The proteomics literature has seen a proliferation of publications that seek to apply the rapidly improving technology of 2D gels to study various biological systems. However, there is a dearth of systematic studies that have investigated appropriate statistical approaches to analyse the data from these experiments.Comparison of the effects of statistical pre-processing on the results of two sample t-tests suggests that the results of 2D gel experiments and by extension the conclusions derived from these experiments are not independent of the statistical protocol used.This study suggests that there is a need for well-conducted validation studies to establish optimal statistical techniques to be used on such data sets. The effort to produce an index of all human proteins (the human protein index, or HPI) began over twenty years ago. This project pre-dates the human genome project by more than a decade. However, the complexity of the task of creating this index was underestimated and the relative simplicity of the human genome with four known nucleic acids arranged in a linear coding order allowed the process of the sequencing of the human genome to progress exponentially . The sucTwo-dimensional electrophoresis is an extremely powerful tool for the analysis of complex protein mixtures. Proteins carry both positive and negative charges. The pH of the medium they are in determines their net charge. The pH that gives a zero net charge is the isoelectric point of the protein (pI). In Isoelectric Focusing (IEF), protein mixtures are electrophoresed in a gel containing a pH gradient. The proteins in the mixture migrate according to charge density until they reach the part of the gel that corresponds to their pI. At this point, their net charge is zero, and migration stops. This is the first dimension of separation in a 2D gel experiment. The electrophoresed gel is then layered on top of a polyacrylamide gel and electrophoresed once again. The proteins now move from top to bottom depending on molecular weight. The distance covered by a protein is inversely proportional to its size. This is the second dimension in a 2D gel. 2D is an effective method for identifying qualitative and quantitative differences between proteins expressed in various tissues or between tissues exposed to different experimental treatments. Although the number of proteins displayed by 2D is much lower than the estimated number of genes in a particular tissue, 2D is currently the only available technique that enables the isolation and separation of thousands of the individual proteins that constitute a tissue proteome . AndersoImages of 2D gels are acquired into a database using an image scanner. Image analysis software converts the gel image into a digitised image in a computer, matches gels and spots on gels across the different groups and creates a database with information about spot intensity and spot location. As mentioned above, the two variables – the pI representing net charge of the protein and the molecular weight of the protein – are not correlated. In geometric terms this suggests that the two dimensions are orthogonal to each other. The two dimensions in a two-dimensional gel thus can be thought of as the two axes in a two dimensional graph. The coordinate on the x-axis is a measure of the isoelectric point (pI) of the protein, and the coordinate on the y-axis is a measure of the molecular weight of the proteinThe information in the database includes a gel identification variable, a spot identification variable, the x and y coordinates of a protein spot and its intensity measured by the amount of light transmitted by the spot. Depending on the software package, one can obtain other parameters in the database, including a measure of the quality of the spot to various measures associated with spot intensity, such as volume, area, peak height, etc.The intensity of a protein spot is assumed to be directly related to the amount of protein in the particular tissue under investigation at that given time point. Changes in protein intensity are therefore approximated by changes in the intensities of protein spots in gel images. Changes in protein structure associated with post-translational modifications such as phosphorylation, oxidative modification or glycosylations may result in changes in the pI or molecular weight of the protein and are manifested in the gel by a change in the vertical or horizontal position. The object of 2D gel experiments is to detect differences in protein intensity/complexity between two groups of gels.A number of recent publications -10 have Classification is the process of assigning objects to a category. An interest in classification permeates many scientific studies . There aBoth types of classification problems have three stages, input, algorithm and output. Most published literature concentrates on the second of these. However, careful thought about what variables to use and how to characterize or summarize them as inputs into an algorithm are very important issues . It is eIn order to conduct a systematic analysis of 2D gel data, one has to pre-process the data set. Pre-processing in the case of 2D gel analysis includes: 1) normalizing intensities to remove effects of differential loading and staining; 2) transformation of outcome variables to normally distributed variables; and 3) imputing values for missing spot intensities. We are not aware of any other study that has looked systematically at the effect of pre-processing 2D gel data on the results of subsequent statistical analysis of the data. In this paper, we present a protocol for the analysis of 2D gel data and examine the effect of statistical pre-processing of 2D gel datasets.The effect of using two different formulas for normalizing versus not normalizing, log-transformation versus no log-transformation and single value imputation versus multiple value imputation, and averaging spot intensities across replicates versus keeping the replicate information separate are compared using the results of two sample t-tests. We also compare the results of two-sample t-tests provided by the image analysis software PDQUEST to results obtained after following the protocol described in Figure The data used in this study is from an experiment that looked at the effect of a diet enhanced with grape seed extract on the proteome of whole brain homogenates of Sprague-Dawley rats . There wThe resolution of protein spots in a 2D gel is highly variable. It can differ considerably between technical replicates of the same biological sample. Samples 6, 7, 8, 9, and 10 were the five biological replicates in the treatment group. Samples 22, 23, 24, 25, and 26 were the five biological replicates in the control group. Table Table In Tables ® and most data sets produced by the software PROGENESIS®. Only one out of seven 2D gel data sets analysed by us so far did not respond well to this transformation. A closer examination of this data showed it had a large number of saturated spots, and thus needed to be rerun. The transformation of the distribution of the 201 spots also worked reasonably well at the level of individual spot intensities. This was important to confirm since the two sample tests were done at individual spot level. In cases where there was a considerable skew in the distribution, e.g., SSP 1733, the log transformation made the distribution of spot intensities normal. The Anderson-Darling test for normality for SSP 1733 in the control group, has a p-value = 0.03 before the transformation, and p-value = 0.456 after the transformation. In general, this was true of most spots we examined.Figures Table Table In Table Given the lack of association between technical replicates, we used replicate information in two ways: 1) Spot intensities were averaged across the replicates so that the t-tests compared average spot intensities in the five treatment samples versus five control samples, and 2) The replicate gels of each sample were treated as independent gels, and the t-tests compared spot intensities in the fifteen treatment gels to the intensities in the fifteen control gels. Columns 1 and 2 in Table Differential sample loading and stain absorption and other process variables can contribute to variability in measured protein intensity. In order to ensure that the detected differences in protein intensity are not due to a "technical" variability introduced by the process of gel creation, spot intensities are "normalized." Dividing the intensity of each protein on a gel by the total protein intensity of that gel is a widely used technique to reduce the "individual gel effect" on protein intensities . NormaliTo the best of our knowledge, none of the image analysis software packages available to date provide the tools necessary test for the distribution of the data. All of them provide t-tests or the non-parametric Wilcoxon rank sum test or ANOVA to test for differences in individual spot intensities. The probability values  for differences between groups are based on the assumptions of the normality of the distribution of spot intensities and equal variances. In order to make an informed judgement about the validity of the p-values of the tests above, it is important to know if these assumptions are met ,16. If oDespite the fact that 2D-gel technology offers many advantages, one of the pitfalls associated with this technology is the need for several replicates for proper validation of results. There are instances where one does not observe reproducible spot patterns or individual proteins even in replicate gels of the same sample. Missing spot intensities are commonly observed in 2D gel datasets. Multivariate techniques such as Principal Component Analysis and Discriminant Analysis (DA) are ideal tools to use on databases that have multiple outcome variables (protein intensities). However, SAS or any other statistical software that is used to analyse the data using multivariate techniques such as PCA and DA requires data sets with non-missing values. Gels with missing spot information will thus be dropped from the analysis. Since all gels will have some spot information missing, this will result in no gels being available for data analysis. To the best of our knowledge, all image analysis software packages substitute zeroes for missing intensity values. Missing intensities may be caused by the fact that a protein spot truly does not exist in one group compared to the other or because the spot intensity is so low that it is not detected by the image analysis software. In this study we treated all missing spots as undetectable spots. The question we were trying to answer was "If a spot exists but is 'undetectable', what is the best 'detectable' value to substitute as its intensity?" The most intuitive value was the smallest "detectable" intensity in the experiment hence we used the lowest intensity value in the experiment for the single value imputation. However, substituting a single value for all missing spot intensities would skew the distribution of spot intensities considerably. Thus the second option was to use a random process to substitute missing spot intensities with a plausible set of "detectable" intensities. We created a set of lowest 'detectable' intensities from the lowest intensity on each of the thirty gels. These values were used to impute missing intensity values as described in the methods section. Although we have used the terms "imputation" and "multiple imputation", these terms are not to be equated with multiple imputation advocated by Rubin . Rubin'sThe issue of missing protein intensities is one that has not been addressed at all in the literature describing 2D gel studies. In this study we treated all missing intensities as the same. However, all missing intensities in 2D gels are not equal. Some missing intensities are missing because they truly do not exist in one group versus the other, whereas others are missing because of the inherent variability in the process of creating 2D gels. This suggests that one needs to approach the filling of missing values differently based on the probability of a spot being a truly missing protein or one that is missing due to the process of gel creation. One way to do this would be to assume that spots that occur in two out of three replicates (or three out of four replicates) of a sample are true spots. These missing intensities would then be replaced by the mean value of the remaining two (three) spots. On the other hand, the assumption that those proteins that are missing in all control gels or all treatment gels are proteins that are turned on or off is justifiable. This in turn suggests that for this set of proteins the random imputation of missing values with a set of plausible minimum intensity values, acting as placeholders so that the non-missing data can be used in analyses, can also be justified.In the last few years, published reports of 2D gel analysis have concluded that heart failure was associated with protein modifications in three cellular systems , identifEscherichia coli lysate "spiked" with varying amounts of four different known proteins to test a novel experimental design that exploits the sample multiplexing capabilities of DIGE by including a standard sample in each gel. Rabilloud et al [An intuitively appealing way to do this is to create 2D gels with serially diluted quantities of commercially available proteins, establishing the relationship of protein quantity to spot intensity and then proceeding to compare different statistical pre-processing techniques to the datasets acquired from these gels. Since it is a known fact that commercially bought proteins may not necessarily be pure and may be present in multiple modified forms due to the process of isolation, the experiment described above may be enhanced by using controlled biological samples from a cell culture with at least a hundred resolved spots. Once again, the sample could be loaded in on gels in known concentrations and the process described above would be repeated. Clearly, the set of pre-processing techniques that picks up differences that come closest to the true differences would be chosen as the optimal techniques. Some recent publications have used similar techniques to establish the validity of emerging proteomic technology. Alban et al  used an ud et al  comparedIn this study we have used the proteins that were subsequently identified by MALDI-TOF spectroscopy as a measure of how well particular statistical protocols perform. We concede that there is an inherent bias in that the spots that were identified by MALDI-TOF were selected on the basis of the protocol described in this paper. The described protocol therefore will seem to perform much better than others in this comparison. This however does not diminish the main thrust of this paper, which is that statistical protocols affect the conclusions drawn from a 2D gel experiment.This study has demonstrated that the pre-processing of the data from 2D gel experiments can have a significant impact on the results of statistical tests. The purpose of the study was not to identify the particular statistical protocol used in this study as the optimal protocol, but rather to demonstrate that the results and conclusions from a biological experiment are not independent of the statistical protocol. The study has in effect looked at three different statistical protocols. The one described in Figure We used data from an experiment that compared the protein expression in the whole brain homogenate of rats that were fed a diet with 5% of grape seed extract to that of a normal rat diet. This experiment will be described in a separate article. The data from this experiment was exported to a database, which was saved as a text file. The database was imported into SAS V-9.0 , which was then used to do all the statistical analysis.The intensity of each protein spot was divided by the total protein content  in the experiment.The intensity of each spot in each group was then normalized to the median intensity of its group, i.e. the intensity of each spot was converted into the intensity it would have had if the gel it was in had a total intensity equal to the median total intensity of the group. This is described by the following formula:This normalization reduces intra group variability, but maintains the inter group variability.Fold change is a common metric used in articles describing gene array and proteomic experiments. Fold change measures the degree of change in protein intensity in the treatment group, compared to the control group. This is measured by dividing the average spot intensity in the treatment group by the average spot intensity in the control group. In order for this ratio to be a true comparison of the intensities in the two groups, the intensities need to be expressed as proportions of the same quantity, i.e. they need to be divided by the same quantity. Normalization 1 has this property built into the formula. In normalization technique 2, the normalized intensities were divided by the total protein content in the experiment in order to make a fold change comparison meaningful.A spot was considered present only if it was present in both replicates of a sample if there were only two replicates, in two out of three replicates if there were three replicates of a sample, and three out of four replicates if there were four replicates of a sample. We also included spots that were present in all controls and absent from all treatments, or present in all treatment gels but absent from all controls. Using these criteria, we had 201 spots that were available for statistical analysis from the GSE database.Given the criteria used to subset spots for the final analysis, there was a large number of missing spot intensities. We examined the effect of filling in the missing spots in three different ways.1. Missing spot intensities were first replaced with the lowest value of log-transformed intensities. In this case the value was -17.28. The effect of the replacement of this single value for all missing spot intensities is the same as the effect of replacing non-log transformed spot intensities with zero.2. We created 1000 separate data sets that randomly selected one of the 15 lowest spot intensity values from the 15 gels in the control group to replace each of the missing spot intensities in the controls, and repeated the process for the 15 treatment gels.3. In the third imputation method, each missing spot intensity was replaced with a randomly selected value from the 30 lowest values from each gel in the experiment without regard to whether the value chosen was from a control or treatment gel. The two methods of random imputation of spot intensities were replicated 1000 times. two sample t-tests were repeated with each replication.HK is the PI of the project that provided the data for this experiment. JD, HK's Research Associate, created the 2D gels, conducted the image analysis and created the database. SM designed this study and performed the statistical analyses.


Recurrent dislocation of the patella in patients with below knee amputation is a known entity. Abnormally high-riding patella  and medial patellofemoral ligament insufficiency in these patients predisposes them to patellar instability. The established treatment of this problem is surgical realignment.A 25 year old male patient with bilateral below knee amputation presented with bilateral recurrent dislocation of the patella while walking on knees on uneven ground. Clinical and radiographic studies showed patella alta. A simple shoe modification was used to treat this patient.A simple shoe modification can be used to treat such a condition which is otherwise treated surgically. Recurrent dislocation of the patella can follow a violent initial dislocation, but occur more often in knees with one or more underlying anatomic abnormalities that predispose the patella to dislocation or subluxation. In these knees, less trauma is needed for dislocation to occur. The underlying pathologic condition causes an abnormal excursion of the extensor mechanism over the femoral condyles. High-riding patella  and a damaged medial patellofemoral ligament at the time of first episode of dislocation leads to such an abnormality and leads to recurrent dislocation of the patella -4. PatelA 25 years old bilateral below knee amputee presented with recurrent dislocation of the patella while walking on knees in emergent situations on uneven ground without the prosthesis. Amputation was performed at the age of 15 years as a result of train accident. Since then he has been using patellar tendon bearing (PTB) type below knee prosthesis on both sides. First episode of dislocation occurred after 5 years of amputation. The patient used to walk on his knees without using the prosthesis for in-house activities on uneven ground. His patella used to dislocate whenever there was an unnoticed pressure on the medial side of the knee. On clinical examination, patella alta and positive apprehension test were noted on both sides. The ratio of patellar length to patellar tendon length was 0.8 on both sides demonstrating relative elongation of the patellar tendon. The normal ratio is 1.0 [High-riding patella  leads to patellar instability . In the The patient described here used to walk on knees for certain emergent and short mobility in-house activities e.g., going to toilet especially at night, going to kitchen etc. As most of rural houses in developing countries do not have cemented floors, walking on uneven ground cannot be prevented. Walking on knees in such conditions produces eccentric forces on the patella. Sudden laterally directed forces which might result when the knee strikes some elevated surface on medial side can lead to dislocation of patella in patients with patellar instability.Walking on knees is usually discouraged in below knee amputees as it can lead to flexion contractures of the knee joint. In cases of bilateral amputees it is not always possible for the patients to wear prosthesis on both sides in emergency situations as discussed above. Considering these points, the occasional walking on knees could not be avoided in this patient. Surgical correction of instability would not have helped much as walking on knees on uneven ground could have nullified the results of surgery. A modified well moulded shoe was given to the patient for walking on knees that prevented any eccentric forces and protected the patella against dislocation.6 months follow-up results were excellent in this patient with no episode of dislocation on either side. The patient was very comfortable in using the shoes as it takes him only 15 seconds to wear them. Hence a simple low cost shoe modification has been used to treat a condition which is mostly treated surgically.Recurrent dislocation of the patella in cases of below knee amputees using patellar tendon bearing prosthesis is very rare and the usual treatment is surgical realignment. To our knowledge no case of bilateral recurrent dislocation of the patella in below knee amputees has been described in the literature. A simple shoe modification can be used in difficult situations to treat such a condition.The author(s) declare that they have no competing interests.SB was the principle surgeon who planned the treatment protocol of this patient, in addition to conceptualizing and drafting the article.RK guided the designing of the shoe.PL was the prosthetist who designed and made the shoe.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


This paper explores the geographical accessibility of health services in urban and rural areas of the South West of England, comparing two measures of geographical access and characterising the areas most remote from hospitals.Straight-line distance and drive-time to the nearest general practice (GP) and acute hospital (DGH) were calculated for postcodes and aggregated to 1991 Census wards. The correlation between the two measures was used to identify wards where straight-line distance was not an accurate predictor of drive-time. Wards over 25 km from a DGH were classified as 'remote', and characterised in terms of rurality, deprivation, age structure and health status of the population.2>0.93). The greatest differences were found in coastal and rural wards of the far South West. Median straight-line distance to GPs was 1 km (IQR = 0.6–2 km) and to DGHs, 12 km (IQR = 5–19 km). Deprivation and rates of premature limiting long term illness were raised in areas most distant from hospitals, but there was no evidence of higher premature mortality rates. Half of the wards remote from a DGH were not classed as rural by the Office for National Statistics. Almost a quarter of households in the wards furthest from hospitals had no car, and the proportion of households with access to two or more cars fell in the most remote areas.The access measures were highly correlated (rDrive-time is a more accurate measure of access for peripheral and rural areas. Geographical access to health services, especially GPs, is good, but remoteness affects both rural and urban areas: studies concentrating purely on rural areas may underestimate geographical barriers to accessing health care. A sizeable minority of households still had no car in 1991, and few had more than one car, particularly in areas very close to and very distant from hospitals. Better measures of geographical access, which integrate public and private transport availability with distance and travel time, are required if an accurate reflection of the experience those without their own transport is to be obtained. The UK National Health Service has always aimed to provide health care for all. Although the importance of "fair access for all" (independent of the ability to pay, age, sex or area of residence) has been highlighted in recent policy documents , the meaAlthough 'fair access' can be characterised simply as 'providing the right service at the right time in the right place', it is aAlthough there is some evidence that increasing distance from health services inhibits the use of primary  and secoOne assumption which is commonly made is that geographical inaccessibility of health services is essentially a rural problem, but there is little evidence demonstrating the differences in accessibility between rural and other areas. In any area, the greatest disadvantage is likely to be experienced by individuals without access to a car (including members of one-car households without daytime access). With the declining availability of public transport, it is likely that a private car is the only convenient way to travel in rural Britain . AlthougIn this paper, we aim to determine the geographical accessibility of health services, and the demographic and health related factors associated with it. We compare two measures of geographical access: straight-line distance and modelled drive-time along the road network to primary and secondary care throughout South West England. We investigate whether the most geographically inaccessible populations are in rural areas, describe the relationship between geographical access to hospital and population health, and investigate whether the populations furthest from health services have a greater need for health care due to age or deprivation.The study area is the former South West Region, comprising the counties of Avon, Cornwall and the Isles of Scilly, Devon, Dorset, Gloucestershire, Hampshire, the Isle of Wight, Somerset and Wiltshire. As defined in 1991 this area has a population of about 6 million, with a low proportion from ethnic minorities, and a higher than average proportion living in rural areas.The straight-line and drive-time measures were highly correlated for both GP and hospital services figure . Areas wDistances to GPs were low, with a median distance of just 1 km to the closest practice (IQR 0.6 – 2.2). The calculation was repeated excluding branch surgeries (which tend to have limited opening hours), but this made little difference to the outcome, with a median distance to a main surgery of just 1.2 km. 95% of wards (98% of the population) were under 4.4 km, or 6.3 minutes, from their closest GP. The maximum distance to a GP was just 9.4 km (13.7 minutes). The median distance to a DGH was just less than 12 km (IQR 5.4 – 19.0), with a maximum of 50 km, corresponding to an estimated 13 and 48 minutes drive-time  remote from primary care. These areas have just 3% of the regional population. Of these the majority (63%) were ONS 'rural' areas. There were 162 wards which we classified as remote from hospitals . All had drive-times to hospital of over 21 minutes; 81 (51%) were urban by the ONS classification, 69 (43%) were rural areas and the remaining eight (5%) were rural fringe. Four wards had no ONS urban / rural classification , we conclude that caution should be exercised when evaluating and responding to poor access to health services, a high proportion of which occurs outside areas traditionally considered to be remote.In this study, we found no clear threshold at which need becomes greater or health status sharply declines. If anything, the converse is true with worse health status and greatest need in areas close to health services. Distance to health care was not associated with a high proportion of elderly or very young residents, but was related to deprivation. We found high deprivation in areas close to hospitals, relative affluence in more distant areas and an increase in deprivation in the most remote wards. Deprivation indices have been criticised for failing to represent deprivation in rural areas  and the Although the highest rates of morbidity and mortality were found in the areas closest to hospitals, there was some evidence of increasing rates in more remote areas. Rates of LLTI, particularly for those under 64, show an upwards trend in more remote areas. This supports previous findings that LLTI is higher in rural wards with the most dispersed populations , but it There are a number of important limitations to our study. We have explored only one region of England, a relatively affluent area with a very small ethnic minority population and an unusual 'peninsular' geography. Our findings need to be reproduced in other areas. We have limited our definition of access to simple geographical measures. Other aspects of accessibility include the quantity and quality of health services, and financial and cultural barriers to their use, and have not been explored here. The choice of SMRs and LLTI rates as health outcome indicators may have resulted in our inability to observe stronger relationships between geographical access and health: even over a six year period absolute numbers of deaths were low. More research is needed including the young and elderly and using a wider range of health status measures. Finally, the inter relationship between use of health care, need and access has been insufficiently explored.This paper has provided a population-based estimate for access to both primary and secondary health care in South West England. We have shown that although geographical access to health services is generally good, remoteness from health services is an issue which affects both urban and rural areas. Studies concentrating purely on rural areas are therefore likely to underestimate the extent of geographical barriers to accessing health care. Areas which were furthest from hospitals did not have an especially old or young population, but there was some evidence of higher rates of LLTI and of deprivation in the most remote wards, indicating higher need for services in the areas furthest from them. Finally, the fact that almost a quarter of households in the decile of wards most remote from hospital services had no car in 1991 indicated a large number of people for whom travel is likely to be more difficult than implied by current measures of geographical access.Our understanding of the effect of distance on the use of services and on health outcomes is far from complete. Both the measurement of access and the understanding of need and deprivation require further exploration. The development of web-based public transport information systems may supply the data needed to enhance currently available measures of access by adding public transport travel times, likely to be relevant to access for the poorest and most deprived populations and the introduction of the Indices of Multiple Deprivation 2000 in England may present a clearer picture of the need for health care than traditional census-based indices . This inA Geographical Information System (Arc/Info) and custom written programs were used to calculate two measures of access to health services. Access was calculated from all residential postcodes to primary care services  surgeries) and secondary care services ) in the region. Data on main and branch GP surgeries (n = 1469) were obtained from all Family Health Services Authorities in 1998. Acute DGHs (n = 39) were defined as hospitals with general medicine and general surgery facilities and an Accident and Emergency department. DGHs were identified using the hospital year-books (1992–97) and hospitals were contacted to clarify their status in 1997 as necessary.The first access measure calculated was a widely used measure: the shortest straight-line distance between every residential postcode, the closest GP (both main and branch) and the closest DGH. A more complex measure of access was the shortest drive-time from each residential postcode to the closest GP and the closest DGH. This was modelled using estimated road-network travel speeds along the Bartholomew digital road network and associating these with residential postcode locations by the use of a travel time (drive-time) surface model. While including provision for congestion and slow travel through urban areas, this measure does not include any estimates for parking times or transfers between car and surgery or hospital. The methods are described in detail elsewhere .Proxy measures of the need for health care were calculated. The Townsend score, a widely used indicator of material deprivation, was calculated from 1991 census data. The variables used in the score are the percentage of economically active people over the age of 16 who are unemployed; the percentage of households which are overcrowded; the percentage of households with no car and the percentage of households not owning their own home. A log transformation is applied to the overcrowding and unemployment variables. The logged variables and the car ownership and owner occupation variables are standardised by creating z-scores for each value, and the four z-scores are summed to provide the final Townsend score. Scores are standardised to give a mean of zero for England and Wales: any scores greater than zero indicate relative deprivation, any less than zero represent relative affluence. The proportions of the population over 65 and under 5 years old – were taken from the 1991 Census Small Area Statistics (SAS). Health status was assessed using indirectly standardised rates of all-cause mortality and Limiting Long Term Illness (LLTI) for all those under 65 . Data on LLTI were taken from the 1991 Census. The Office for National Statistics (ONS) provided data on all-cause mortality for the years 1991–1996. Data were aggregated over the six years, to minimise problems due to small numbers of cases in some wards.Postcodes were allocated to 1991 Census wards using the 1991 and subsequent postcode to enumeration district directories. Travel times and distances were calculated for all residential postcodes and aggregated to ward level for analysis. The resident population of each ward was used to weight individual postcode times and distances to create a population-weighted average, as demonstrated in table To investigate if straight-line distance was a valid proxy for the more complex drive-time measure, the two were compared using Pearson correlation coefficients and a regression analysis of drive-time against straight-line distance. Areas where straight-line distance appeared to underestimate the drive-time more than expected were identified and mapped to investigate the extent of geographical clustering. Access to primary and secondary health services was described using median distances and inter-quartile ranges for both measures.To investigate the assumption that it is the residents of rural areas who are most disadvantaged by poor geographical access to health services, we first had to define poor access. Standard estimates of 'remoteness' from health services have not been established – there is no a priori definition of the distance regarded as 'remote from health services' and no consensus has been established in the literature on access to health services. The proportion of rural, rural fringe and urban wards which were 'remote' from health services under the definition of a straight-line distance of three, five or seven kilometres to a GP and 20, 25, 30 or 35 km to a hospital was therefore calculated Table . We usedWe then identified the proportion of remote wards that were rural under the ONS classification. To investigate relationships between distance to health services and the need for health care, straight-line distance to hospital was used to group wards into deciles and the deprivation score and the age profile of the population in each decile was described. Standardised rates for premature all-cause mortality and LLTI were used to indicate health outcomes for each decile of wards, and car ownership (as reported in the 1991 census) was used to indicate how easy travel would be for the population in each group.HJ carried out the analyses and drafted the manuscript. HJ, PR, and DM collaborated in the formation of the research questions, the management of the study and the development of the paper. HJ and SB collected the data and calculated measures of deprivation, health and accessibility. DM designed the drive-time access measure. All authors read and approved the final manuscript.


Previous studies showing a strong relationship between Cheyne-Stokes respiration and the severity of left ventricular systolic dysfunction have usually been done in selected patient populations with lower age and a higher proportion of males than the "typical" in-hospital patient with heart failure. The purpose of the present study was test the strength of this relationship in unselected patients admitted to hospital due to decompensated chronic heart failure.We evaluated 191 patients (32% women), mean age 73 years, ready for discharge from the heart failure unit in the University Hospital of Malmö, Sweden. The patients underwent echocardiography for determination of left ventricular ejection fraction and left ventricular inner diastolic diameter. A respiratory investigation during sleep was performed the last night before discharge.We found that 66% of the patients had Cheyne-Stokes respiration more than 10% of the total recording time. Only 7 (3.6%) of the patients had predominantly obstructive apnoeas. There was a significant but very weak relationship between left ventricular ejection fraction and left ventricular inner diastolic diameter on one hand and Cheyne-Stokes respiration on the other. Age was a stronger determinant of Cheyne-Stokes respiration than any of the cardiac or other clinical variables.Although presence of Cheyne-Stokes respiration indicates left ventricular dysfunction, its severity seems only weakly related to the severity of heart failure. Age was found to be a stronger determinant, which may reflect the underlying age-dependency found also in healthy subjects. Due to age restrictions or other selection criteria, the importance of age may have been underestimated in many previous studies on factors associated with Cheyne-Stokes respiration. Cheyne-Stokes respiration (CSR) during sleep, is common in patients with heart failure ,2. CheynMost previous studies have included selected patients with impaired left ventricular systolic function. A large proportion of heart failure patients are elderly and have relatively preserved left ventricular systolic function. However, elderly heart failure patients have often been excluded from sleep studies. Such studies are not representative of the everyday clinical spectrum of heart failure patients, including the increasing number of elderly patients usually seen in general medical wards. The proportion of women, with predominantly diastolic heart failure, may also be greater in this group.The aims of the present study were to test the strength of the relationship between left ventricular dysfunction and nocturnal Cheyne-Stokes respiration in unselected patients admitted to hospital due to decompensated chronic heart failure and to determine the correlation between Cheyne-Stokes respiration and other clinical variables to provide source material for subsequent analyses of quality of life and survival.All patients admitted to the Heart Failure Unit at the Department of Cardiology, Malmö University Hospital from January 1996 to november 1999 primarily due to decompensated chronic heart failure were eligible for inclusion. Malmö University Hospital serves as the main hospital for the whole population of Malmö (population 250,000) and is the only hospital in the city. Patients were excluded only if unable to comply to the study protocol due to some other condition, to complete the study questionnaire or to provide informed consent. Heart failure was diagnosed according to European Society of Cardiology guidelines for the diagnosis of heart failure . All patAll patients provided informed consent to participate in the study, which was performed in accordance with the principles of the Declaration of Helsinki and approved by the Medical Ethics Committee at Lund University.Ischaemic heart disease was diagnosed based on findings from previous coronary angiography, documented myocardial infarction or typical signs of ischaemia at exercise testing. Hypertension  was diagnosed if blood pressure was >150/95 mmHg or the patient was receiving drug therapy for hypertension. Diabetes was diagnosed if fasting blood glucose levels were >7 mmol/l or the patient was treated with oral anti-glycaemic medication or insulin. The diagnosis of other concomitant diseases was based on patient history and/or patient records.Echocardiographic examinations were performed using a Hewlett-Packard Sonos 2000 (Andover, Mass, USA). Parasternal and apical views were obtained with the patient in a left lateral recumbent position. Measurements were acquired during silent respiration or end-expiratory apnoea. Left ventricular systolic function was assessed by determination of the mean left atrioventricular plane displacement (AVPD), global qualitative assessment and/or single plane ellipse (modified Simpson's rule) -11.We recorded oronasal airflow by thermocouples, electrocardiogram, chest wall movement by electrical impedance, and finger pulse oximetry using the EdenTrace II Plus Multirecording System (EdenTech Corp, Eden Prairie, MN, USA) ,13. The The records were scored manually for Cheyne-Stokes respiration  ,15 and fSince CSR% was not normally distributed, we used Spearmans rank correlation test to relate CSR% to the continuous variables age, BMI, LVEF and LVIDD. For analysis of CSR% with respect to the categorical variables gender, cerebrovascular disease, ischaemic heart disease, NYHA class, atrial fibrillation and habitual snoring, we used the Mann-Whitney U-test.There are no data in the literature to allow categorisation of patients as normal and abnormal according to any specific level of CSR%. In table Two hundred and three patients were included, however final analysis only included 191 patients (32% women). Three patients were excluded because of total recording time less than two hours. Eight patients were excluded due to the poor quality of their recordings and one patient was excluded because of an abnormal irregular breathing pattern that could not be categorized as Cheyne-Stokes or obstructive sleep apnoea.The aetiology of the heart failure was ischaemic heart disease in 60%, dilated cardiomyopathy in 3%, hypertension in 14%, valvular disease in 8% and other or unknown reason in 15%. Half of the patients had had heart failure diagnosis for more than a year. All but eight patients were prescribed diuretics, 43% digitalis, 70% ACE inhibitors and 28% beta-blockers. Almost half (48%) of the patients used hypnotics .The clinical characteristics of the study patients are presented in Table Overall 26% had heart failure due to left ventricular diastolic dysfunction (ejection fraction >45%). Female patients were older Table  and moreThe average recording time was 424 (SD 75, median 444, interquartile 400–472) minutes and average CSR% 35 (SD 30, median 28, interquartile 6–59). Predominantly obstructive apnoeas were found in seven patients (3.7%) and CSR (arbitrarily defined as CSR% >10%) in 126 patients (66%). Sixty (31%) of the patients had CSR more than 50% of the recording time (Table 2 0.06 vs 0.03 and 0.03). The severity of CSR was also greater in patients with ischaemic heart disease. There were no relationships between medication (e.g. betablockers or benzodiazepines) and CSR%.Univariate rank correlation analysis showed that CSR% was most strongly correlated to age (R = 0.24, p < 0.01, figure Seven patients (3.6%) were found to have predominantly obstructive sleep apnoea (average AHI 16.0, SD 7.0, median 16.2, interquartile range 8.7 – 21.8). Excessive snoring, obesity or male gender were not overrepresented in this group.We have shown that CSR is common in elderly patients hospitalised due to decompensated chronic heart failure and that age was a stronger determinant of CSR than any of the cardiac or other clinical variablesMalmö University Hospital serves a population of 250,000 inhabitants. In general all patients primarily admitted to hospital due to decompensated chronic heart failure are treated at the Heart Failure Unit until discharge from hospital. Due to this and to the liberal inclusion criteria we believe that the study patients are representative of the general population of patients with heart failure. Many other studies suffer from the disadvantages of a selection bias by excluding older patients, including only men or including only patients specifically referred for sleep studies  or for eOne major difference between our study design and that of the majority of other studies is that we included patients immediately after an episode of decompensation, whereas most other authors have studied patients 1–3 months after discharge. We do not, however, know to what extent this approach affects nocturnal breathing patterns. The results reported by Tremel et al  suggest At the time of the study, the most convenient and easy manageable system available at our department was the EdenTrace system. The semiquantitative nature of chest wall impedance measurement may have reduced the sensitivity of our measurements. However, by combining impedance measurement of respiratory movements, recording of oronasal airflow with thermocouples and finger pulse oxymetry with a careful visual analysis af the traces by an experienced physician (SB), we postulate that the distinction between central and obstructive events is sufficiently accurate. Furthermore that the bias introduced, would tend to underestimate, rather than overestimate the prevalence of sleep disordered breathing in the studied population.Whereas there are widely accepted standards for analysis of sleep apnoea the definition of CSR is less precise, and there is no accepted standard method for its quantification. We quantified the severity of CSR as percentage of total recording time. This approach is suggested by Ancoli-Israel et al  and is aThe overall prevalence of CSR in this material was 66%, using an arbitrary limit of CSR% >10%. This figure is in the same magnitude as the 50–60% previously reported in stable heart failure outpatients ,19 and cWe found 28 patients with mixed apnoeas  has been claimed to be an important factor for the development of CSR . ImpaireAtrial fibrillation is a third factor that has been claimed to be associated with CSR ,18,22, bPatients with a history of stroke are usually excluded from studies on CSR and heart failure. This may be a limitation of the external validity of such studies, since many patients with heart failure  also have a history of minor or major cerebrovascular disease. We found however, quite unexpectedly, that this was not associated with a higher occurrence of CSR, thereby corroborating the findings of Blackshear et al . NeitherAlthough presence of Cheyne-Stokes respiration indicates presence of left ventricular dysfunction, its severity seems only weakly related to severity of heart failure. Age was a stronger determinant, which may reflect the underlying age-dependency found also in healthy subjects. Follow-up of the current patient cohort will be performed, but from the present data, we cannot conclude if Cheyne-Stokes respiration is of clinical importance or not.LM coordinated and performed the study which was designed by CC and BM. LE and CC were responsible for the cardiac investigations and SB and BM for the interpretation of the nocturnal respiratory recordings.


HIV infection and progression to AIDS is characterized by the depletion of T cells, which could be due, in part, to apoptosis mediated by the extra-cellular HIV-encoded Tat protein as a consequence of Tat binding to tubulin. Microtubules are tubulin polymers that are essential for cell structure and division. Molecules that target microtubules induce apoptosis and are potent anti-cancer drugs. We studied the effect on tubulin polymerization of three Tat variants: Tat HxB2 and Tat Eli from patients who are rapid progressors (RP) and Tat Oyi from highly exposed but persistently seronegative (HEPS) patients. We compared the effect on tubulin polymerization of these Tat variants and peptides corresponding to different parts of the Tat sequence, with paclitaxel, an anti-cancer drug that targets microtubules.We show that Tat, and specifically, residues 38–72, directly enhance tubulin polymerization. We demonstrate that Tat could also directly trigger the mitochondrial pathway to induce T cell apoptosis, as shown in vitro by the release of cytochrome c from isolated mitochondria.These results show that Tat directly acts on microtubule polymerization and provide insights into the mechanism of T cell apoptosis mediated by extra-cellular Tat. AIDS is due to human immunodeficiency virus HIV-1) infection of CD4 T cells and is characterized by the cell death of HIV-infected lymphocytes and uninfected bystander cells  infectio,6. Tat wVinca.alkaloids. The MDAs inhibit microtubule dynamics in living cells and lead to apoptosis after cell cycle disturbance [c, leads to the caspase cascade activation and thus to apoptosis [in vitro by the release of cytochrome c from isolated mitochondria [Another mechanism was proposed that involves microtubules to induce Tat-mediated apoptosis . We showturbance -24. Theyturbance ,26. The poptosis . Moreovechondria .The aim of this study was to evaluate the consequence of Tat binding to microtubules and the correlation with the mitochondria-mediated T cell apoptosis. We used three Tat variants with sequences from 99 to 101 that have the size of the main Tat variants found in the field . The HIVThe minimal concentration of tubulin (Cr) necessary to obtain tubulin polymerization without drug was 7 μM in our buffer conditions . We measWhen the temperature of the samples was decreased to 10°C, we observed a complete depolymerization for tubulin alone (line 1) and tubulin with 4 μM and 8 μM Tat (lines 2 and 4 respectively). Even at the highest concentrations of Tat (16 μM and 20 μM), at 10°C the turbidity decreased to the original values (data not shown). By contrast, in the same conditions, we did not observe any depolymerization in the presence of paclitaxel (line 3). However, full depolymerization in the presence of paclitaxel was obtained with a longer incubation at 4°C (data not shown). The reversibility of following an incubation at 10°C or 4°C strongly suggests that turbidity enhancement is not related to precipitation or aggregation but to microtubule formation.We also observed that when Tat concentration is increased to stoichiometric quantities with tubulin, the turbidity values increased dramatically to an absorbance greater than 2.These values are superior to the absorbance obtained in the same conditions using the stoichiometric quantity (15 μM) of paclitaxel, which induces effective tubulin polymerization. Turbidity is known to be a function of the total weight concentration of scattering particles only when the particles have small diameters compared with the wavelengths of the incident light ,35. ThusWe evaluated the ability of different Tat variants to modify tubulin polymerization. In our conditions, when 15 μM of tubulin are incubated at 37°C with 8 μM Tat HxB2, Eli or Oyi, the turbidity plateau values at 350 nm are 1.01, 1.61 and 0.80 respectively (Table To identify the sequence(s) region(s) implicated in tubulin, peptides overlapping the sequence of Tat HxB2 and Tat Oyi were synthesized. The sequences of peptides 38–72 and 73–99 derived from both Eli and Oyi are indicated in figure p = 0.037) using one-sided ANOVA. The percentages of apoptotic cells in the presence of 1 μM Tat Oyi were similar to the control cells (0.1 %)  staining. This method allows the quantification of apoptotic cells by the observation of the characteristic nuclear fragmentation. After 20 hours treatment, the percentage of apoptotic cells in the presence of 10 μM Tat HxB2 (11 %) and 10 μM Tat Eli (12.5 %) were higher than those obtained with 10 μM Tat Oyi 3.5 %) Fig. . These d.5 % Fig. %) Fig. . Thus, aPaclitaxel stabilizes the microtubule network by inducing the formation of pseudoasters in mitotic cells and the formation of bundles, structures that correspond to strongly associated microtubules, in interphasic cells . Considei.e. 48 h) in the presence of paclitaxel leads to 90 % of cell death (data not shown). After 20 hours treatment, the percentage of the G2/M cells in the presence of 1 μM Tat HxB2 or 10 μM Tat Oyi were 28 % and 32 % respectively and similar to the 33 % observed in the untreated cells  whereas the level of cytochrome c with 0.2 μM of Tat Eli (lane T0.2) was comparable to the untreated mitochondria (lane T-). In parallel, cytochrome c levels decreased slightly in mitochondria treated with Tat, as revealed by Western Blot of the pellets  with FASTMoc chemistry according to the method of Barany and Merrifield  on 4-hyd275 nm = 1.07 L·g-1·cm-1 in 0.5% SDS in neutral aqueous buffer or ε275 nm = 1.09 L·g-1·cm-1 in 6 M guanidine hydrochloride.Lamb brain tubulin was purified by ammonium sulfate fractionation and ion-exchange chromatography, stored in liquid nitrogen and prepared for use as described ,46. DuriIn vitro studies have been derived from experiments that show that the variation of temperature permits the disassembly/reassembly cycles of tubulin in the presence of GTP and Mg2+ [2, and 3.4 M glycerol, pH 6.5. The various concentrations of Tat or paclitaxel were mixed with tubulin at 4°C. The reaction was started by warming the samples to 37°C in a 0.2 × 1 cm cells, and the mass of polymer formed was monitored by turbidimetry at 350 nm using a Beckman DU7400 spectrophotometer thermostated. Depolymerization occurs when the temperature is lowered to 10°C. The data showed in fig 350 nm values correspond to values without the buffer. The microtubule mass was determined by centrifugation assay. Samples were taken at the turbidity plateau at 37°C. They were ultra-centrifuged at 50,000 rpm for 15 min at 37°C using fixed angle rotor TLA 100.2 in a TL100 Beckman apparatus. In these conditions, microtubules precipitate in the pellet and non- polymerised tubulin remains in the supernatant. Pellet and supernatant were loaded separately on 10 % acrylamide SDS-PAGE, colored with Coomassie blue after migration  containing 10 % fetal bovine serum (BioWhittaker), 2 mM glutamine (BioWhittaker, Verviers, Belgium), 1 % penicillin and streptomycin (BioWhittaker, Verviers, Belgium). Exponentially growing cells (105 cells/ml) were seeded 3 days before paclitaxel or Tat treatment.The lymhoblastoid Jurkat cell line was cultured in RPMI 1640 supplemented with 2 mM ultraglutamine (BioWhittaker, Verviers, Belgium), penicillin (100 U/ml), streptomycin (100 μg/ml), and 10 % heat-inactivated fetal calf serum (BioWhittaker, Verviers, Belgium). Human neuroblastoma SK-N-SH cells were routinely maintained at 37°C and 5 % COc antibody  was used at 1/1000. Secondary antibody was goat antimouse monoclonal antibody conjugated with peroxidase (Jackson ImmunoResearch Laboratories, USA). The antibodies used for immunofluorescence microscopy were anti-α-tubulin (clone DM1A Sigma, Saint Louis, USA) and FITC mouse anti-goat IgG (Jackson ImmunoResearch Laboratories, USA).Stock solution of paclitaxel (Sigma) was prepared in DMSO. The final concentration of DMSO used in cell culture was less than 0.05 %. For Western blotting, anti-VDAC antibody (anti-porin, 31 HL France Biochem, Meudon, France) was used at 1/500 and anti- cytochrome 5 cells/ml were incubated in the absence or presence of Tat or paclitaxel at different concentrations . The proportion of hypodiploid cells was used as an estimate of apoptosis. Cell death was further analysed using fluorescence microscopy of cells stained with DAPI. 5 × 105. Jurkat cells/ml were incubated at 37°C for 20 hours with various Tat or paclitaxel in Lab-Tek chamber slides. . After centrifugation for 10 min at 3500 rpm, spin cells were fixed with 3.7 % formaldehyde, permeabilized with 0.1 % saponin and treated with PBS containing 10 μg/ml 4'6-diamidino-2-phenylindole (DAPI) (Sigma). The morphology of the cell nuclei was observed with a fluorescence microscope using an excitation wavelength of 350 nm. Nuclei were considered to have the normal phenotype when glowing bright and homogenously. Specific well drawn line up chromosome were seen in equatorial plates for cells in metaphase. Apoptotic nuclei were identified by the condensed chromatin gathering at the periphery of the nuclear membrane or a total fragmented morphology of nuclear bodies. Cells were counted and the percentage of normal., mitotic and apoptotic nuclei determined. Data indicated in the figure Jurkat cells at a concentration of 5 × 10see Fig. . After 26 Jurkat cells/ml were treated for 6 hours with 10 μM Tat HxB2 or 1 μM paclitaxel and were processed for tubulin indirect immunofluorescence staining as previously described [10escribed . After i7 cells were suspended in a sucrose buffer (250 mM sucrose, 1 mM dithiothreitol, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1.5 mM MgCl2, phenylmethylsulfonyl fluoride, protease inhibitors, 20 mM Hepes, pH 7.4) at 4°C, homogenized with 50 strokes in a glass homogenizer (Kimble Kontes, Vineland, NJ), and centrifuged twice at 800 g for 10 min. The supernatants were then centrifuged at 15,000 g for 10 min at 4°C. Mitochondrial pellets were immediately washed three times in the sucrose buffer. Isolated mitochondria were aliquoted and incubated for 2 hr at 37°C with various Tat concentrations. After centrifugation (15,000 g for 10 min at 4°C), pellets and supernatants were carefully separated. Mitochondrial pellets were lysed in lysis buffer  and loaded in parallel with the supernatants on a reducing 15 % SDS-PAGE gel. The gel was processed for immunoblotting and revealed using antibodies against VDAC and cytochrome c. Anti-VDAC antibody  was used to ensure that equal amounts of mitochondrial proteins were loaded on the gels and that no mitochondria or lyzed mitochondria were present in the supernatant. Secondary antibodies were added and visualization was carried out using an enhanced chemiluminescence detection kit (ECL, Amersham, Aylesbury, United Kingdom). The results presented are representative of four independent experiments.Mitochondria were isolated by cell fractionation from the neuroblastoma SK-N-SH cell line as previously described . 9 × 107HIV, human immunodeficiency virusMDA, microtubule damaging agentVDAC, voltage-dependent anion selective channel.The author(s) declare that they have no competing interests.JdM carried out most of the experimental work, performed the experimental design and drafted the manuscript. MC performed part of the cell, mitochondrial and immunofluorescence study directed by DB. PB performed tubulin purification, part of tubulin experiments on polymerization, quantification of microtubules and electron microscopy directed by VP. GRC, SL, SO, DE and JW synthezised the peptides and characterized them biochemically and biophysically. CP performed FACS studies. EPL participated in the design of the study, the redaction of the manuscript and funded the studies.


Reports exist of transmission of culture in nonhuman primates. We examine this in a troop of savanna baboons studied since 1978. During the mid-1980s, half of the males died from tuberculosis; because of circumstances of the outbreak, it was more aggressive males who died, leaving a cohort of atypically unaggressive survivors. A decade later, these behavioral patterns persisted. Males leave their natal troops at adolescence; by the mid-1990s, no males remained who had resided in the troop a decade before. Thus, critically, the troop's unique culture was being adopted by new males joining the troop. We describe (a) features of this culture in the behavior of males, including high rates of grooming and affiliation with females and a “relaxed” dominance hierarchy; (b) physiological measures suggesting less stress among low-ranking males; (c) models explaining transmission of this culture; and (d) data testing these models, centered around treatment of transfer males by resident females. A unique less-aggressive suite of behaviors that affects the overall structure and social atmosphere of a wild baboon troop potentially represents an intergenerational transfer of social culture A goal of primatology is to understand the enormous variability in primate social behavior. Early investigators examined interspecies differences, e.g., that pair-bonding is more common among arboreal than terrestrial primates . Attenti(Pan troglodytes) across Africa demonstrate variability in 39 behaviors related to tool use, grooming, and courtship  related to the overall structure and social atmosphere of the troop. We also document physiological correlates of this troop atmosphere, the transmission of relevant behaviors past their originators, and possible mechanisms of transmission.Nearly all such cases of nonhuman culture involve either technology (for example, the use of hammers for nut cracking by chimpanzees), food acquisition, or communication. In this paper, we document the emergence of a unique culture in a troop of olive baboons In the early 1980s, Forest Troop slept in trees 1 km from a tourist lodge. During that period, an open garbage pit was greatly expanded at the lodge. This attracted an adjacent baboon troop, Garbage Dump Troop, which slept near the pit and foraged almost exclusively there.By 1982, many Forest Troop males went to the garbage pit at dawn for food. While such refuse eaters did not differ in age distribution (data not shown) or average dominance rank from non–refuse eaters, they were more aggressive ; such agIn 1983, an outbreak of bovine tuberculosis occurred, originating from infected meat in the dump. From 1983 to 1986, most Garbage Dump animals died, as did all refuse-eating Forest Troop males ; no other Forest Troop animals died .These deaths greatly altered Forest Troop composition, such that there were fewer adult males and more adult females; this more than doubled the female:male ratio . By 1986Because of these events, observations of the troop were stopped, and only censusing was done until 1993. Research was begun on Talek Troop, approximately 50 km away.In 1993, informal observation of Forest Troop indicated that the behavioral features seen by 1986 had persisted. Critically, by 1993, no adult males remained from 1983–1986; all current adult males had joined the troop following 1986. Thus, the distinctive behaviors that emerged during the mid-1980s because of the selective deaths were being carried out by the next cohort of adult males that had transferred into the troop. Focal sampling on Forest Troop recommenced in 1993, in order to document this phenomenon. Data from Forest Troop 1993–1996 (henceforth, F93–96) were compared with two other data sets that served as controls: observations from 1993–1998 on the Talek Troop (henceforth T93–98), and observations of Forest Troop itself prior to the deaths . These two control data sets did not differ significantly from each other and were combined, henceforth T93–98/F79–82.Males of F93–96 and T93–98/F79–82 had similar rates of approach–avoidance dominance interactions (data not shown). Moreover, dominance stability did not differ, as measured by the percentage of approach–avoidance interactions which represented a reversal of the direction of dominance within a dyad of males of adjacent rank (16% ± 5% and 20% ± 5% for F93–96 and T93–98/F79–82, respectively, n.s.). There was also no difference in the average tenure length of the highest-ranking male (approximately a year).Despite those similarities, dominance behavior in F93–96 differed from the two control cases in ways that, arguably, made for less stress for low-ranking males. A first example concerns approach–avoidance dominance interactions between males more than two ranks apart in the hierarchy. The overwhelming majority of such interactions were won by the higher-ranking individual. Because a male is rarely seriously threatened by an individual more than two ranks lower in the hierarchy, interactions between individuals that far apart typically represent harassment of or displacement of the subordinate by the higher-ranking male, rather than true competition. In T93–98 and F79–82, approximately 80% of approach–avoidance interactions were between males more than two ranks apart in the hierarchy. In contrast, a significantly smaller percentage of approach–avoidance interactions were soin F93–96 A. InsteaPatterns of aggression also differed between F93–96 and T93–98/F79–82 in a way that suggested a less stressful environment for subordinates in F93–96. The troops had similar overall rates of aggressive interactions . HoweverWe examined the data for reconciliative behavior (i.e., affiliative behaviors between pairs following aggressive interactions ) in F93–Quantitative data on affiliative behaviors were not available for F79–82. However, F93–96 males socially groomed more often than did control T93–98 males A this di2R of correlation between rank and reproductive success: 0.25 ± 0.25 and 0.54 ± 0.10, respectively, n.s.).Sexual behavior did not differ between F93–96 and T93–98/F79–82. The percentages of nonpregnant, nonlactating females in estrus per day did not differ (27% ± 7% and 30% ± 4%, respectively, n.s.). Moreover, the relationship between male rank and reproductive success did not differ , hypertension, and decreased levels of high density lipoprotein cholesterol, growth factors, and circulating lymphocytes . We testThis was the case A. In F79Subordinate F93–96 males were spared another stress-related physiological marker. Experimental anxiety was induced by darting males, intramuscularly, with β-carboline-3-carboxylic acid, a benzodiazepine receptor antagonist which induces behavioral and physiological indices of anxiety in primates . Males wβ-carboline-3-carboxylic acid had no effect on behavior in high-ranking males in T93–98 or F93–96 B.The druThus, in the more typical F79–82 and T93–98 troops, subordination had distinctive stress-related physiological correlates. In contrast, F93–96 males lacked these rank-related differences.A decade after the deaths of the more aggressive males in the troop, Forest Troop preserved a distinct social milieu accompanied by distinct physiological correlates. Critically, as noted, no adult males in F93–96 had been troop members at the end of the tuberculosis outbreak. Instead, these males had subsequently transferred in as adolescents, adopting the local social style. A number of investigators have emphasized how a tolerant and gregarious social setting facilitates social transmission e.g., , exactly(Macaca mulatta) housed with stumptail macaques (M. artoides) assume the latter's more conciliatory style ((Papio anubis) and hamadryas baboons (P. hamadryas) differ in social structure, and females of either species experimentally transferred into a group of the other adopt the novel social structure within hours  or aversive ones (e.g., supplantation or attack) contingent upon their assimilating the troop tradition. In such models, a key question is who “instructs.” Much as with the term “culture” being used with respect to animal behavior, the use of the term “instruction” has also generated some controversy, with some preferring the concept of “active behavioral modification” by others bringing about the change. As a striking example of that, when young male cowbirds learn to produce their local song, they initially produce an undifferentiated repertoire of songs, and females react to the production of appropriate dialect with copulation solicitation displays, thus providing positive reinforcement and shaping those behaviors .In “observational models” applied to chimpanzee tool use, young learn nut cracking by observing and copying adults. As applied to F93–96, transfer males would model behavior upon that of resident males.In “facilitation models” of the chimpanzee example, proximity to adults and their hammers increases the likelihood of the young experimenting with hammers and deriving the skill themselves. As applied to the baboons, male F93–96 behaviors would be an implicit default state where, in the absence of the more typical rates of male aggression , females broadly tend to become more affiliative, and in the context of more affiliative female behavior, transfer males broadly tend to become less aggressive. As perhaps a way of stating the same, the default state may emerge because of the atmosphere of a troop with a high female:male ratio .Finally, a “self-selection model” may apply to the baboons, in which particular kinds of males were more prone to transfer into such a troop .We assessed these models by analyzing cases where adolescent males transferred on known dates and were observed for at least 2–6 mo afterward. Thus, we searched for behavioral patterns involving new transfer males that might differ between F93–96 (five such transfers) and T93–98/F79–82 (12 transfers).Many interactions involving new transfer males did not differ . TransfeWe then examined affiliative interactions between females and new transfer males, and found striking differences between F93–96 and T93–98/F79–82, in that F93–96 females treated new transfer males in the same affiliative manner that they treated resident males. F93–96 transfer males had a shorter latency until first being groomed by or presented to by a female than did T93–98/F79–82 transfer males A. The dB. While These data allow some insight as to the mechanisms of social transmission in F93–96 . The lack of contingency in the treatment of transfer males by residents argues against instruction; commensurate with this, there is relatively little evidence for “instruction” in nonhuman primate cultural transmission . The simThe data instead support either observational or facilitative/default models. Insofar as resident males in all troops interacted with transfer males similarly, transmission in F93–96 could have involved observation only if such observations were of how resident males interacted with females or each other. Some, but not all, studies support observational models of social transmission in other primates ; there aThis analysis raises the possibility that there is no social transmission, but that the F93–96 pattern is merely the emergent outcome of the 2:1 female:male ratio. To test this, we analyzed the five available studies of baboon troops with adult female:male ratios of 2 or more which contained quantitative data comparable to the present data . The keyThe unconditional  nature of the default model is puzzling, in that it requires that females be relatively affiliative to recent transfer males who, nonetheless, are initially aggressive to them. This seems counter to the long-standing emphasis in primatology on individual relations . Precedent for this unexpected implication comes from the social epidemiology literature concerning “social capital,” in which health and life expectancy increase in a community as a function of communitywide attributes that transcend the level of the individual or individual social networks .In summary, we have observed circumstances that produced a distinctive set of behaviors and physiological correlates in a troop of wild baboons. Moreover, these behaviors were taken on by new troop members; while obviously not conclusive, the data suggest that this most likely occurs through observational or facilitative/default models. Finally, somewhat uniquely in nonhuman primate studies, these findings concern the intergenerational transfer of social, rather than material culture.These findings raise some issues. There appear to be adverse health consequences of the stress-related physiological profile of subordination in typical baboon troops . The disA converse issue concerns circumstances that might destroy the F93–96 culture. The culture might be destroyed if numerous males transfer into the troop simultaneously, or if a male transfers in who, rather than assuming the F93–96 culture, instead takes advantage of it. Game theory suggests that F93–96 would be vulnerable to such “cheating.” Another issue concerns the fate of natal males from F93–96 when they transfer elsewhere. Reciprocal altruism models  suggest Finally, these findings raise the issue of their applicability to understanding human social behavior and its transmission. Human history is filled with examples of the selective loss of demographic subsets of societies . The present data suggest that demographic skews may have long-term, even multigenerational consequences, including significant changes in the quality of life in a social group.(Papio anubis) living in the Masai Mara Reserve of Kenya. Olive baboons live in multimale troops of 30–150 animals, with polygamy and considerable male–male aggression. Males change troops at puberty and, as adults, achieve ranks in somewhat fluid dominance hierarchies. In contrast, females remain in their natal troop, inheriting a rank one below that of their mother.Subjects were a troop, Forest Troop, of olive baboons Subjects were observed each summer from 1978–1986, and continuously since 1993. An additional troop, Talek Troop, was observed continuously since 1984. Behavioral data were collected as 20-min focal samples . During Reproductive success was indirectly estimated from frequencies of matings and consortships . The value of any given consortship or mating was adjusted by the probability of a fertile mating occurring that day .Endocrine data were collected under circumstances allowing for measures of basal steroid hormone levels . Subject


Journal of Experimental & Clinical Assisted Reproduction is an Open Access, online, electronic journal published by BioMed Central with full contents available to the scientific and medical community free of charge to all readers. Authors maintain the copyright to their own work, a policy facilitating dissemination of data to the widest possible audience without requiring permission from the publisher. This Open Access publishing model is subsidized by authors (or their institutions/funding agencies) in the form of a single £330 article processing charge (APC), due at the time of manuscript acceptance for publication. Payment of the APC is not a condition for formal peer review and does not apply to articles rejected after review. Additionally, this fee is waived for authors whose institutions are BioMed Central members or where genuine financial hardship exists. Considering ordinary publication fees related to page charges and reprints, the APC at Journal of Experimental & Clinical Assisted Reproduction is comparable to costs associated with publishing in some traditional print journals, and is less expensive than many. Implementation of the APC within this Open Access framework is envisioned as a modern research-friendly policy that supports networking among investigators, brings new research into reach rapidly, and empowers authors with greater control over their own scholarly publications. Journal of Experimental & Clinical Assisted Reproduction is a scientific and clinical journal established in September 2004, offering rapid peer review of research of the advanced reproductive technologies. Content is administered by two chief editors with offices in New York and Atlanta, with peer review supported by an international editorial board. Importantly, all published manuscripts are freely available to a global audience in full text format to facilitate sharing of investigative insights, laboratory methods, and surgical techniques. No other journal presently has these objectives. The journal considers submissions of the following types: Original research, Reviews, Book reviews, Case reports, Commentaries, Debate articles, Hypotheses, Methodology articles and Short reports. All manuscripts published by Journal of Experimental & Clinical Assisted Reproduction are included by PubMed at the National Library of Medicine (United States) [ States) , as well States) , and FraCreation of the "Open Access" publishing model represented a watershed moment in how research manuscripts are processed, paid for, provided, and protected. Historically, it was the end consumer (the library or reader) who was charged for access to medical, scientific and technical literature. Such payment was traditionally in the form of subscriptions or by access fees levied in exchange for controlled access to particular articles via internet. In contrast, Open Access publishing asks the author to offset the cost of access.The advent of internet technology facilitated the indexing of medical literature with a speed and degree of sophistication impossible before the world wide web. Nevertheless, once an article was located via computer database search, generally only the 250 word abstract could be viewed – even though full text was often required. As no library can offer each issue of every journal, the literature collection/review process increasingly has become a multi-institutional endeavor. Unfortunately the high cost of maintaining multiple subscriptions has actually caused some libraries to provide fewer journals for users .With journal holdings at some libraries trending down, smaller academic publishers were early casualties of the reduced demand. Many were lost to consolidation. This resulted in a once diverse publishing community being controlled by only a few organizations with little incentive to change. Meanwhile the number of specialty journals multiplied to keep pace with the growing complexities of medical research. Even as modern research became more multidisciplinary, academic publishers did not make it easy to share findings across dissimilar journals without the reader paying a price. Thus, a paradoxical albeit unintended contraction of access to published medical research resulted. Within the world of academic publishing, limited competition may have registered profits for some but the worldwide impact on readers' access to scholarly literature has been distinctly negative .It was against this "restricted access" background that the Open Access model of academic publishing was conceived, with a view to maximize rapid collaboration among researchers using a relatively new resource with tremendous potential – the world wide web. Open Access publishing incorporates internet technology which frees the investigator from the limitations of a particular institution's journal holdings as well as the delay of the interlibrary loan.Implementation of the Open Access publishing model has several important benefits for the scientific community. First, published works are available full text via internet thus making them rapidly accessible to a global audience. Since authors maintain the copyright to their own work, they are free to reproduce it on their own website, link it to related sites, or distribute it according to their own needs without obtaining the publisher's permission first. The only requirement is to acknowledge the article's source. "Reprint requests" are obviated since full text manuscripts are available for free via internet. Such networking has already been shown to boost article citations and impact since these manuscripts are easy to find ,9. SeconJournal of Experimental & Clinical Assisted Reproduction, it is hoped that the £330 charge will be affirmed as a good value, especially since subsequent costs associated with reprint fees or page charges assessed by many traditional print journals can easily exceed this amount.The Open Access publishing model is subsidized by authors (or their institutions/funding agencies) in the form of a single £330 article processing charge (APC), collected at the time of manuscript acceptance for publication. Payment of the APC is never a condition for any manuscript's formal peer review and does not apply to articles rejected after peer review. Additionally, this fee is not assessed for authors whose institutions are BioMed Central members or where genuine financial hardship exists. Such waiver requests are considered by the chief editors on a case-by-case basis. Since the journal exists as an internet resource there is no page limit to the number of color photographs, diagrams, or figures attached to a particular article. Movie files (<10 MB) accompanying a manuscript may also be published using the journal's electronic publishing platform, a feature particularly suited for articles demonstrating new operative or laboratory techniques. Given the full range of publishing possibilities at While several journals have moved to offer free internet access to selected articles, this should not be confused with Open Access . Free acAPC = article-processing charge.The author(s) declare that they have no competing interests.


A fascinating case, with much to learn about diagnosis and treatment of patients with abnormal CSF results. After learning from the case, take our online quiz A 17-year-old woman, who was born in Bangladesh, presented to an accident and emergency department in the United Kingdom with a history of being unwell for 24 hours. She had a headache and fever, and was vomiting. On questioning, there was no photophobia or neck stiffness. She lives in the United Kingdom and had been for a holiday to Bangladesh 4 months previously. The family members were all well, and none had similar symptoms. The woman had no previous medical history and was on no medication.9 per millilitre , with 84% neutrophils. Her erythrocyte sedimentation rate was 15 mm in the first hour , and her C-reactive protein was less than 5 mg/l . A malaria screen was negative, and renal and liver function tests were normal.On examination, she looked unwell, with a temperature of 38.5 °C, pulse 96 beats per minute, blood pressure 112/52 mm Hg, and a respiratory rate of 16 breaths per minute. She had a Glasgow Coma Score of 14, there was no neck stiffness, and her ocular fundi were said to be normal. There were no other significant findings on examination. Immediate investigations showed a normal blood count apart from a raised white blood cell (WBC) count of 11.1 × 10Haemophilus antigens, and bacterial culture was subsequently negative. The patient was given 2 g of ceftriaxone intravenously immediately, and ceftriaxone treatment was continued following the CSF results. Aciclovir IV, at a dose of 10 mg/kg every 8 hours, was added to cover the possibility of herpes encephalitis.A clinical diagnosis of probable viral meningitis was made pending the results of further investigations. Chest X ray was normal, as was an unenhanced CT brain scan. Lumbar puncture showed a normal cerebrospinal fluid (CSF) opening pressure, and the CSF was not blood stained. Laboratory analysis showed 1,606 WBC/ml , of which 60% were lymphocytes with protein of 1.08 g/l  and glucose of 2.9 mmol/l , with a corresponding plasma glucose of 5.7 mmol/l . No organisms were seen on Gram stain. The CSF was negative for pneumococcal, meningococcal, and recurrent episodes of apparent aseptic meningitis.The differential diagnosis was now viral, bacterial, tuberculous (TB), fungal, or malignant meningitis, or sarcoidosis. Additional investigations were requested, including polymerase chain reaction (PCR) on CSF for viruses and tuberculosis. There were no risk factors for HIV infection (HIV seroconversion can present with meningitis). Mollaret’s meningitis (recurrent aseptic meningitis associated with herpes simplex virus) was a possibility , though The following morning the patient’s temperature returned to normal at 37 °C, and she felt better. Referral was made to the Infectious Disease Service. Twenty-four hours after admission, she was afebrile but clinically worse, with marked headache and vomiting. A day later she spiked a fever of 39 °C. No organisms were cultured from the CSF, urine, or blood. CSF bacterial antigens, cryptococcal antigen, and CSF auramine stain (for mycobacteria) were also all negative.The following day the patient complained of further headache, nausea, blurred vision, and photophobia. In addition, she was noted to have bilateral large pupils, which did not react to light, and very pink optic nerves. Papilloedema was thought likely, and no other neurological signs were detected. The original CSF sample was negative on PCR for tuberculosis and herpes simplex virus, C-reactive protein remained at less than 5 mg/l, and a further head CT scan with contrast was normal.The diagnosis was revised to meningoencephalitis. Other agents—such as listeria, tuberculosis (a systematic review found that PCR has a sensitivity of only 56% [95% CI, 46%–66%] for detecting TB meningitis ), and viThe fixed, dilated pupils were of major concern, and urgent ophthalmic review was requested. Examination by the ophthalmologist showed reduced vision at 6/60 right eye and 6/36 left eye. On testing with Ishihara charts, the patient had severely reduced colour vision. Her pupils were large and non-reactive to light or accommodation in both eyes. The eyes were inflamed, with cells present in the aqueous and vitreous humours. The optic nerves were swollen and very pink , but theThe combination of the clinical symptoms, signs, and ocular features was characteristic of Vogt-Koyanagi-Harada (VKH) syndrome . All antThis young patient presented acutely with a fever and some signs suggestive of meningitis. She was initially treated as having viral meningitis, but the CSF findings indicated that other aetiologies needed to be considered. In particular, in a woman who previously lived in and recently visited Bangladesh, with a lymphocytic meningitis and borderline CSF glucose, tuberculosis had to be considered. Initially the ocular symptoms and signs were not a prominent feature, but the signs were likely to have been present when the patient was first seen.The typical CSF changes associated with meningitis of different aetiologies are shown in There is a wide range of infectious causes of meningitis worldwide. The likely infecting organism will be determined by the age and immune status of the patient plus the situation in which the infection was contracted. Thus, in an immunocompetent adult in the UK, enteroviruses are the commonest cause of viral meningitis, with meningococcus and pneumococcus the commonest bacterial agents. Tuberculosis is more common in people who have lived in a highly endemic area. In other parts of the world, the differential diagnosis may include viral infections such as West Nile virus in the continental United States and Japanese B encephalitis in Asia, or other pathogens such as rickettsiae, borrelia (Lyme disease), and protozoa. In the immunocompromised host, listeria must be considered, and there is an increased risk of fungal infection and tuberculosis. Finally, it is important to consider sexual exposure, as both secondary syphilis and HIV seroconversion may present with meningitis.It is important, therefore, in the evaluation and management of patients presenting with a meningoencephalitis that the differential diagnosis be continually reviewed if the patient is not responding to therapy . When apVKH syndrome ,5,6 is aVKH syndrome is usually bilateral, but occasionally the eyes can be affected asymmetrically so that one is very mildly involved. The syndrome is accompanied by marked intraocular inflammation, and there are choroidal infiltrates  associatThe disorder is caused by an immune response to melanin and affects parts of the body where melanin is found. The initiating stimulus for this response is unknown, but T-cells sensitised to melanin-associated antigens are found in the peripheral blood. In the ear, the melanocytes of the inner ear are the target, and the inflammatory response here results in hearing loss and balance problems. In longstanding untreated cases, depigmentation may occur in other sites such as skin .Consider meningitis in the differential diagnosis of a patient presenting with fever and headache.CSF analysis is essential to confirm meningitis and as part of establishing the cause.Consider non-infectious causes when a patient does not respond rapidly to therapy.Blurring of vision must be investigated and may help in determining the underlying diagnosis or the presence of papilloedema.SutlasPNUnalAFortaHSenolSKirbasD2003Tuberculous meningitis in adults: Review of 61 casesInfection31387391Early suspicion and appropriate long-term anti-tuberculosis therapy together with corticosteroids may reduce mortality and morbidity in patients with TB meningitis14735380RedingtonJJTylerKL2002Viral infections of the nervous systemArch Neurol59712718This review is an update on diagnosis and treatment12020250ThomsonRBJrBertramH2001Laboratory diagnosis of central nervous system infectionsInfect Dis Clin North Am1510471071This paper discusses conventional tests, such as culture, and others such as antigen testing and PCR11780267RotbartHA2000Viral meningitisSemin Neurol20277292The virology, pathogenesis, epidemiology, clinical manifestations, diagnostic studies, and established and potential antiviral therapies for viral meningitis are discussed. A differential diagnosis of the aseptic meningitis syndrome is provided11051293NegriniBKelleherKJWaldER2000Cerebrospinal fluid findings in aseptic versus bacterial meningitisPediatrics105316319Polymorphonuclear cell predominance in the CSF does not discriminate between aseptic and bacterial meningitis10654948KamondiASzegediAPappASeresASzirmaiI2000Vogt-Koyanagi-Harada disease presenting initially as aseptic meningoencephalitisEur J Neurol7719722This paper describes the neurological and eye signs in VKH syndrome11136362SeehusenDAReevesMMFominDA2003Cerebrospinal fluid analysisAm Fam Physician6811031108Lumbar puncture is frequently performed in primary care, and this review outlines the interpretation of the clinical and laboratory findings14524396ShahKHEdlowJA2002Distinguishing traumatic lumbar puncture from true subarachnoid hemorrhageJ Emerg Med236774The purpose of this article is to assist emergency physicians in distinguishing traumatic lumbar punctures from subarachnoid hemorrhage12217474


Atrial septal defect closure is now routinely performed using a percutaneous approach under echocardiographic guidance. Centrally located, secundum defects are ideal for device closure but there is considerable morphological variation in size and location of the defects. A small proportion of atrial septal defects may have multiple fenestrations and these are often considered unsuitable for device closure. We report three cases of multiple atrial septal defects successfully closed with two Amplatzer septal occluders. Atrial septal defect (ASD) closure is now commonly performed using a transcatheter, percutaneous approach and with the Amplatzer septal occluder, large defects can be safely closed ,2. DevicThe majority of patients require a single device for closure of the ASD but a small proportion of patients may have more than one defect in the atrial septum. This can be difficult to diagnose using transthoracic echocardiography (TTE) as abnormal colour flow obscures the origins of the shunt, particularly if the second defect is situated inferiorly. We report three cases of patients referred for ASD closures that were found to have multiple ASDs and the techniques used to close these defects.2) was 400. Repeat TTE continued to demonstrate left to right shunting and the patient was readmitted for a further device closure six months later. At cardiac catheterisation, the second defect was identified low in the secundum septum between the fossa ovalis and the mouth of the coronary sinus. The defect was successfully closed with a 9 mm Amplatzer septal occluder with no evidence of obstructed flow in either the coronary sinus or the inferior vena cava. Equivalent chest x-ray radiation dose for the second procedure was 187. The patient remained well with no evidence of residual shunt six months following the procedure.A 34-year old woman was referred for consideration of percutaneous ASD closure. The ASD had been diagnosed when the patient was 12 years old and TTE had suggested that the right ventricle was dilating. At cardiac catheterisation there were mildly elevated right ventricular systolic pressures and a pulmonary to systemic flow ratio of over two. The secundum ASD was estimated to be 15 mm wide using TTE with aneurysmal formation of the interatrial septum. The patient was admitted for percutaneous ASD closure and underwent uncomplicated placement of a 17 mm Amplatzer septal occluder. Transesophageal echocardiography (TEE) during the procedure revealed the presence of a second ASD near the inferior vena cava and a small post-procedure shunt. The septal occluder did not completely cover both defects. Equivalent chest x-ray radiation dose  but it likely that simultaneous deployment will increase the procedure risk ,6. When There is considerable variation in atrial septal defect anatomy. A small proportion of patients with an ASD have more than one defect and these can be closed using conventional septal occluders under transoesophageal echocardiography guidance. The use of intracardiac echocardiography should allow more accurate device positioning, particularly defects located low in the atrial septum.