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 (age 3.8 ± 0.5 months, weight 2.3 ± 0.1 kg) (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 (Stephanie®, Radiometer Corp., Copenhagen, Denmark). 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 (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) (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 (Druck Ltd. Transducer, Leicestershire, UK) and, like Paw, was calibrated with a water manometer. Arterial blood pressure and heart rate were measured using the same type of transducer (Druck Ltd. Transducer, Leicestershire, UK) 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 (Recorder 330P, Hellige AG, Freiburg, Germany).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 (Digitimer 130® and Spike Trigger NL 200, Digitimer Research Instrumentation Inc., Welwyn Garden City, Hertforshire, UK) 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 (Tektronix Inc., Portland, Oregon, USA).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 (Windaq Pro+®, Dataq Instruments Inc., Akron, OH, USA). 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 (37.5°C, 30 mL/kg) 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 (PIP/PEEP 3.2/0.5 kPa, RR 60/min, Ti 0.33, FiO2 1.0). 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 (Dataq Instruments, Inc., Akron, OH, USA) was used to review the recorded signals. Analysis was done by means of Windaq Playback® and Excel® (Office 2000, Microsoft Corporation, USA). For statistical evaluation, Sigmastat® (SPSS Inc, IL, USA) 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 (i.e., there is no genetic variance). 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 [.The better way to address these contentious issues is to rely on quantitative genetic analyses devised to partition phenotypic variation into genetic and environmental components . Environvidovitz . The othThe third process involved in the control of phenotypic variability is plasticity, which can be defined as the ability of an individual to express one phenotype under one set of environmental circumstances and another phenotype under another set. The expressed phenotypes can be discontinuous thus eliciting discrete morphs (i.e., polyphenism), or there can be a continuous range of potential phenotypes (i.e., reaction norm). The reaction norm is thus a property of the genome: genetic canalization and phenotypic plasticity are not mutually exclusive and can combine to form canalized reaction norms ,17. PlasDrosophila is regarded as an excellent model system to investigate those problems [Drosophila is well understood [Drosophila melanogaster is quite resistant to developmental temperature. Conversely, in D. subobscura there are changes in wing proportions along a latitudinal size cline mediated by cell area [Within the framework of recently developed geometrically based methods for the statistical analysis of size and shape variation (collectively referred to as geometric morphometrics ,22), theproblems ,24. Wingderstood , and thederstood ). When fderstood . This plderstood . Howeverderstood  concludeell area ,31. Thesell area ,33.D. subobscura. We consider the consequences of inbreeding and temperature on the two components of developmental homeostasis , and the relationship between them. The remainder of the paper is planned as follows. First, we provide a short account of the inversion polymorphism in D. subobscura and the experimental settings. Then, based on the well balanced data set rendered by the experimental design we used the standard least-squares (ANOVA) method to decompose sources of variation for wing size and shape into causal components at the core of further analyses. Furthermore, because the underlying assumption to use FA as a measure of DS is that left – right-side variation has not heritable basis, the genetic and environmental components of bilateral asymmetry were partitioned. As a result, our approach is unusual in studies of DS in providing estimates of the two components of special environmental effects (co-) variance under different genetic backgrounds and general environmental settings. We also present some evidence for the presence of genetic variation in directional asymmetry (DA) but not in FA. Next, we test whether or not the vectors describing variation of landmark position for fly vein traits are concordant, and finally we discuss the main findings in relation with the evolution of buffering mechanisms and the putative adaptive value of natural wing shape variation in D subobscura.Here we report on the effects of clinal genetic variation (inversion polymorphism) on wing form (size and shape) and bilateral asymmetry using isochromosomal lines of D. subobscura is a particularly inversion-rich species, with up to 38 natural chromosomal arrangements already reported for the largest chromosome O  were used in the present experiments.nogaster ) for whiaryotype ). In nat derived ) and are derived . On the  pattern . Therefo) with 18 crosses in total, or outbred  with 36 (18 + 18) crosses in total. The six lines with a given gene arrangement were crossed to produce the three different outbred homokaryotypes (i.e., ). The three kinds of heterokaryotypic flies were similarly obtained but using lines with different gene arrangements (i.e., ). Since all isochromosomal lines were homogeneous for the same genetic background , maternal effects were not considered to be critically important. Anyhow, experimental flies were randomly derived form reciprocal crosses for all outbred combinations. Two developmental temperatures were used in the experiment: optimal (18°C) and warm (23°C).The experimental flies were obtained from 54 crosses, which will be referred to as inbred  differences of centroid size did not significantly departure from normality in any case . In addition, none of the regressions of centroid size FA on average wing size was statistically significant (ranging from β = -0.045 (95% C.I.: -0.091, 0.001) for inbred females at 18°C to β = 0.030 (-0.005, 0.064) for inbred females at 23°C), thus suggesting independence between size and size FA.Signed left-right  ranged from 0.235  to 0.602 . 3+4 flies were always the biggest within inbred lines  = 9.78, P = 0.002; 23°C F(1,195) = 9.19, P = 0.003; males: 18°C F(1,195) = 1.84, P = 0.176; 23°C F(1,195) = 4.23, P = 0.041), but interactions of dominance effects were observed in all samples with discernible heterosis in Ost/O3+4 lines when compared to their homokaryotypic counterparts.No significant size differences were generally detected among karyotypes for average CS, although Ones Fig. . On the st isochromosomal lines [Drosophila wing vein has dorsal and ventral components that come together after the apposition of the dorsal and ventral surfaces, but each vein protrudes only in one wing surface ("corrugation") [χ2 = 0.16, 1 df, P = 0.691); in males the corresponding figures were 383 (59.1%) and 401 (61.9%), respectively (χ2 = 1.05, 1 df, P = 0.306). Potential biasing effects when measuring wings; namely, dorsal or ventral Bitmap images or possible differences between left and right wings when Bitmap images are captured from the top or bottom of the microscope slide, were checked from a subset of 75 females and 75 males. An additional set of two images for each wing were taken in the same session from the top and bottom of the slide and digitized once. The centroid size differences between the averages of both measurements was apparently random with respect to digitizing procedure and always lower than 0.07%, whereas left wings were 0.26% bigger than the right ones in females and 0.34% in males. We are, therefore, quite confident that the fairly subtle DA for wing CS is not an experimental artifact but a real phenomenon.In concert with some independent preliminary results using a set of Oal lines  a quite gation") . When wi2).In addition to DA, there was subtle but significant FA in all crosses , but results were qualitatively identical without a log-transformation) as the dependent variable, with karyotype, temperature and inbreeding as fixed effects, and crosses nested within karyotypes, clearly indicated inbreeding depression together with temperature by inbreeding interaction , but no karyotype by temperature interaction was detected. These results confirm that wing size is not a purely additive trait in D. subobscura, in agreement with the previous observation that heterokaryotypes were bigger than homokaryotypes in outbred crosses .F(1,856) = 9.46, P = 0.002).Both inbreeding and  temperature effects were also apparent in females when overall size FA index FA1) was used as the dependent variable in three-way factorial ANOVAs, with no differences among karyotypes. On the other hand, no statistically significant effects were detected for males, basically because inbred crosses performed approximately equal at both temperatures  values in Tables (CS) estimates in outbred crosses within rearing temperature.It is worth mentioning here that in outbred crosses overall size FA was about the same for homokaryotypes and heterokaryotypes: the only significant effect was again an increase in FA at the sub-optimal temperature  and development at the optimal temperature of 18°C. However, no positive association was found between DS and chromosomal heterozygosity in outbred crosses.Two-way MANOVA analyses to quantify inter- and intra-individual variation in wing shape are shown in Tables st isochromosomal lines, where DA for some landmarks (e.g. those defining the position of the anterior crossvein) but not for overall wing shape was detected [Similarly to what had been found for CS, differences between left and right wings were also highly significant ("sides" effect), thus indicating that DA was present for overall wing shape. This finding is contrary to our previous claim from a subset of Odetected . After pP = K + C + E as a simple multivariate extension of the two-level nested ANOVAs, where P is the phenotypic covariance matrix and K, C, and E are, respectively, the covariance matrices for karyotypes, crosses within karyotypes, and the residuals.As has been forcefully stressed  shape isK matrices showed the clearest trend to quickly decrease after the first PC. Permutation tests indicated that matrix correlations (MCs) between K and C matrices were generally higher at 18°C  than at 23°C , but none of the MCs was statistically significant. On the other hand, VCV matrices were correlated across rearing temperatures  covariance matrix onto the total variance of the phenotypic covariance matrix was lower at 18°C in both sexes . A caveat: these ratios cannot be interpreted as estimates of shape heritability [Fig. rocedure ). A closrocedure . Thus, ttability .Drosophila development [sonic hedgehog) have recently been discovered. In Drosophila, Ligoxygakis et al. [Drosophila obviously require further study.MANOVA results in Tables elopment  because elopment . Actuallelopment ) distings et al.  were the) differences, but notice that for the multivariate case it is not equivalent to calculate the absolute  differences of all Procrustes coordinates.Following  a multivMANOVA analyses of these "unsigned" shape asymmetries in outbred crosses did not detect any genetic variation for shape FA at 18°C or 23°C Tables , 10. Howvs. outbred homokaryotypic flies) as the categorical predictors and CS (as loge (pixels)) as the covariate. Temperature effects were only significant in males, but inbreeding and temperature × inbreeding interaction effects were highly significant in both sexes (results not shown), which suggests a strong effect of the categorical predictors on the nonallometric component of shape. Size effects were also found to be significant  = 2.496, P < 0.001; males: Wilks' λ = 0.915, F(22,405) = 1.715, P = 0.024), but the allometric effect on shape remained relatively consistent at both temperatures in females (size × temperature interaction: Wilks' λ = 0.930, F(22,405) = 1.395, P = 0.111) but not in males (Wilks' λ = 0.853, F(22,405) = 3.165, P <0.001). The association between size and temperature , which indicates that the effects of temperature and size on wing shape could be effectively separated.To investigate allometric and nonallometric temperature effects on overall wing shape we performed a multivariate analyses of covariance (MANCOVA) of the Procrustes coordinates (after averaging both sides and the two replicated measurements per side) considering temperature and inbreeding  on wing shape FA were tested from the ratio between the traces of the corresponding "individual × side" VCV matrices. Notice that the traces of these interaction matrices are equal to the respective mean squares of the Procrustes ANOVA as implemented by Klingenberg and McIntyre [ .Inbreeding effects (isogenic McIntyre , and arex FA4 in ) for eacPrincipal component analyses were only implemented for the outbred crosses since they are more representative of the natural situation. The percentages of total shape variation, together with the features of variation associated with the dominant PCs, are graphically plotted in Figs. P < 0.01; Table P = 0.0355 ; males MC = 0.494, P = 0.1066 ; statistical significance vanishes after the Bonferroni procedure).Permutation tests indicated that VCV matrices were mostly correlated for FA and measurement error effects within samples , which reflects the similarity due to landmarks 3, 6 and 7. However, the first three PCs for interindividual variation were generally distinct to those of FA: the only clear correspondences were between the PC1s for females at 18°C (α = 21.5°), and the PC2 of interindividual variation with the PC1 of FA for males at 18°C (α = 11.8°).  Overall, these results seem to suggest that canalization and DS do not generally share the same underlying regulatory mechanisms (but see below).The angles between the PC1s for FA and measurement error were very much alike  and measurement error . For the interindividual variation the correlations between PC1s were significant only in males ; however, the PC1 vector describing the joint interindividual variation of landmark position in females at 18°C matched the PC2 of the interindividual covariance matrix at 23°C .Between rearing temperatures the congruence of PC1 eigenvectors was also very high for FA  it is important here to asses the patterns of joint displacements of landmarks for each of the causal components of wing shape variation Figs. , 9, 10. D. subobscura had been previously identified in an outbred stock [st dose is also clear here when considering the six karyotypes  do not affect the longitudinal veins; however, some mutants that affect the longitudinal veins also influence the crossveins . Intra-nts (see ). HoweveThe matrix permutation tests Table  indicateα = 85.8°, males α = 77.3°; 23°C: females α = 75.6°, males α = 78.5°). The only matches were between PC2 of karyotypes and PC1 of FA for females at 18°C (α = 13.0°) and males at 23°C (α = 31.2°). The PC1s of crosses and FA were also poorly correlated; the only exception being females a 23°C (α = 43.3°). These results clearly support the hypothesis that genetic canalization and DS are not functionally the same mechanism.In addition, the PC1s of karyotypes and FA were nearly at right angles .  Together with the overall comparisons of the covariance matrices  and FA were relatively small and highly significant  environmental canalization  would be FA because the two sides share the same genome  and nearly the same environment, so differences between sides are likely to be small. Under stabilizing selection this lower limit is obviously associated with higher fitness. However, this "canalization limit" would hardly ever be observed because of unavoidable additional sources of environmental variance . A similar logic than the one used in this work has been applied to distinguish between intrinsic and extrinsic stochastic variation in gene expression: intrinsic noise can be separated by contrasting the levels of gene expression in a construct with two identically regulated but fluorescently distinguishable ironment ,53.D. subobscura. The main findings can be summarized as follows: (i) for the analysis of overall size, DS was positively correlated with levels of heterozygosity (i.e., inbred vs. outbred homokaryotypes) and development at the optimal temperature; however, no positive association was found between DS and chromosomal heterozygosity in outbred crosses; (ii) there was detectable genetic variation  for the directional component of morphological asymmetry (i.e., DA) but not for FA, which likely reflects variation due to stochasticity in development; (iii) for analyses of shape, the patterns of covariation for FA and measurement error were highly concordant in all samples, which also provides strong reasons to conclude that FA is generated by random perturbations of developmental processes ; (iv) the inter- and intraindividual variation patterns were generally poorly correlated, which supports the hypothesis that canalization and DS are distinct mechanisms; however, (v) the patterns of variation due to the (micro-) environmental component of canalization  were quite similar to those observed for FA; (vi) the lack of a significant within-group correlation between the VCV matrices associated with the interindividual genetic components of canalization and FA, as well as the low similarity between the corresponding vectors describing variation of landmark position, strongly suggest that genetic and environmental canalization are not similar mechanisms.This study applied the methods of geometric morphometrics in the context of quantitative genetics of wing form variation using isochromosomal lines of D. subobscura developed in North America after ~20 years since colonization, but males were clearly lagging behind females [In addition, (vii) a discrepancy between sexes was observed in some situations; e.g. overall size FA increased with inbreeding and  temperature effects mainly in females, and the allometric effect on wing shape at both experimental temperatures was similar in females but not in males. It is also interesting to note here that wing size  and (vi) above) give some credence to that conjecture. Genetic canalization on wing shape does not seem to arise as a by-product of environmental canalization and, therefore, canalization is not a single mechanism to buffer any source of variation as has been suggested [However, it is still an open question whether or not natural wing shape changes in pe (e.g. ), contrape e.g. ,59). Man. ManDrosme e.g. ,61), and, andDrosbobscura  can be lbobscura ,33, and bobscura  speculatuggested .P(permutation) = 0.0001) but not for males (MC = 0.064, P(permutation) = 0.485). The angles between the PC1s also reflect this discrepancy between samples . When considered together, these results clearly suggest that FA and genetic variation for DA may or may not be functionally linked.According to Graham et al.  the clasD. subobscura isochromosomal lines for the O chromosome in an otherwise homogeneous genetic background were derived from an outbred stock collected at Puerto Montt  in November 1999 as previously indicated [ch-cu marker strain, which is homozygous for the morphological recessive markers on the O chromosome cherry eyes (ch) and curled wings (cu) and fixed for the gene arrangement O3+4. A single  / Ocu + ch  + male from the offspring was backcrossed to ch-cu females, and its arrangement on the wild-type chromosome was identified after four generations of backcrosses. Followed by at least another backcross to ch-cu females, a single male from each line carrying the wild chromosome was crossed to two virgin females from the Va/Ba balanced marker stock. This strain was derived from the ch-cu stock and carries the dominant lethal genes Varicose (Va) and Bare (Ba) on the O chromosome. The isochromosomal lines were established from the final crosses ♀♀ OVa ch + cu /  × ♂♂ OVa ch + cu / . All lines used here had a quasi-normal viability according to the recorded proportions of wild-type flies raised in the final crosses to obtain the isochromosomal lines [A large number of ndicated . Brieflyal lines . The linAs previously indicated the experimental flies were obtained from 54 crosses. Reciprocal crosses were made for all outbred combinations by mating one-week virgin females and males. After three days the males were discarded and equal numbers of females from each reciprocal cross were placed together in a plastic chamber with a spoon containing non-nutritive agar with a generous smear of live yeast for egg collections. To standardize the experimental conditions, eggs from the inbred (isogenic) crosses were also obtained in a similar way; namely, after mating the flies in bottles and transferring the females to plastic chambers. Eggs were placed in six 2 × 8 cm vials with 6 mL of food ; three vials were kept at 18°C  and the other three at 23°C . Within each experimental temperature the vials were randomly placed on the same incubator shelf. As a result, the total experiment consisted of 324 vials , and all eggs were sampled on the same day. Emerging flies (not less than 2 or 3 days old) were stored in Eppendorf tubes with a 3:1 mixture of alcohol and glycerol at 4°C before wing measurements.2 anesthesia on flies not less than 6 h after eclosion.All fly handling was done at room temperature using COx and y coordinates of 13 morphological landmarks  connected to a PC computer with MGI VideoWave software and mounted on a compound microscope (Zeiss Axioskop, Jena, Germany), using a 2.5 × objective. Calibration of the optical system was checked at each session. The images were stored on a Dell Workstation PWS350. To quantify and minimize measurement error all wings were digitized two times at different sessions as follows: images of both the left and right wings were captured during a given session and after an entire round on all individuals the same process was repeated again. A similar procedure was also used to record the Geometric morphometrics precisely separates morphological variation (i.e., variation in form) into size and shape components ,22. Sizex coordinate of every landmark for configurations from the right side. The reflection, scaling, and superposition steps were performed for all wings within each cross and temperature simultaneously, which allows contrasting wing shapes between different lines or crosses. The final iteration to minimize the sum of the squared distances between the landmarks of all wings in the sample was done without additional scaling and, consequently, we performed a partial Procrustes fit according to Dryden and Mardia [ρ) [The shape of an original configuration of landmarks is the geometrical information that is invariant to uniform scaling (variation in size), translation (differences in position), and rotation (differences in orientation). In contrast to size, shape is an inherently multidimensional space and we used Procrustes superimposition to characterize shape variation. This method allows comparing configurations of landmarks by optimally superimposing (according to a least-squares criterion) homologous landmarks in two or more specimens to achieve an overall best fit .Because d Mardia . Given trdia [ρ)  would hap – 4, where p is the number of landmarks [The landmark coordinates after Procrustes superimposition are amenable to standard multivariate analyses. However, it is important to remember that the removal of size, position (in two dimensions), and orientation reduces the dimensional space to 2andmarks . Thus, fandmarks ,67: (i) Quantitative genetic studies of directional and fluctuating asymmetry obviously require measures from individuals that can be grouped into families or independent lines. Our final data set was a fully balanced design, comprising 54 crosses × 3 vials per cross × 2 females per vial × 2 males per vial × 2 sides per fly × 2 measurements per wing × 2 temperatures = 5,184 wing landmark configurations in total. Within each sex and temperature, least-squares (ANOVA) estimates of variance components (i.e. CS) can be easily obtained from the linear model:Yijkl = μ + κi + lj(i) + νk(ji) + εl(kji),μ is the overall grand mean, κi is the effect of the ith karyotype, lj(i) is the random effect of the jth cross within karyotype i, νk(ji) is the random effect of the kth vial within cross j and karyotype i, and εl(kji) is the residual error associated with the trait  of the lth individual within vial k, cross j, and karyotype i. Since there was no genetic variation within crosses, the residual error provides an estimate of the total special environmental effects variance (i.e. ). Variation among the three replicated vials was generally negligible (results not shown) and, therefore, we have conveniently reduced the previous model to a two-level nested ANOVA after grouping flies across vials.where )] 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 [Neisseria meningitidis, that causes high death rates in African communities. The agent is highly contagious, and person-to-person aerial transmission occurs through respiratory and throat secretions [MCM is an infection of the meninges, caused by the bacteria cretions . Interaccretions .Among favorable conditions for the resurgence and then dispersion of the disease, climatic conditions may be important as environmental forces inducing periodic fluctuations of disease incidence. Recent findings concerning the population dynamics of some infectious diseases have clearly identified the importance of climate as a major driver ,4. MCM oThe winter climate causes damage to the mucous membranes of the oral cavity through dry air and strong dust winds, and creates propitious conditions for the transmission of the bacteria responsible for MCM; low absolute humidity and dust may enhance meningococcal invasion by damaging the mucosal barrier directly or by inhibiting mucosal immune defenses. In contrast, higher humidity during both the spring and summer seasons strongly reduces disease risk by decreasing the transmission capacity of the bacteria ,8, and MMost studies thus far have focused on very small spatial scales, and the need remains to discriminate between local properties and potential large-scale effects in disease patterns, to go beyond data heterogeneities and idiosyncratic details in order to identify important disease patterns influenced by large-scale forces such as climate variability (see Methods). The aim of the present work is thus to document the climatic context of MCM disease outbreaks and population dynamics in a highly affected Sahelian country, that is, Mali, and to show, if it exists, the presence of a correlation between climate and seasonal resurgence of disease. The idea behind the present study was to explain MCM disease dynamics in Mali in a statistically robust way, which will permit us to propose some tools for predicting disease risks for the benefit of public health.f such that:Recent findings concerning the population dynamics of some infectious diseases have clearly identified the importance of climate as a major driver ,4. With The aggregation of local data in the scaling-up approach is a simple way to go beyond data heterogeneities and idiosyncratic details so that only the important disease effects of large-scale forces remain. That is the rationale for our study: to show that large-scale phenomena such as the seasonal Harmattan winds over the whole of Mali can contribute to the periodic resurgence of MCM and its variation in time on a national scale, even if this aggregate analysis for the entire country is not able to capture variations at smaller space scales.The diagnosis of MCM is based on both physical examination and on evaluation of the cerebrospinal fluid (CSF) from a lumbar puncture. As the disease is characterized by a sudden onset of intense headache, fever, nausea, vomiting, photophobia, and stiff neck, in association with neurological symptoms (lethargy, delirium, coma, and convulsions), the WHO  recommenThis case definition allows the detection of cases of meningococcal septicemia.a.Suspected case of acute meningitis Sudden onset of fever  with stiff neck. In patients under 1 y of age, a suspected case of meningitis occurs when fever is accompanied by a bulging fontanelle.b.Probable case of bacterial meningitis Suspected case of acute meningitis as defined above with turbid CSF.b.Probable case of MCM Suspected case of either acute or bacterial meningitis as defined above with Gram stain showing Gram-negative diplococcus or ongoing epidemic or petechial or purpural rash.c.Confirmed case Suspected or probable case as defined above with either positive CSF antigen detection for N. meningitides or positive culture of CSF or blood with identification of N. meningitides.aOften the only diagnosis that can be made in dispensaries .bDiagnosed in health centers where lumbar punctures and CSF examination are feasible (intermediate level).cDiagnosed in well-equipped hospitals .M(w) of the number of cases asIn this study, an epidemic is defined in terms of population dynamics following Anderson and May  and Grenyand yσ represent, respectively, the mean and the standard deviation of the 54 weekly values of cases yX(w) for the year y, and N = 9 represents the number of years for the 1994–2002 period.where X̄yX(w), we computed yU(w) asWe determined the week of the onset of the epidemic for each year by characterizing a breaking slope in the annual cycle of the number of cases. The dates of the breaking slope have been determined objectively by using the Mann–Whitney–Pettitt test , which iw ≤ 54 and yU(1) = yV(1), where yV(w) is defined bywhere 2 ≤ y is the point for which the valuey|U(w)| is maximized. The probability yP(w) of a given week being a change point is defined byThen the the most significant change point of the year T = 54 (the length of the time series in weeks).where The National Centers for Environmental Prediction (NCEP) and the National Center for Atmospheric Research (NCAR) have completed a reanalysis project with a current version of the Medium Range Forecast model . This dath PC time series αψ can thus be obtained by a linear combination of the initial variables throughThe principal component analysis (PCA)  is a muluα is the αth eigenvector of the correlation matrix C associated with the eigenvalue λα.where αth spatial pattern is the correlation map between the initial wind fields and the αth PC time series. The examination of the different spatial patterns and PC time series (not shown here but previously discussed in [The ussed in ) revealsWo”) as determined by a breaking slope in the annual cycle of the number of cases. The dates of breaking slope have been determined objectively by using the Mann–Whitney–Pettitt test [The weekly records of the WHO's Department of Communicable Disease Surveillance and Response of cases and deaths due to MCM for the 1994–2002 period allowed us to describe the seasonal evolution of MCM epidemics in Mali. Two important parameters were used: the date of the onset of the epidemic and the date of the seasonal maximum number of cases. We determined for each year the week of the onset of the epidemic ; this low number is the main limitation of the present analysis.Using this atmospheric index, we defined the date of “winter maximum” as the first minimum of the wind index for each year of the period 1994–2002. The winter maximum thus corresponds to the week where wind speed is the strongest. The mean date of winter maximum is around the sixth week, with a standard deviation of 2 wk, corresponding to the week when the Intertropical Front is located at its southern latitude. The Harmattan wind index shows a temporal pattern very similar to the that of the number of cases of MCM, with a clear breaking slope at the sixth week, on 15 February, corresponding to the onset of the epidemic and to the winter maximum, and with a recession of the disease at the 16th week concomitant with the onset of the wet season in the south part of Mali in early May. It is interesting to note that although they are determined from two different datasets, the mean weeks of winter maximum and of the onset of the epidemic are identical, 7–15 February. This coherence is reinforced by a very strong correlation between the two dates (0.92) for the years 1994 to 2002. In this paper and a previous publication of ours [17], 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. (In Y chromosome, we only identified 12 testis-specific genes and 7 other genes.) 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 (25 K, 50 K, 75 K and 100 K), 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 (COPD, asthma, arthritis, angina or heart insufficiency). 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 (77 ex-smokers, 19 smokers). 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 (p = 0,05) 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 (rho = 0,64), 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.