . also the ffq showed that women generally consumed more f&v than men did . consumption was lowest among respondents with 0.5 h self - reported pa / week ( p0.001 ) , as well as among men born in sweden ( p=0.006 ) . f&v were consumed in almost equal amounts , and fresh f&v were most popular . intake of berries and cooked f&v was relatively low.conclusionthe present study shows a relatively high f&v consumption close to the recommended five portions per day . gender differences still exist . also pa and country of birth were significantly associated with f&v consumption .

osteoporosis is a disease characterized by the structural deterioration of bone and low bone mass . the world health organization defines osteoporosis as a bone mineral density ( bmd ) value of 2.5 standard deviations or more below the young adult mean.1 an estimated 10 million patients in the united states have osteoporosis , and its prevalence is expected to continue to rise in the world population.2 degenerative spondylolisthesis and spinal stenosis , as well as vertebral compression fractures , have been reported at an increased rate in patients with osteoporosis,3 and the disease requires special consideration for any older patient undergoing spine surgery . obtaining optimal fixation in the osteoporotic spine is technically challenging ; osteoporosis may also be associated with a poor fusion rate , with rates as low as 56% in elderly patients.4 however , for appropriately selected patients , the presence of osteoporosis is not a contraindication to surgical intervention . very little available literature exists specifically evaluating the clinical and radiographic outcomes in patients with osteoporosis undergoing transforaminal lumbar interbody fusion ( tlif ) . in fact , to our knowledge , no previous study has examined the effect that osteoporosis may have on rates of interbody cage subsidence , migration , pedicle screw loosening , or iatrogenic fractures in patients undergoing instrumented tlif . therefore , we sought to compare the radiographic and clinical outcomes of tlif in patients with and patients without osteoporosis . after institutional review board approval , we retrospectively reviewed all patients aged 50 years or older undergoing instrumented tlif for any indication at one of three institutions between july 2004 and june 2014 . only patients with immediate postoperative and follow - up radiographs and computed tomography ( ct ) scans available for evaluation of the interbody cage were included in the study . we excluded all patients without at least a 6-month follow - up ct scan . utilizing a technique similar to that described by lee et al,5 we measured ct hounsfield units ( hus ) on the lumbar vertebrae for all included patients using preoperative ct scans . patients were then stratified as either osteoporotic or nonosteoporotic based on average lumbar vertebral body hu values . the hu cutoff values were previously determined from a separate study at our institution and found to correlate with bmd obtained from dual - energy x - ray absorptiometry ( dexa ) scans ( p.m. formby et al , personal communication ) . patients with hu 112.4 were considered osteoporotic , and those with hu > 112.4 were considered nonosteoporotic . plain radiographs and ct scans were evaluated for evidence of implant subsidence , migration , interbody fusion , iatrogenic fracture , or loosening of posterior pedicle screw fixation . patient medical records were then reviewed for postoperative symptoms , which were further subdivided into persistence of symptoms or recurrence of symptoms . we used the student t test to evaluate continuous variables and mid - p exact tests for dichotomous data , and p values 0.05 were considered statistically significant . twenty - five ( 19.5% ) of these patients were designated as osteoporotic based on the postoperative ct hu measurements versus 103 ( 80.4% ) patients without osteoporosis . we excluded 40 patients who did not have at least a 6-month follow - up ct imaging , leaving 18 ( 20.5% ) patients with osteoporosis and 70 ( 79.5% ) patients without osteoporosis . these patients had a mean radiographic follow - up of 35.8 27.9 months . the mean age was significantly higher in the osteoporotic group ( 65.2 years ) versus the nonosteoporotic group ( 56.9 years , p < 0.0001 ) . males comprised 50 and 64.3% of the osteoporotic and nonosteoporotic patient groups , respectively , with a combined mean age of 58.5 7.9 years . we found no significant differences between the osteoporotic and nonosteoporotic groups with regard to sex ( p = 0.28 ) , body mass index ( 27.6 versus 29.9 , p = 0.12 ) , number of identified medical comorbidities ( 2.3 versus 1.8 , p = 0.15 ) , total number of vertebral levels fused ( 1.9 versus 1.8 , p = 0.56 ) , or fusion rate ( 83.3 versus 88.6% , p = 0.56 ) . there were no statistically significant differences between groups for gross implant migration in the coronal or sagittal planes ( 11.1 versus 1.4% , p = 0.11 ) , pedicle screw loosening ( 22.2 versus 8.6% , p = 0.14 ) , revision surgery ( 16.6 versus 14.3% , p = 0.78 ) , or postoperative symptoms ( 44.4 versus 50.0% , p = 0.69 ; tables 1 , 2 ) . there were no statistically significant differences between the osteoporotic and nonosteoporotic groups in persistent or recurrent symptoms , though the rate of recurrence in the nonosteoporotic group was three times higher ( persistent symptoms : 38.9 versus 30.0% , p = 0.47 , respectively ; recurrent symptoms : 5.6 versus 18.6% , p = 0.18 , respectively ) . abbreviations : bmi , body mass index ; ca / vit d , calcium / vitamin d ; dexa , dual - energy x - ray absorptiometry ; hu , hounsfield units . note : the only significant differences between the osteoporotic and nonosteoporotic groups were for mean patient age and hu measurements . note : the rates of interbody cage subsidence , iatrogenic fracture , and overall complication rates were higher in the osteoporotic group . significantly higher rates of subsidence ( 5.6 2.4 [ 72.2% ] versus 5.2 1.9 [ 45.7% ] , p = 0.05 ) and iatrogenic fractures ( 16.7 versus 1.4% , overall , the osteoporotic population had significantly higher radiographic complication rates compared with patients without osteoporosis ( 77.8 versus 48.6% , p = 0.03 ; table 2 ) . as the average life expectancy continues to increase in the united states , spine surgeons should expect to manage a greater proportion of elderly patients with degenerative spinal conditions . concomitant with increasing age is an associated increase in the prevalence of osteopenia and osteoporosis . in a study of patients over the age of 50 years undergoing spinal surgery in south korea , chin et al found osteopenia and osteoporosis rates of 41.4 and 51.3% , respectively , based on dexa.3 despite the growing population of elderly patients , however , few studies have evaluated the role that low lumbar bmd may have in the complication rates and overall outcomes of patients undergoing lumbar interbody fusion . we found that a minority of patients ( 21.5% ) undergoing tlif had any dexa information available at the time of surgery , despite advancing age and clinical risk factors for osteoporosis . utilizing hu measurements to assess lumbar vertebral body bmd , we found that of the patients with hu values consistent with osteoporosis , only 27.8% had any workup for osteoporosis prior to or following their tlif . this finding is concerning , particularly because we found a higher overall radiographic complication rate in patients with osteoporosis undergoing tlif , and it suggests that patients are undergoing spinal fusion surgery without being appropriately evaluated for low bmd . although there were few patients with osteoporosis in this study , we found an increased incidence and amount of cage subsidence ( fig . 1 ) , a higher rate of iatrogenic fractures , and more overall radiographic complications in patients with osteoporosis . though our data did not reach statistical significance , we also found a greater propensity for implant migration and posterior pedicle screw loosening . ( a ) immediate postoperative and ( b ) 2-year follow - up sagittal computed tomography ( ct ) scans of a 53-year - old man who underwent l4l5 transforaminal lumbar interbody fusion . the patient had hounsfield unit ( hu ) measurements on immediate postoperative ct scan consistent with osteoporosis ( average l4 hu = 105.7 ) . interbody cage subsidence into the superior end plate of l5 with interspace collapse is evident ( 9.9 mm ) , with evidence of increased fusion mass in the anterior column . however , at 26-month clinical follow - up , he had no recurrence of symptoms . however , it is important to note that the increased overall radiographic complication rate did not necessarily portend worse clinical outcomes for patients with osteoporosis . patients with osteoporosis and patients without osteoporosis underwent similar rates of revision surgery ( 16.7 versus 14.3% , respectively ) , and patients without osteoporosis actually had a higher prevalence of postoperative symptoms following tlif , particularly when stratifying the constellation of symptoms based on persistence versus subsequent recurrence of symptoms : we found that the rate of recurrent symptoms was higher in the nonosteoporotic group ( 18.6 versus 5.6% , respectively ) , although this finding also did not reach statistical significance . it is unclear , however , why this trend was observed , and it may be related to the fact that we only included patients with six - month or later postoperative ct scans in our study , which may have biased our clinical data with an increased proportion of patients with postoperative symptoms . despite a higher interbody cage subsidence rate in the osteoporotic group , which in turn would theoretically lead to loss of foraminal height and eventual recurrence of radiculopathy , there did not appear to be any association between osteoporosis and recurrence of symptoms . we are unaware of any other studies specifically evaluating postoperative radiographic complications in patients with osteoporosis undergoing tlif . previous studies have drawn conflicting conclusions regarding whether elderly patients are at increased risk for postoperative complications following lumbar fusion . carreon et al found a 79.6% rate of postoperative complications in patients 65 years old at the time of posterior lumbar instrumented fusion.6 however , cassinelli et al found a much lower rate of major complications ( 3% ) and minor complications ( 31% ) in a similar cohort of patients 65 years undergoing instrumented and noninstrumented lumbar spine arthrodesis.7 similarly , cavagna et al evaluated patients > 65 years undergoing instrumented lumbar fusions and found good clinical outcomes and no serious postoperative complications or reoperations in this cohort.8 however , they did find a 10.3% rate of implant failure ( two screws and two rods failed ) at 2-year or later follow - up . glassman et al investigated clinical outcomes of patients 65 years old compared with those < 65 years old undergoing single - level instrumented posterolateral lumbar arthrodesis ; the authors found that the older population had similar clinical and health - related quality - of - life improvements when compared with the younger cohort.9 however , the older cohort experienced significantly increased rates of serious ( 38 versus 17% ) and overall adverse postoperative events ( 56 versus 36% ) , although there was no increased risk of revision or reoperation between these groups . likewise , okuda et al did not find major differences in clinical outcomes between patients 70 years old and those < 70 years old undergoing instrumented lumbar interbody fusion.10 these authors found no increased risk or instrument failure between groups , although they did find lower rates of fusion and higher rates of collapsed union and delayed union in the elderly group.10 inherent to any small retrospective study , the possibility of type ii statistical error limits the generalizability of our analysis . the proportion of patients with osteoporosis in our study was low , which could be a source of bias . our study was also limited by the quality of the medical records , as well as available radiographs and ct scans . in addition , despite a minimum age of 50 years for inclusion in this study , our patients were still relatively young and healthy , with few comorbidities and a mean age of only 58.5 years . we looked at a heterogeneous group of patients with disparate preoperative diagnoses undergoing tlif for any reason , which could contribute to bias in this study . the use of hu for measurement of bone density has not been validated in the literature , with few reports of its use to date . further prospective studies are needed to evaluate the role that osteoporosis plays in complication rates and overall clinical outcomes in patients undergoing lumbar fusion surgery . in conclusion , we found significantly increased rates of cage subsidence , iatrogenic fractures , and overall radiographic complications in patients with osteoporosis undergoing tlif . however , these radiographic complications did not predispose patients with osteoporosis to worse clinical outcomes , and larger , prospective studies are needed to further evaluate lumbar fusion surgery in patients with osteoporosis .

study design retrospective review . objective to compare clinical outcomes after transforaminal lumbar interbody fusion ( tlif ) in patients with and patients without osteoporosis . methods we reviewed all patients with 6-month postoperative radiographs and computed tomography ( ct ) scans for evaluation of the interbody cage . ct hounsfield unit ( hu ) measurements of the instrumented vertebral body were used to determine whether patients had osteoporosis . radiographs and ct scans were evaluated for evidence of implant subsidence , migration , interbody fusion , iatrogenic fracture , or loosening of posterior pedicle screw fixation . medical records were reviewed for persistence of symptoms or recurrence of symptoms . results the final data analysis included 18 ( 20.5% ) patients with osteoporosis and 70 ( 79.5% ) patients without osteoporosis . males comprised 50% of patients with osteoporosis , and 64.3% of patients without osteoporosis . the mean age was significantly higher in the osteoporotic group ( 65.2 years ) versus the nonosteoporotic group ( 56.9 years ; p < 0.0001 ) . we found significantly higher rates of subsidence ( 72.2 versus 45.7% , p = 0.05 ) and iatrogenic fractures ( 16.7% versus 1.4% , p = 0.03 ) in the osteoporotic group . in addition , the osteoporotic group had significantly higher radiographic complication rates compared with the nonosteoporotic group ( 77.8 versus 48.6% , p = 0.03 ) . there was no difference between groups for revision surgery ( 16.6 versus 14.3% , p = 0.78 ) or postoperative symptoms ( 44.4% versus 50.0% , p = 0.69 ) . conclusions our data demonstrated significantly increased rates of cage subsidence , iatrogenic fracture , and overall radiographic complications in patients with osteoporosis . however , these radiographic complications did not predispose patients with osteoporosis to an increased risk of surgical revision or worse clinical outcomes .

in recent years , morbidity and mortality of nosocomial infections have increased , at least partly due to the increased frequency of antibiotic resistance in both gram - positive and gram - negative organisms . in the past , gram - negative aerobes were the most frequently reported pathogens , but staphylococcus aureus has been increasing in frequency , particularly methicillin - resistant s. aureus ( mrsa ) in intensive care units.1 over 70% of s. aureus isolates are resistant to methicillin in the middle east and with > 85% of s. aureus pneumonia cases being mrsa pneumonia in the arabian peninsula.2 in saudi arabia , 76% of health - care workers have tested positive for mrsa colonization despite being asymptomatic.3 on a separate note , a systematic review on the epidemiology of s. aureus in the middle east indicates high incidence of community - acquired mrsa carried by healthy individuals , with positive mrsa samples obtained from random wound , throat , and nasal specimens.4 glycopeptide antibiotics , vancomycin and teicoplanin , have been considered drugs of choice in treatment for infections due to suspected or documented mrsa.58 burgeoning glycopeptide prescribing is believed to have led to the emergence of vancomycin - resistant enterococci and , more recently , s. aureus with reduced glycopeptide susceptibility . treatment resistance of clinical mrsa has also been shown in this region against both -lactam and non--lactam antibiotics ( erythromycin , clindamycin , ciprofloxacin , and tetracycline ) , chloramphenicol , gentamicin , and sulfamethoxazole trimethoprim.4 even though there is a recent debate surrounding the use of vancomycin,911 it is still a common antibiotic for the treatment of nosocomial pneumonia and is recommended in the most recent guideline endorsed by the american thoracic society and the infectious disease society of america.1 despite guidelines , real - world practice patterns are known to vary substantially from those recommended . furthermore , little is known in low- and middle - income countries of the middle east regarding practice patterns and resource associated with mrsa pneumonia . the objectives of this study were to describe the microbiologic profile of mrsa pneumonia in lebanon and saudi arabia and assess the treatment received by these patients , in terms of drug selection , dosing , timing , and sensitivity of the infection pathogen , and to document hospital resource utilization and clinical outcomes among patients with this infection . this study is a retrospective , observational medical chart review that took place at a total of five study sites in saudi arabia ( two sites ) and lebanon ( three sites ) . per the study protocol , the study investigator ( local physician responsible for data collection ) assessed the need for ethics / irb application at each site , irb waiver or approval was completed at all sites based on local requirements . specifically , approval was obtained from national guard health affairs , king abdalaziz medical city- jeddah and riyadh , kingdom of saudi arabia ; ain wazein hospital irb ; and makassed general hospital , riad el - solh beirut , lebanon . in order to be included , patients had to have suspected or culture - proven mrsa pneumonia with appropriate respiratory symptoms . in the absence of culture results , suspected cases needed to have at least two of the following symptoms for inclusion : fever ( body temperature 38.5c ) , hypothermia ( body temperature 35.5c ) , respiratory rate ( > 30 breaths / min ) , systolic hypotension ( < 90 mmhg ) , heart rate ( > 120 beats / min ) , elevated peripheral white blood cell count ( > 10,000/mm ) , leukopenia with total white blood cell count ( < 4,500 cells / mm ) , and elevated inflammatory markers ( eg , procalcitonin or c - reactive protein ) . patients were excluded if they had inadequate data on the treatments and outcomes of interest or hospital resource utilization for the study , as judged by the investigators . data were collected from patient medical records by hospital - based infectious disease specialists , medical microbiologists , internal medicine specialists , surgeons , and intensivists . all data collection instruments were translated into local language and , prior to roll out , pilot tested to ensure relevance and clarity of data points to be collected . extracted chart data included demographics and comorbid conditions ; antibiotic treatment patterns , including initial and subsequent inpatient antibiotic regimens prescribed and antibiotic therapy prescribed at discharge ; health - care resource utilization , including diagnostic procedures , laboratory tests , and length of hospital stay ; and clinical outcomes , including clinical response at discharge and rehospitalization . pneumonia was classified into one of the four categories by the abstracting physician as follows : community - acquired pneumonia : symptom onset prior to hospital admission but with no known patient history of 1 ) another hospital visit , 2 ) visit to another health - care institution , or 3 ) residence in a nursing home within 90 days prior to the present hospital admissionhealth - care - associated pneumonia ( hcap ) : symptom onset during 1 ) another hospital visit , 2 ) visit to another health - care institution , or 3 ) residence in a nursing home within 90 days prior to the present hospital admissionnosocomial pneumonia : symptom onset at least 48 hours after hospital admissionunknown community - acquired pneumonia : symptom onset prior to hospital admission but with no known patient history of 1 ) another hospital visit , 2 ) visit to another health - care institution , or 3 ) residence in a nursing home within 90 days prior to the present hospital admission health - care - associated pneumonia ( hcap ) : symptom onset during 1 ) another hospital visit , 2 ) visit to another health - care institution , or 3 ) residence in a nursing home within 90 days prior to the present hospital admission nosocomial pneumonia : symptom onset at least 48 hours after hospital admission antibiotics received during hospitalization were categorized as mrsa active and not mrsa active . mrsa - active antibiotics as defined by guidelines included vancomycin ( intravenous only ) , teicoplanin ( intravenous only ) , clindamycin , doxycycline , trimethoprim / sulfamethoxazole , linezolid , fusidic acid , rifampin , and tigecycline.12 antibiotics categorized as not mrsa active included beta - lactams ( by definition ) , and in the absence of institution - specific antibiograms , fluoroquinolones and carbapenems were only categorized as mrsa active if culture results indicated sensitivity to the respective antibiotic . unless otherwise listed , binary and categorical end points were summarized in terms of percentages in each category , and continuous variables were presented using number of observations ( when less than the entire sample ) , arithmetic mean , and standard deviation ( sd ) . this study is a retrospective , observational medical chart review that took place at a total of five study sites in saudi arabia ( two sites ) and lebanon ( three sites ) . per the study protocol , the study investigator ( local physician responsible for data collection ) assessed the need for ethics / irb application at each site , irb waiver or approval was completed at all sites based on local requirements . specifically , approval was obtained from national guard health affairs , king abdalaziz medical city- jeddah and riyadh , kingdom of saudi arabia ; ain wazein hospital irb ; and makassed general hospital , riad el - solh beirut , lebanon . in order to be included , patients had to have suspected or culture - proven mrsa pneumonia with appropriate respiratory symptoms . in the absence of culture results , suspected cases needed to have at least two of the following symptoms for inclusion : fever ( body temperature 38.5c ) , hypothermia ( body temperature 35.5c ) , respiratory rate ( > 30 breaths / min ) , systolic hypotension ( < 90 mmhg ) , heart rate ( > 120 beats / min ) , elevated peripheral white blood cell count ( > 10,000/mm ) , leukopenia with total white blood cell count ( < 4,500 cells / mm ) , and elevated inflammatory markers ( eg , procalcitonin or c - reactive protein ) . patients were excluded if they had inadequate data on the treatments and outcomes of interest or hospital resource utilization for the study , as judged by the investigators . data were collected from patient medical records by hospital - based infectious disease specialists , medical microbiologists , internal medicine specialists , surgeons , and intensivists . all data collection instruments were translated into local language and , prior to roll out , pilot tested to ensure relevance and clarity of data points to be collected . extracted chart data included demographics and comorbid conditions ; antibiotic treatment patterns , including initial and subsequent inpatient antibiotic regimens prescribed and antibiotic therapy prescribed at discharge ; health - care resource utilization , including diagnostic procedures , laboratory tests , and length of hospital stay ; and clinical outcomes , including clinical response at discharge and rehospitalization . pneumonia was classified into one of the four categories by the abstracting physician as follows : community - acquired pneumonia : symptom onset prior to hospital admission but with no known patient history of 1 ) another hospital visit , 2 ) visit to another health - care institution , or 3 ) residence in a nursing home within 90 days prior to the present hospital admissionhealth - care - associated pneumonia ( hcap ) : symptom onset during 1 ) another hospital visit , 2 ) visit to another health - care institution , or 3 ) residence in a nursing home within 90 days prior to the present hospital admissionnosocomial pneumonia : symptom onset at least 48 hours after hospital admissionunknown community - acquired pneumonia : symptom onset prior to hospital admission but with no known patient history of 1 ) another hospital visit , 2 ) visit to another health - care institution , or 3 ) residence in a nursing home within 90 days prior to the present hospital admission health - care - associated pneumonia ( hcap ) : symptom onset during 1 ) another hospital visit , 2 ) visit to another health - care institution , or 3 ) residence in a nursing home within 90 days prior to the present hospital admission nosocomial pneumonia : symptom onset at least 48 hours after hospital admission antibiotics received during hospitalization were categorized as mrsa active and not mrsa active . mrsa - active antibiotics as defined by guidelines included vancomycin ( intravenous only ) , teicoplanin ( intravenous only ) , clindamycin , doxycycline , trimethoprim / sulfamethoxazole , linezolid , fusidic acid , rifampin , and tigecycline.12 antibiotics categorized as not mrsa active included beta - lactams ( by definition ) , and in the absence of institution - specific antibiograms , fluoroquinolones and carbapenems were only categorized as mrsa active if culture results indicated sensitivity to the respective antibiotic . unless otherwise listed , binary and categorical end points were summarized in terms of percentages in each category , and continuous variables were presented using number of observations ( when less than the entire sample ) , arithmetic mean , and standard deviation ( sd ) . chart - level data were collected for 93 patients with mrsa pneumonia , 50 in saudi arabia and 43 in lebanon . of the 93 patients in the sample , 20% of the mrsa infections were community acquired , 32% health - care associated , 35% nosocomial , and 12% of unknown origin . however , the most common comorbidities among these patients were diabetes ( 39% ) and congestive heart failure ( 30% ) . ninety of the 93 patients in the sample ( 97% ) had at least one culture taken , and 82 of these ( 91% ) had at least one culture positive for mrsa . as would be expected , only one - third of the isolates tested were sensitive to ciprofloxacin and only 15% were sensitive to beta - lactams . patients most frequently had positive cultures from pulmonary ( 87% ) and blood ( 27% ) samples ; nasal sites were also culture positive in 16% of patients . table 2 shows the susceptibility profile for mrsa isolates from patients with pneumonia . all isolates tested were sensitive to vancomycin ( n=80 ) and teicoplanin ( n=32 ) . only ten isolates were tested for linezolid sensitivity , and all were found to be sensitive . drug selection for mrsa pneumonia , expressed as the percentage of patients receiving each antibiotic ( with potential for multiple antibiotics per line of therapy ) , is summarized in figure 1 . overall , 79 patients ( 85% ) received at least one antibiotic considered to be mrsa active . the 93 mrsa pneumonia patients received a total of 140 antibiotic orders designated by study investigators to be first - line therapy ( as some regimens consisted of multiple antibiotics ) . of these , 28% were beta - lactams ( thus representing inactive therapy ) , and the most frequently prescribed mrsa - active antibiotic was vancomycin ( 22% of orders ) . among the 48 patients ( 52% ) who were prescribed a total of 66 second - line antibiotic orders , vancomycin remained the most commonly prescribed antibiotic ( 39% of orders ) , followed by teicoplanin ( 12% ) and linezolid ( 9% ) . of the eleven third - line drugs received by eight patients , linezolid was the most commonly used ( 36% of orders ) , followed by vancomycin ( 27% ) and ciprofloxacin ( 18% ) . overall , the mean total length of stay ( los ) was 32.4 days , with the majority ( 20.4 days ) spent in general wards ( figure 2 ) . thirty - six patients ( 39% ) required mechanical ventilation , for a mean sd of duration of 18.724.4 days per patient . four patients ( 4% ) required hemodialysis , for a mean of 9.8 11.9 days per patient . of these patients , two patients were reported to have moderate - to - severe renal disease on admission , but whether these patients were receiving dialysis prior to admission is unknown . other interventions required among patients with mrsa pneumonia included peripheral catheters ( 52% ) , central venous catheters ( 52% ) , and peripherally inserted central catheters ( 6% ) . over one - fourth of the patients were considered treatment failures at the time of discharge ( table 3 ) . the overall in - hospital mortality rate was 30% , with nearly one - third of deaths being mrsa related . of the 28 patients who died , treatment response at discharge was failure in 24 ( 86% ) cases , improved in one ( 3.5% ) , cure in two ( 7% ) , and indeterminate in one ( 3.5% ) . relapse of mrsa pneumonia ( 3% ) and rehospitalization for mrsa pneumonia ( 2% ) were rare ; however , all - cause rehospitalization occurred in 20% of patients . chart - level data were collected for 93 patients with mrsa pneumonia , 50 in saudi arabia and 43 in lebanon . of the 93 patients in the sample , 20% of the mrsa infections were community acquired , 32% health - care associated , 35% nosocomial , and 12% of unknown origin . however , the most common comorbidities among these patients were diabetes ( 39% ) and congestive heart failure ( 30% ) . ninety of the 93 patients in the sample ( 97% ) had at least one culture taken , and 82 of these ( 91% ) had at least one culture positive for mrsa . as would be expected , only one - third of the isolates tested were sensitive to ciprofloxacin and only 15% were sensitive to beta - lactams . patients most frequently had positive cultures from pulmonary ( 87% ) and blood ( 27% ) samples ; nasal sites were also culture positive in 16% of patients . all isolates tested were sensitive to vancomycin ( n=80 ) and teicoplanin ( n=32 ) . only ten isolates were tested for linezolid sensitivity , and all were found to be sensitive . only one - third of the isolates tested were sensitive to ciprofloxacin . drug selection for mrsa pneumonia , expressed as the percentage of patients receiving each antibiotic ( with potential for multiple antibiotics per line of therapy ) , is summarized in figure 1 . overall , 79 patients ( 85% ) received at least one antibiotic considered to be mrsa active . the 93 mrsa pneumonia patients received a total of 140 antibiotic orders designated by study investigators to be first - line therapy ( as some regimens consisted of multiple antibiotics ) . of these , 28% were beta - lactams ( thus representing inactive therapy ) , and the most frequently prescribed mrsa - active antibiotic was vancomycin ( 22% of orders ) . among the 48 patients ( 52% ) who were prescribed a total of 66 second - line antibiotic orders , vancomycin remained the most commonly prescribed antibiotic ( 39% of orders ) , followed by teicoplanin ( 12% ) and linezolid ( 9% ) . of the eleven third - line drugs received by eight patients , linezolid was the most commonly used ( 36% of orders ) , followed by vancomycin ( 27% ) and ciprofloxacin ( 18% ) . overall , the mean total length of stay ( los ) was 32.4 days , with the majority ( 20.4 days ) spent in general wards ( figure 2 ) . thirty - six patients ( 39% ) required mechanical ventilation , for a mean sd of duration of 18.724.4 days per patient . four patients ( 4% ) required hemodialysis , for a mean of 9.8 11.9 days per patient . of these patients , two patients were reported to have moderate - to - severe renal disease on admission , but whether these patients were receiving dialysis prior to admission is unknown . other interventions required among patients with mrsa pneumonia included peripheral catheters ( 52% ) , central venous catheters ( 52% ) , and peripherally inserted central catheters ( 6% ) . over one - fourth of the patients were considered treatment failures at the time of discharge ( table 3 ) . the overall in - hospital mortality rate was 30% , with nearly one - third of deaths being mrsa related . of the 28 patients who died , treatment response at discharge was failure in 24 ( 86% ) cases , improved in one ( 3.5% ) , cure in two ( 7% ) , and indeterminate in one ( 3.5% ) . relapse of mrsa pneumonia ( 3% ) and rehospitalization for mrsa pneumonia ( 2% ) were rare ; however , all - cause rehospitalization occurred in 20% of patients . this real - world burden and treatment patterns study of mrsa pneumonia in the middle east indicates a resource - intensive disease with a high los and mortality , consistent with other studies in high - income countries . inappropriate / inactive therapy for mrsa nosocomial pneumonia was prescribed more than a quarter of the time over the entire course of therapy , and in first - line treatment was prescribed 42% of the time . the relatively high mortality rate in this study may have been influenced by the high proportion receiving suboptimal therapy as first - line treatment . in a multinational observational study in asia and a randomized clinical trial in canada , the frequency of prescribing inadequate initial antibiotic therapy in pneumonia may be > 50% , and this significantly increases mortality rates.13,14 mrsa - targeted antibiotics were more frequently prescribed in second - line ( vancomycin 54% ) and third - line ( linezolid 51% ) treatments . potential reasons for the administration of inappropriate antibiotics may be due to lack of physician knowledge , delay in receiving culture results , or availability of medications that cover mrsa . while the exact reason for prescribing inappropriate antibiotics can not be determined from this study , a body of literature has focused on inadequate initial therapy as a driver of added morbidity and mortality.14,15 administration of inappropriate empiric antibiotic therapy can increase mortality in patients with hospital - acquired pneumonia ( defined in this study as occurring within 48 hours of admission ) and ventilator - associated pneumonia ( vap , defined as occurring within 48 hours of endotracheal intubation ) . results from a prospective surveillance study conducted through the asian network for surveillance of resistant pathogens ( ansorp ) found that of the 15.8% of patients with s. aureus hospital - acquired pneumonia or vap , 50.4% received discordant therapy.13 patients receiving discordant therapy had higher all - cause ( 44% vs 35% ) and pneumonia - related ( 32% vs 21% ) mortality than patients who received concordant therapy . multivariate analysis results showed that discordant therapy was a risk factor for pneumonia - related mortality ( or , 1.542 ; 95% ci , 1.1272.110 ; p=0.007 ) . administration of inappropriate empiric antibiotic therapy can also increase mortality in patients with hcap , defined as pneumonia in a patient admitted from a nursing home or other long - term care setting , who was hospitalized within the last 12 months , receiving dialysis or infusion therapy requiring regular visits to a hospital - based clinic , or who is immunocompromised . in a us - based study , patients with hcap compared to community acquired pneumonia had higher mortality ( 24.6% vs 9.1% , p<0.001 ) and were more likely to receive inappropriate antimicrobial treatment ( 28.3% vs 13.0% , p<0.001).16 mortality was higher for patients who received inappropriate compared to appropriate antibiotic therapy ( 32.2% vs 15.7% , p<0.001 ) . inappropriate initial antimicrobial therapy was identified as an independent risk factor for hospital mortality ( or , 2.19 ; 95% ci , 1.273.78 ; p<0.005 ) . results from separate studies conducted at the same institution found that treatment escalation did not decrease mortality in patients with hcap treated with inappropriate empiric antibiotic therapy17 and that initial therapy for hcap should include an antibiotic that targets mrsa.16 the resource use findings were quite disparate between countries in this study where the mean los was 30 days longer in saudi arabia than in lebanon . it is unclear whether this difference results from an underlying disparity in the severity of infection between groups , or a difference in health - care infrastructure . a surveillance study in saudi arabia looking at 2,800 patients admitted to the medical - surgical intensive care unit suggests that long los is not uncommon for patients that are severely ill . they found that patients with vap had a longer mean los of 85 days versus 61 days in those without vap.18 this population had primarily gram - negative pneumonias , but 20% were attributed to gram - positive infection . the study is naturally limited by its retrospective design and reliance on data that are documented in the medical chart . since inclusion criteria required that the patients were diagnosed with mrsa pneumonia , we do not have details on other patients that may have been colonized and not developed active infection . however , the information gathered from the charts represents a snapshot of the real - world treatment patterns and supplements the limited data we have in this indication and region of the world . mrsa pneumonia in saudi arabia and lebanon may be treated with antibiotics that are inactive against mrsa in a majority of patients for first - line therapy , resulting in an added resource use burden and mortality . the information gathered in this middle east population provides important real - world perspectives on the current treatment patterns and opportunities to improve care . future studies should more closely examine the appropriateness of initial therapy in mrsa pneumonia patients .

objectivesthe objective of this study is to describe the real - world treatment patterns and burden of suspected or confirmed methicillin - resistant staphylococcus aureus ( mrsa ) pneumonia in saudi arabia and lebanon.methodsa retrospective chart review study evaluated 20112012 data from hospitals in saudi arabia and lebanon . patients were included if they had been discharged with a diagnosis of mrsa pneumonia , which was culture proven or suspected based on clinical criteria . hospital data were abstracted for a random sample of patients to capture demographics ( eg , age and comorbidities ) , treatment patterns ( eg , timing and use of antimicrobials ) , hospital resource utilization ( eg , length of stay ) , and clinical outcomes ( eg , clinical status at discharge and mortality ) . descriptive results were reported using frequencies or proportions for categorical variables and mean and standard deviation for continuous variables.resultschart-level data were collected for 93 patients with mrsa pneumonia , 50 in saudi arabia and 43 in lebanon . the average age of the patients was 56 years , and 60% were male . the most common comorbidities were diabetes ( 39% ) , congestive heart failure ( 30% ) , coronary artery disease ( 29% ) , and chronic obstructive pulmonary disease ( 28% ) . patients most frequently had positive cultures from pulmonary ( 87% ) and blood ( 27% ) samples . all isolates were sensitive to vancomycin , teicoplanin , and linezolid , and only one - third of the isolates tested were sensitive to ciprofloxacin . beta - lactams ( inactive therapy for mrsa ) were prescribed 21% of the time across all lines of therapy , with 42% of patients receiving first - line beta - lactams . fifteen percent of patients did not receive any antibiotics that were considered to be mrsa active . the mean hospital length of stay was 32 days , and in - hospital mortality was 30%.conclusionthe treatment for mrsa pneumonia in saudi arabia and lebanon may be suboptimal with inactive therapy prescribed a substantial proportion of the time . the information gathered from this middle east sample provides important perspectives on the current treatment patterns .

india , yoga combines specific postures ( asanas ) , breathing techniques ( pranayama ) , meditative techniques ( dhyana ) , chants ( mantras ) , and wisdom teachings ( sutras ) to encourage health and relaxation . following the worldwide commercial success of yoga , industry reports an increase in yoga participation since 2005 with over 30 million people practicing yoga for health benefits daily . as the number of yoga practitioners and yoga schools in the west increases , interpretations of the practice of yoga vary with several applications of spiritual , cultural , and therapeutic knowledge . the national institutes of health ( us ) and national health services ( uk ) describe yoga as a safe and effective intervention to increase strength , flexibility and balance , and treatment for high blood pressure , heart disease , aches and pains , depression , stress , and potentially asthma . the purpose of this study is to investigate the prevalence of yoga research by subject and type in the context of a western healthcare database . specifically , as the popularity of yoga practice for health in western populations continues to increase , do publication trends in yoga research indicate a similar surge in interest ? a service offered by the national library of medicine ( nlm ) , pubmed contains publication information of over 21 billion articles for biomedical literature with citations that include fields of biomedicine , behavioral sciences , chemical sciences and bioengineering . this database is most commonly used to source relevant articles for health professionals and academics in the western hemisphere , primarily because of its subject focus , abundance of material and free - access to medical abstracts in the english language . the annual statistics of pubmed results for yoga - related research is generated from an automated online tool and key - term search strategy ( appendix a ) . a preliminary analysis indicates the first recorded yoga article in pubmed dates to 1948 authored by e. abegg with an unknown title , while the first full - text yoga article appears to be written for 1964 . therefore , to ensure a comprehensive and pragmatic search any yoga - related publication from the years 1950 to 2012 is included . any citation that is not included on the pubmed search tool will be excluded and considered a limitation of the research . a service offered by the national library of medicine ( nlm ) , pubmed contains publication information of over 21 billion articles for biomedical literature with citations that include fields of biomedicine , behavioral sciences , chemical sciences and bioengineering . this database is most commonly used to source relevant articles for health professionals and academics in the western hemisphere , primarily because of its subject focus , abundance of material and free - access to medical abstracts in the english language . the annual statistics of pubmed results for yoga - related research is generated from an automated online tool and key - term search strategy ( appendix a ) . a preliminary analysis indicates the first recorded yoga article in pubmed dates to 1948 authored by e. abegg with an unknown title , while the first full - text yoga article appears to be written for 1964 . therefore , to ensure a comprehensive and pragmatic search any yoga - related publication from the years 1950 to 2012 is included . any citation that is not included on the pubmed search tool will be excluded and considered a limitation of the research . the total number of yoga - related articles listed on the pubmed database from 1950 to 2012 is 2,099 . of these titles , 498 ( 24% ) include yoga trials and 324 ( 15% ) systematic reviews [ figure 1 ] . yoga titles by type on pubmed two yoga - related articles are published in 1950 , with a cumulative total of seven titles until 1960 . in 1961 , seven further articles are added with annual additions of two to 13 articles until 1973 . this trend of less than 20 new articles per year continues until 1991 with the exception of 3 years ( 1973 - 1975 ) , [ figure 1 ] . the number of yoga publications in pubmed more than doubles between 1995 and 2000 from 17 to 39 . the rate of publication following 2000 is sporadic until a surge in 2007 when 114 yoga titles are added to pubmed in one year . a steady increase in yoga research is indicated since 2007 with an increased rate of publication by at least 24 articles per year . from 2010 to 2012 , 189 , 229 and 274 new yoga - related articles are added per year . of all yoga - based literature on pubmed the results of the key term search indicate a vast majority ( 90.3% ) of all yoga research is associated with 10 health conditions . the most prevalent health conditions for yoga study are stress / anxiety ( 343 ) , pain ( 276 ) , depression ( 207 ) , cardiovascular disease ( 200 ) , and blood pressure / hypertension ( 198 ) . respiratory conditions , cancer , body weight , hormones , and diabetes also indicate high association with yoga research [ figure 2 ] . sleep , addiction , prenatal care , infection , and injury appear to have less association with yoga research ( cumulative < 5% ) . the total number of yoga - related articles listed on the pubmed database from 1950 to 2012 is 2,099 . of these titles , 498 ( 24% ) include yoga trials and 324 ( 15% ) systematic reviews [ figure 1 ] . two yoga - related articles are published in 1950 , with a cumulative total of seven titles until 1960 . in 1961 , seven further articles are added with annual additions of two to 13 articles until 1973 . this trend of less than 20 new articles per year continues until 1991 with the exception of 3 years ( 1973 - 1975 ) , [ figure 1 ] . the number of yoga publications in pubmed more than doubles between 1995 and 2000 from 17 to 39 . the rate of publication following 2000 is sporadic until a surge in 2007 when 114 yoga titles are added to pubmed in one year . a steady increase in yoga research is indicated since 2007 with an increased rate of publication by at least 24 articles per year . from 2010 to 2012 , 189 , 229 and 274 new yoga - related articles are added per year . of all yoga - based literature on pubmed the results of the key term search indicate a vast majority ( 90.3% ) of all yoga research is associated with 10 health conditions . the most prevalent health conditions for yoga study are stress / anxiety ( 343 ) , pain ( 276 ) , depression ( 207 ) , cardiovascular disease ( 200 ) , and blood pressure / hypertension ( 198 ) . respiratory conditions , cancer , body weight , hormones , and diabetes also indicate high association with yoga research [ figure 2 ] . sleep , addiction , prenatal care , infection , and injury appear to have less association with yoga research ( cumulative < 5% ) . the first recorded yoga article in western medical research dates to 1948 , authored by e. abegg with an unknown title . the first full - text article found ( 1964 ) explores oxygen consumption during yoga - type breathing patterns . yoga research in the first 30-years of study includes diverse topics such as metabolism , arterial blood gases , body composition bronchial asthma and hypertension . the body of literature still comprises largely of 10 health conditions , thought the recent 5-year trend indicates a shift in focus . stress / anxiety , pain , and depression remain the most prevalent , with cancer now exceeding the number of articles of hypertension , respiratory conditions , and cardiovascular disease [ figure 3 ] . in 2012 publications , 53% of all publications relate to stress / anxiety ( 21% ) , pain conditions ( 17% ) , and depression ( 15% ) , while 37 new articles in cancer ( 12% ) indicates an area of emerging interest . 5-year publication trends on pubmed by health condition this bibliometric analysis employs a gross method to observe the annual trends of yoga citations in one medical database . as the results of yoga and health conditions are not investigated , no conclusions in terms of the direction of correlation can be drawn from this study . the first recorded yoga article in western medical research dates to 1948 , authored by e. abegg with an unknown title . the first full - text article found ( 1964 ) explores oxygen consumption during yoga - type breathing patterns . yoga research in the first 30-years of study includes diverse topics such as metabolism , arterial blood gases , body composition bronchial asthma and hypertension . the body of literature still comprises largely of 10 health conditions , thought the recent 5-year trend indicates a shift in focus . stress / anxiety , pain , and depression remain the most prevalent , with cancer now exceeding the number of articles of hypertension , respiratory conditions , and cardiovascular disease [ figure 3 ] . in 2012 publications , 53% of all publications relate to stress / anxiety ( 21% ) , pain conditions ( 17% ) , and depression ( 15% ) , while 37 new articles in cancer ( 12% ) indicates an area of emerging interest . this bibliometric analysis employs a gross method to observe the annual trends of yoga citations in one medical database . as the results of yoga and health conditions are not investigated , no conclusions in terms of the direction of correlation can be drawn from this study . a surge of yoga 's popularity in western culture in 2005 is matched with academic interest in 2007 . there are now over 2,000 yoga titles on the database , with a rate of more than 200 additions per year . systematic reviews and yoga trials are increasing , indicating a potential increase in quality of evidence . three conditions show consistently high correlations with yoga research : stress / anxiety , pain , and depression . a significant rise in the number of cancer publications suggests an area of emerging research . investigation into the quality and direction of these findings would assist in understanding the potential impact of yoga on several predominant health conditions .

context : the promotion of yoga practice as a preventative and treatment therapy for health outcomes in the western hemisphere is increasing rapidly . as the commercial success of yoga burgeons in popular culture , it is important to investigate the trends of yoga as a therapeutic intervention in academic literature . the free - access search engine , pubmed is a preeminent resource to identify health - related research articles published for academics , health practitioners and others.aims:to report the recent yoga - related publications in the western healthcare context with particular interest in the subject and type of yoga titles.materials and methods : a bibliometric analysis to describe the annual trends in publication on pubmed from january 1950 to december 2012.results:the number of yoga - related titles included in the pubmed database is limited until a marked increase 2000 and steady surge since 2007 . bibliometric analysis indicates that more than 200 new titles are added per annum since 2011 . systematic reviews and yoga trials are increasing exponentially , indicating a potential increase in the quality of evidence . titles including pain management , stress or anxiety , depression and cancer conditions are highly correlated with yoga and healthcare research.conclusions:the prevalence of yoga research in western healthcare is increasing . the marked increase in volume indicates the need for more systematic analysis of the literature in terms of quality and results .

there is a lot of epidemiological evidence to suggest that vitamin d has a role in glucose homeostasis , especially in maintaining normal glucose homeostasis and protecting against type 2 diabetes [ 1 , 2 ] . however , controlled supplementation trials with vitamin d or its analogues have not been able to produce similar results . in a recent systematic review and meta - analysis of 15 trials lasting from two months to 7 years two trials in subjects with impaired glucose tolerance ( igt ) did find a positive effect on glucose homeostasis with vitamin d3 supplementation [ 47 ] , whereas three trials in subjects with igt and one in subjects with normal glucose tolerance found no effects [ 810 ] . insulin resistance , metabolic syndrome , and type 2 diabetes are associated with a systemic , chronic inflammation , where , for example , circulating levels of proinflammatory cytokines and c - reactive protein are elevated [ 11 , 12 ] . although vitamin d has immunomodulatory and anti - inflammatory properties , especially based on experimental studies , bodies of evidence from human studies in the prediabetic state assessing the role of moderate to large dose supplementation of vitamin d in inflammation and glucose metabolism are still relatively few . as more evidence is clearly needed , we decided to study the placebo - controlled effect of 5-month 40 g / d ( 1,600 iu ) or 80 g / d ( 3,200 iu ) oral vitamin d3 supplementation on glucose homeostasis in an eastern finnish population with either impaired fasting glucose ( ifg ) or igt during winter time when serum vitamin d3 concentrations drop without supplementation . furthermore , we assessed the anti - inflammatory effect of the supplementation by measurements of serum high - sensitive c - reactive protein ( hscrp ) , plasma soluble tumor necrosis factor receptor type ii ( pstnfrii ) , plasma interleukin-6 ( pil-6 ) , plasma interleukin-1 receptor antagonist ( pil-1ra ) , and plasma interleukin-1 beta ( pil-1 ) concentrations . the glucose metabolism effects of vitamin d supplementation in prediabetes ( vitdmet ) study was a 5-month randomized , double - blind , placebo - controlled supplementation trial that was conducted in winter 2011/2012 in eastern finland . the primary aim was to assess the effects of vitamin d3 supplementation on glucose homeostasis in subjects with ifg or igt . for inclusion the subjects needed to be 60 years of age with evidence of disturbed glucose homeostasis , that is , either ifg ( fasting plasma glucose concentration 5.6 to 6.9 mmol / l ) or igt ( oral glucose tolerance test ( ogtt ) 120 min plasma glucose concentration 7.8 to 11.1 mmol / l ) , but not type 2 diabetes ( fasting plasma glucose concentration 7.0 mmol / l or ogtt 120 min plasma glucose concentration 11.1 mmol / l ) , and to be overweight ( bmi > 25 ) , but not severely obese ( bmi < 35 ) . furthermore , subjects needed to have their serum 25(oh)d3 concentration < 75 nmol / l . exclusion criteria were any disease that could be affected by vitamin d , such as sarcoidosis , or a condition that could affect vitamin d metabolism , such as kidney disease . the adult voluntary subjects were recruited by advertisements in regional newspapers and after a prescreening on the phone for inclusion and exclusion criteria ( self - reported elevated blood glucose , age , height , weight , and diseases ) were invited for the first screening at the study clinic ( visit 1 ) . a written informed consent was collected , hba1c was measured with a point - of - care device , the subjects were interviewed , and their self - administered questionnaires were checked . if the subject 's hba1c was 5.57.0% , fasting venous blood samples were drawn and analyzed for plasma glucose and serum 25(oh)d3 concentrations . those fulfilling the inclusion criteria were scheduled for the ogtt ( visit 2 ) , after which those fulfilling all criteria were randomized to receive either placebo , 40 g / d , or 80 g / d vitamin d3 daily . each dose comprised a combination of placebo and 20 g tablets , two in the morning and two in the evening . randomization was carried out by a statistician , in the order of entry after visit 2 . study nurse distributed the containers according to the statistician - generated list and neither the nurse nor a participant knew the allocation group . all tablets and containers were physically identical , the allocation arm code hidden in the serial number on the container label . the average time between visit 2 and the beginning of the supplementation was approximately one week . during the 5-month supplementation period between october 2011 and april 2012 , the subjects visited the study site three times : 1 and 3 months after the start of the supplementation and at the end of the study ( figure 1 ) . we aimed to recruit 102 participants , 34 into each of the three supplementation groups , but due to lower than expected public interest to participate and generally higher than anticipated serum 25(oh)d3 concentrations in the base population , at closing of the recruitment window we had 73 randomized subjects in the three supplementation arms ( 63 men and 10 women ) . according to power computations , to reach 80% power ( i.e. , = 0.20 ) at = 0.05 , with a difference of 1 sd to 0.75 sd in the effect measure between the groups , there needed to be 16 to 28 subjects per group . 1 sd equals an effect size of 10% to 50% in most of the outcome parameters . the research ethics committee of the northern savo hospital district has approved the study protocol . serum 25(oh)d3 concentration was measured from venous blood samples by a high performance liquid chromatography with coulometric electrode array ( hplc - cead ) , as described . the interassay cv% of the method is < 8.0% and the intra - assay cv% is < 7.3% . plasma glucose concentration was assayed by the photometric hexokinase method ( konelab 20xt , thermo fischer scientific , vantaa , finland ) and plasma insulin concentration by the chemiluminescence assay ( diasorin liaison , diasorin , dietzenbach , germany ) . plasma glucose and plasma insulin concentrations were measured at three time points : at start ( i.e. , fasting ) , at 30 min , and at 120 min . homeostatic modeling assessment ( homa2 ) indices were computed according to the nonlinear function presented by wallace and coworkers . the insulinogenic index ( igi ) was computed as follows : igi = ( 30 min insulin 0 min insulin)/(30 min glucose 0 min glucose ) in the ogtt , as described by phillips and coworkers , and the insulin sensitivity index ( isi ) was computed as follows : isi = 10000/sqr[(fasting glucose fasting insulin ) ( mean glucose mean insulin ) ] , as described by matsuda and defronzo . hba1c was measured with a point - of - care device from a capillary blood sample as instructed by the manufacturer ( dca vantage , siemens healthcare diagnostics , deerfield , illinois , usa ) . serum liver enzymes gamma - glutamyl transpeptidase ( sggt ) and alanine aminotransferase ( salat ) were determined with enzymatic photometric ifcc methods , kidney function marker serum creatinine ( screa ) with an enzymatic photometric test , and serum total calcium ( sca ) with colorimetric arsenazo iii test at site with a clinical chemistry analyzer ( konelab 20xt , thermo fisher scientific , vantaa , finland ) . serum parathyroid hormone ( spth ) and serum thyroid stimulating hormone ( stsh ) were assayed by chemiluminescence methods ( diasorin liaison , diasorin , dietzenbach , germany ) . total blood count ( tbc ) and inflammatory cytokines hscrp , pstnfrii , pil-6 , pil-1ra , and pil-1 were measured in eastern finland laboratory centre , kuopio , finland . plasma il-6 , il-1ra , soluble tnfrii , and il-1 were measured with elisa from r&d systems ( minneapolis , mn , usa ) . body weight was measured to 0.1 kg with subjects wearing light underwear , with a recently calibrated vetek ti-1200 scale ( vetek , sweden ) , and height in a frankfurt position to 0.5 cm by a wall - mounted device ( kawe , germany ) . body mass index ( bmi ) was computed as weight / height squared ( kg / m ) . blood pressure was measured after a 5 min rest from the upper arm at a supine position , using a recently calibrated semiautomatic device ( omron intellisense m7 , omron healthcare , netherlands ) . systolic and diastolic blood pressure values were computed as the mean of the last two of three measurements . participant compliance in taking supplements as instructed was assessed by counting the study supplements not consumed at the last study visit . from the statistical analyses we excluded two subjects who had an increase in their bmi to > 35 kg / m between the randomization and the start of the supplementation ( visit 3 ) , two subjects who reported at the end of the study that they had started to use a high - dose ( 50 g / day and 125 g / day ) vitamin d3 supplement in addition to the study supplements during the study period , and one subject due to low compliance . the subject with the low compliance in the 80 g / day group had used only 17% of the study tablets . after the exclusions , the number of subjects in the analyses with the baseline data was 68 ( table 1 ) and in the analyses with the change variables 66 ( table 2 ) , after the two dropouts ( figure 1 ) were excluded . glucose homeostasis was assessed by fasting glucose , insulin , and hba1c measurements and ogtt glucose and insulin measurements . the derived variables to be tested were the change in the chosen variables over the supplementation period . these were as follows : serum hba1c ( concentration and % ) , fasting , 30 min and 120 min glucose and insulin concentrations , homa2 insulin resistance ( homa2-ir ) , insulin sensitivity ( homa2-is ) and beta cell function ( homa2-b% ) , igi , and isi . inflammatory effects were assessed by analyzing the change in the hscrp , pstnfrii , pil-6 , pil-1ra , and pil-1 concentrations over the supplementation period . physiological and safety indicators were changes in the 25(oh)d3 , spth , sca , screa , sggt , salat , and stsh . as a large proportion of the variables did not follow the normal distribution , we used the kruskal - wallis test ( pk - w ) to test the intergroup differences , mann - whitney u test ( pm - wu ) to test the pairwise intergroup differences ( placebo versus 40 g / d and placebo versus 80 g / d ) , and the jonckheere - terpstra test ( pj - t ) for trend across the groups . related - samples wilcoxon signed rank test ( pwsr ) was used to study the intragroup change over time . a two - sided p < 0.05 was considered statistically significant for the kruskal - wallis test , a 1-sided p < 0.05 for the jonckheere - terpstra test , and a two - sided p < 0.025 for the mann - whitney u test ( i.e. , with the bonferroni correction ) . all statistical analyses were computed with the spss statistical package for windows , version 21.0 ( armonk , ny : ibm corp . ) . the supplementation effect analyses were conducted per protocol ; as for the dropouts there were no data available at the end of the study to compute the change variables . despite the moderately small sample size , the randomization yielded reasonably identical groups with regard to vitamin d and glucose metabolism - related parameters . characteristics of the 68 study subjects at baseline are presented in table 1 . the median age of the subjects at entry was 65.7 ( interquartile range 62.7 to 69.7 ) years , and the median serum 25(oh)d3 concentration was 57.2 ( interquartile range 47.2 to 67.2 ) the arithmetic mean 25(oh)d3 concentration was 57.0 nmol / l with a standard deviation of 11.0 nmol / l . of the 68 subjects that entered the supplementation period , 66 completed the study ( figure 1 ) . there was a marked dose - dependent increase in the average serum 25(oh)d3 concentration in the three study groups , from 55.3 to 59.0 nmol / l , from 57.7 to 85.4 nmol / l , and from 58.1 to 103.1 nmol / l , in the placebo , 40 g / d , and 80 g / d groups , respectively ( mean ranks for change 17.1 , 35.4 , and 47.8 , resp . , pk - w < 0.001 ; see table 2 ) . at baseline , one of the subjects in the 80 g / day group had serum 25(oh)d3 > 75 nmol / l . at the end of the study , 3/21 ( 14% ) subjects in the placebo group , 16/24 ( 67% ) subjects in the 40 g / d group , and 18/21 ( 86% ) subjects in the 80 g / d group had serum 25(oh)d3 > 75 concomitant with the increase in the serum 25(oh)d3 concentrations , there was a dose - dependent decrease in the serum pth concentrations ( table 2 ) . changes in the tested glucose homeostasis parameters over the supplementation period are given in table 2 . the only statistically significant effect between the groups was an increase in the 120 min plasma glucose concentration , that is , opposite to expected ( pk - w = 0.039 , pj - t = 0.021 ) , although only the pairwise group difference for the placebo versus 40 g / d was statistically significant after bonferroni correction ( p = 0.022 ) , and a decreasing trend in the hba1c concentration ( pj - t = 0.024 ) and in the 30 min insulin concentration ( pj - t = 0.030 ) . borderline statistically significant trend was observed in the igi ( pj - t = 0.063 ) . in the tested inflammation markers , shown in table 2 , the only borderline statistically significant finding was a decreasing trend in the plasma il-1ra concentration ( pj - t = 0.070 ) . there were detectable concentrations of pil-1 only for four subjects at entry and for two subjects at the end of the study and therefore the p values can not be considered reliable . no statistically significant changes were observed in sca ( table 2 ) or in the parameters of liver or kidney function or in the tbc ( data not shown ) . no statistically significant differences between groups were observed in changes in waist circumference or bmi , in blood pressure , or in circulating lipids ( data not shown ) . we repeated the analyses by including also those with bmi > 35 at the start of the supplementation period , those who started their own high - dose vitamin d supplementation during the trial , or the one with low compliance ( n = 71 ) . the results were generally similar to those with the exclusions , except for the p for trend for il1-ra , which was statistically significant ( pj - t = 0.027 ) . our study showed that oral supplementation with daily 40 g or 80 g doses of vitamin d3 for five months markedly increases the circulating concentrations of 25(oh)d3 , even over the winter months that usually are associated with a decrease in the 25(oh)d3 in this population . we also showed that the 80 g dose is more potent than the 40 g dose in increasing serum 25(oh)d3 and that a considerably large proportion of the subjects in both supplementation groups reached serum 25(oh)d3 concentrations above 75 nmol / l . furthermore , we showed that the supplementation also resulted in a decrease in serum pth concentration but did not induce a rise in serum total calcium concentration . vitamin d3 supplementation , even at 80 g per day , was also well tolerated and safe . the primary outcome of the study , body glucose homeostasis , remained for the most part practically unchanged , thus speaking against the role of vitamin d3 as an important regulator of glucose homeostasis in an ageing finnish population with prediabetes . among the 13 glucose homeostasis indices analyzed , the 120 min plasma glucose was the only index that was statistically significant also in the pairwise analyses . we believe that other changes observed ( 30 min insulin , hba1c ) may be related to random fluctuation of glucose metabolism in individuals with impaired glucose metabolism . among the interventional studies on glucose homeostasis indicators in populations with prediabetes , an open - label study by nazarian et al . showed an improvement in insulin sensitivity in 11 ifg subjects after a 4-week 10,000 iu / d ( 250 g / d ) vitamin d3 intervention . in an earlier post , there was a lower rise in fasting plasma glucose concentration and lower increase in homa - ir in 3 years in the group taking 700 iu ( 17.5 g ) vitamin d3 plus 500 mg calcium citrate daily as compared with subjects taking placebo . glucose homeostasis was also improved in the study by mitri et al . , where 16-week vitamin d3 supplementation of 2,000 iu / d ( 50 g / d ) improved -cell function and attenuated the rise in hba1c . in an open - label study by dutta et al . in subjects with igt or ifg , vitamin d3 supplementation of 60,000 iu ( 1500 g ) once per week for 8 weeks and then monthly along with 1250 mg of calcium carbonate / d resulted in lower fasting blood glucose and improvement in insulin resistance during an average follow - up of 28 months , when compared with subjects taking only calcium carbonate . in a recent placebo - controlled study by gagnon et al , vitamin d3 upplementation for six months with a dose aimed at increasing the serum 25(oh)d3 to 75 nmol / l ( 20006000 iu / d , 50150 g / d ) improved insulin sensitivity in subjects with prediabetes , but not in subjects without prediabetes . in contrast , in a study by harris et al . in 89 overweight or obese african american subjects , 12-week supplementation with 4,000 iu / d ( 100 g / d ) of vitamin d3 , compared with placebo , led to reduced insulin sensitivity and increased insulin secretion . in the 12-month supplementation trial by davidson et al . in 117 latino and african american subjects in california with hypovitaminosis d3 ( i.e. , serum 25(oh)d3 concentration < 30 ng / ml ( 75 nmol / l ) ) , the average weekly dose of 88,865 iu ( 2,222 g , or about 317 g / day ) of vitamin d3 had no effect on insulin secretion , insulin sensitivity , or development of type 2 diabetes , as compared with placebo . our results support these null findings on glucose homeostasis in finns , a caucasian population at northern latitudes , even though we have earlier observed vitamin d3 sufficiency to associate with the lower risk of type 2 diabetes in an observational study of the same source population . the observed increase in the 120 min plasma glucose is somewhat unexpected , but one explanation may simply be a chance finding due to a largish number of tests that were carried out . the increase was larger in the 40 g group than in the 80 g group , which supports that the finding occurred by chance as we believe is the case with other variables we monitored , that is , 30 min insulin and hba1c . taken together , the evidence from the observational studies supports the association between low vitamin d status and impaired glucose metabolism , but the results from the experimental studies have not found improvements in glucose metabolism with vitamin d supplementation . a recent mendelian randomisation study that looked into the association between body vitamin d status and glucose homeostasis and type 2 diabetes with four snps and four glycaemic traits showed an association in two snps near genes related to 25(oh)d synthesis with fasting insulin , but not in any of the other eleven tests conducted . however , if a limitation is to be pointed out , many of the trials have been conducted in subjects with healthy glucose homeostasis or in subjects with diabetes , and only few have been carried out in subjects with prediabetes , the subjects that would probably show any effect on glucose homeostasis most easily . therefore , well - designed randomized trials with large sample sizes and in people with insufficient vitamin d status may be needed to elucidate the role of vitamin d supplementation in glucose homeostasis and in prevention of type 2 diabetes . we found supplemental vitamin d3 to exert some anti - inflammatory action , when a borderline statistically significant decrease in the il-1ra concentration was found . il-1ra is regarded as one of the most sensitive markers of inflammation , as it is readily secreted by many cell types and plasma levels are high enough to be reliably measured . elevated values of il-1ra have also been shown to predict the onset of type 2 diabetes [ 2224 ] . the results from previous studies of the anti - inflammatory effects of vitamin d supplementation have been inconsistent . some studies have found vitamin d supplementation to improve the levels of certain inflammatory markers , such as il-6 , il-10 , crp , or tnf- [ 6 , 10 , 2527 ] , whereas others have found no effects on any of the studied markers [ 19 , 2830 ] . however , only a few studies have been conducted in subjects with prediabetes [ 6 , 19 , 31 ] and , to our knowledge , no study thus far has investigated the impact of vitamin d supplementation on il-1ra . our study was conducted during october to april , the period of low uvb light exposure from the sun in finland , to allow as unbiased supplemental vitamin d effect as possible . strengths of the study were the homogenous study population and use of two different , sufficiently large vitamin d3 doses , which enabled studying possible dose response . a limitation of the study was the unbalanced gender distribution , especially the low number of females . another limitation is the lower than planned total number of study subjects , which was due to the need to close recruitment to allow a sufficiently long supplementation during the low uvb exposure period , and the higher than anticipated average 25(oh)d3 concentration of the study population at the start of the study . however , the fairly consistent results of our study do not suggest a marked change in observed effects even if some more subjects would have been included , or if intention - to - treat analysis had been carried out instead of per - protocol analysis , given that per - protocol tends to exaggerate the treatment effects , if anything . in conclusion , our study does not support the role of winter - time relatively high - dose vitamin d3 supplementation as a means to improve glucose homeostasis in a general ageing population with prediabetes but suggests a modest anti - inflammatory effect .

epidemiological evidence suggests a role for vitamin d in type 2 diabetes prevention . we investigated the effects of vitamin d3 supplementation on glucose metabolism and inflammation in subjects with prediabetes . a 5-month randomized , double - blind , placebo - controlled intervention with three arms ( placebo , 40 g / d , or 80 g / d vitamin d3 ) was carried out among sixty - eight overweight ( bmi 2535 ) and aging ( 60 years ) subjects from finland , with serum 25-hydroxyvitamin d3 [ 25(oh)d3 ] < 75 nmol / l and either impaired fasting glucose or impaired glucose tolerance . analyses included 66 subjects who completed the trial . glucose metabolism was evaluated by fasting and 2-hour oral glucose tolerance test - derived indices and glycated hemoglobin . inflammation was evaluated by high - sensitive c - reactive protein and five cytokines . although a dose - dependent increase in serum 25(oh)d3 over the supplementation period was observed ( p trend < 0.001 ) , there were no other statistically significant differences in changes in the 13 glucose homeostasis indicators between the study groups other than increase in the 120 min glucose concentration ( p trend = 0.021 ) and a decreasing trend both in 30 min plasma insulin ( p trend = 0.030 ) and glycated hemoglobin ( p trend = 0.024 ) concentrations . a borderline statistically significant decreasing trend in interleukin-1 receptor antagonist concentration was observed ( p = 0.070 ) . vitamin d3 supplementation does not improve glucose metabolism in ageing subjects with prediabetes but may have modest anti - inflammatory effects .

sole aim and the prerequisite for dentistry is an effective pain control during dental procedures . to achieve this goal local anesthesia is being used since long . in 1943 , lofgren synthesized the first modern anaesthesia.1 it is lidocaine , which was an amide derivative of diethyl amino acetic acid . it is because of the high density and the firm adherence of palatal mucosa to the underlying bone . twenty - five years after lidocaine , articaine was first synthesized by muschaneau in 1969 . it has a thiophene ring in its molecule instead of usual benzene ring.2,3 this is most commonly used in germany . commercially articaine for dental use is available in 4% solution with epinephrine 1:200000 or 1:100000 . it also contains maximum 0.6 mg na - sulfite in 1.0 ml and sodium chloride . once injected , absorption starts from the site of injection into the vascular compartment.1 the unbound local anesthetic is distributed throughout all the body tissue . due to the presence of thiophene ring , it is inactivated in the liver as well as by hydrolyzation in the tissue and blood . the aim of the study was to evaluate and compare the efficacy of 4% articaine hydrochloride and 2% lignocaine hydrochloride for the orthodontic extraction . the study was carried out on 50 patients at outpatient department of oral and maxillofacial surgery who needed bilateral maxillary premolar extractions for orthodontic purpose . patients included in this study were in the age group of 15 - 25 years , both genders and systemically healthy . bleeding disorders , hypertensive , diabetic , pregnant , allergic to local anesthetics , reluctant and medically compromised patients were excluded from the study . all the patients were explained about visual analog scale ( vas ) before injecting local anesthesia . experimental sites ( group 1 ) were injected with 0.5 - 1 ml of 4% articaine hcl containing 1:100000 adrenaline , incrementally in the buccal vestibule . no palatal anesthesia was injected , but the desired anesthetic effect was achieved with the above . on the other hand , control sites ( group 2 ) were injected with 0.8 - 1 ml of 2% lignocaine hcl containing 1:100000 adrenaline , incrementally in the buccal vestibule . when the objective symptoms were checked , it was found that palatal anesthesia was absent hence additional 0.5 ml was injected to obtain a desired result . after assessing the signs and symptoms of obtaining complete anesthesia all the parameters , i.e. , drug volume , time of onset , duration of anesthesia and pain rating were recorded for entire patients . the mean administered volume of articaine and lignocaine were 0.779 0.1305 and 1.337 0.2369 respectively . it should be noted that the articaine volume administered was almost half of the lignocaine ( table 1 ) . the mean onset time of lignocaine anesthesia was 1.337 0.2369 , whereas in articaine group the mean time was 1.012 0.2058 min . this indicates that onset time of articaine was significantly less than lidocaine ( p < 0.0005 ) ( table 2 ) . pain rating showed that there was no significant difference in pain score in articaine palatal and buccal group ( p > 0.8892 ) , whereas a significant difference was noted in lignocaine palatal and buccal group ( tables 3 and 4 ) . duration of pain in group 1 was 69.08 18.247 and 55.66 6.414 in group 2 patients . duration of anesthesia is articaine group is more than the lignocaine group . in the entire study , it goes biotransformation in both liver and plasma and hence gets cleared much quickly . recent studies have shown that articaine carries lot of advantages over other anesthetic agents.4 in this study , we observed that the palatal infiltration was required in approximately 98% of cases when lignocaine was used , whereas in articaine group palatal anesthesia was never required . thiophene ring in its molecular structure , which makes it more lipophilic and this accounts for its diffusion properties across all the tissues.5 articaine is metabolized in the liver , tissues and blood and hence it is cleared out very fast from the body . this is the only anesthetic agent , which is inactivated from our body in two ways . zlkowska et al . has reported that like all other anesthetic agents articaine is safe in epileptic patients.6 this study showed no adverse effects and no complications . this study is in accordance with study by malamed et al . suggesting 4% articaine with 1:100000 adrenaline is safe and have a low risk of toxicity.2 statistical analysis in this study showed no significant difference in extraction pain on vas for test and control sites . this inference relates to the study done by fan et al.7 oertel et al.8 uckan et al.9 and evans et al.10 when articaine is injected the local concentration of active drug is nearly twice of that obtained with lignocaine . oertel et al . in his study showed this by determining the concentration of 4% articaine and 2% lidocaine in alveolar blood using high - performance liquid chromatography.11,12 thiophene derivative articaine blocks ionic channels at lower concentration than benzene derivative lidocaine.13 potocnik et al . in vitro study on rat surap nerve concluded that 2% and 4% of articaine is more effective than 2% and 4 % of lidocaine in depressing compound action potential of the a fibres.14 - 16 this efficacy and safety factors are observed in this study too . it is a well - known fact that palatal anesthesia is a very painful experience even though surface anesthesia is used . hence , if articaine is used , patients can be relieved from the painful palatal anesthesia without compromising with safety and efficacy . certain added advantages like shorter time of onset , longer duration of action and greater diffusion property makes it an ideal anesthetic agent to be used in dentistry .

background : articaine in an anesthetic agent , which is used less frequently in dentistry . it differs from other agents due to the presence of a thiophene ring in its molecular structure . few groups of researchers claim that it is superior to lignocaine . hence , the purpose of this study was to compare the efficacy of 4% articaine hydrochloride and 2% lignocaine hydrochloride in the orthodontic extraction.materials and methods : the study was carried out in 50 patients who needed the orthodontic extraction in the age group from 15 to 25 years . experimental sites were injected with 0.5 - 1 ml of 4% articaine hcl containing 1:100000 adrenaline , incrementally in the buccal vestibule without palatal anaesthesia . control sites were injected with 0.8 - 1 ml of 2% lignocaine hcl containing 1:100000 adrenaline , incrementally in the buccal vestibule . all the parameters , that is volume , duration , time of anesthesia and pain rating were noted and statistically compared.result:when statistically compared mean volume of articaine ( 0.779 0.1305 ) was less than lignocaine ( 1.337 0.2369 ) . mean time of onset of articaine was 1.012 0.2058 min , whereas that of was 1.337 0.2369 . pain rating showed not much difference , but in the lignocaine group palatal anesthesia was required in all the patients . finally , the mean duration of anesthesia in articaine group was 69.08 18.247 , whereas in the lignocaine group was 55.66 6.414.conclusion:articaine has proved its usefulness in all regards . literatures have proved its usefulness . like other anesthetic , it is safe and more effective . it surpasses the need of additional palatal anesthesia . rapid inactivation in liver and plasma reduces the risk of the drug overdose . all the above factors make it an ideal anesthetic agent to be used in dentistry .

pyoderma gangrenosusm ( pg ) is a chronic , rare , inflammatory cutaneous disorder and is manifested by the development of painful ulcers , bulla , pustules and rarely vegetating lesions and histologically by predominantly neutrophilic infiltration . we report a case of severe pg in a girl since her 4 months of age . the difficulty in diagnosis and methodical approach in such cases is highlighted . a 6-month - old hindu girl presented with progressive , extensive , and painful ulcers since her 4 months of age . ulcers developed from erythematous papules and vesicles , had purple red undermined margin , raw or crusted surface , merged with other ulcers in the neighborhood and formed large ( even > 8 cm ) lesions with annular , polycyclic or crescent shape . they were distributed over scalp , face , ear , trunk ( less over anterior trunk ) , buttocks , thigh , legs , dorsum of hands and feet without any mucosal involvement [ figure 1 ] . ( a ) face , ( b ) arm , ( c ) dorsum of hand , ( d ) leg , ( e ) thigh , ( f ) back , ( g ) encircled site where intradermal normal saline was injected , ( h ) same site after 48 h showing pustulation ( positive pathergy test ) the child was playful with normal physical and intellectual growth . there was mild fever , moderate pallor without any cyanosis , clubbing , organomegaly , lymphadenopathy , or joint abnormality . pustule developed following intradermal injection of normal saline on normal skin ( positive pathergy test ) [ figure 1 ( g , h ) ] . there was low hemoglobin ( 8.9 gm / dl ) , leucocytosis ( 22,600/cmm ) , neutrophilia ( 70% ) , and elevated sedimentation rate ( 69 mmhg ) . chest x - ray , mantoux test , dna - pcr for tuberculosis , immunoglobulin level , ra factor , ana ( hep-2 cell ) , vdrl , c - anca , hiv- elisa , pus culture , and colonoscopy of rectal mucosa revealed no abnormality . histopathology of the skin biopsies with h and e stain from the margin of the ulcer from two different sites showed epidermal ulceration and dense dermoepidermal collection of inflammatory exudates consisting predominantly of neutrophils with occasional multinucleated giant cells [ figure 2 ] . one of the biopsies also showed foci of granulomatous reaction [ figure 2 , inset ] . z - n stain ( for afb ) and pas stain ( for fungus ) were negative . photomicrograph showing ulceration of epidermis and collection of inflammatory exudates along with occasional giant cells ( h and e , 100 ) . inset showing ill formed granuloma with giant cells ( h and e , 400 ) it was diagnosed as a case of pyoderma gangrenosum . the patient responded satisfactorily to oral prednisolone ( 2 mg / kg / day ) . however , there were signs of relapse with dose less than 1 mg / kg / day . at that time , the patient became irregular in follow - up and finally lost to follow up . in most of the cases , pg is associated with some systemic diseases like inflammatory bowel diseases ( ibd ) , rheumatoid arthritis , myeloproliferative disease , etc . extensive distribution of the disease was unusual for an infant with pg who generally have ulcers on head and buttocks . onset at 4 months of age was extremely rare and possibly not reported so far . due to much atypicality , diagnosis was difficult and many possibilities were considered like pg , tuberculous , atypical mycobacterial infection , fungal , malignant and vasculitic ulcers , wegener 's granulomatosis , sweet 's syndrome , and epidermolysis bullosa . violaceous undermined margin with erythematous halo , pain , characteristic histological appearance and positive pathergy test were helpful for diagnosis . multiple site colonoscopic biopsies from even normal rectal mucosa could have increased the diagnostic yield for any subclinical evidence of ibd . the case

a female child developed multiple , progressive , therapy - resistant , painful large ulcers , vesicles , and pustules since her 4 months of age . the ulcers were large , some even measured more than 8 cm ; most had violaceous undermined margin with surrounding erythematous halo , raw and crusted surface and were distributed extensively over scalp , face , ear , trunk , buttocks , thigh , legs , dorsum of hands , and feet without any mucosal involvement . after detail clinical examination and investigation , it was diagnosed as a case of pyoderma gangrenosum . extensive search did not reveal any systemic abnormality or detect any infective etiology . the case highlights the problems of diagnosis of multiple ulcers at very early age .

primary human schlemm 's canal cell lines were grown to confluence in six - well plates ( bd falcon , franklin lakes , nj , usa ) containing dulbecco 's modified eagle 's medium ( dmem ; life technologies , grand island , ny , usa ) ; 10% fetal bovine serum ( mediatech , manassas , va , usa ) ; and 1% penicillin / streptomycin ( life technologies ) in 5% co2 at 37c . confluent cells were washed twice with phosphate - buffered saline and incubated in serum - free dmem for 24 hours to synchronize the growth potential of the cells . cells were incubated with latanoprost ( de - esterified free - acid form at 100 nm final concentration ; cayman chemical , ann arbor , mi , usa ) or vehicle ( ethanol , final dilution 1:1000 ) in dmem containing 1% penicillin / streptomycin . primary schlemm 's canal cells were grown to confluence , treated with 100 nm latanoprost for 6 hours , and harvested . total rna was extracted using an rna isolation kit ( rneasy total rna isolation kit ; qiagen , hilden , germany ) . approximately 250 ng of total rna was reverse transcribed into cdna using a synthesis kit ( iscript cdna ; bio - rad laboratories , inc . , hercules , ca , usa ) . quantitative real - time polymerase chain ( qpcr ) reaction using stc-1 ( forward , 5-aggcggagcagaatgactc-3 ; reverse , 5-gttgaggcaacgaaccactt-3 ) and glyceraldehyde 3-phosphate dehydrogenase ( gapdh : forward , 5-cctctgacttcaacagc-3 ; reverse , 5-gctgtagccaaattcgt-3 ) primers were performed on a pcr system using a master mix ( roche light cycler 480 with sybr green i ; roche , indianapolis , in , usa ) . we performed qpcr amplification with a predenaturation step at 95c followed by 45 cycles of denaturation at 95c , annealing at 63c , and extension at 72c . primary schlemm 's canal cells were grown to confluence , treated with 100 nm latanoprost for 15 minutes , 1 , 2 , 4 , or 6 hours , and harvested . schlemm 's canal cell pellets were suspended in ice - cold lysis buffer ( 50 mm tris ph 8.0 , 0.5% sodium dodecyl sulfate , 0.5% triton x-100 , 137 mm nacl , 3 mm kcl , 8 mm na2hpo4 - 7h2o , 1 mm kh2po4 , protease inhibitors ; roche ) and passed repeatedly through a 21-gauge needle for homogenization . lysate was centrifuged at 13,000 g for 10 minutes , and total protein was quantified by the bradford assay . cell lysates containing 20 g total protein were mixed with reducing lane marker sample buffer ( thermo fisher scientific , waltham , ma , usa ) containing 15% 2-mercaptoethanol ( sigma - aldrich corp . , st . louis , mo , usa ) , heated and separated on a 4% to 15% sds - page gradient gel ( bio - rad laboratories , inc . ) . proteins were transferred to polyvinylidene difluoride membrane ( millipore corp . , billerica , ma , usa ) in 1x transfer buffer ( 50 mm tris , 384 mm glycine , 0.01% sds , 20% methanol ) . membranes were blocked in 20 mm tris ( ph 7.5 ) , 150 mm nacl , 0.05% tween-20 , and 2% instant nonfat dry milk . blots were probed with rabbit monoclonal anti - human stc-1 ( novus biologicals , littleton , co , usa ) and mouse monoclonal anti - human gapdh ( novus biologicals ) . secondary antibodies used were horseradish peroxidase linked anti - rabbit or anti - mouse , respectively ( ge healthcare , piscataway , nj , usa ) . antibody / antigen complexes were detected using ecl western blot signal detection reagent ( ge healthcare ) . chemiluminescence film ( biomax xar ; eastman kodak , rochester , ny , usa ) was used to visualize protein signals . each film was digitized with a photographic scanner ( epson perfection 2400 ; epson america , inc . , long beach , ca , usa ) . the band intensities for western blot analysis were quantified using imagej software ( http://rsb.info.nih.gov/ij/index.html in the public domain by the national institutes of health , bethesda , md , usa ) and normalized to gapdh . all animal studies and treatment protocols were approved by the mayo clinic ( rochester , mn , usa ) institutional animal care and use committee and adhered to the arvo statement for the use of animals in ophthalmic and vision research . we obtained stc-1 and littermate wild - type mice from the sheikh - hamad laboratory , baylor college of medicine , and bred at mayo clinic . a handheld rebound tonometer ( icare tonolab ; colonial medical supply , franconia , nh , usa ) was used to measure iop in conscious mice . for iop measurements , the tonometer was held perpendicular to the cornea according to the manufacturer 's instructions . the tonometer records six readings from the same eye , discards the highest and lowest values , and shows the average of the remaining four values as a single iop reading . three independent measurements were obtained daily at similar time points and were averaged to obtain the daily iop value for each eye . after 1 week of baseline iop measurements , stc-1 mice were treated with latanoprost ( n = 10 ) or rho kinase inhibitor y27632 ( enzo life sciences , farmingdale , ny ; n = 10 ) . congenic wild - type controls were treated with latanoprost ( n = 8) or y27632 ( n = 10 ) . treatments were daily in one eye for 7 consecutive days with 5 l of latanoprost - free acid ( 100 m dissolved in 1:1000 dmso in pbs ) or 10 mm y27632 ( dissolved in phosphate - buffered saline ) . in the contralateral eye , vehicle was added daily in the same proportion as the treated eye for 7 consecutive days . additionally , wild - type mice ( n = 7 ; charles rivers laboratories , wilmington , ma , usa ) were treated with 5 l of topically administered recombinant human stc-1 ( 0.5 mg / ml ; biovendor research & diagnostic products , asheville , nc , usa ) or vehicle ( phosphate - buffered saline ) daily for 7 days to examine the effect of stc-1 on iop . in all animals , the right eye served as the vehicle control eye while the left eye received study drug ( latanoprost , y27632 , or stc-1 ) . we recorded iop in both eyes three times daily at 1 , 4 , and 23 hours following treatment . anterior segments from human donor eyes ( age 75.5 17.5 years , range : 51 to 98 years ; n = 8) were perfused in culture with dmem within 10.2 4.4 hours of death as previously described . after achieving a stable baseline pressure , one anterior segment from each pair received recombinant human stc-1 at concentrations of 5 , 50 , or 500 ng / ml ( dissolved in h2o ) , while the fellow eye received vehicle and served as the control . we added stc-1 and vehicle using a gravity - driven constant pressure method of anterior chamber exchange followed by continuous perfusion . hourly pressure readings were obtained from the average of 60 , one - minute pressure measurements using a custom - designed software program . the experimental eye was typically the right eye and the control was the left eye . for human anterior segments , selected wedges of tissue 180 apart that included the trabecular meshwork and schlemm 's canal were isolated and fixed with 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.2 ) . tissue wedges were dehydrated in a series of ascending ethanol concentrations , cleared with 100% acetone , infiltrated and embedded in epon araldite , and sectioned at 0.5 m . eye tissue sections were stained with toluidine blue and examined using a light microscope ( nikon corp . , additional tissue wedges were sectioned at 100 nm , placed on copper film grids , and stained with uranyl acetate and lead citrate . tissue sections on copper film grids were examined using a transmission electron microscope ( jeol-1400 ; jeol usa , inc . , peabody , ma , usa ) . for mouse eye histopathology eyes were placed in 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.2 ) overnight , and processed for light microscopy as described above for human anterior segment wedges . additional 100-nm sections were placed on copper film grids , and stained with uranyl acetate and lead citrate . processed sections were obtained following examination under a light microscope ( nikon corp . ) and a transmission electron microscope ( jeol usa , inc . ) . prior to initiation of statistical analysis , all data sets were evaluated for distribution assessment using the shapiro - wilk test . for animal studies , significance of iop change was assessed between experimental and vehicle - treated control eyes using student 's paired t - test for data sets with normal distribution and wilcoxon sign - rank test for nonparametric data sets . variations in daily iop are graphically presented as the mean daily iop of the vehicle and treated eyes . statistical calculations were performed using a statistical software package ( jmp ; sas institute , inc . , cary , nc , usa ) . for human anterior segment studies , the effect of latanoprost was expressed as the change in outflow facility ( c ) for each anterior segment . results from each pair of anterior segments were combined into a group mean for each drug , and statistical significance was analyzed using a student 's 2-tailed paired t test . primary human schlemm 's canal cell lines were grown to confluence in six - well plates ( bd falcon , franklin lakes , nj , usa ) containing dulbecco 's modified eagle 's medium ( dmem ; life technologies , grand island , ny , usa ) ; 10% fetal bovine serum ( mediatech , manassas , va , usa ) ; and 1% penicillin / streptomycin ( life technologies ) in 5% co2 at 37c . confluent cells were washed twice with phosphate - buffered saline and incubated in serum - free dmem for 24 hours to synchronize the growth potential of the cells . cells were incubated with latanoprost ( de - esterified free - acid form at 100 nm final concentration ; cayman chemical , ann arbor , mi , usa ) or vehicle ( ethanol , final dilution 1:1000 ) in dmem containing 1% penicillin / streptomycin . primary schlemm 's canal cells were grown to confluence , treated with 100 nm latanoprost for 6 hours , and harvested . total rna was extracted using an rna isolation kit ( rneasy total rna isolation kit ; qiagen , hilden , germany ) . approximately 250 ng of total rna was reverse transcribed into cdna using a synthesis kit ( iscript cdna ; bio - rad laboratories , inc . , hercules , ca , usa ) . quantitative real - time polymerase chain ( qpcr ) reaction using stc-1 ( forward , 5-aggcggagcagaatgactc-3 ; reverse , 5-gttgaggcaacgaaccactt-3 ) and glyceraldehyde 3-phosphate dehydrogenase ( gapdh : forward , 5-cctctgacttcaacagc-3 ; reverse , 5-gctgtagccaaattcgt-3 ) primers were performed on a pcr system using a master mix ( roche light cycler 480 with sybr green i ; roche , indianapolis , in , usa ) . we performed qpcr amplification with a predenaturation step at 95c followed by 45 cycles of denaturation at 95c , annealing at 63c , and extension at 72c . primary schlemm 's canal cells were grown to confluence , treated with 100 nm latanoprost for 15 minutes , 1 , 2 , 4 , or 6 hours , and harvested . schlemm 's canal cell pellets were suspended in ice - cold lysis buffer ( 50 mm tris ph 8.0 , 0.5% sodium dodecyl sulfate , 0.5% triton x-100 , 137 mm nacl , 3 mm kcl , 8 mm na2hpo4 - 7h2o , 1 mm kh2po4 , protease inhibitors ; roche ) and passed repeatedly through a 21-gauge needle for homogenization . lysate was centrifuged at 13,000 g for 10 minutes , and total protein was quantified by the bradford assay . cell lysates containing 20 g total protein were mixed with reducing lane marker sample buffer ( thermo fisher scientific , waltham , ma , usa ) containing 15% 2-mercaptoethanol ( sigma - aldrich corp . , st . louis , mo , usa ) , heated and separated on a 4% to 15% sds - page gradient gel ( bio - rad laboratories , inc . ) . proteins were transferred to polyvinylidene difluoride membrane ( millipore corp . , billerica , ma , usa ) in 1x transfer buffer ( 50 mm tris , 384 mm glycine , 0.01% sds , 20% methanol ) . membranes were blocked in 20 mm tris ( ph 7.5 ) , 150 mm nacl , 0.05% tween-20 , and 2% instant nonfat dry milk . blots were probed with rabbit monoclonal anti - human stc-1 ( novus biologicals , littleton , co , usa ) and mouse monoclonal anti - human gapdh ( novus biologicals ) . secondary antibodies used were horseradish peroxidase linked anti - rabbit or anti - mouse , respectively ( ge healthcare , piscataway , nj , usa ) . antibody / antigen complexes were detected using ecl western blot signal detection reagent ( ge healthcare ) . chemiluminescence film ( biomax xar ; eastman kodak , rochester , ny , usa ) was used to visualize protein signals . each film was digitized with a photographic scanner ( epson perfection 2400 ; epson america , inc . , long beach , ca , usa ) . the band intensities for western blot analysis were quantified using imagej software ( http://rsb.info.nih.gov/ij/index.html in the public domain by the national institutes of health , bethesda , md , usa ) and normalized to gapdh . primary human schlemm 's canal cell lines were grown to confluence in six - well plates ( bd falcon , franklin lakes , nj , usa ) containing dulbecco 's modified eagle 's medium ( dmem ; life technologies , grand island , ny , usa ) ; 10% fetal bovine serum ( mediatech , manassas , va , usa ) ; and 1% penicillin / streptomycin ( life technologies ) in 5% co2 at 37c . confluent cells were washed twice with phosphate - buffered saline and incubated in serum - free dmem for 24 hours to synchronize the growth potential of the cells . cells were incubated with latanoprost ( de - esterified free - acid form at 100 nm final concentration ; cayman chemical , ann arbor , mi , usa ) or vehicle ( ethanol , final dilution 1:1000 ) in dmem containing 1% penicillin / streptomycin . primary schlemm 's canal cells were grown to confluence , treated with 100 nm latanoprost for 6 hours , and harvested . total rna was extracted using an rna isolation kit ( rneasy total rna isolation kit ; qiagen , hilden , germany ) . approximately 250 ng of total rna was reverse transcribed into cdna using a synthesis kit ( iscript cdna ; bio - rad laboratories , inc . , hercules , ca , usa ) . quantitative real - time polymerase chain ( qpcr ) reaction using stc-1 ( forward , 5-aggcggagcagaatgactc-3 ; reverse , 5-gttgaggcaacgaaccactt-3 ) and glyceraldehyde 3-phosphate dehydrogenase ( gapdh : forward , 5-cctctgacttcaacagc-3 ; reverse , 5-gctgtagccaaattcgt-3 ) primers were performed on a pcr system using a master mix ( roche light cycler 480 with sybr green i ; roche , indianapolis , in , usa ) . we performed qpcr amplification with a predenaturation step at 95c followed by 45 cycles of denaturation at 95c , annealing at 63c , and extension at 72c . primary schlemm 's canal cells were grown to confluence , treated with 100 nm latanoprost for 15 minutes , 1 , 2 , 4 , or 6 hours , and harvested . schlemm 's canal cell pellets were suspended in ice - cold lysis buffer ( 50 mm tris ph 8.0 , 0.5% sodium dodecyl sulfate , 0.5% triton x-100 , 137 mm nacl , 3 mm kcl , 8 mm na2hpo4 - 7h2o , 1 mm kh2po4 , protease inhibitors ; roche ) and passed repeatedly through a 21-gauge needle for homogenization . lysate was centrifuged at 13,000 g for 10 minutes , and total protein was quantified by the bradford assay . cell lysates containing 20 g total protein were mixed with reducing lane marker sample buffer ( thermo fisher scientific , waltham , ma , usa ) containing 15% 2-mercaptoethanol ( sigma - aldrich corp . st . louis , mo , usa ) , heated and separated on a 4% to 15% sds - page gradient gel ( bio - rad laboratories , inc . ) . proteins were transferred to polyvinylidene difluoride membrane ( millipore corp . , billerica , ma , usa ) in 1x transfer buffer ( 50 mm tris , 384 mm glycine , 0.01% sds , 20% methanol ) . membranes were blocked in 20 mm tris ( ph 7.5 ) , 150 mm nacl , 0.05% tween-20 , and 2% instant nonfat dry milk . blots were probed with rabbit monoclonal anti - human stc-1 ( novus biologicals , littleton , co , usa ) and mouse monoclonal anti - human gapdh ( novus biologicals ) . secondary antibodies used were horseradish peroxidase linked anti - rabbit or anti - mouse , respectively ( ge healthcare , piscataway , nj , usa ) . antibody / antigen complexes were detected using ecl western blot signal detection reagent ( ge healthcare ) . chemiluminescence film ( biomax xar ; eastman kodak , rochester , ny , usa ) was used to visualize protein signals . each film was digitized with a photographic scanner ( epson perfection 2400 ; epson america , inc . the band intensities for western blot analysis were quantified using imagej software ( http://rsb.info.nih.gov/ij/index.html in the public domain by the national institutes of health , bethesda , md , usa ) and normalized to gapdh . all animal studies and treatment protocols were approved by the mayo clinic ( rochester , mn , usa ) institutional animal care and use committee and adhered to the arvo statement for the use of animals in ophthalmic and vision research . we obtained stc-1 and littermate wild - type mice from the sheikh - hamad laboratory , baylor college of medicine , and bred at mayo clinic . mice , aged 5 to 8 months , were utilized in the experiments . a handheld rebound tonometer ( icare tonolab ; colonial medical supply , franconia , nh , usa ) was used to measure iop in conscious mice . for iop measurements , . the tonometer records six readings from the same eye , discards the highest and lowest values , and shows the average of the remaining four values as a single iop reading . three independent measurements were obtained daily at similar time points and were averaged to obtain the daily iop value for each eye . after 1 week of baseline iop measurements , stc-1 mice were treated with latanoprost ( n = 10 ) or rho kinase inhibitor y27632 ( enzo life sciences , farmingdale , ny ; n = 10 ) . congenic wild - type controls were treated with latanoprost ( n = 8) or y27632 ( n = 10 ) . treatments were daily in one eye for 7 consecutive days with 5 l of latanoprost - free acid ( 100 m dissolved in 1:1000 dmso in pbs ) or 10 mm y27632 ( dissolved in phosphate - buffered saline ) . in the contralateral eye , vehicle was added daily in the same proportion as the treated eye for 7 consecutive days . additionally , wild - type mice ( n = 7 ; charles rivers laboratories , wilmington , ma , usa ) were treated with 5 l of topically administered recombinant human stc-1 ( 0.5 mg / ml ; biovendor research & diagnostic products , asheville , nc , usa ) or vehicle ( phosphate - buffered saline ) daily for 7 days to examine the effect of stc-1 on iop . in all animals , the right eye served as the vehicle control eye while the left eye received study drug ( latanoprost , y27632 , or stc-1 ) . we recorded iop in both eyes three times daily at 1 , 4 , and 23 hours following treatment . anterior segments from human donor eyes ( age 75.5 17.5 years , range : 51 to 98 years ; n = 8) were perfused in culture with dmem within 10.2 4.4 hours of death as previously described . after achieving a stable baseline pressure , one anterior segment from each pair received recombinant human stc-1 at concentrations of 5 , 50 , or 500 ng / ml ( dissolved in h2o ) , while the fellow eye received vehicle and served as the control . we added stc-1 and vehicle using a gravity - driven constant pressure method of anterior chamber exchange followed by continuous perfusion . hourly pressure readings were obtained from the average of 60 , one - minute pressure measurements using a custom - designed software program . the experimental eye was typically the right eye and the control was the left eye . for human anterior segments , selected wedges of tissue 180 apart that included the trabecular meshwork and schlemm 's canal were isolated and fixed with 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.2 ) . tissue wedges were dehydrated in a series of ascending ethanol concentrations , cleared with 100% acetone , infiltrated and embedded in epon araldite , and sectioned at 0.5 m . eye tissue sections were stained with toluidine blue and examined using a light microscope ( nikon corp . , additional tissue wedges were sectioned at 100 nm , placed on copper film grids , and stained with uranyl acetate and lead citrate . tissue sections on copper film grids were examined using a transmission electron microscope ( jeol-1400 ; jeol usa , inc . , peabody , ma , usa ) . for mouse eye histopathology , whole eyes were enucleated from euthanized mice following termination of the experiment . eyes were placed in 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.2 ) overnight , and processed for light microscopy as described above for human anterior segment wedges . additional 100-nm sections were placed on copper film grids , and stained with uranyl acetate and lead citrate . processed sections were obtained following examination under a light microscope ( nikon corp . ) and a transmission electron microscope ( jeol usa , inc . ) . prior to initiation of statistical analysis , all data sets were evaluated for distribution assessment using the shapiro - wilk test . for animal studies , significance of iop change was assessed between experimental and vehicle - treated control eyes using student 's paired t - test for data sets with normal distribution and wilcoxon sign - rank test for nonparametric data sets . variations in daily iop are graphically presented as the mean daily iop of the vehicle and treated eyes . statistical calculations were performed using a statistical software package ( jmp ; sas institute , inc . , cary , nc , usa ) . for human anterior segment studies , the effect of latanoprost was expressed as the change in outflow facility ( c ) for each anterior segment . results from each pair of anterior segments were combined into a group mean for each drug , and statistical significance was analyzed using a student 's 2-tailed paired t test . preliminary findings from confluent primary human schlemm 's canal cell lines treated with latanoprost identified stc-1 , a secreted phosphoglycoprotein hormone with multiple functions , as a gene consistently upregulated following latanoprost treatment . quantitative pcr studies in human schlemm 's canal cells showed an 18.1 4.3-fold ( n = 3 ) induction . cell lysates isolated from latanoprost - treated primary human schlemm 's canal cells at various time points showed an increase of stc-1 protein expression of 1.9-fold at 2 hours and approximately 5.0-fold at 4 and 6 hours , correlating with increased mrna expression levels identified by qpcr ( fig . 1 ) . induction of stc-1 in schlemm 's canal cells following treatment with latanoprost . because of its multifunctional properties and ability to act in an autocrine and paracrine fashion similar to prostaglandin analogs , we examined the role of stc-1 in downstream signaling following latanoprost treatment in vivo using stc-1 mice and congenic wild - type controls . assessment of baseline iops for 6 days showed no significant difference between stc-1 mice ( 16.6 0.5 , n = 10 ) and wild - type controls ( 16.2 0.3 , n = 8 ; p = 0.1 ) . histologic examination of stc-1 mice by light and transmission electron microscopy showed a normal - appearing ocular anatomy , an open angle with appropriate trabecula within the trabecular meshwork , and intact inner and outer walls of schlemm 's canal , all similar to wild - type controls ( fig . , we proceeded to assess the effect of latanoprost treatment on iop in these mice . topical eye treatment with latanoprost ( 100 nm ) reduced iop in wild - type controls by 3.8 mm hg ( p < 0.001 , n = 8) , which correlated to a 22.0% 1.9% decrease in iop when compared to the fellow contralateral eyes treated with vehicle alone ( fig . consistent iop change was identified throughout the once daily dose as determined by iop pressure monitoring at 1 , 4 , and 23 hours after treatment ( table ) . in contrast , stc-1 mice did not show any significant reduction in iop after topical latanoprost treatment ( 0.5% 0.7% , p = 0.34 , n = 10 ) at any time point throughout the 7-day treatment regime . to determine if this was unique to latanoprost , we treated stc-1 with y27632 , a rho kinase inhibitor that increases outflow through both the uveoscleral and the conventional outflow pathway . treatment of either wild - type ( n = 10 ) or stc-1 ( n = 10 ) mice with y27632 resulted in significant reductions of iop , 12.5% 1.2% and 13.1% 2.8% , respectively ( p < 0.0001 , n = 10 ; fig . 4 ; table ) . these data suggest that stc-1 has a unique and key role in latanoprost signaling mediated iop reduction . top : light microscopy images of whole eye sections from wild - type and stc-1 mice . wild - type and stc-1 mice have similar - sized lens with both wild - type and stc-1 mice showing similar retinal morphology . middle : light microscopy images of wild - type and stc-1 mice showing conventional outflow pathway with open angles and similar number of trabecula , trabecular meshwork ( tm ) cells , and schlemm 's canal ( sc ) volume . bottom : transmission electron micrographs of wild - type and stc-1 mice showing similar appearing trabecular meshwork and intact schlemm 's canal inner walls ( iw ) and outer walls ( ow ) . the daily iop was recorded as the average of iop measurements at 1 , 4 , and 23 hours following latanoprost treatment . while congenic control mice show iop reduction with latanoprost , stc-1 do not , suggesting stc-1 is an important effector molecule for latanoprost - induced iop reduction . intraocular pressure in wild - type and stc-1 mice following treatment with latanoprost and rho kinase inhibitor rho kinase inhibitor y27632 reduces iop in stc-1 mice . n = 10 ) and congenic controls ( n = 10 ) were treated once daily with 10 mm y27632 . the daily iop was recorded as the average of iop measurements at 1 , 4 , and 23 hours following y27632 treatment . both stc-1 mice and congenic controls showed iop reduction following treatment with y27632 . to determine if stc-1 would influence iop reduction by itself , we perfused human anterior segments with several concentrations of recombinant stc-1 for 24 hours . perfusion with either 5 ng / ml ( 0.15 0.04 to 0.15 0.04 l / min / mm hg , n = 2 , p = 0.20 ) or 50 ng / ml ( 0.14 0.05 to 0.18 0.07 l / min / mm hg , n = 4 , p = 0.20 ) had no significant effect on outflow facility . however , all anterior segments perfused with stc-1 at 500 ng / ml had decreased pressure and increased outflow facility ( 0.15 0.03 to 0.27 0.09 l / min / mm hg , n = 5 , p = 0.02 ) compared to baseline ( fig . 5 ) . paired controls treated with vehicle showed no change in outflow facility from their baseline values . to determine whether the changes in pressure could be secondary to morphologic changes morphologic analysis showed viable and healthy cells in the trabecular meshwork and schlemm 's canal of control and treated eyes ( fig . no major disruptions of the juxtacanalicular tissue or the basement membrane of schlemm 's canal inner and outer walls were observed . these results indicate that stc-1 may be a molecule within the latanoprost signaling pathway that can be therapeutically targeted to lower iop . ( a ) outflow facility of human anterior segments ( n = 8) following perfusion with 5 , 50 , or 500 ng / ml of recombinant human stc-1 . ( b ) representative graph of an eye pair perfused with 500 ng / ml stc-1 . ( a , b ) representative sections ( 3 m ) of recombinant stc-1 and vehicle - treated eyes that were stained with toluidine blue . ( c , d ) transmission electron micrographs showing ultrastructure of recombinant stc-1 and vehicle - treated eyes . recombinant human stc-1 and vehicle - treated eyes had similar morphology and ultrastructural appearance suggesting no apparent detrimental side effects of recombinant stc-1 treatment . ac , anterior chamber ; jct , juxtacanalicular region ; tm , trabecular meshwork ; sc , schlemm 's canal . to determine if stc-1 has ocular hypotensive activity in vivo , wild - type c57bl/6 mice were treated with recombinant stc-1 ( 0.5 mg / ml ) or vehicle daily for 7 days ( fig . stanniocalcin-1 reduced iop by 15.2% 3.0% when compared to vehicle - treated contralateral eyes ( range of iop reduction from 5.1% 5.2% these results suggest that stc-1 has iop - lowering properties when used as a standalone agent . wild - type mice ( n = 10 ) were treated topically with recombinant stc-1 ( 0.5 mg / ml ) or vehicle ( phosphate buffered saline ) . the daily iop was recorded as the average of iop measurements at 1 , 4 , and 23 hours following stc-1 treatment . preliminary findings from confluent primary human schlemm 's canal cell lines treated with latanoprost identified stc-1 , a secreted phosphoglycoprotein hormone with multiple functions , as a gene consistently upregulated following latanoprost treatment . quantitative pcr studies in human schlemm 's canal cells showed an 18.1 4.3-fold ( n = 3 ) induction . cell lysates isolated from latanoprost - treated primary human schlemm 's canal cells at various time points showed an increase of stc-1 protein expression of 1.9-fold at 2 hours and approximately 5.0-fold at 4 and 6 hours , correlating with increased mrna expression levels identified by qpcr ( fig . 1 ) . induction of stc-1 in schlemm 's canal cells following treatment with latanoprost . because of its multifunctional properties and ability to act in an autocrine and paracrine fashion similar to prostaglandin analogs , we examined the role of stc-1 in downstream signaling following latanoprost treatment in vivo using stc-1 mice and congenic wild - type controls . assessment of baseline iops for 6 days showed no significant difference between stc-1 mice ( 16.6 0.5 , n = 10 ) and wild - type controls ( 16.2 0.3 , n = 8 ; p = 0.1 ) . histologic examination of stc-1 mice by light and transmission electron microscopy showed a normal - appearing ocular anatomy , an open angle with appropriate trabecula within the trabecular meshwork , and intact inner and outer walls of schlemm 's canal , all similar to wild - type controls ( fig . , we proceeded to assess the effect of latanoprost treatment on iop in these mice . topical eye treatment with latanoprost ( 100 nm ) reduced iop in wild - type controls by 3.8 mm hg ( p < 0.001 , n = 8) , which correlated to a 22.0% 1.9% decrease in iop when compared to the fellow contralateral eyes treated with vehicle alone ( fig . consistent iop change was identified throughout the once daily dose as determined by iop pressure monitoring at 1 , 4 , and 23 hours after treatment ( table ) . in contrast , stc-1 mice did not show any significant reduction in iop after topical latanoprost treatment ( 0.5% 0.7% , p = 0.34 , n = 10 ) at any time point throughout the 7-day treatment regime . to determine if this was unique to latanoprost , we treated stc-1 with y27632 , a rho kinase inhibitor that increases outflow through both the uveoscleral and the conventional outflow pathway . treatment of either wild - type ( n = 10 ) or stc-1 ( n = 10 ) mice with y27632 resulted in significant reductions of iop , 12.5% 1.2% and 13.1% 2.8% , respectively ( p < 0.0001 , n = 10 ; fig . 4 ; table ) . these data suggest that stc-1 has a unique and key role in latanoprost signaling mediated iop reduction . top : light microscopy images of whole eye sections from wild - type and stc-1 mice . wild - type and stc-1 mice have similar - sized lens with both wild - type and stc-1 mice showing similar retinal morphology . middle : light microscopy images of wild - type and stc-1 mice showing conventional outflow pathway with open angles and similar number of trabecula , trabecular meshwork ( tm ) cells , and schlemm 's canal ( sc ) volume . bottom : transmission electron micrographs of wild - type and stc-1 mice showing similar appearing trabecular meshwork and intact schlemm 's canal inner walls ( iw ) and outer walls ( ow ) . the daily iop was recorded as the average of iop measurements at 1 , 4 , and 23 hours following latanoprost treatment . while congenic control mice show iop reduction with latanoprost , stc-1 do not , suggesting stc-1 is an important effector molecule for latanoprost - induced iop reduction . intraocular pressure in wild - type and stc-1 mice following treatment with latanoprost and rho kinase inhibitor rho kinase inhibitor y27632 reduces iop in stc-1 mice . stc-1 mice ( n = 10 ) and congenic controls ( n = 10 ) were treated once daily with 10 mm y27632 . the daily iop was recorded as the average of iop measurements at 1 , 4 , and 23 hours following y27632 treatment . to determine if stc-1 would influence iop reduction by itself , we perfused human anterior segments with several concentrations of recombinant stc-1 for 24 hours . perfusion with either 5 ng / ml ( 0.15 0.04 to 0.15 0.04 l / min / mm hg , n = 2 , p = 0.20 ) or 50 ng / ml ( 0.14 0.05 to 0.18 0.07 l / min / mm hg , n = 4 , p = 0.20 ) had no significant effect on outflow facility . however , all anterior segments perfused with stc-1 at 500 ng / ml had decreased pressure and increased outflow facility ( 0.15 0.03 to 0.27 0.09 l / min / mm hg , n = 5 , p = 0.02 ) compared to baseline ( fig . 5 ) . paired controls treated with vehicle showed no change in outflow facility from their baseline values . to determine whether the changes in pressure could be secondary to morphologic changes morphologic analysis showed viable and healthy cells in the trabecular meshwork and schlemm 's canal of control and treated eyes ( fig . 6 ) . no major disruptions of the juxtacanalicular tissue or the basement membrane of schlemm 's canal inner and outer walls were observed . these results indicate that stc-1 may be a molecule within the latanoprost signaling pathway that can be therapeutically targeted to lower iop . ( a ) outflow facility of human anterior segments ( n = 8) following perfusion with 5 , 50 , or 500 ng / ml of recombinant human stc-1 . ( b ) representative graph of an eye pair perfused with 500 ng / ml stc-1 . p 0.05 . histologic analysis of human anterior segment ocular tissue following treatment with recombinant stc-1 . ( a , b ) representative sections ( 3 m ) of recombinant stc-1 and vehicle - treated eyes that were stained with toluidine blue . ( c , d ) transmission electron micrographs showing ultrastructure of recombinant stc-1 and vehicle - treated eyes . recombinant human stc-1 and vehicle - treated eyes had similar morphology and ultrastructural appearance suggesting no apparent detrimental side effects of recombinant stc-1 treatment . ac , anterior chamber ; jct , juxtacanalicular region ; tm , trabecular meshwork ; sc , schlemm 's canal . to determine if stc-1 has ocular hypotensive activity in vivo , wild - type c57bl/6 mice were treated with recombinant stc-1 ( 0.5 mg / ml ) or vehicle daily for 7 days ( fig . stanniocalcin-1 reduced iop by 15.2% 3.0% when compared to vehicle - treated contralateral eyes ( range of iop reduction from 5.1% 5.2% [ day 1 ] to 21.4% 5.3% these results suggest that stc-1 has iop - lowering properties when used as a standalone agent . wild - type mice ( n = 10 ) were treated topically with recombinant stc-1 ( 0.5 mg / ml ) or vehicle ( phosphate buffered saline ) . the daily iop was recorded as the average of iop measurements at 1 , 4 , and 23 hours following stc-1 treatment . prostaglandin analogs like latanoprost are a first - line medical therapy for iop reduction due to their once daily dosing , greater response rates , and greater amount of iop reduction compared to other classes of pressure - lowering medications . identifying the critical effector molecules in the signaling pathway of latanoprost will provide insights into additional molecules to target for iop reduction , potentially with fewer side effects . in the current study , we identified stc-1 as a transcript and protein that is highly induced following treatment of human schlemm 's canal cells with latanoprost . topical latanoprost significantly reduced iop in wild - type controls while stc-1 mice demonstrated no iop reduction to topical latanoprost . in contrast , rho kinase inhibitor y27632 demonstrated iop reduction in both wild - type and stc-1 mice , indicating that stc-1 is a unique and important downstream signaling molecule necessary for the ocular hypotensive properties of latanoprost . additionally , recombinant stc-1 by itself also increased outflow facility in human anterior segments and reduced iop in vivo in wild - type mice . together , these results suggest that stc-1 is a critical and unique effector molecule for the latanoprost signaling pathway that is necessary for latanoprost - induced iop reduction and that stc-1 by itself can act as an ocular hypotensive agent . stanniocalcin was first described in fish as a 50-kda homodimeric glycoprotein that is secreted from the corpuscles of stannius into the bloodstream in the setting of hypercalcemia to regulate calcium excretion at the gills and gut . in mammals , two homologues of stc stc-1 is the most studied of the two mammalian forms , having a 50% amino acid homology with its fish counterpart . stc-1 is a secreted , homodimeric phosphoglycoprotein that has preserved protein structure similarity between mammals and fish including the conservation of 11 cysteine residues . it is expressed in a wide variety of tissues most notably bone , skeletal muscle , heart , thymus , and spleen . functionally , stc-1 has been associated with calcium uptake , hypoxic preconditioning , and antioxidative stress properties through suppression of reactive oxygen species . additionally , stc-1 has also been shown to be neuroprotective for neurons , photoreceptors , and retinal ganglion cells , and has been linked to anti - inflammatory effects by inhibiting macrophage chemotaxis , modulating transendothelial migration of leukocytes and reducing t cell infiltration . in treating wild - type mice with topical latanoprost , we observed a reduction in iop by approximately 22.0% 1.9% , consistent with previous reports , while the stc-1 mice showed no response ( 0.55% 0.7% ) . mice that were stc-1 were responsive to rho kinase inhibitor y27632 , which confirmed that stc-1 mice have normal functioning outflow pathways , but that elimination of stc-1 expression in these mice renders topical latanoprost ineffective . the current study adds schlemm 's canal cells to the list of cells that express stc-1 and shows that its expression is highly influenced by latanoprost . this is an important finding as little is known about the molecular events that connect latanoprost treatment with iop reduction . latanoprost has been shown to phosphorylate myosin light chain kinase in the iris , indicating a role for latanoprost in cell relaxation . however , stc-1 is the first individual molecule that has been identified as a key downstream effector of latanoprost signaling . in addition to its variety of functional activities , our results also suggest that stc-1 has ocular hypotensive properties , since addition of recombinant stc-1 to human anterior segment cultures decreased pressure and increased outflow facility , and when applied topically to wild - type mice reduced iop in vivo . despite the benefits of latanoprost in lowering iop , long - term treatment with latanoprost can often cause significant side effects , which can be contraindicative for its prolonged usage . identification of stc-1 as a downstream signaling molecule through which latanoprost executes its iop - lowering effects , and the fact that recombinant stc-1 shows ocular hypotensive properties , makes this an attractive therapeutic target . utilization of a specific downstream molecule such as stc-1 within the latanoprost signaling cascade may eliminate some of the major side effects while providing a specialized therapeutic strategy . in summary , additionally , stc-1 demonstrates ocular hypotensive properties when used by itself , mimicking the effect of latanoprost . given its novel role in latanoprost - mediated iop reduction , stc-1 may be considered a promising candidate molecule for devising future therapeutic regiments to reduce iop .

purposeto identify downstream signaling molecules through which intraocular pressure ( iop ) is lowered following treatment with the prostaglandin analog latanoprost.methodstotal rna and protein isolated from primary human schlemm 's canal cells ( n = 3 ) treated with latanoprost ( free acid ; 100 nm ) were processed for quantitative pcr and western blot analysis . iop was evaluated in stanniocalcin-1 ( stc-1/ ) and wild - type mice following treatment with latanoprost or rho kinase inhibitor y27632 . human anterior segment pairs ( n = 8) were treated with recombinant stc-1 ( 5 , 50 , or 500 ng / ml ) and pressure was recorded using custom - designed software . the effect of recombinant stc-1 ( 0.5 mg / ml ) on iop was evaluated in wild - type mice . tissue morphology was evaluated by light and transmission electron microscopy.resultsincreased stc-1 mrna ( 4.0- to 25.2-fold ) and protein expression ( 1.9- to 5.1-fold ) was observed within 12 hours following latanoprost treatment . latanoprost reduced iop in wild - type mice ( 22.0% 1.9% ) , but had no effect on stc-1/ mice ( 0.5% 0.7% ) . in contrast , y27632 reduced iop in both wild - type ( 12.5% 1.2% ) and in stc-1/ mice ( 13.1% 2.8% ) . human anterior segments treated with stc-1 ( 500 ng / ml ) showed an increase in outflow facility ( 0.15 0.03 to 0.27 0.09 l / min / mm hg ) while no change was observed in paired vehicle - treated controls . recombinant stc-1 reduced iop in wild - type mice by 15.2% 3.0% . no observable morphologic changes were identified between treatment groups when evaluated by microscopy.conclusionslatanoprost-induced reduction of iop is mediated through the downstream signaling molecule stc-1 . when used by itself , stc-1 exhibits ocular hypotensive properties .