Inhibition of phrenic nerve activity (PNA) can be achieved when alveolar ventilation is adequate and when stretching of lung tissue stimulates mechanoreceptors to inhibit inspiratory activity. During mechanical ventilation under different lung conditions, inhibition of PNA can provide a physiological setting at which ventilatory parameters can be compared and related to arterial blood gases and pH.To study lung mechanics and gas exchange at inhibition of PNA during controlled gas ventilation (GV) and during partial liquid ventilation (PLV) before and after lung lavage.t), PIP, oesophageal pressure and arterial blood gases were measured at inhibition of PNA. One way repeated measures analysis of variance and Student Newman Keuls-tests were used for statistical analysis.Nine anaesthetised, mechanically ventilated young cats  (mean ± SD) were studied with stepwise increases in peak inspiratory pressure (PIP) until total inhibition of PNA was attained before lavage (with GV) and after lavage (GV and PLV). Tidal volume  and Vt before than after lung lavage. After lavage, inhibition of inspiratory activity was achieved at the same PIP, Ptp and Vt during GV and PLV, but occurred at a higher PaCO2 during PLV than during GV in cats with surfactant-depleted lungs. This could indicate that PLV induces better recruitment of mechanoreceptors than GV.Inhibition of inspiratory activity occurs at a higher PaCO Partial liquid ventilation (PLV) combines liquid ventilation and gas ventilation (GV). Perfluorocarbon is administered to the trachea in a volume equivalent to the pulmonary functional residual capacity, and ventilation is maintained with conventional gas ventilation of the liquid-filled lung . The imp2 and a higher pH will be needed [During breathing of gas the rate and depth of breathing is influenced by mechanoreceptors in the lung -6, and be needed .No studies have been presented concerning PNA during PLV, but it has been shown in studies of animals that spontaneous breathing can take place during PLV with beneficial physiological effects ,14. InhiThis study was therefore undertaken to determine whether inhibition of PNA can be achieved at the same airway or transpulmonary pressures during GV and PLV and to find out at what levels of arterial blood gases and pH inhibition occurs with these modes of ventilation in cats with healthy and surfactant-depleted lungs.®, F. Stephan Biomedical Inc., Gackenbach, Germany) and the animal was placed on assist control (A/C) ventilation during the surgical procedures with the following settings: peak inspiratory pressure (PIP) 1.0 kPa, positive end-expiratory pressure (PEEP) 0.3 kPa, inspiratory time (Ti) 1 sec, respiratory rate (RR) 30/min.Juvenile cats  were anaesthetised with chloroform, placed in a supine position and endotracheally intubated (tube 4.0 mm inner diameter). The tube was then connected to an infant ventilator . The cat's core body temperature, measured as deep rectal temperature, was maintained at 38°C by a heating blanket and an overhead warmer.The right femoral vein and artery were dissected and catheters were inserted with the tip of each catheter placed in the thorax close to the heart. Anaesthesia was continued with 0.72% α-chloralose  (50 mg/kg) and maintained at regular intervals via the venous line. A continuous infusion of 10% glucose (2/3) and 5% 0.6 M sodium bicarbonate (1/3) was given at a rate of 6.4 mL/kg/h (7.15 mg/kg/min of glucose) through the venous catheter throughout the experiment. The arterial line was used for continuous monitoring of blood pressure and intermittent determination of blood gases  was inserted into the distal part of the oesophagus and a ligature was softly tied around the oesophagus to avoid air entrance into the stomach . Both ph® infant ventilator. This sensor is a pneumotachometer with the dynamic properties of an original Fleisch 00 pneumotachograph, but with less dead space (0.6 ml) and resistance (1.1 kPa/l/s at 5 L/min) [®, Timeter Instrument Corporation, Lancaster, PA, USA). Airway pressure (Paw) was measured at the connector of the endotracheal tube. Oesophageal pressure (Poes) was recorded from the oesophageal balloon catheter by a pressure transducer  and, like Paw, was calibrated with a water manometer. Arterial blood pressure and heart rate were measured using the same type of transducer  connected to the arterial catheter with the tip of the catheter at the same level as the transducer. Continuous recordings of arterial blood pressure and heart rate were made with a polygraph recorder .Airflow was measured by a sensor placed between the endotracheal tube connector and the Y connector of the tubing circuit of the Stephanie5 L/min) . Airflow® . The rectified nerve signal was fed to a spike trigger to produce spikes of uniform amplitude  and subsequently integrated by a resistance-capacitance low-pass filter with a leak (time constant 250 ms), providing a moving time average of PNA. Monitoring of the signals was achieved by means of an oscilloscope .PNA was amplified, filtered and rectified with a Neurolog systemaw were obtained directly from the analogue outlets of the ventilator. Together with signals of Poes and the PNA they were transferred to an analogue-digital converter and recorded on disk at a sampling rate of 10 kHz per channel by a data acquisition system . Compliance and resistance values were given by the ventilator.Signals of airflow and PThe cats were kept ventilated with air using A/C ventilation and the ventilation was adjusted so that normal arterial blood gases were achieved. The cats were then treated with endotracheal continuous positive airway pressure with 0.3 kPa PEEP in order to monitor and record the spontaneous breathing activity of each cat. Pressure-controlled mechanical ventilation with sinusoidal inspiratory waveform was then initiated with the following settings: RR 60/min; Ti 0.33 sec; PIP 0.8 to 1.0 kPa using a PEEP of 0.5 kPa. PIP was adjusted so that blood gas values were in a normal range. The fraction of inspired oxygen was kept at 0.21. PIP was then gradually increased until rhythmic PNA disappeared. Three breaths after inhibition of PNA, data from 20 consecutive breaths were recorded and arterial blood gases were analysed.2 1.0), ventilation was increased until PNA was inhibited. Airway pressures were then recorded and arterial blood gases and pH were measured again.Thereafter lung lavage was performed by filling the lungs with warmed saline solution  through a funnel connected to the endotracheal tube. Very gentle chest compressions were performed to allow the saline to be well distributed, before it was removed by suctioning. This procedure was repeated 7 to 8 times and mechanical ventilation was provided in between the lavage procedures. After a 30-minute period of stabilisation on mechanical ventilation  was instilled into the endotracheal tube via an adapter with a side port. Instillation of PFC into the lung was performed within 10 minutes during pressure-controlled ventilation . Sufficient filling was ascertained by disconnecting the endotracheal tube from the ventilator circuit at the end of the filling procedure and observing to see that a meniscus was present in the endotracheal tube at end-expiration. If no meniscus could be observed prior to recording, additional PFC was instilled. After a stabilisation period of 10 minutes, the cats were studied with the same protocol during PLV as during GV, but with an FiO2 of 1.0 and a PIP adjusted to blood gases in the normal range.In the next step a bolus of 30 ml/kg prewarmed (38°C) perfluorocarbon (PFC) .® Software  was used to review the recorded signals. Analysis was done by means of Windaq Playback® and Excel® . For statistical evaluation, Sigmastat®  was used.Windaq PlaybackThe amplitude of the integrated PNA was monitored and inhibition of spontaneous breathing activity was defined as total disappearance of PNA, i.e. total inhibition of inspiratory activity.aw and Poes were measured at peak inspiratory pressures. The airflow signal was integrated to obtain tidal volumes (Vt) at different PIPs. Transpulmonary pressure (Ptp) was calculated as Paw – Pes. Lung compliance (CL) is given as the ratio of Vt over Ptp. In three cats an endotracheal tube leak of > 20% of the tidal volume was found, and in those cats no volume values were therefore calculated and consequently no compliance values can be given.Gas flow, PAfter inhibition of PNA, the 20 breaths were evaluated at the three settings studied, i.e. during GV with a normal lung, and during GV and PLV after surfactant depletion. Data are presented as mean ± SD or median and 25th and 75th percentiles. One way repeated measures analysis of variance (ANOVA) or RM ANOVA on ranks was performed to test for differences between the three groups. Student-Newman-Keuls tests were applied for comparisons between two groups when a difference was detected by ANOVA. The level of significance was set at p < 0.05 in all tests. An a posteriori power analysis revealed that the study had a power of 99% to detect a difference in PIP between healthy and surfactant-depleted lungs during GV, and of 100% to detect such a difference between healthy and liquid-filled lungs (n = 9). The power values for detecting differences in tidal volume between the same groups were 98% and 61% respectively (n = 6).Inhibition of PNA could be achieved in all cats during GV and PLV both before and after lavage at the applied ventilatory frequency of 60/min, insufflation time 0.33% of the period time and PEEP of 0.5 kPa. Figure tp and lower tidal volumes  filled lung (1). PLV has been shown to decrease the alveolar surface tension mainly in dependent parts of the lung, resulting in alveolar recruitment and reduced ventilation-perfusion mismatch, thereby improving gas exchange and lung mechanics . These bIn the present study a ventilatory strategy of a moderate PEEP (0.5 kPa) and positive pressure ventilation at 60/min was chosen in a model of surfactant depletion to simulate a relevant clinical situation in which lung recruitment and possibly low tidal ventilation could be promoted. The point of inhibition of PNA represents the time point at which central inspiratory activity ceases and at which all spontaneous breathing activity has disappeared completely. It has been used as a point of comparison between different ventilatory patterns .Lung compliance did not differ between PLV and GV in surfactant-depleted lungs, but resistance was higher during PLV, as reported elsewhere . This miIn this study all experiments were performed in the same order and time sequence, i.e. first GV in the healthy lung, and then GV and PLV in that order in the surfactant-depleted lung. We avoided randomisation of the order of GV and PLV in the surfactant-depleted lung, as that approach would have meant a much longer period of mechanical ventilation in the group randomised to PLV as the first part of the protocol, to allow evaporation of the perfluorocarbon.In cats with normal lungs the pulmonary stretch receptor (PSR) activity is similar during GV and PLV, indicating that there is no extensive stretching of the lung during PLV . On the 2 during PLV than during GV. This possibility is supported by the finding that administration of surfactant increases the activity of mechanoreceptors in surfactant-depleted animals [2 during PLV influences the respiratory drive.In animals with surfactant-depleted lungs, which may be partially atelectatic, mechanoreceptors in some well-ventilated areas may be active, whereas other receptors in atelectatic areas may not give any impulses. In the present study all receptors which were active during GV were also active during PLV. The study showed that during GV inhibition of PNA occurred at much higher airway pressures after than before lung lavage, but at similar arterial blood gases, findings which might be due to an altered stretch receptor input from, for example, areas that are surfactant-depleted and/or partially atelectatic. As instillation of perfluorocarbon might exert an effect similar to that of surfactant on lavaged lungs, increased mechanoreceptor discharge during PLV due to increased stretch receptor activity might explain why PNA inhibition occurs at a higher arterial PCO animals . It is u2 during PLV than during GV in animals with surfactant-depleted lungs, which might be explained by recruitment of pulmonary stretch receptors during PLV. This may be a reason why inhibition of spontaneous breathing is more easily achieved during PLV than during GV in animals with surfactant-depleted lungs.Higher airway pressures are needed to achieve inhibition of inspiratory activity during GV in animals with surfactant-depleted lungs than in animals with normal lungs. After surfactant depletion, inhibition of inspiratory activity during PLV occurs at about the same peak inspiratory and end-expiratory pressures and tidal volume as during GV. Inhibition of inspiratory activity occurs at a lower arterial pH and a higher arterial PCOPNA, phrenic nerve activityGV, gas ventilationPLV, partial liquid ventilationPIP, peak inspiratory pressuretV, tidal volumeHFPPV, high frequency pressure ventilationPEEP, positive end-expiratory pressureA/C ventilation, assist/control ventilationTi, inspiratory timeRR, respiratory rateawP, airway pressureoesP, oesophageal pressuretpP, transpulmonary pressureLC, lung complianceRM-ANOVA, one way repeated measures analysis of varianceThe authors declare that they have no competing interests.ERF participated in designing the study, was involved in the preparation and care of the animals, was responsible for the acquisition and analysis of the data and drafted the manuscript. RS participated in the design of the study, was responsible for the preparation of the animals, was involved in the acquisition and analysis of the data, and revised the manuscript. AJ participated in the design of the study, was responsible for the preparation of the animals and for the neurophysiological recordings, and revised the manuscript. AS made substantial contributions to the data collection and their interpretation, and revised the manuscript. GS conceived of the study and its design, performed the lavage and PFC instillation procedures, helped to interpret the data, and revised the manuscript. All authors read and approved the final manuscript.


Many properties of organisms show great robustness against genetic and environmental perturbations. The terms canalization and developmental stability were originally proposed to describe the ability of an organism to resist perturbations and to produce a predictable target phenotype regardless of random developmental noise. However, the extent to which canalization and developmental stability are controlled by the same set of genes and share underlying regulatory mechanisms is largely unresolved.Drosophila subobscura. For the analysis of overall size, developmental stability was positively correlated with levels of heterozygosity and development at the optimal temperature. For analyses of shape, the overall comparisons by matrix correlations indicate that inter- and intraindividual variation levels were poorly correlated, a result also supported when comparing the vectors describing patterns of variation of landmark position. The lack of similarity was basically due to the discrepancy between the genetic and environmental components of the interindividual variation. Finally, the analyses have also underscored the presence of genetic variation for directional asymmetry.We have analyzed the effects of clinal genetic variation (inversion polymorphism) on wing asymmetry by applying the methods of geometric morphometrics in the context of quantitative genetics using isochromosomal lines of Drosophila populations is loosely related to individual fitness.The results strongly support the hypothesis that environmental canalization and developmental stability share underlying regulatory mechanisms, but environmental and genetic canalization are not functionally the same. A likely explanation for this lack of association is that natural wing shape variation in  Phenotypic robustness refers to the invariance of the specified target phenotype given the genetic makeup and environmental conditions. Whereas the presence of naturally occurring phenotypic variation is at the core of evolutionary biology, developmental geneticists have traditionally considered it as a nuisance. Instead, they have relied on the study of single or multiple mutant combinations to reveal the generation of phenotypic patterns (e.g. ). A resuThree major processes are involved in the control of phenotypic variability : canali) and special (micro) environmental effects : the first refer to influential factors (e.g. temperature) that are shared by groups of individuals, whereas the latter are residual deviations from the phenotype that would be specified on the basis of genotype and general environmental effects. Such deviations are unique to individuals and are largely unpredictable. The variance associated with special environmental effects can be estimated when experiments are performed on completely inbred lines . In bilaterally symmetrical organisms it is also feasible to estimate the two sources that contribute to those special environmental effects: among-individual  and within-individual variance . If the only real cause of asymmetry is variation due to stochasticity in development, then FA can be taken as an estimated of . Therefore, FA is only one source of the phenotypic variation within environments , contrarily to the arguments in Nijhout and Davidovitz  have a mean of zero, the interaction term is a measure of fluctuating asymmetry  provided that there is no genetic variation for DA [To first partition the total phenotypic variation into interindividual, intraindividual and measurement error components, we used the conventional mixed model, two-way ANOVA  for the study of left-right asymmetries . In thisn for DA , and the; there is no genetic variance within crosses). An estimated of the among-fly special environmental effects variance (i.e. ) is therefore obtained by subtracting the individual × side interaction effect (which includes  plus measurement error) as the appropriate error term. However, when genetic variation for DA is present the unbiased within-fly special environmental effects variance (i.e. FA) is estimated after partitioning the individual × side interaction effect into its causal components [We now digress slightly to point out some inconsistencies in the literature on what is the appropriate error term to test for the "interindividual" effect in the mixed model, two-way ANOVA  outbred crosses the karyotype covariance components were calculated as  , the cross covariance components as , and the among-fly special environmental effects covariance components as .The SSCPIS matrix into their causal components [Similarly, genetic effects for DA can be investigated after partitioning the SSCPmponents . for i ≠ j,  for i = j; superscript 'denotes transposition) in an n-dimensional space.Within each sex and temperature, principal component analyses  of the Vx- and y-coordinates  since covariance matrices are symmetrical, and statistical significance was assessed using permutation tests designed to maintain the associations between pairs of  columns ,15); othA second test examined the congruence of the landmark displacements corresponding to each emergent PC for the different effects within groups. Because the PCs correspond to directions in the multivariate shape space, correlations can be obtained by angular comparisons of component vectors. Statistical significance of these correlations was then assessed by comparing those observed values to a null distribution of absolute angles between 100,000 pairs of 22-dimensional random vectors . The 0.1) [) against mean centroid size .The occurrence of antisymmetry  for cen) . The indP-values > 0.10) and, therefore, no size corrections were necessary.Scatter plots of left-right differences for each landmark after Procrustes superimposition were visually checked to see whether or not there was evidence for clustering of these vectors that would have argue for the occurrence of AS ,15. No iThe computer programs used for statistical data analyses were MATLAB (V.6. ) togetheMS conceived the study, carried out extraction of O chromosomes, experimental crosses, egg collections, statistical analyses, and drafted the final manuscript. PFI carried out extraction of O chromosomes, experimental crosses, egg collections, wing measurements, and preliminary statistical analyses and drafts of results. WC read all salivary gland squashes for gene arrangement identification and mounted the wings on microscope slides. All authors read and approved the final manuscript.


Every year West African countries within the Sahelo-Sudanian band are afflicted with major meningococcal meningitis (MCM) disease outbreaks, which affect up to 200,000 people, mainly young children, in one of the world's poorest regions. The timing of the epidemic year, which starts in February and ends in late May, and the spatial distribution of disease cases throughout the “Meningitis Belt” strongly indicate a close linkage between the life cycle of the causative agent of MCM and climate variability. However, mechanisms responsible for the observed patterns are still not clearly identified.By comparing the information on cases and deaths of MCM from World Health Organization weekly reports with atmospheric datasets, we quantified the relationship between the seasonal occurrence of MCM in Mali, a West African country, and large-scale atmospheric circulation. Regional atmospheric indexes based on surface wind speed show a clear link between population dynamics of the disease and climate: the onset of epidemics and the winter maximum defined by the atmospheric index share the same mean week  and are highly correlated.This study is the first that provides a clear, quantitative demonstration of the connections that exist between MCM epidemics and regional climate variability in Africa. Moreover, this statistically robust explanation of the MCM dynamics enables the development of an Early Warning Index for meningitis epidemic onset in West Africa. The development of such an index will undoubtedly help nationwide and international public health institutions and policy makers to better control MCM disease within the so-called westward–eastward pan-African Meningitis Belt. An analysis of winds and yearly meningitis epidemics in West Africa shows a surprisingly close correlation that could help to predict disease outbreaks Meningococcal meningitis (MCM) has affected Sahelian Africa for centuries and became endemic over the past 25 y. During the 1980s, the World Health Organization (WHO) registered between 25,000 and 200,000 disease cases per year, with about 10% of them resulting in death, and with the highest infection rates observed in younger children , by using the weekly number of cases of MCM disease in Mali and large-scale fields of surface wind speed, we clearly identify a strong relation between climate and the seasonal pattern of MCM cases in Mali. It is shown that the onset of disease outbreak is characterized by a clear breaking slope in the seasonal cycle of the number of cases at the sixth week of the year, that is, 15 February. The computation of an atmospheric index based on surface wind speed over Mali points out that this abrupt shift is also present in the atmospheric signal, corresponding to the winter wind maximum, when Harmattan winds are the strongest in Sahelo-Sudanian Africa. The similarity in the seasonal patterns of both Harmattan winds and MCM disease cases is obvious, with a strong correlation between the week of winter maximum and that of the onset of epidemic. Similar results, not illustrated here, have been obtained by using surface temperatures and specific humidity for the computation of atmospheric indexes, attesting to the robustness of the analysis.However, whatever the climatic index is used, this analysis does not allow us to link the intensity of the “epidemic”  to the intensity of winter in terms of absolute humidity and surface wind speed. This lack of a relation may be due to the time series length, with an insufficient number of years to study interannual variations, or it may imply that the climatic influence is limited to explaining the occurrence of the seasonal cycle of the epidemic and its geographical range distribution, but not its intensity. Although they fail to forecast epidemic intensity, such climatic indexes, with their correlation with the onset and the seasonal course of the epidemic in Sahel, provide an important means of disease monitoring and prediction in Africa. Indeed, the seasonal pattern of humidity and Harmattan winds can be easily tracked, thus promoting the emergence of an Early Warning Index (EWI) for the onset of MCM epidemics. The seasonal forecast of this EWI based on Harmattan winds could thus be implemented routinely by using comprehensive coupled models of the atmosphere, oceans, and land surface that provide a degree of predictability of climate fluctuations with a seasonal lead time in many parts of the world . The abiThis EWI parameter, in association with other environmental parameters implicated in disease resurgence , could hClimate is known to be one of the factors that can affect when and how epidemics occur; for example, floods often increase the risk of waterborne disease. However, there are many more subtle climatic changes that might also be important in affecting when and how diseases occur.Neisseria meningitides). These researchers had previously published some detailed work on the local climate in a French journal. In this paper they have focussed more on the aspects that deal with disease. They found out that over several years the onset of the epidemic coincided with the peak of the winds.They looked at the relationship between a recurring epidemic of a disease called meningococcal meningitis in Mali in West Africa and the local climatic conditions, especially the winds. Meningococcal meningitis is a serious infection of the lining of the brain and spinal cord by a bacterium (called People who would find these results useful are those who plan for epidemics. Such information will allow them to plan in advance, and even predict whether an epidemic will occur at all. However, these results were based on only the years between 1994 and 2002, and so will need to be confirmed in more years.


West African Journal of Medicine [In this perspective, Andrew Browning of the Fistula Hospital in Addis Ababa discusses a study on obstetric fistula in Ilorin, Nigeria. The study was originally published in the Medicine . With th Obstetric fistula causes women enormous distress and social isolation in developing countries like Nigeria. What do we know about the condition? The obstetric urogenital fistula has caused women misery ever since they first started delivering children. It was once common worldwide, but with the advent of safe obstetric care during the early part of the last century, the condition has become rare in rich countries. Urogenital fistulae do still occur in developed countries, but unlike in the developing world, they are usually a complication of a difficult pelvic surgery, cancer, or radiation .Obstetric fistula—a urogenital fistula from obstructed labour—is now only encountered in countries where health resources are scarce. The shame associated with incontinence drives affected women further into a life of poverty and begging. Many women with fistula either do not know that they can get medical help, or if they do, they are unable to pay.Furthermore, very little scientific research has been published about obstetric fistula and its management, partly because the people treating patients with this condition are working in remote areas, often with very limited resources for research. What has been written consists largely of personal case series and a few epidemiological studies .To date there has been only one randomised trial in the developing world, involving 79 women operated on by a single surgeon in Benin, which found that intra-operative intravenous antibiotics did not reduce the risk of failed surgical repair or of objective incontinence . There hWhat we do know about the obstetric urogenital fistula is that the women who have these injuries are young, usually illiterate, and of a lower socioeconomic background. They are more often primiparous and short in stature, and they have an average length of labour of some 3.9 days. The labour is usually unattended, or if attended, it is by someone unskilled. The women inevitably deliver a stillborn child. About half of the women with fistula are divorced as a direct result of their incontinence ,5,6,7.The initial injury that leads to a fistula results from ischaemic necrosis of the soft tissues of the pelvis due to an impacted presenting part during the long labour.The ischaemia then affects the bladder and vagina (and sometimes the rectum and vagina), resulting in a fistula. The process also affects other pelvic structures. These include the nerves of the sacral plexus, resulting in foot drop and hamstring compartment weakness . Bony abnormalities are common, separating or obliterating the symphysis pubis.Up to half of patients develop upper renal tract abnormalities: scarring of the ureter can cause obstructive uropathies . Up to tThe continual leakage of urine over the perineal skin can cause local and painful irritation, termed ‘urine dermatitis’. Bladder stones can occur, as women affected by fistula often drink less to try and pass less urine and the concentrated urine can form calculi. The obstetric injury has been termed a ‘field injury’, as the pathology is broad rather than isolated . The resA recently published retrospective case note review provides new data on obstetric urogenital fistula in northern Nigeria. Ijaiya and Aboyeji reviewed 34 cases of fistula managed at the University of Ilorin Teaching Hospital over a two-year period . During ‘[Obstetric fistula] usually occurs when a young, poor woman has an obstructed labour and cannot get a Caesarean section when needed. The obstruction can occur because the woman's pelvis is too small, the baby's head is too big, or the baby is badly positioned. The woman can be in labour for five days or more without medical help. The baby usually dies. If the mother survives, she is left with extensive tissue damage to her birth canal that renders her incontinent.’www.unfpa.org/fistula/about.htm).Source: UNFPA Campaign to End Fistula: “What is Fistula?”  and the International Federation of Gynecology and Obstetrics (www.figo.com) are endeavouring to help. UNFPA has already sponsored training workshops on fistula surgery for surgeons and fledgling fistula units in Bangladesh and some parts of Africa.Resource-rich countries were able to eradicate the obstetric fistula almost 100 years ago, but the challenge to resource-poor countries is enormous. There are an estimated 2 million women with fistula in the world, with anywhere between 100,000 and 500,000 new cases developing each year . At the The obstetric fistula is an entirely preventable condition. Several strategies have been proposed to eradicate this condition in developing countries , just as‘Postpone marriage and pregnancy for young girls‘Increase access to education and family planning services for women and men‘Provide access to adequate medical care for all pregnant women and emergency obstetric care for all who develop complications‘Repair physical damage through medical intervention and emotional damage through counselling' www.unfpa.org/fistula/facts.htm).Source: UNFPA Campaign to End Fistula: “Fast Facts” (Even if such centres are established, women will need to be convinced of the importance of seeking help without delay for a difficult labour. And then, to be able to receive that help, roads need to be built, transport systems need to be put in place, and communications need to be improved. The obstacles are clearly huge, and with currently very little money and very few professionals available, women with obstetric fistula will sadly be with us for many more years to come.Text S1Full Text of Ijaiya and Aboyeji's Study  (234 KB Click here for additional data file.


Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens. In order to further investigate the mode of tissue-specific gene clustering in higher eukaryotes, we have performed a genome-scale analysis of positional clustering of the mouse testis-specific genes.Genes are not randomly distributed on a chromosome as they were thought even after removal of tandem repeats. The positional clustering of co-expressed genes is known in prokaryotes and recently reported in several eukaryotic organisms such as Our computational analysis shows that a large proportion of testis-specific genes are clustered in groups of 2 to 5 genes in the mouse genome. The number of clusters is much higher than expected by chance even after removal of tandem repeats.Our result suggests that testis-specific genes tend to cluster on the mouse chromosomes. This provides another piece of evidence for the hypothesis that clusters of tissue-specific genes do exist. Saccharomyces cerevisiae [Drosophila melanogaster [Caenorhabditis elegans [Homo sapiens [Positional clustering of co-expressed genes is mainly due to operons in prokaryotes. Genes located within the same operon are transcribed together and thus co-regulated. In general, operons are missing from eukaryotes. Eukaryotic genes appear to be transcribed individually and are thought to be scattered on chromosomes without apparent organization by function or positional expression except for tandem duplication. However, positional clustering has recently been reported in several eukaryotes such as revisiae ,23, Drosnogaster ,5, Caeno elegans , and Hom sapiens ,8.et al. first showed clustering of co-expressed yeast genes on a genome-scale. They found that about 25% of genes with cell-cycle-dependent expression pattern were adjacent to those induced in the same phase of the cell cycle [et al. [et al. computationally analyzed the whole-genome gene expression using the same dataset. They consider two ORFs to be adjacent if they occur on the same chromosome and if there are no other ORFs or functional elements such as Ty element, long terminal repeats, tRNAs, snRNAs between them. They observed that adjacent or nearby non-adjacent pairs of genes showed correlated expression independent of their orientation in the yeast genome using chromosome correlation maps that display correlations between the expression patterns of genes on the same chromosome. In addition, they also showed that genes with similar functions tend to occur in adjacent positions along the chromosomes.Cho ll cycle . This fa [et al. . Cohen eet al. [Drosophila melanogaster and these clustered genes account for over 20% of the total assayed genes. By mapping Expressed Sequence Tags (EST) back to the Drosophila genome, Boutanaev et al. [Spellman et al.  found thv et al.  observedCaenorhabditis elegans. Although operon and tandem duplication are major mechanisms for the observed co-expression gene clusters in the worm, co-expression gene clustering is still evident after exclusion of these two causes [Similarly, positional clustering of co-expressed genes was also reported in o causes ,7.et al. [Region of IncreaseD Gene Expression). However, these RIDGES seem to consist mainly of housekeeping genes [et al. [et al. [Expressed Sequence Tag), they showed positional clustering of these genes. These studies suggest that clusters of tissue-specific genes do exist, and might be more frequent than initially thought.Clusters of highly co-expressed genes were also revealed in the human and other mammalian genomes ,8,5,12. ng genes . Gabriel [et al.  performe [et al.  investigHere we performed a genome-wide analysis of testis-specific genes in the mouse genome using a method similar to one proposed in . We usedTo further investigate testis-specific gene clusters in the mouse genome, we used the full-length cDNA sequences publicly available in the Fantom 2.1.1 database to identify differently expressed genes. After mapping 60,770 cDNA sequences back to the genome, we obtained the gene expression profile by assembling overlapping cDNA sequences into a single gene model. Testis-specific genes were selected by using only sequences from libraries where the tissue type included the keyword "testis" and not the keyword "embryo". Again, our statistical analysis shows the existence of testis-specific gene clusters along the mouse chromosomes even after removal of tandem duplicated genes. This suggests that considerable clusters of tissue-specific genes do exist in higher eukaryotes. Our results provide further support for the hypothesis that chromatin domain model may be involved in the regulation of gene expression in higher eukaryotesThe testis-expressed profile is a global view of gene expression and provides a foundation for understanding how the tissue functions in molecular level. We assume that most of the testis-specific genes are expressed after embryo phases in mouse. Therefore, we selected only testis genes models that exclude any expression in embryo for the construction of the testis-specific gene profile, which reveals positional clustering along chromosomes. Here, we analyzed the cluster distribution on all the chromosomes except for chromosome Y.  Based on our study, we obtain the following observations.The testis-specific gene expression profile along the mouse genome shows that testis-specific genes are distributed on all the chromosomes with different gene density. The ratio (R) of the number of the observed testis-specific genes (O) to the expected number (E) is used to measure the gene density, where E is calculated according to the chromosome's size on assumption that the testis-specific genes are uniformly distributed throughout the genome. The lowest gene density appears on chromosomes 1 (R = 0.834), 15 (R = 0.893), 16 (0.855), and 18 (R = 0.767) except for sex chromosomes. But, chromosomes 9 (R = 1.145), 11 (R = 1.374), 14 (R = 1.207) and 19 (R = 1.48) have rather high gene density. Within a chromosome, the distribution of testes-expressed genes also shows diverse density.We observed numerous groups of physically neighboring testis-specific genes that shared strikingly similar expression levels. In eukaryotes, it is clear that the activity of a promoter can be modified by transcription factors binding to DNA sequence, which are located from hundreds to thousands of base pairs from the promoter. Recently, increasing evidences show that genes may also be regulated in a group. For example, when transgenes are removed from their local environment and reinserted elsewhere in the same genome, they tend to show variant levels of expression . More coWe consider two genes are adjacent if there are no other genes between them on the same chromosome. We observed that 1170 testis-specific genes appear in adjacent pairs, which account for 31.8% of the testis-specific genes after removal of tandemly duplicated genes. There are 350 adjacent pairs, 86 adjacent triplets, 23 adjacent quadruplets, and 4 quintuplets . We obtained 3679 testis-specific genes on all the chromosomes except for Y after removal of tandemly duplicated genes. We conducted statistical analysis with 4 different values of D: 25 K, 50 K, 75 K, and 100 K .When D = 25 K, we obtained 288 two-gene, 21 three-gene, and 2 four-gene clusters. In total, 18% of the testis-specific genes are contained in these clusters. By incorporating the variance of gene density in different regions on a chromosome, we conclude that 45 two-gene, 7 three-gene and 2 four-gene clusters are significant with p-value less than 4.9·10-2, 3.1·10-2 and 1.2·10-2 respectively. The most significant one is a two-gene cluster on chromosome 18 .When D = 50 K, we obtained 392 two-gene, 56 three-gene, 19 four-gene and 1 five-gene clusters. In total, 28% of the testis-specific genes are in these clusters. By incorporating the variance of gene density in different regions on a same chromosome, we conclude that 14 two-gene, 6 three-gene and 2 four-gene clusters are significant with p-value less than 4.9·10-2, 3.1·10-2 and 4.9·10-2 respectively. The most significant one is a two-gene cluster on chromosome 16 .When D = 75 K, we obtained 454 two-gene, 79 three-gene, 28 four-gene, 4 five-gene and 1 six-gene clusters. In total, 35% of the testis-specific genes are in these clusters. By incorporating the variance of gene density in different regions on a chromosome, we conclude that 10 two-gene, 2 three-gene and 3 four-gene clusters are significant with p-value less than 4.1·10-2, 4.8·10-2 and 3.2·10-2 respectively. The most significant one is a two-gene cluster on chromosome 16 .When D = 100 K, we obtained 463 two-gene, 114 three-gene, 46 four-gene, 7 five-gene and 2 six-gene clusters. In total, 41% of the testis-specific genes are in these clusters. By incorporating the variance of gene density in different regions even on the same chromosome, we conclude that 12 two-gene, 4 three-gene and 1 four-gene clusters are significant with p-value less than 4.9·10We also observed that there are more significant gene clusters on chromosomes 5, 7, 12 X than any other chromosomes.It seems that in most eukaryotes, the transcription factor machinery is sufficient for ensuring co-regulation so that co-localization of genes in a genome is not necessary. However, there could be some degree of selection for keeping co-regulated genes in clusters on a chromosome, for instance, to make them available to transcription more efficient as a group.Clustering of co-expressed genes could be due to tandem duplication. Tandem duplication and subsequent divergence of amplified copies might result in an array of paralogous genes that may retain common regulatory element. Such a type of clustering is exemplified by the Hox gene family . From thIn the most of chromosomes except for chromosomes 2, 5, 7, 10, no more than 15 duplicated genes were identified. On chromosomes 2, 5, 7, 10, we identified 18, 21, 19, 16 duplicated genes respectively. Since, for each of distance threshold value , we obtained more than 300 gene clusters, we conclude that duplication has no obvious effect on the positional clustering of testis-specific genes in the mouse genome.Eukaryotic genes have not only promoters, but also one or more enhancers. An enhancer is a DNA sequence that influences transcription of neighboring genes. Enhancers are different from promoters in their ability to act over thousands of base pairs upstream or downstream of a gene. While a promoter is responsible for initiating low levels of transcription and determining the transcription start site, enhancers are responsible for increasing or "enhancing" transcription levels, as well as for regulating cell- or tissue-specific transcription.There are presently two models for non-duplicated gene clustering. One is the incidental expression mode. In this model, co-regulation within an expression neighborhood is due to incidental interactions between transcriptional enhancers and promoters . When trAn alternative model of clustering is that gene clusters may correspond to regions of active chromatin. This explanation is called the chromatin domain model. In mammalian genomes, gene clusters are on a much larger scale (Mb rather than 5–20 kb) and may involve long-range chromatin interactions ,19. The Our findings can most be explained as regulation at the level of chromatin structure for the following reasons. First, the regions showing similarities in expression are quite large; each gene presumably has its own core promoter. Second, one or two genes in a group often displays a high level of differential expression. If the chromatin in a region that contains many genes was 'opened' so that a single target gene could be expressed, it might increase the accessibility of the promoters and enhancers of other genes to transcriptional machinery, leading to corresponding increases in their expression. Such an effect could account for the observations we have made. Although the organization of neighborhoods along a chromosome indicates that there must be cis-acting structures, no known structure correlates with the blocks. The structure basis of positional gene clustering is still under investigation.After removal of tandemly duplicated genes, significant clusters of co-expressed testis-specific genes were still found on all the chromosomes. However, the mechanism responsible for this observation is still unknown.We revealed the positional clustering of testis-specific genes in the mouse genome by using EST profiling. Our analysis implies that the mouse genome is enriched with clusters of testis-specific genes. A significant trend for large clusters at least 3 genes) was discernible. Positional clustering of co-expressed, non-homologous genes has also been reported in nematode  genes wa. This inFrom this fascinating starting point we expect further insight into the significance of gene clustering and the mechanisms that generate them as more genomes are sequenced and more expression patterns are studied in the coming years.A total of 60,770 full-length cDNA sequences are available in the Fantom  2.1.1 database. These sequences were aligned back to the mouse genome using BLAT  .Recall that we define clusters using the distance between two neighboring testis-specific genes along the chromosome. Two testis-specific genes are in a cluster if and only if there is a series of testis-specific genes between them such that the distance between two consecutive genes in the series is less than a specified threshold (L (2000 Kbp here) so that genes on such a segment are roughly uniformly distributed and analyze the significance of a cluster within the segment according to the number N of all the genes (not just testis-specific genes) in the segment and the values of parameters L and D.To incorporate the variance of gene density in different regions on a chromosome, we divide a chromosome into segments of length x, x + D) in the segment with probability D/L. Thus, the number of genes that fall in this interval has a binomial distribution with mean ND/L. In our case, D/L is smaller than 0.1. This distribution is approximately Poisson with mean ND/L. Here we use this approximation in our analysis. The Poisson approximation implies that the number of the genes that falls in a randomly chosen interval of length D has a Poisson distribution with mean m = ND/L, and so the probability that at least one gene falls in this interval is 1 - em-.Since we assume the (start) position of a gene is uniformly distributed in a segment of length L, it falls in an interval  pn-1, n = 0, 1, 2, …. Hence, the p-value of a cluster with n genes is the probability that a cluster has more than n genes, which is pn.Suppose page 10, ). This iD = 25 K, 50, 75 K, 100 K. Actually, the distance between two successive testis-specific genes in the most of observed clusters is much smaller than D in each case. Hence, the real p-value for each cluster could be much smaller than one reported in the result section.In our analysis, To remove the effect of non-uniform distribution of genes on analysis, we adopt an idea from order statistics. Here, we focus on the position rank of a gene rather than its specific position by ordering all the genes according to their positions on a chromosome. We also treat the set of the testis-specific genes and the set of all the other genes as two types of identical objects. In this way, a specific gene distribution on a chromosome is modeled as a 0–1 string in which 0 represents a test-specific gene and 1 a non-testis-specific gene.M genes in a chromosome and T of them are testis-specific genes, where T ≤ M. Then, there are  possible gene arrangements on the chromosome and the number of arrangements with r adjacent testis-specific gene clusters is . Thus, the probability that a random arrangement has r adjacent testis-specific gene clusters isAssume there are Using above formula, we can obtain that the mean number of adjacent testis-specific gene clusters in a random arrangement is. Take chromosome 4 for example. We have M = 2060, T = 220, we obtain that the mean number is 196.6 and the standard deviation is 4.4. However, we only observed 182 adjacent gene clusters (including singletons) on the chromosome.and the standard deviation is BL and QL carried out the software development, sequence analysis, and the design of the study. LXZ conceived of the study, performed the statistical analysis, and participated in its coordination. All authors read and approved the final manuscript.


The development and validation study of the Clinical Chronic Obstructive Disease (COPD) Questionnaire (CCQ) has recently been published in this journal. The CCQ is the first questionnaire that incorporates both clinician and patient guideline goals in the clinical control evaluation of patients with COPD in general clinical practice. The aim of this study is the validation of the CCQ questionnaire in Italian, in specific pulmonary disease clinical practice.1, FVC, BMI and functional dyspnoea). Test-retest reliability was tested by re-administering the CCQ after 2 weeks. Responsiveness was tested by re-administering the CCQ after three weeks of hospital pulmonary rehabilitation. Distance walked and Borg breathlessness rating were measured at the end of the six-minute walking test (6 MWT), before and after rehabilitation.Validity was tested on a population of healthy subjects and patients with COPD, using the Italian validated version of the Short Form Health Survey (SF-36) and guideline recommended routine measurement in COPD patients . Cronbach's alpha was high (0.89). The CCQ scores in patients were significantly worse than in healthy subjects. The CCQ total score in patients with COPD was significantly worse in those with BMI < = 21. Significant correlations were found between the CCQ total score and domains of the SF-36 (rho = -0.43 to rho = -0.72). The correlation between the CCQ and FEV1 % predicted was rho = -0.57. The correlation between the CCQ and MRC was rho = 0.63. Test-retest reliability was determined in 112 subjects over a period of two weeks (Intra Class Coefficient = 0.99). Forty-six patients with COPD showed significant improvement in CCQ scores, distance-walked and Borg breathlessness rating after 3 weeks of pulmonary rehabilitation, indicating CCQ responsiveness.The CCQ is self-administered and has been specially developed to measure clinical control in patients with COPD. Data support its validity, reliability and responsiveness in Italian and in specific pulmonary disease clinical practice. The American Thoracic Society (ATS) and European Respiratory Society (ERS) have jointly proposed standards  for the 1) and forced vital capacity (FVC), body mass index (BMI) and functional dyspnoea .The Clinical COPD questionnaire (CCQ)  is the fHealthy subjects were selected in social meeting places. Subjects were asked, individually, to answer a simple questionnaire after the study had been explained to them. Only subjects over 40 years of age were interviewed. We excluded subjects with any disease symptoms, or any limitation in daily activities for any reason, or who mentioned suffering from disabling chronic diseases . All subjects gave their informed written consent for baseline spirometry and questionnaires administration, as approved by the local Medical Ethics Committee. We enrolled 55 subjects, 52 non-smokers and 3 ex-smokers. Subject data are shown in Table 1/FVC <= 0.70) were classified as mild (FEV1 post-bronchodilator (pb) >= 80% predicted), moderate (FEV1 pb >= 50% predicted), severe (FEV1 pb >= 30% predicted) and very severe (FEV1 pb < 30% predicted). We excluded COPD patients with: a) significant improvement of FEV1 pb (>= 15 % and/or 200 ml) compared with baseline, b) disease exacerbation in the previous four weeks, c) asthma, chronic heart failure, obstructive sleep apnoea syndrome, cancer or other disabling diseases except COPD. We enrolled 120 patients . In 1999, the local health service authority approved the standard evaluation procedures used in our outpatient clinic for patients with COPD. The patients' data are shown in Table Patients with COPD were consecutively enrolled in the outpatient section of our Division during medical consultation. According to the guidelines ,2, COPD 1 pb 46 % predicted (range 18–68). In 1999, the local health service authority approved our continuous pulmonary rehab program for patients with COPD.Forty-six patients with COPD (exclusion criteria as mentioned above) were enrolled in a continuous pulmonary rehabilitation program, 31 males, 30 ex-smokers, 6 smokers, 13 in long-term oxygen therapy (LTOT), 2 in home-assisted mechanical ventilation during the night (HMV), median age 72 (range 41–83), median FEVThe CCQ was administered to all subjects. They were instructed to recall their experiences during the previous week. The CCQ is self-administered and contains only 10 items, subdivided into three domains: symptom (item 1–2–5–6), functional state (item 7–8–9–10) and mental state item 3–4). Subjects responded to each question using a 7 point scale from 0 = asymptomatic or no-limitation, to 6 = extremely symptomatic or totally limited. The overall clinical COPD control score and the score of the three domains was calculated by adding all the scores together and dividing the sum by the number of questions. The Italian translation of the copyrighted questionnaire and permission for use was obtained from T. van der Molen [–4. SubjeLung function (FEV1 and FVC) was measured according to ERS guidelines  using a 2), for all subjects.The copyrighted Italian validated version  of the 31 60 % predicted (range 19–117). We tested the CCQ responsiveness in patients with COPD undergoing continuous pulmonary rehabilitation. Patients were treated in four successive groups in our hospital following a standard three-week protocol. According to guidelines [The CCQ was re-administered after 2 weeks (where there was no variation of the previous therapy or introduction of new therapy) in 112 subjects , 75 males, median age 71 years range 41–84), median FEV4, medianWe applied the same analysis undertaken in the original English validation study , taking The distributions for all domains and the overall scores were skewed. In the population study, 12 subjects (7%) scored optimally (= 0) in the total score, whereas 87 subjects (50%) scored optimally in the mental state domain. In the COPD group (120 subjects), 3% of the patients scored optimally in the total score, whereas 35% scored optimally in the mental state.Cronbach's alpha was 0.89 for the total score. Internal consistencies of symptom, functional state and mental state were 0.71, 0.88 and 0.80, respectively.1 pb >= 80% predicted). At the same time, this small group did not differ significantly  from the group (34 patients) with moderate COPD (FEV1 pb < 80% and >= 50% predicted). For this reason, Table Healthy subjects had significantly lower (better) CCQ score only in the symptom domain  compared with the small number (6 subjects) of patients with mild COPD , symptom domain (p = 0.000), mental state domain (p = 0.005), except functional state domain. Patients with mild-moderate COPD had better CCQ values compared with patients with severe COPD, with respect to total score (p = 0.041), functional state (p = 0.017), mental state (p = 0.037), except symptom domain. Patients with severe COPD had lower CCQ scores than patients with very severe COPD, with respect to total score (p = 0.007), functional state domain (p = 0.003), symptom domain (p = 0.032) except mental state domain.The healthy subjects group had a significantly (p = 0.003) lower (better) MRC score than patients with mild-moderate COPD. Patients with severe COPD had a significantly (p = 0.003) higher (worse) MRC score than patients with mild-moderate COPD. Patients with very severe COPD had a significantly (p = 0.000) worse MRC score compared with those with severe COPD Table .1 pb % predicted, age and MRC score. CCQ scores were higher in both low and high BMI groups, with respect to the acceptable BMI range group. CCQ scores did not indicate any significant difference between acceptable-range and high-range groups, except in the CCQ mental state domain (p = 0.02). On the other hand, there was a statistically significant difference between low-range BMI and acceptable-range BMI groups for CCQ total (p = 0.01), CCQ symptom (p = 0.01), CCQ mental state (p = 0.04) except CCQ functional state.We did not find Table  any signThe CCQ score showed significant correlations with all SF-36 components except the pain component, functional state , symptom (rho = 0.52) and mental state (rho = 0.44).No significant correlation was found between BMI and all the CCQ scores.The intra-class correlation coefficient was 0.99 for the overall CCQ score.1 pb %. After two successive weeks of individualized home rehabilitation there was a worsening of CCQ scores compared with the scores when hospital discharge took place. Nevertheless, CCQ scores were still significantly better than in baseline condition  for total (p = 0.01), functional state (p = 0.01), symptom (p = 0.02) and mental state (p = 0.03).In table 1 was used to measure divergent validity with the same a priori assumption behind the original English version (range from -0.20 to -0.4). The correlation was stronger than expected also in the Italian version, concerning the whole population study and the COPD population alone.The validated Italian version of SF-36 was used as an instrument to measure the convergent validity of the clinical COPD questionnaire. Moderate to high correlations were found in the present study supporting the convergent validity in the Italian version, reflecting the original English development and validation study [1, also MRC functional dyspnoea has proved to be useful in predicting outcomes in patients with COPD, thus MRC functional dyspnoea measurement is recommended in the routine handling and evaluation of these patients [In addition to the FEV1 falls below 50% predicted, i.e., as a result of the transition from mild-moderate to severe airways obstruction in patients with COPD.We found no significant difference, as far as the CCQ functional state is concerned, between healthy subjects and mild-moderate patients with COPD. This reflects COPD guidelines , which sBMI calculation has also proved to be useful in the routine handling of patients with COPD . In the We have been able, by means of the CCQ scores, to detect significant changes in response to the inpatient portion of a comprehensive and continuous standard pulmonary rehabilitation program for patients with COPD. Disease control improvement is also documented with independent outcome measurements of variables at the end of 6 MWT. It is a well-known fact that improvements in clinical disease control and health status occur with pulmonary rehabilitation, despite a minimal effect on pulmonary function measurement, i.e., FEV1 % predicted ,11. The The comparison of our data with the results presented in the original CCQ article  show simIn our study, a separate comparison between mild and moderate COPD was impossible, since patients with mild COPD seldom refer to our specialized outpatient clinic. Furthermore, in our clinical setting, we have been unable to find any subject presenting symptoms of COPD in the absence of airways obstruction (subjects who risk developing COPD). We believe such patients are more typical in a general practice setting, as is the case in the original CCQ development and validation study .The clinical COPD questionnaire is the first to have been specifically developed and validated to measure clinical control in patients with COPD in general practice . The valSD designed the study, analyzed the results, performed the statistical analysis and drafted the manuscript. SP participated to the study design and organization, collected and elaborated the SF-36 data and helped to draft the manuscript. CB-VF-CR-RR-FR participated in the organization of the study, in the individual subjects clinical selection and in the results discussion. All Authors read and approved the final manuscript.


Numerous features distinguish prokaryotes from eukaryotes, chief among which are the distinctive internal membrane systems of eukaryotic cells. These membrane systems form elaborate compartments and vesicular trafficking pathways, and sequester the chromatin within the nuclear envelope. The nuclear pore complex is the portal that specifically mediates macromolecular trafficking across the nuclear envelope. Although it is generally understood that these internal membrane systems evolved from specialized invaginations of the prokaryotic plasma membrane, it is not clear how the nuclear pore complex could have evolved from organisms with no analogous transport system. Here we use computational and biochemical methods to perform a structural analysis of the seven proteins comprising the yNup84/vNup107–160 subcomplex, a core building block of the nuclear pore complex. Our analysis indicates that all seven proteins contain either a β-propeller fold, an α-solenoid fold, or a distinctive arrangement of both, revealing close similarities between the structures comprising the yNup84/vNup107–160 subcomplex and those comprising the major types of vesicle coating complexes that maintain vesicular trafficking pathways. These similarities suggest a common evolutionary origin for nuclear pore complexes and coated vesicles in an early membrane-curving module that led to the formation of the internal membrane systems in modern eukaryotes. Structural similarities between the proteins of a nuclear pore subcomplex and proteins comprising vesicle coating complexes indicate a common origin for nuclear pore complexes and coated vesicles The ability to sharply curve membranes was a defining event in the evolution of early eukaryotes, allowing the formation of endomembrane systems . In mode(Saccharomyces) , which form pores between the inner and outer NE membranes by stabilizing a sharply curved section of connecting pore membrane. NPCs are approximately 50-MDa octagonally symmetric cylinders that function as the only known mediators of nucleocytoplasmic exchange; while permitting the free flow of small molecules, they restrict macromolecular trafficking to selected cargoes that are recognized by cognate transport factors. NPCs are found in all eukaryotic cells and are composed of a broadly conserved set of proteins, termed nups (reviewed in romyces)  and vertromyces) , there iThe yNup84/vNup107–160 subcomplex has a molecular weight of approximately 600 kDa and has been shown in yeast to be flexible , presentWe support our fold assignments using four considerations . First, Finally, we provide direct biochemical evidence in support of our fold assignments, using proteolytic mapping of domain boundaries and loop locations in the seven nups see . Tagged Arabidopsis) homologs. All seven nups from both human and Arabidopsis yielded identical domain fold assignments to their yeast counterparts  and plant  is as components either of coated vesicles or of the yNup84/vNup107–160 subcomplex. Thus, the clathrin heavy chain, a major component of clathrin-coated vesicles, appears remarkably similar in domain architecture  to both In clathrin-coated vesicles, clathrin is attached via its amino-terminal domain to an adaptin complex. There are four types of adaptin complexes, all made of two large subunits that wrap around two small subunits. The bulk of each large subunit is made of an α-solenoid trunk  Collins. SimilarOur analyses showed that the yNup84/vNup107–160 subcomplex and all three major classes of vesicle coating complexes can be linked together through their common architecture. As summarized in The common architecture of the yNup84/vNup107–160 subcomplex and all three major classes of vesicle-coating complexes suggests that all of these complexes have common function in curving membranes. There is, in fact, circumstantial evidence for a role of the yNup84/vNup107–160 subcomplex in the establishment and maintenance of pore membrane curvature. Members of this complex, when disrupted in yeast, cause the uniformly distributed NPCs to cluster into patches in the plane of the NE , suggestAs shown here, protein structure modeling is particularly useful in uncovering potential evolutionary and functional relationships that are refractory to classical approaches based on comparison of protein sequences alone. Our results show that clathrin/adaptin complexes, COPI complexes, COPII complexes, and the yNup84/vNup107–160 subcomplex all share a common molecular architecture. This commonality could have arisen by either convergent or divergent evolutionary pathways.In a convergent pathway, β-propeller and α-solenoid folds could have been independently utilized by both NPCs and vesicle-coating complexes at different stages of eukaryotic evolution. This possibility is supported by the high abundance of both fold types in eukaryotic genomes   and the In a divergent pathway, NPCs and vesicle-coating complexes share these folds because both complex types could have originated from a common ancestor. In this scenario, a single “protocoatomer” would have been the progenitor for numerous vesicle coating complexes, as well as the yNup84/vNup107–160 subcomplex. Several lines of evidence support this latter hypothesis. First, the most confident models of the yNup84/vNup107–160 subcomplex proteins are based on structures of coated vesicle proteins . Second,The lack of detectable sequence similarity between the proteins in the yNup84/vNup107–160 subcomplex and the coated vesicles is not surprising. Sequence comparisons of α-solenoid- and β-propeller-containing proteins suggest that these folds arose just before or around the time of the origin of eukaryotes, then rapidly duplicated and diversified . Both foThe current protocoatomer hypothesis posits that a simple coating module containing minimal copies of the two conserved folds evolved in protoeukaryotes as a mechanism to bend membranes into sharply curved sheets and invaginated tubules . The abiIn conclusion, we suggest that the progenitor of the NPC arose from a membrane-coating module that wrapped extensions of an early ER around the cell's chromatin. In this primitive NE, the coating modules would have originally formed the sharply curved membrane, creating large and freely permeable pores . These pOnly two domains in the seven nups have their folds assigned by sequence comparison to proteins of known structure . TherefoFor each of the seven yeast nups and representative homologs, potentially related known structures were detected by the mGenThreader  and FUGUThe matches obtained in the previous step provided an operational definition of a domain. They were either accepted or refined by manual and automated alignment. Manual realignment relied on sequence conservation and secondary structure predictions by PROF  and PSI-For each alignment, an all-atom model was built by comparative modeling based on satisfaction of spatial restraints as implemented in MODELLER .The fold assignment, alignment, and model building were repeated by varying the domain boundaries, target sequences for modeling, template structures, and their alignments. The aim was to improve model assessment by statistical potentials of ProsaII  and DFIRTo search for proteins that resemble the domain architecture of clathrin, we queried MODBASE , our rel2, 1 mM DTT, 1:500 protease inhibitor cocktail ) were added to cells and homogenized at 4 °C with a Polytron . The cell lysate was clarified by centrifugation . The magnetic beads were added to the extract to a ratio of about 8 × 109 beads per g of cells. After incubation for 1 h at 4 °C, the beads were magnetically recovered. The beads were washed, resuspended in 50 μl of reaction buffer (according to the manufacturer's specifications), and Asp-N  or Lys-C  proteinase was added to give a weight ratio of 1:200 of proteinase to the tagged nup. After incubation at different time points at 37 °C, bead aliquots were removed and washed, and tagged fragments were eluted with 0.5 M NH4OH containing 0.5 mM EDTA. The eluant was vacuum-dried, resuspended in SDS-PAGE sample buffer, and separated on a 4%–12% bis-Tris gel . Proteins were then either transferred electrophoretically to nitrocellulose or PVDF and probed with HRP-rabbit IgG , or analyzed by amino-terminal Edman sequencing  were conjugated to rabbit IgG  according to the manufacturer's instructions. Yeast cells carrying PrA-tagged versions of nups were grown and harvested as described previously . Cell pequencing .Figure S1The graphs plot the assessment score of the model (Melo Z-score)  versus t(87 KB DOC).Click here for additional data file.Figure S2http://salilab.org/damien/NPC/).The secondary structure predicted from sequence by PROF  and PSI-(47 KB DOC).Click here for additional data file.Protocol S1(42 KB DOC).Click here for additional data file.Table S1(491 KB DOC).Click here for additional data file.Table S2(101 KB DOC).Click here for additional data file.Table S3(115 KB DOC).Click here for additional data file.Table S4(132 KB DOC).Click here for additional data file.Table S5(124 KB DOC).Click here for additional data file.Table S6(93 KB DOC).Click here for additional data file.Table S7(144 KB DOC).Click here for additional data file.http://www.pir.uniprot.org) for proteins discussed in this paper are as follows. Yeast: ySeh1 (P53011), ySec13 (Q04491), yNup84 (P52891), yNup85 (P46673), yNup120 (P35729), yNup133 (P36161), and yNup145C (P49687). Human: vSec13 l (Q96EE3), vSec13R (P55735), vNup107 (P57740), vNup75 (Q9BW27), vNup160 (Q12769), vNup133 (Q8WUM0), and vNup96 (P52948).Uniprot  accessio


How does the brain convert sounds and phonemes into comprehensible speech? In the present magnetoencephalographic study we examined the hypothesis that the coherence of electromagnetic oscillatory activity within and across brain areas indicates neurophysiological processes linked to speech comprehension.Amplitude-modulated  auditory verbal and nonverbal stimuli served to drive steady-state oscillations in neural networks involved in speech comprehension. Stimuli were presented to 12 subjects in the following conditions (a) an incomprehensible string of words, (b) the same string of words after being introduced as a comprehensible sentence by proper articulation, and (c) nonverbal stimulations that included a 600-Hz tone, a scale, and a melody. Coherence, defined as correlated activation of magnetic steady state fields across brain areas and measured as simultaneous activation of current dipoles in source space (Minimum-Norm-Estimates), increased within left- temporal-posterior areas when the sound string was perceived as a comprehensible sentence. Intra-hemispheric coherence was larger within the left than the right hemisphere for the sentence (condition (b) relative to all other conditions), and tended to be larger within the right than the left hemisphere for nonverbal stimuli (condition (c), tone and melody relative to the other conditions), leading to a more pronounced hemispheric asymmetry for nonverbal than verbal material.We conclude that coherent neuronal network activity may index encoding of verbal information on the sentence level and can be used as a tool to investigate auditory speech comprehension. One key function of the cerebral cortex involves the integration of elements into a percept that separates them from the background. In this process, changes in cortical networks are formed and modified by experience through the simultaneous excitation of groups of neurons -3. TheseEvent-related brain responses, traditionally used in the study of cognitive processes, have been found to result from regional perturbations in ongoing brain activities in a self-organizing system rather than constituting a response set from an otherwise silent system. For example, Makeig and coworkers -7 showedOn a macroscopic level, oscillatory coupling between large neuronal populations can be examined by externally driving the nervous system using oscillatory stimulation and then measuring the regional coherence of the electromagnetic activity . Amplitucomprehension of speech was related to the activation of neuronal assemblies in the left hemisphere, then we should see increased coherence in this region with the recognition of a meaningful sentence as compared to an incomprehensible string of sounds. We further hypothesized that meaningful verbal stimuli should be processed differently from musical melodies. That is to say, verbal material should affect the coherence of electromagnetic signals more in the left than in the right hemisphere whereas listening to a nonverbal complement of a meaningful sentence like a melody will activate more right- than left-hemispheric neuronal networks and influence coherence patterns involving the right hemisphere. Given that language and music share components, we assumed only a relative dominance in the interconnection of networks toward left- or right-hemispheric activity.The present study investigated coherence patterns of the auditory evoked magnetic Steady-State-Field (SSF), specifically coherence among SSF-generators within and across hemispheres, as a measure of neural networks involved in speech comprehension. If, as we hypothesized, the The present study studied co-activated cortical networks involved in speech comprehension by using auditory steady-state (41.5-Hz amplitude modulated) stimuli and measuring the coherence of generator activity of the magnetic steady state response. Steady-state stimulus modulation were used for a sentence, which – following a German play-of-words – was first presented as an incomprehensible string of sounds, but became a comprehensible sentence after the sentence's meaning was explained to the subjects and was properly articulated. In addition to verbal stimuli, non-verbal stimuli were also studied which included a 600-Hz tone, a scale, and a melody-like combination of the scales' tones. The present analysis of SSF coherence in the source space (see methods) extended previous approaches , which eFigure MNE amplitudes were most pronounced in auditory areas of both hemispheres, with a varying degree of laterality. For the Laterality Index  = 3.72, p < 0.05, ε = 0.46): As illustrated in Fig. Intra-hemispheric Given that the major goal was to depict network signatures specifically involved in sentence comprehension, we applied an ANOVA to compare the coherence measure of the two verbal conditions. These were identical with respect to the physical stimulation, but differed in meaningful comprehension. For intra-hemispheric coherence a significant interaction involving CONDITION × HEMISPHERE × GRADIENT = 7.37, p < 0.05) reflected a relatively higher coherence in the left-posterior area after the string of words had been made comprehensible by explaining the sentence's meaning as opposed to the higher coherence in the right-posterior area for the incomprehensible word string. Profiles of intra- and inter-hemispheric coherence were similar, thereby resulting in similar statistical power for the CONDITION effect. This cannot be explained simply by a reduced signal-to-noise ratio in the verbal conditions, because normalized values show the same effect. We rather assume that increased laterality varies with decreased inter-hemispheric communication (coherence).Inter-hemispheric coherence between dipoles located in the left  and right  auditory cortex and the remaining dipole sites are characterized Fig.  by largeConsidering coherent activity, i.e., synchronized oscillations between spatially distributed maps, as the representation of a percept, we followed Makeig et al. ,7 who diWhereas Scott et al.  reportedFinally, there is the possibility that the order of stimulus presentations may have affected the results. While counterbalancing was not possible for the specific verbal stimulus condition (see methods), we would not have expected order effects to be large since similar temporal dynamics were not observed for the nonverbal conditions. However, an effect of time cannot be ruled out as steady state responses and their generator activity were largest for a simple 600-Hz tone which was presented first.SSF were larger for the nonverbal conditions  than for the verbal material, particularly in the right hemisphere. While right-hemispheric processing of tonal perception has frequently been reported -31, the Still, greater right- over left-hemispheric generator activity asymmetry was found in the nonverbal conditions and less asymmetry found in the verbal conditions. Moreover, intra-hemispheric coherence patterns showed distinct, hemisphere-specific patterns for verbal (more pronounced left-hemispheric) and nonverbal (more pronounced right-hemispheric coherence) processing. When lateralized coherence patterns were examined by a laterality index, the clearest left-hemispheric coherence focus emerged for the comprehensible sentence and the clearest right-hemispheric coherence focus emerged for the tone. While we had expected a melody induced dominant right-hemispheric activation, a more bilateral activation was found for the melody-like tone sequence. For the scale, there was a shift towards left-hemispheric asymmetry of coherence. An explanation for this finding might be that the 'melody' was constructed to include the tones of the scale which may have resulted in a melody-like tone sequence even though it did not resemble common melodies or songs. This processing of an unfamiliar 'melody' might have activated temporal (left) and spectral (right) processing, as suggested by ,29, resuIn sum, the present study demonstrates that the analysis of the synchronization of evoked magnetic steady-state fields in the source space can map neuronal networks (co-)activated during speech comprehension. Our techniques add spatial information to evidence on left-hemispheric areas involved in language processing, and support co-activation or synchronization within complex neuronal networks as a cortical substrate of integration in perception – like speech comprehension.Data of twelve German native speaking subjects  were included in the analysis.  It was ascertained by interview that the subjects did not suffer from any language, audiological or neurological dysfunction. Right-handedness was assessed by a modified version of the Edinburgh handedness questionnaire  to be 97All stimuli were amplitude modulated at 41.5 Hz  with a modulation depth of 90%. Verbal stimuli consisted of words composing a sentence. Nonverbal stimuli consisted of tones forming a scale or a tune or a simple tone. A German play-on-words served as the template for the two verbal conditions. In the first case a sentence is spoken without spacing between words and without accents which creates an incomprehensible word string. The German sentence 'Mähn Äbte Heu? Heu mähn Äbte nie! Äbte mähn Gras' means in English 'Do abbots cut hay? Abbots never cut hay, abbots mow lawns'. If pronounced as a string 'MähnÄbteHeuHeumähnÄbtenieÄbtemähnGras' this utterance, due to a lack of non-phonetic context , sounds In each subject, the hearing threshold was assessed by presenting short 600 Hz beeps with ascending and descending intensity. For each subject and ear the mean hearing threshold was determined from the ascending and descending sequence.The three nonverbal conditions comprised of a 600 Hz sinusoidal tone, a descending major scale , and an arrangement of the same tones (C5 E5 G5 C6 A5 F5 D5 B5). All stimuli of all conditions were adjusted to the length of the sentence and lasted for 4419 ms , and each of the five conditions comprised 15 repetitions that were separated by inter-stimulus intervals of 4419 ms. This long inter-stimulus interval allowed the same signal-to-noise ratio for the baseline and the stimulus conditions which should prevent habituation effects on the SSF. Stimuli were adjusted to have the same average loudness by normalizing to root-mean square (RMS) and were presented at 50 dB above the individually assessed hearing threshold balanced for both ears. During the experiment, which lasted about 45 minutes, the subject was seated in a supine position. Subjects were asked to listen carefully to the stimuli, while fixating a point at the ceiling of the chamber in order to avoid head and eye movements. They were further informed that they would be asked questions about the stimuli during the experimental session, and that they should reply by saying 'yes' or 'no'.th presentation, whether he/she understood the meaning of the speech (None of them could).All stimuli were presented in blocks with 15 repetitions. Conditions were separated by breaks of about 5 min each. For every subject the experimental session started with the 600-Hz sinus tone , followed by the word string (condition 2). After 5 repetitions, the subject was asked whether he/she understood what he/she was hearing and could reproduce the meaning of the speech. (None of the subjects could.) Subsequently, the stimulus presentation was continued, and the subject was asked again after the 10th and the 15Then, the experimenter entered the room and pronounced the sentence properly and slowly, so that its meaning became clear. Each subject was asked to reproduce the sentence, in order to ascertain that it was properly understood. After the experimenter had left the subject chamber, the experiment continued with condition 3, which comprised the identical physical stimulation as condition 2 differing only in that the subject now listened to the string of words knowing its meaning, Again, the subjects were asked after 5 repetitions, if they could reproduce the meaning of the sentence, which now they all could. Given that once the sentence's meaning is obvious, one can easily grasp the sentence, the sequence of condition 2 and 3 could not be reversed and thus, the sequence of presentation could not be randomized across subjects.Condition 4  and 5 (melody-like tone sequence) were arranged in a similar way, in that the subject was asked after 5 repetitions each, whether or not s/he perceived the sequence of tones as a melody. Eleven of the twelve subjects indicated that the tone sequence sounded like a melody and one was not sure about it. None of them perceived the scale or the simple tone as melodic.® 2500WH, 4D Neuroimaging, San Diego, USA) installed in a magnetically shielded room . Data were recorded continuously with a sampling-rate of 1017.25 Hz and a 0.1–100 Hz band-pass filter. The electrooculogram (EOG) and the electrocardiogram (EKG) were recorded and stored together with the MEG-data for offline artifact control. Silver-silverchloride electrodes were placed on the outer canthi for the monitoring of horizontal eye movements, and above and below the right eye for vertical eye movements. EKG electrodes were placed on the right collarbone and below left costal arch.The magnetoencephalogram (MEG) was recorded with a 148-channel whole head system  and averaged across epochs separately for each condition . Epochs were visually inspected for EOG and EKG artifacts and epochs with magnetic fields greater than 5 pT were rejected. A minimum 13  epochs per subject were available for further analyses.The steady state field (SSF) in response to the 41.5-Hz amplitude modulated stimuli was extracted using a moving average procedure. A window of 5 cycles (120.5 ms) of the 41.5 Hz Steady-State signal was shifted 179 times cycle-by-cycle (24.5 ms) across averaged epochs . The resulting moving-average epoch was detrended. Figure The generators of the SSF were determined in the source space for each epoch using the minimum norm estimate, MNE -37 using2 = min). This solution is obtained by multiplying the pseudo-inverse of the leadfield matrix L with the data. Given the high number of sensors and the presence of noise, spatial regularization is performed with the factor λ. This algorithm allows sources to be omitted, if they do not contribute to the measured magnetic field. A priori information about the number or locations of cortical sources is not required. Following Hauk et al. [2, was determined as mean vector length of both tangentially orientated dipole-components across 5 cycles.The MNE is an inverse method reconstructing the primary current that underlies an extracranially recorded time-locked magnetic field. The procedure is based on the assumption that the data vector d, which contains the recorded magnetic activity at given sensor sites, can be described as the product of the leadfield matrix L, which specifies the sensor's sensitivity to the sources, the source current vector j  and a nok et al. ,36, who Co-activation of generators was evaluated by all possible pair-wise combinations of the MNE at all dipole locations, according to the algorithm :Spectral coherence is a function of frequency with values between 0 and 1 that indicate how well the input x (in the present study MNE at dipole location x) corresponds to the output y (MNE at dipole location y) as a function of frequency (in the present study 41.5 Hz). This algorithm estimates the coherence of two vectors x and y by computing the ratio of the squared cross power spectra (Pxy), divided by the product of the power spectra for each vector (Pxx.*Pyy), where Pxy(f) is the cross power spectrum estimate, Pxx(f) is the power spectrum of the time series at location x, Pyy(f) is the power spectrum estimate of time series at location y and f is the frequency index.x -dpy). As a measure of co-activation or coherence, the first order coherence between a region of interest (ROI) covering 7 locations over Heschl's gyrus and all other 77 locations was determined.The vectors of 4495 points in length were subdivided into 8 overlapping segments of 613 points (603 ms), each of which was submitted to Hanning windowing. For each vector, the power spectra were obtained as the product of the Discrete Fourier Transforms and its complex conjugate, scaled by the number of points used for x and y, and averaged across segments . For croEffects of the five conditions on the distribution of MNE amplitudes and on the coherence measure were evaluated by means of repeated measurement analyses of variance (ANOVA) with the factors CONDITION, HEMISPHERE , and GRADIENT . For inspection of the hemispheric asymmetry of MNE, the ANOVA was performed on the Laterality Index . For the evaluation of intra-hemispheric coherence, the first order coherence between the respective left- or right-hemispheric ROI and the other 34 locations of the respective hemisphere entered the ANOVA (comparing conditions), for the evaluation of inter-hemispheric coherence, coherence between the left-hemispheric ROI and all other 34 locations of the right hemisphere and between the right-hemispheric ROI and all other 34 locations of the left hemisphere was submitted to the ANOVA comparing conditions. A separate ANOVA of the two verbal conditions with the factors CONDITION, HEMISPHERE and GRADIENT probed the hypothesis of a change in coherence-topography induced by sentence comprehension. Where appropriate, significance levels are reported with Greenhouse-Geisser correction adjusted degrees of freedom. Interactions were verified by planned posthoc comparisons (two-tailed paired t-tests), and displayed in t-maps without additional alpha correction.ANOVA: Analysis of varianceEEG: ElectroencephalogramMEG: MagnetoencephalogramMNE: Minimum Norm EstimateRMS: Root-mean squareSSF: Steady-State- (magnetic) FieldSSR: Steady-State-ResponseMH developed the experimental design, carried out the experimental study and developed and accomplished the data analyses, BR supervised the study and composed the paper, AK advised and assisted the SS-design and SSF analysis, CW supervised the MEG measurements and advised the coherence analyses, TE provided the experimental idea, advised the experimental design, the MEG methods and analyses.


Certain conditions characterised by blood vessel occlusion or vascular spasm have been found to cluster together in epidemiological studies. However the biological causes for these associations remain controversial. This study used a classical twin design to examine whether these conditions are linked through shared environmental exposures or by a common underlying genetic propensity to vasospasm.We investigated the association between hypertension, migraine, Raynaud's phenomenon and coronary artery disease in twins from a national register. Phenotype status was determined using a questionnaire and the genetic and environmental association between phenotypes was estimated through variance components analysis.Responses were obtained from 2,204 individuals comprising 525 monozygotic and 577 dizygotic pairs. There was a significant genetic contribution to all four traits with heritabilities ranging from 0.34 to 0.64. Multivariate model-fitting demonstrated that a single common genetic factor underlies the four conditions.We have confirmed an association between hypertension, migraine, Raynaud's phenomenon and coronary artery disease, and shown that a single genetic factor underlies them. The demonstration of a shared genetic factor explains the association between them and adds weight to the theory of an inherited predisposition to vasospasm. A number of conditions characterised by blood vessel occlusion and/or vascular spasm have been found to be associated in both clinical and epidemiological studies. These include hypertension (HPT), Raynaud's phenomenon (RP), migraine (MIG) and coronary artery disease (CAD) -5. WhethSubjects for this study were twins enlisted with the St Thomas' UK Adult Twin Registry . These mClassification of the traits HPT, RP, MIG and CAD was based on standard, validated criteria where possible. HPT was classified by asking about a doctor's diagnosis of "high blood pressure" when not pregnant. Questions to determine presence of migraine were based on the UCSD Migraine Questionnaire   corrected to take into account the paired nature of the data. The odds ratios were adjusted for possible confounding by age, smoking and body mass index (BMI). Evidence for a genetic contribution to each trait was examined by estimating casewise concordance . This meWhere data suggested a genetic influence, a quantitative measure of genetic contribution was estimated using structural equation modelling (Mx software ). This sModeling was then extended to consider the genetic and environmental associations between all four variables simultaneously . Three m1) the Cholesky decomposition  the independent pathway model  the common pathway model  with that of the fullest model, the Cholesky. The AIC represents the balance between model fit and the number of parameters (parsimony), with lower values of AIC indicating the most suitable model. Parameter estimates from the most appropriate model were used to calculate the genetic and environmental correlation between variables.Complete data for analysis were available from 1,102 pairs of female twins, comprising 525 MZ and 577 DZ pairs. The mean (± SD) age of the twins was 48.5 (± 8.2) years. The prevalence of each trait is shown in Table Four of the six combinations of traits showed significant phenotypic association after adjusting for age, smoking and BMI  declare that they have no competing interests.FMKW analysed the data and drafted the manuscript. LFC designed the questionnaires, analysed the data and assisted with the manuscript. TDS collected the twins on the Twin Register and assisted with the manuscript. AJM conceived of the study, collected the twins on the Twin Register, analysed the data and co-drafted the manuscript.The pre-publication history for this paper can be accessed here: