Melanerpes lewis) has experienced population declines in both Canada and the United States and in 2010 was assigned a national listing of threatened in Canada. We conducted a two-year study (2004–2005) of this species at its northern range limit, the South Okanagan Valley in British Columbia, Canada. Our main objective was to determine whether the habitat features that influenced nest-site selection also predicted nest success, or whether other factors  were more important. Nest tree decay class, density of suitable cavities and total basal area of large trees were the best predictors of nest-site selection, but these factors were unrelated to nesting success. Estimates of demographic parameters (mean ± SE) included daily nest survival rate , nest success (0.52±0.08), clutch size (5.00±0.14 eggs), female fledglings per successful nest (1.31±0.11), and annual productivity . Although higher nest survival was associated with both early and late initiated clutches, early-initiated clutches allowed birds to gain the highest annual productivity as early clutches were larger. Nests in deep cavities with small entrances experienced lower predation risk especially during the peak period of nest predation. We concluded that nest-site selection can be predicted by a number of easily measured habitat variables, whereas nest success depended on complicated ecological interactions among nest predators, breeding behaviors, and cavity features. Thus, habitat-based conservation strategies should also consider ecological factors that may not be well predicted by habitat.Lewis's Woodpecker ( Understanding habitat selection and its influence on reproductive success is crucial to species recovery planning. Habitat protection has proven one of the most effective measures to rescue and recover threatened and endangered species Lewis's Woodpecker populations have been declining over the last 40 years because of the loss of suitable habitat due to fire suppression, forest cutting, agricultural development, water management practices and urbanization Previous studies on Lewis's Woodpeckers have examined either nest-site selection or effects of habitat on reproductive success. Nest-site selection appears to be influenced by features both of the landscape [e.g. vegetation type and fire history; Our objectives are two-fold. First, we examine which habitat features determine nest-site selection by this species near its northern range limit. Previous studies of habitat and nest-site selection by Lewis's Woodpecker are centered on the western U.S. [e.g. Colaptes auratus), and tends to return to the same nest-sites in subsequent years Pinus ponderosa) forest, riparian woodland dominated by black cottonwood  or aspen (Populus tremuloides) and partially logged or burned pine forest Lewis's Woodpecker, a short distance migratory bird, tends to form long-term pair bonds Pinus ponderosa), but also included semi-arid grassland, shrub steppe, black cottonwood open forest, mixed coniferous and broadleaved open woodland, and Douglas-fir (Pseudotsuga menziesii) forest , included six areas ranging in elevation from 320–1100 m, each with one to nine pairs of Lewis's Woodpeckers/site/year: Chopaka Grassland Protected Area (∼500 ha), Spotted Lake Grassland Protected Area (∼430 ha), Vaseux Lake Ecological Reserve (∼2400 ha), Kilpoola Grassland Protected Area (∼800 ha); Sun Oka Provincial Park (40 ha) and surrounding areas (∼1500 ha). All sites were dominated by ponderosa pine  forest . BecauseFrom early May to late August, we conducted four rounds of intensive surveys to search for nests and adults, with consistent effort among all the study areas and years. Once a nest was found, we visited it at 3–4 day intervals until either fledging or failure. During each nest visit, we first observed adult breeding behavior for one hour . When feasible, we accessed the nest using a climbing rope and documented the contents of the nest with a Sony DSC-U20 digital camera inserted into the nest hole. We were able to estimate nest age for most nests through comparisons of digital photographs taken over time, plus behavioral observations of parent birds. We defined nest age as time (in days) from the laying of the first egg. We considered a nest successful if at least one young fledged. When it was not feasible to access the nest cavity, we used our observation data to assign nesting status .We anticipated that nest-site selection and nest survival would be based on habitat features linked to cavity availability and predation risk. Because weak cavity excavators like Lewis's Woodpeckers mainly use pre-existing cavities Mustela spp.), and snakes In addition to these habitat features, we also considered cavity features and time of season. The degree to which cavity-nesting birds evade nest predators depends on cavity features such as cavity shape or height above ground We conducted vegetation surveys at 50 nesting sites (involved 57 nesting attempts over both years) and 28 random sites after the breeding season . We sampIn each site, we measured seven habitat variables. In the 50 m-radius plots we recorded elevation (m above sea level) at the central tree, live tree canopy cover , density of suitable cavities , and basal area of large trees per ha . We visually estimated shrub cover (%) and grass cover (%) in the 10 m and 5 m-radius plots, respectively. We averaged grass and shrub cover in the subplots to provide an estimate of the average cover for each in the entire 50 m plot. Finally, we recorded decay class of the central tree as 1 = live tree, 2 = dead-top tree, 3 = dead tree with all branches present, 4 = rotten dead tree with >50% branches missing For all nest trees, we measured the following features of the cavity : height (m) of the nest cavity above the ground measured with a clinometer and the ratio of vertical hole depth to entrance size . We used a plumb line to measure the vertical cavity depth from the bottom of the cavity to the lower edge of the entrance. We transformed this ratio into a unitless value >0.01 by multiplying by 100 mm.We estimated daily nest survival rate, nest success, clutch size, female fledglings per successful nest, and annual productivity for the entire study period using all nests. We estimated daily nest survival rate using a null model (i.e. a model fitted with just a constant) of Mayfield logistic regression We used mixed logistic regression models to test how the habitat around a tree, and created by the tree, affects the probability of that tree being selected for nesting by Lewis's Woodpecker Rather than test all possible subsets of the seven habitat variables, we used an information-theoretic approach whereby we constructed candidate models which seemed most probable based on previous work, most parsimonious given the system, and most biologically meaningful. Previous work on woodpeckers has shown that the most important determinant of population density is often the number of pre-existing cavities or trees suitable for cavity excavation 0β represents the intercept; jβ represents the jth parameter; xji denotes the ith observation of the jth covariate. 0β and jβ are parameters to be estimated. We used mixed Mayfield logistic regression to investigate impacts of habitat features, cavity features, and temporal factors on daily nest survival rate We outlined two competing general hypotheses in the form of global models: (1) the same habitat variables found to be important in nest-site selection are also important for daily nest survival rate, (2) cavity features and temporal factors determine daily nest survival rate. We first separately assessed the importance of habitat variables and temporal/cavity models, and then compared simultaneously the two global models and all associated candidate models. For the habitat variables, we used an identical global model and associated candidate models to those used for nest-site selection. The other global model included cavity features  and temporal factors . Here we again used the literature to identify two core variables - nest age and clutch initiation date - that are consistently shown to be correlated with daily nest survival in other species For analysis of daily nest survival rate, we assumed that if a nest failed between two nest visits, the failure occurred in the midpoint of the interval. Previous work Because nest survival over each day is assumed to be independent between days in Mayfield logistic regression, we can estimate nest success as a consecutive accumulative product of daily nest survival rate from nest age one through to fledgling. In our study, daily nest survival rate (Si) was modeled as a function of habitat and cavity features (X), nest age (NA) and clutch initiation date (C). Thus, nest success can be estimated as:n = 18 nests followed from first egg to fledge date). This nesting cycle was composed of a 6–9 day laying period, a 14–15 day incubation period, and a 30–34 day nestling period.We used a nest age of 54 days to estimate nest success  has been exhaustively studied, and does not differ significantly from 1∶1 at the hatching stage, although it starts to be very slightly male-biased (54∶46) by fledging M. formicivorus may be related to cooperative breeding Annual productivity depends on (a) the probability of nest success (estimated by iP (C) represents the probability to produce i fledglings as a function of clutch initiation date (C). Multiplying Here, Here, X denotes a series of covariates. We assigned 10 May (the earliest clutch initiation date in our study) as day one.All data are presented as means ± SE unless otherwise specified and the significance level for all tests was set at α<0.05. Statistical analysis was performed using Stata version 10.1 (StataCorp 2009).In total, we found 57 nests, comprising 36 nests in 2005 and 21 in 2004 . OverallSites with nests differed significantly from sites centered on randomly-selected trees in most habitat variables . In the Successful and failed nests did not differ in most habitat attributes, save grass cover . Furtherth worst model out of all 41 models examined, more than 22 AICc units worse than the best cavity/temporal model, and substantially poorer fit than even the null model (by 5.5 AICc units). The best model for nest survival included quadratic effects of clutch initiation date and nest age, and linear effects of cavity depth∶ entrance ratio , and this resulted in fewer female fledglings per successful nest . No habitat variables except elevation influenced clutch size or fledglings per successful nest: Lewis's Woodpeckers nesting at higher elevations started laying later , and thus had smaller clutches . Annual productivity was greatest for nests initiated mid-season in deep cavities with small entrances , and nuthatches [Sitta sp., In the South Okanagan Valley, we found that Lewis's Woodpeckers preferentially chose decayed nest trees surrounded by trees with suitable cavities. As in As found in our study, Lewis's Woodpeckers in Colorado, Wyoming, and California avoid dense forests for breeding, favoring open habitat with low ground cover Tamiasciurus hudsonicus) may switch to searching for cavity nests when they are most plentiful (mid-season) after which other foods may become available. However it is also possible that the factors responsible for the variation in initiation date also determined temporal variation in nest survival. A number of factors may be responsible for the variation in clutch initiation date, including characteristics of the individual birds , annual variation, and habitat characteristics. For example, we found that birds at higher elevation initiated clutches later. It is not possible to evaluate bird quality or annual variability without a much longer-term study involving marked individuals. Only one other study on Lewis's woodpecker has considered within-season temporal patterns, and found that daily nest survival decreased monotonically with initiation date and increased with daily maximum temperatureIn this study, nest success of Lewis's Woodpeckers was primarily determined by clutch initiation date, nest age and cavity shape. Clutches initiated both early and late in the breeding season had higher daily survival than those initiated in mid-June. Nest survival was lowest 18 days after initiation (mid to late incubation). It is possible that both temporal patterns are related to changes in predation risk as generalist nest predators such as red squirrels (Glaucomys sabrinus) The most successful nests occurred in cavities with small entrances and deep holes. Nest predation was the major cause of nest failure in Lewis's Woodpeckers, and deep cavities with small entrances may be the best shape to exclude access by predators such as weasels, red squirrels and northern flying squirrels , Northern Flickers, and/or American Kestrels . In several cases, we observed aggressive interactions with and nest usurpation by starlings; Lewis's Woodpeckers tended to initiate breeding later than starlings, possibly to reduce this conflict A second explanation is that Lewis's Woodpeckers do prefer nest sites that maximize their reproductive success, but these preferences are rarely realized because of interspecific competition for nest sites A third explanation is that nest success is limited, in part or completely, by ecological factors that are uncorrelated to habitat. Indeed, we found nest success to be primarily determined by clutch initiation date and cavity shape (cavity shape was uncorrelated with the habitat around the nest tree). As discussed earlier, both clutch initiation date and cavity shape may affect the likelihood of predation, and clutch initiation date may also be a stand-in for bird quality. Other studies have also found time-related factors to be more important than habitat features in predicting nest success In addition to the above explanations that address ultimate causes for why habitat features predicting nest-site selection were not correlated with nest success, a fourth suite of explanations suggests that methodological limitations prevented a real correlation from being detected. For example, our ability to detect nest-site selection effects on nest survival depends on some birds utilizing suboptimal nest sites. If nest-site selection is sufficiently strong, if optimal sites are not limited, or if population size has declined, there may be no nests in the poorest quality habitat to monitor for variation in survival, restricting the range of habitat variables we can evaluate Poecile rufescens) nest survival rates between years despite stable squirrel densities (20% vs 80%) Our study adds to the growing list of studies that report mismatches between nest-site selection and reproductive success Such mismatches between nest-site selection and nest success have important management implications. Until the reasons for these apparent paradoxes are resolved, a precautionary approach is to manage not only for preferred habitat but also to consider community wide processes such as potential interactions with predators or nest-site competitors. We recommend such an approach for Lewis's Woodpecker.Table S1Contribution of each study area and year to estimates of nest-site selection and nest success in Lewis's Woodpecker, in terms of number of random sites at which vegetation was recorded, number of nests found, fate of each nest, and Mayfield estimate of nest success (mean ± S.E.). All the study areas were mosaics of forest and grassland. The dominant forest vegetation at each site is abbreviated as OPP = Open ponderosa pine; DF = Douglas fir; RPP = riparian ponderosa pine; BCW = black cottonwood.(DOCX)Click here for additional data file.Table S2Correlation coefficient matrix for the seven explanatory variables . None of the correlation coefficients exceeded 0.8, the level that may result in a collinearity issue in a regression model. The associated P-value is shown in parentheses. Habitat variables include EL = elevation; TC = live tree canopy cover (%); SC = shrub cover (%); GC = grass cover (%); BA = total basal area of large trees (m2 per ha); ND = nest tree decay class; DS = density of suitable cavities (per ha).(DOCX)Click here for additional data file.Table S3Full ranking of habitat-based models that predict Lewis's Woodpecker's nest-site selection (n = 73). This is an expanded version of c = Akaike's Information Criterion for small samples; ΔAICc = adjusted AICc relative to the top model; Wi = AICc weight; EL = elevation; TC = live tree canopy cover; SC = shrub cover; GC = grass cover; BA = total basal area of large trees; ND = nest tree decay class; DS = density of suitable cavities.(DOCX)Click here for additional data file.Table S4Full ranking of cavity and time-based models that predict Lewis's Woodpecker's daily nest survival. This is an expanded version of (DOCX)Click here for additional data file.Table S5Full model ranking for habitat-based models that predict Lewis's Woodpecker daily nest survival analysis. Global and candidate models are identical to those used to predict nest site selection ((DOCX)Click here for additional data file.Table S6Full model ranking for Lewis's Woodpecker daily nest survival analysis when the global and candidate models listed in  Models in bold are the cavity/temporal variable models listed in (DOCX)Click here for additional data file.


Crocodylus suchus, which occur in isolated groups, usually less than five individuals, along the mountains of Mauritania (West Africa). We used both invasive and non-invasive sampling methods and a combination of mitochondrial DNA (12 S and ND4) and microsatellite markers (32 loci and a subset of 12 loci). Our results showed high genetic differentiation and geographic structure in Mauritanian populations of C. suchus. We identified a metapopulation system acting within four river sub-basins (high gene flow and absence of genetic structure) and considerable genetic differentiation between sub-basins (FST range: 0.12–0.24) with rare dispersal events. Effective population sizes tend to be low within sub-basins while genetic diversity is maintained. Our study suggests that hydrographic networks  allow C. suchus to disperse and maintain metapopulation dynamics within sub-basins, which attenuate the loss of genetic diversity and the risk of extinction. We highlight the need of hydrographic conservation to protect vulnerable crocodiles isolated in small water bodies. We propose C. suchus as an umbrella species in Mauritania based on ecological affinities shared with other water-dependent species in desert environments.The maintenance of both spatial and genetic connectivity is paramount to the long-term persistence of small, isolated populations living in environments with extreme climates. We aim to identify the distribution of genetic diversity and assess population sub-structuring and dispersal across dwarfed desert populations of  Crocodylus suchusHabitat connectivity is important for populations living in climate-extreme environments, which are naturally patchy and have temporally variable resources gueltas), located upstream of mountain valleys, and floodplains , located at the foothills of the mountains gueltas (0.001–1.0 ha) restricts their carrying capacity, thus they usually harbour less than five adult individuals gueltas and tâmoûrtsC. suchus may constitute a metapopulation. Analyses of molecular markers are needed to corroborate field observations and interpretations about the potential connectivity, possibly enabling gene flow between crocodile populations and confirming the existence of a dynamical metapopulation. Assessing genetic structure and gene flow among populations, as well as identifying landscape features that have an impact on gene flow are key to identify dispersal corridors as targets for conservation efforts. The genetic patterns of these relict crocodile populations may help understand possible metapopulations dynamics in desert landscapes and contribute to the prioritization of conservation actions on a species that could serve as a model for connectivity conservation, i.e. strategies for protecting and restoring dispersal corridors In Mauritania, crocodile populations are known from 81 small rocky pools  detect the occurrence of contemporary gene flow among gueltas/tâmoûrts and between sub-basins; and 3) quantify genetic diversity within each sub-basin. Taking into account isolated populations and small group size within each locality; we predict genetic structuring, reduced gene flow, low levels of genetic diversity and signs of inbreeding. However, the dispersal ability of crocodiles during the rainy season might counteract the negative effects of their isolation.Using a combination of mitochondrial DNA (mtDNA) and microsatellite markers, we aim to: 1) assess population sub-structuring across isolated populations of Fieldwork was developed with permission from the Ministére Délégué auprès du Premier Ministre Chargé de l'Environnement, Nouakchott (Permit: 460/MDE/PNBA). This permit was valid for the entire country and no specific permissions were required for any specific locality. Analyses were done at a CITES registered laboratory: 13PT0065/S. Field collection and handling practices were approved by the Committee of Animal Experimentation of the University of Porto  under the Directive 2010/63/EU of the European Parliament. No animal was sacrificed and there were no animal husbandry, experimentation and care/welfare concerns.gueltas; 2) the Gorgol el Abiod that results from water draining from the southern Tagant and northern Assaba; 3) the Gorgol el Akhdar and 4) Garfa sub-basins that are formed through water run-offs from the western Assaba; 5) the Karakoro that flows between the Assaba and the Afollé, although it is mostly fed by run-offs from the later mountain, and is characterized by low water availability; and 6) the Kolimbiné that flows east-ward of the Afollé. With the exception of the Gabbou, the other five sub-basins flow to the Senegal River and are composed of gueltas in mountain slopes and seasonal tâmoûrts at foothills of the mountains The study area encompasses three southern Mauritanian mountains: Tagant, Assaba and Afollé . These mC. suchus from fieldwork expeditions from 2007 to 2011 ca. 25% and 5% of the total population of crocodiles in the Tagant/Assaba and Affolé mountains respectively. We captured live crocodiles by hand or using hand nets, and then immediately released them at the point of capture after clipping a 5 mm piece of tail tissue for genetic analysis. Team members obtained the tissue samples following ethical guidelines for use of live reptiles. We used samples obtained from carcasses of C. suchus from Guinea-Bissau and C. niloticus from Malawi as outgroups in the analyses . We followed Frantz et al., We amplified two mtDNA fragments of 421 bp and 878 bp from 12 S and ND4, respectively, for fresh and near fresh samples in 10 µl reaction volumes, containing 5 µl of master mix , 0.5 µM of each primer and approximately 1 µl of genomic DNA , thus we excluded one from the dataset. We performed four PCR replicates for each sample (discarding samples that failed to have a good amplification rate) after a maximum likelihood evaluation of error rates  using Pedant 1.0 Na), and observed (Ho) and expected (He) heterozygosity values) within each geographic groups (sub-basins) identified in population structure analyses (see below) and using the 12 loci dataset to maximize the number of samples. We calculated allelic richness per sub-basin using the rarefaction algorithm implemented in ADZE 1.0 xyr implemented in IDENTIX 1.1 rxy for each river sub-basin and compared to their expected distribution, generated by 1,000 permutations of genotypes among individuals, under the null hypothesis of no relatedness.Two out of 35 samples that we successfully analyzed exhibited identical genotypes  ranging from 1 to 7, a burn-in period of 1×106 and a run length of 5×106 iterations. We implemented an admixture model with uncorrelated allele frequencies and without prior information on sample population membership. We used Structure Harvester 0.6.93 K by the highest value of log probability of data L(K) K) K-value. We calculated pairwise FST values P - values with the sequential Bonferroni correction.We used the Bayesian genotype clustering method implemented in STRUCTURE 2.3.4 To identify spatial genetic patterns we also performed multivariate analyses using the spatial Principal Component Analyses (sPCA) m) between sub-basins. BIMR uses assignment tests to estimate the proportion of genes derived from migrants within the last generation and assuming linkage equilibrium and allowing for deviation from Hardy-Weinberg equilibrium. We used a burn-in period of 1×105 and a run length of 5×105 iterations for each of the 10 replicate runs, and then averaged pairwise migration rates between and within sub-basins across runs.We used BIMR C. suchus haplotypes and diverged by 53 mutational steps from the second network containing all C. niloticus haplotypes, whereas a third network represented by a single haplotype from Malawi differed in 19 mutational steps from the C. niloticus main network. Mauritanian C. suchus exhibited low genetic diversity (only three haplotypes), with a common haplotype (Mauritania A) widespread across the whole study area (three mountains and main river sub-basins) and a second haplotype  that is restricted to four samples from the Karakoro-Kolimbiné sub-basin . Haplotype network reconstruction using statistical parsimony in TCS resulted in three haplotype networks not connected under the 95% connection probability criteria . One netub-basin . A thirdP<0.05 after Bonferroni correction) for the 12 loci dataset. As this deviation occurred only at Karakoro-Kolimbiné and was not observed for the 32 loci dataset, all loci were retained in the analyses. No evidence of linkage disequilibrium was apparent in any pair of loci/population after adjusting the significance level for multiple comparisons. Mean values of allelic dropout and false alleles amplification across loci  and across samples (7.7% and 0.0%) were low, supporting the robustness of the selected loci.All loci showed low to moderate polymorphism when considering the entire dataset. The number of alleles per locus ranged from three (CUD68) to seven (CpP302). One locus deviated from Hardy–Weinberg equilibrium at Karakoro-Kolimbiné sub-basin  with the exception of the Gorgol el Akhdar-Garfa sub-basin that showed the lowest level of inbreeding (xyr≈0.5) were found within three sub-basins at isolated gueltas  as the most probable number of groups within our dataset (estimated at both L(K) and Δ(K) methods). These genetic groups correspond to independent hydrographic sub-basins, Gabbou and Gorgol el Abiod in the Tagant mountain, and to combinations of neighbor sub-basins within mountains, Gorgol el Akhdar-Garfa in Assaba and Karakoro-Kolimbiné in Afollé (K = 2) clearly shows a partition between sub-basins in the Tagant and Assaba mountains  from sub-basins in the Karakoro-Kolimbiné in the Afollé mountains (K), suggesting that increasing the number of markers slightly improve the resolution of population structure (K = 3), sub-basins within Tagant and Assaba mountains are split (results not shown).STRUCTURE analyses identified four genetic demes , but no local structure  has been detected (vs. Afollé mountains). To investigate deeper genetic structuring in these two groups, we selected the second best number of clusters (K = 5) in which DAPC recovered the four clusters obtained in STRUCTURE and further subdivided the Karakoro-Kolimbiné in two clusters  to 0.24 (between Gorgol el Abiod and Karakoro-Kolimbiné) (Pairwise limbiné) . Estimatlimbiné)  and consC. suchus in Mauritania is consistent with the recent findings of Hekkala et al. C. suchus. However, relict populations or individuals of C. niloticus present in West Africa cannot be excluded since the sample sizes and geographic areas surveyed are still limited The presence of mtDNA haplotypes of FST) across relict populations in Mauritania, suggesting that geographic isolation might have started since the beginning of the arid phase. In addition to recent geographic isolation, we also note that demographic factors and drift FST values (0–15–0.24) between sub-basins of Afollé and sub-basins from both Tagant and Assaba together with the first split of these geographic groups identified with STRUCTURE, suggest that Afollé population may have experienced a longer isolation from the remaining populations. Genetic differentiation and genetic structure of these populations also reflect ecological constraints on dispersal in C. suchus. Pairwise FST values between sub-basins are much higher in comparison to those found in other studies of crocodilians C. suchus can be explained by a combination of geographic isolation, low population sizes and genetic drift. In addition, genetic demes correspond mainly to sub-basins  supporting previously hypothesized metapopulation dynamics in the region gueltas and tâmoûrts during the rainy seasons, while these water bodies act as refugia to relict populations during the dry seasons when they remain isolated by sandy areas. However, individuals from relatively close localities but different sub-basins  and genetic differentiation  considered for demographic independence K = 2; The results obtained from the combination of genetic markers unveiled the development of a metapopulation system of gueltasgueltas might act as dispersers, colonizing other water bodies by overland dispersals. Our study shows evidence of long dispersal between sub-basins for one individual (sample 2642), which leads us to question why and how do crocodiles disperse. Two main benefits of dispersal are the avoidance of kin competition (to limit inbreeding) and the access to better habitats gueltas are unsuitable and individuals would have to move long distances among gueltas, thus risking successful dispersal and eventually their lives. Permanent and temporal rivers are thought to be the main way to disperse for crocodiles, as for other semi-aquatic organisms The reduction of connectivity among fragmented populations is negatively associated with migration rates and gene flow, decreasing the colonization capacity and the rescue effect that ultimately impact both genetic diversity and structure C. suchus, highlighting that individual's dispersal abilities help maintain current populations by diminishing the negative effects associated with stochastic events and genetic drift, and thus preventing local extinctions and increasing the populations' recovery in this harsh and mostly unsuitable environment.Overall, our study confirms genetic connectivity among isolated water bodies within main sub-basins for C. suchus genetic demes , make them vulnerable to demographic and environmental events that may reduce genetic variability. However, our results do not show drastically low genetic diversity for any sub-basin. In fact, heterozygosities in C. suchus in isolated localities as complements to molecular approaches will contribute to understanding population dynamics and dispersal rates. In addition to the use of remote sensing techniques to quantify water availability patterns Future efforts should be focus on identifying landscape features shaping genetic structure and distribution of genetic diversity across crocodile populations in the region. Further fieldwork efforts are needed within these mountains along with integrative spatial/genetic analyses, such as those employed in landscape genetics approaches gueltas are presently overexploited by humans, resulting in water shortage during the dry season, faecal contamination, excessive eutrophication, and increased activities for excavating pools or pumping water C. suchus occur, as these may be considered independent conservation units. Identifying and protecting the species' refugia in temporary freshwater ecosystems can contribute to conservation plans designated to enhance the resilience of freshwater biodiversity To curb local population extinctions it is advisable to increase both the carrying capacity of populations and the facilitation of dispersal among populations. Increasing water quality is needed as many gueltasC. suchus in Mauritania constitutes a good candidate umbrella species, since it's conservation is tied to other aquatic or semi-aquatic organisms , which also live in isolated spots across the Sahara-Sahel. The use of genetic tools to study dispersal in umbrella species has been considered as a realistic approach to implement ecological networks that could assist species protection at the regional level C. suchus provides an initial framework to manage and protect threatened desert populations that depend on permanent or seasonal water bodies.Crocodiles are usually viewed as a conservation flagship species and considered a valuable indicator in ecosystem monitoring and restoration programs Table S1Crocodylus suchus samples analysed in this study. For each sample (ID), we list the country, mountain chain, basin, sub-basin, locality, GPS coordinates of collection sites and their genetic assignment at both mtDNA (haplotype) and microsatellite level (genetic deme).(PDF)Click here for additional data file.Table S2Loci used on this study with primer information, allele ranges, PCR conditions and references for description of primers used both for mtDNA and microsatellite markers.(PDF)Click here for additional data file.Table S3Pairwise relatedness (xyr) among all crocodile individuals.(PDF)Click here for additional data file.Input File S1Input file (Structure format) containing genotype information for 32 microsatellite markers.(TXT)Click here for additional data file.Input File S2Input file (Phylip format) containing sequences of the 12 s mtDNA fragment.(TXT)Click here for additional data file.Input File S3Input file (Phylip format) containing sequences of the ND4 mtDNA fragment.(TXT)Click here for additional data file.


Impaired liver regeneration is associated with a poor outcome in patients with decompensated alcoholic liver disease (ALD). We assessed whether autologous bone marrow mononuclear cell transplantation (BMMCT) improved liver function in decompensated ALD.58 patients  were randomized early after hospital admission to standard medical therapy (SMT) alone (n = 30), including steroids in patients with a Maddrey’s score ≥32, or combined with G-CSF injections and autologous BMMCT into the hepatic artery (n = 28). Bone marrow cells were harvested, isolated and reinfused the same day. The primary endpoint was a ≥3 points decrease in the MELD score at 3 months, corresponding to a clinically relevant improvement in liver function. Liver biopsy was repeated at week 4 to assess changes in Ki67+/CK7+ hepatic progenitor cells (HPC) compartment.Both study groups were comparable at baseline. After 3 months, 2 and 4 patients died in the BMMCT and SMT groups, respectively. Adverse events were equally distributed between groups. Moderate alcohol relapse occurred in 31% of patients. The MELD score improved in parallel in both groups during follow-up with 18 patients (64%) from the BMMCT group and 18 patients (53%) from the SMT group reaching the primary endpoint  in an intention to treat analysis. Comparing liver biopsy at 4 weeks to baseline, steatosis improved (p<0.001), and proliferating HPC tended to decrease in both groups .Autologous BMMCT, compared to SMT is a safe procedure but did not result in an expanded HPC compartment or improved liver function. These data suggest either insufficient regenerative stimulation after BMMCT or resistance to liver regenerative drive in patients with decompensated alcoholic cirrhosis.ISRCTN83972743.Controlled-Trials.com  Decompensated alcoholic liver disease (ALD) in non-abstinent patients, often called alcoholic hepatitis, is a common cause of acute-on-chronic liver failure Impaired liver regeneration seems to be common to situations of ongoing liver failure. Clinically, prolonged periods of severe jaundice, low coagulation factors and clinically significant portal hypertension put these patients at risk of complications. On liver biopsies of patients with ASH, hepatic progenitor cells (HPC) expansion correlates with disease severity and mortality It has been proposed that pluripotent non-haematopoietic stem cells originating from the bone marrow may participate in the repopulation of a damaged liver and may improve its function In order to test whether bone marrow cells mobilized and infused into the liver enhance hepatocyte proliferation and result in improved liver function over a 3-months period, we designed a pragmatic, prospective randomized trial in clinically decompensated ALD, comparing standard medical therapy (SMT) alone versus SMT combined with bone marrow mononuclear cell mobilization and infusion into the hepatic artery, reported as bone marrow mononuclear cell transplantation (BMMCT).From February 2008 to March 2011, all patients admitted to our hospital for decompensated ALD were considered eligible if they met the following criteria: (i) clinical decompensation manifested by ascites and/or jaundice in active drinkers ; (ii) a liver biopsy performed within 7 days of admission; (iii) age 18 to 75 yrs; (iiii) MELD score <26; and (v) written informed consent to participate. Exclusion criteria included pregnancy, hepatitis B, C or HIV, documented hepatocellular carcinoma, biliary tract obstruction, liver biopsy showing causes other than ALD for decompensation, complete portal vein thrombosis, hypersensitivity to G-CSF, severe coagulopathy (platelets <50 G/l+INR >1.5), serum creatinine >150 umol/l, any ongoing infection, recent (<10 days) gastrointestinal bleed, estimated survival <6 months and clinically overt hepatic encephalopathy. High creatinine serum level and severe coagulopathy were considered exclusion criteria due to the risk of, a) further deterioration of renal function with the use of contrast media during angiography, and b) major bleeding during bone marrow aspiration and femoral artery catheterization.The protocol for this trial and supporting CONSORT checklist are available as supporting information; see http://www.controlled-trial.com/ISRCTN83972743) was conducted in a single tertiary care centre , and designed to compare SMT alone or combined with autologous BMMCT to improve liver function in decompensated ALD  unaware of patients’ characteristics and treatment allocation. The entire biopsy specimen was examined by using 20–50 high-power fields (x 400) as previously described We used a double immunohistochemistry staining method on liver tissue specimens to determine the number of proliferating cells in the ductular reaction We used a multiplex suspension array technique based on the sandwich immunoassay method  for multiple measurements on a single plasma sample Patients allocated to the BMMCT arm received a 5-day course of lenograstim  to mobilize bone marrow cells at a dose of 10 µg/kg per day subcutaneously. On day 5 the bone marrow aspiration procedure was performed under propofol sedation. A mean volume of 103±18 ml of bone marrow was collected from the posterior iliac crest in bags containing 2500 IU heparin. Cell suspensions were immediately centrifuged (3000 rpm at 20°C for 7 minutes) over a Ficoll-Hypaque plus  gradient and mononuclear cells (MNC) recovered at the interface. Cells were washed twice with 140 mM NaCl containing 25% of hu-Albumin CSL 5%  and then suspended in 40 ml of human Albumin CSL 5% 1∶1 containing 10 U/ml of heparin, and transferred in a 400 ml infusion pouch. Analytical samples were taken to assess total nucleated cell count, haematopoietic colony forming unit (CFU) frequency, CD34+ cell frequency and mesenchymal stem cell (MSC) frequency. The CFU assay for MSC was read after 14 days of culture in methocult medium , as previously described The follow-up visits were scheduled at 1, 2 and 3 months after randomization. In addition to clinical and biological evaluations, alcohol consumption was monitored using blood ethanol levels at every follow-up visits. All adverse events were graded as mild, average or severe and classified according to Common Terminology Criteria for Adverse Events, version 4.0.A written informed consent was obtained from each patient. The study protocol was in accordance with International Committee for Harmonization/Good Clinical Practice (ICH/GCP) and approved both by our institution Ethics Committee  and by the Swiss regulatory authorities .U-test. The ANOVA repeated measure test with Bonferroni correction for multiple comparisons was used to assess MELD changes in each treatment group for the effect of time. Cytokines values and liver tissue parameters at baseline and at 3 months were compared using the Wilcoxon signed rank test. Correlations between MELD score changes and bone marrow cell subpopulations were made using the Spearman rank test. All analyses were based on the intention-to-treat principles. All tests were two-sided, and the level of significance was set at a p<0.05. The analyses were conducted using Statistical Package for the Social Sciences .To achieve a statistical power of 85% at 5% type I error, with the initial assumption that the primary endpoint will be reached in 60% of BMMCT-treated patients and 20% in patients on SMT alone, a sample size of 26 patients in each arm was calculated. Assuming a 10% of included patients lost to follow-up, a sample of 60 patients was required. The assumptions we made for calculation of the sample size were based on data from several uncontrolled studies reporting significant improvement in liver function tests following bone marrow cell therapy The algorithm of patient selection complying with CONSORT Guidelines is provided in 8/kg, including 0.24±0.11×106 CD34+ cells/kg and 3.39±5.9×105 MSC/kg. No blood products were administered prior to arteriography. The infusion of MNC didn’t result in any thrombosis during or after the procedure.Laboratory procedures were successful in all patients, and the yield of mononuclear bone marrow cells was above 60% after Ficoll-Hypaque gradient centrifugation. The BMMCT procedure could be fully administered in all but 2 patients . The mean number of MNC infused was 0.47±0.15×10The evolution of the MELD score showed similar improvement in both groups . The perAt 4 weeks, a reduction in the hepatic venous pressure gradient could be observed both in the BMMCT group  and in the SMT group .Adverse events were equally distributed between groups . There wThe evolution of steatosis, hepatocyte ballooning and lobular inflammation in paired liver biopsies from 26 patients in each group is given in In the BMMCT group, the changes in the MELD score at 3 months correlated neither with the total number of infused MNC , nor with the sub population of CD34+ cells , nor with MSC .This trial was designed to test the potential of BMMCT to stimulate regeneration in patients with clinically decompensated ALD. In order to obtain mechanistic data on BMMCT on the liver, we repeated a liver biopsy at 4 weeks. We demonstrate that autologous BMMCT combined with SMT is not superior to SMT alone using an improvement in the MELD score as an endpoint.ex vivo expansion of these cells prior to transplantation ex vivo expansion of cellular subpopulations, and selected the hepatic arterial route for stem cell reinfusion, as previously described in several studies This study was designed in accordance with the recommended approaches for trials of bone marrow stem cells in patients with chronic liver diseases We chose a 3-month follow-up with regards to the elevated morbidity and mortality reported in this patient population with decompensated ALD Following acute liver damage and liver cell loss, an increased number of proliferating hepatocytes on liver biopsy is associated with survival 9 cells as compared to_0.47×108/kg in our study) in patients abstinent from alcohol with a moderately decompensated alcoholic cirrhosis Two lines of reasoning may account for the absence of benefit in our patients. Firstly, was the stimulus sufficient to induce proliferation at the site of injury? Secondly, are there any target organ specific features that would make it unresponsive to BMMCT? Although we didn’t expand the bone marrow derived cells in culture, the mean number of infused cells was in the range of previous studies Our BMMCT protocol included a 5-day G-CSF course to mobilize bone marrow stem cell prior to bone marrow aspiration, a procedure reported to increase circulating HGF The second hypothesis relates to the target organ. Cirrhosis is a state characterized by replicative senescence of hepatocytes A third hypothesis for a lack of beneficial effects of therapy on liver function is alcohol relapse in 31% of our patients. Recurrence of alcohol misuse in this population is not rare, reported in approximately 40% of patients after a 3-month follow-up in a recent review Our study has several strengths including well characterized study groups of acutely decompensated alcoholic cirrhotics with similar characteristics at baseline, including a balanced number of patients receiving steroids. However, we didn’t consider a treatment group receiving only G-CSF mobilization therapy in our study design. In fact, we chose to compare SMT alone to a strategy that we believed could be most efficient to promote liver repopulation and regeneration, that is G-CSF combined to autologous BMMCT. In addition, we were not able to study the fate nor the level of engraftment of the infused bone marrow cells into the liver parenchyma. Thus, whether a larger number of MNC are necessary to elicit a proliferative response in this clinical situation remains speculative. Finally, the immunostaining results obtained on the repeat liver biopsy provided only indirect information regarding the effect of treatment.This trial in non-abstinent patients admitted with decompensated ALD is a truly negative study with comparable groups at baseline, without any tendency toward improvement in BMMCT-treated patients during follow-up. The parallel evolution of the MELD score, biological and liver tissue parameters including similar HPC profiles, suggest an apparent refractoriness to the proliferative stimulus. BMMCT, which included G-CSF mobilization therapy, failed to elicit HPC expansion and liver regeneration. Autologous bone marrow derived cell transplantation in patients with decompensated alcoholic cirrhosis is feasible and well tolerated, but cannot be considered as a suitable regenerative therapy. The mechanisms associated with this lack of benefit warrants further studies.Checklist S1CONSORT Checklist(DOCX)Click here for additional data file.Protocol S1Trial Protocol(DOCX)Click here for additional data file.


Neisseria meningitidis was responsible for an increase in meningococcal disease incidence during the 1990s. This increase was accompanied by antigenic shift of three outer membrane proteins, with a decrease in the population that predominated in the early 1990s as a different population emerged later in that decade. To understand factors that may have been responsible for the emergence of serogroup Y disease, we used whole genome pyrosequencing to investigate genetic differences between isolates from early and late N. meningitidis populations, obtained from meningococcal disease cases in Maryland in the 1990s. The genomes of isolates from the early and late populations were highly similar, with 1231 of 1776 shared genes exhibiting 100% amino acid identity and an average πN  =  0.0033 and average πS  =  0.0216. However, differences were found in predicted proteins that affect pilin structure and antigen profile and in predicted proteins involved in iron acquisition and uptake. The observed changes are consistent with acquisition of new alleles through horizontal gene transfer. Changes in antigen profile due to the genetic differences found in this study likely allowed the late population to emerge due to escape from population immunity. These findings may predict which antigenic factors are important in the cyclic epidemiology of meningococcal disease.In the United States, serogroup Y, ST-23 clonal complex  Neisseria meningitidis is a leading cause of bacterial meningitis world-wide N. meningitidis was responsible for an increasing proportion of all meningococcal disease during the 1990s and also for an increased incidence of disease. For example, during 1989–1991, ∼ 2% of invasive meningococcal strains in the U.S. were serogroup Y, whereas by the mid 1990s, over a third of cases were caused by this serogroup. The predominant genetic lineage of serogroup Y isolates from this time period was the ST-23 clonal complex In a previous study, we demonstrated that the emergence and maintenance of ST-23 complex serogroup Y meningococcal disease in Maryland was associated with antigenic shift in three key meningococcal outer membrane proteins (OMPs), PorA, FetA, and PorB. A change in the pulsed field gel electrophoresis (PFGE) profile was also described, indicating that the strain that predominated in the early 1990s (early strain type) was replaced by another serogroup Y ST-23 complex strain that emerged later in the same decade (late strain type) through clonal replacement N. meningitidisN. meningitidis carriage isolates In clonal replacement, one circulating strain is supplanted by another. This phenomenon has been observed in disease-causing strains of N. meningitidis isolates obtained from meningococcal disease cases in Maryland during the 1990s. We hypothesized that an accumulation of antigenic differences within an otherwise stable genome was responsible for the emergence of the late ST-23 complex strain type. The increase in serogroup Y meningococcal disease during this period presented the opportunity to investigate differences in disease-causing isolates in a relatively stable human population, thereby minimizing the confounding effects caused by population differences. The goal of this study was to identify differences that may explain clonal emergence and the cyclical nature of meningococcal disease. In addition, this study identified conserved antigens that could be investigated as potential vaccine candidates. To be responsible for the emergence of the late strain type, the differences found between the sequenced genomes of the early and late strain type must be found in the population of isolates from the time period of emergence, not just in the sequenced genomes. Therefore, we investigated target genes in isolates selected from early and late strain type populations. While some genes showed consistent allelic differences between early and late strain type population isolates, some did not. These results contribute to our understanding of which genes may be important in clonal emergence in N. meningitidis.We used whole genome sequencing to more extensively investigate the genetic differences in early and late strain type serogroup Y ST-23 complex The study isolates were obtained from the Maryland Active Bacterial Core surveillance (ABCs) site, which conducts active, population- and laboratory-based surveillance for meningococcal infection throughout Maryland and is a component of the multistate Emerging Infections Program Network To determine if gene differences between NM220 and NM233 were representative of the wider populations of disease-causing isolates, we selected 8 early strain type isolates and 8 late strain type isolates. Strains were chosen to be temporally similar between the early and late populations.DNA was prepared by phenol-chloroform extraction from overnight cultures of isolated colonies grown on chocolate agar. Pyrosequencing using a GS-20 sequencer followed by assembly using the Newbler program (Roche) were accomplished by 454 Life Sciences and resulted in draft genomes. The average depth of coverage for all contigs was 22 for the early strain type and 27 for the late strain type see . Other gPCR and Sanger sequencing were used as needed for resolution of homopolymer differences and verification of relevant insertions, deletions and point mutations, using primers designed from the whole genome sequences. Primers for amplification and sequencing of gene targets in the early and late strain type populations were obtained from the literature or were designed from whole genome sequences.e.g. protein motifs) using tRNAscan-SE Prediction and annotation of open reading frames (ORFs) was accomplished using a suite of automated tools combining Glimmer gene prediction N. meningitidis genomes Two methods were used to predict orthologous genes across the two serogroup Y genomes and all published http://www.ebi.ac.uk/Tools/msa/clustalw2/) was used for further investigation of nucleotide and amino acid sequence alignments. An unrooted phylogenetic network was created by aligning the genomes with Mugsy, using all of the positions present in all 22 available N. meningitidis genomes  to determine the phylogenetic network with the NeighborNet algorithm using SplitsTree4 ClustalW2 S (number of synonymous changes per synonymous site) and πN (number of non-synonymous changes per non-synonymous site) for all ORFs were calculated using codeml in the PAML package S and πN was calculated for the set of all genes, not just those with differences.πORFs from 20 other available sequenced genomes pilC1 (JN681261), pilC2 (JN681262), pilE/pilS (JN681263); NM233 hpuA (JN681260), pilE/pilS (JN681264).This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AGRQ00000000, AGRR00000000. The version described in this paper is the first version, AGRQ01000000, AGRR01000000. The following additional sequences for these genomes have also been deposited in GenBank: NM220 nadA, tspB, and the hemoglobin receptor gene hmbR. Some representative genes found in a majority of other genomes but missing in the serogroup Y genomes are listed in The two study serogroup Y genomes were similar in size and G+C content to previously sequenced neisserial genomes . DiffereN and πS were low over a majority of the genomes , neisserial heparin binding antigen (NhbA), and NadA have been investigated as targets for vaccines against serogroup B disease N and πS in the early strain type/late strain type comparison . Other antigens with relatively high πN and/or πS included lactoferrin binding protein B (LbpB) and PilV , PorB loops V and VII, and FetA VR The early and late strain type genomes contained 456 shared ORFs which are potential antigens based upon predicted lipoprotein attachment sites and/or predicted signal sequences. Of 73 shared lipoproteins, 27 exhibited amino acid differences . Of the N. meningitidis has type IV pili (Tfp) which are polymers whose major subunit is the protein product of the highly variable pilE gene pilS regions of the early strain type and the late strain type were nearly identical over part of the sequence. However, the early strain type contained fewer pilS cassettes  and the pilS region of the late strain type contained a putative IS1160-like transposase between the fourth and fifth pilS cassettes . The sequence of the pilE gene was similar between the early and late strain types over the C-terminal half but highly dissimilar at the N-terminus. The late strain type transposase was only partially resolved and further sequencing was not undertaken.pilV locus have ≥ 98% nucleotide identity and ≥ 99% amino acid identity between the two strain types, indicating that this gene may have participated in horizontal gene transfer.The predicted pilus adhesin, PilC1 (GenBank accession number JN681261and NMY233_1622) exhibited 84% amino acid similarity between the early strain type and the late strain type . Minor pThe early strain type contained the PglB form of the pilin glycosylation B locus (NMY220_1618) while the late strain type contained PglB2 (NMY233_1594). In the late strain type, PglB2 was immediately followed by a conserved hypothetical protein (NMY233_1595) of unknown function zitB (NMY233_0596), which encodes a predicted cation-efflux facilitator family protein, and a gene encoding a rubredoxin (NMY233_0921).The early strain type and late strain type each contained genes not found in the other genome . The majpglB and pglB2, zitB, and rubredoxin , as were the alleles for pilV. Tandem repeat differences in ag473 were maintained in population isolates, except for one early strain type isolate that had a single tandem repeat, and thus did not match either the early or late strain type. Late strain type population lbpB alleles were identical to the NM233 allele. Among the early strain type isolates, five lbpB alleles were identical to the NM220 allele but NM109, NM115 and NM206 had an allele that differed from both NM220 and NM233. All late strain type tbpB alleles were identical. Early strain type isolates contained three alleles for tbpB, all of which differed from the alleles found in either NM220 or NM233, and two isolates failed to amplify that locus.To determine whether the differences identified in the single sequenced early and late strain type isolates were characteristic of the larger population of isolates, ten gene targets were investigated in eight early strain type and eight late strain type population isolates. The early strain type and late strain type population isolates were consistent with NM220 and NM233 in gene content for pilH, pilI, and pilJ. The population of early strain type isolates contained the late strain type allele except for 2 isolates (isolated in 1998 and 2001), which contained the early strain type alleles.NM220 and NM233 contained different alleles for three pilin-associated genes Some studies have compared whole neisserial genomes in an attempt to elucidate the basis for differences in invasiveness and pathogenicity A phylogenetic tree of genetic relatedness of early strain type, late strain type and 20 available sequenced meningococcal genomes indicates that the serogroup Y strains are most closely related to each other and they cluster in a branch not closely related to other sequenced genomes. The serogroup Y strains in this study differ in gene content from other sequenced genomes in the same way as other sequenced genomes differ from each other, that is, mainly in phage-related genes and in gene encoding hypothetical proteins. Most notably, the serogroup Y genomes are missing the genes located in the putative islands of horizontal transfer PNM1, IHT-C and IHT-E, all of which appear to be of phage origin. It has been suggested that these genes contribute to virulence and pathogenicity, but their actual roles are currently unknown Closely related strains that differ in their ability to cause disease are ideal for investigating the genetic factors that contribute to that ability to cause disease N and πS. The highest value of both πN and πS was found in the gene encoding PorB, which is known to be both immunogenic and highly variable. Other genes with relatively high πN and πS included those encoding PorA, FetA, PilV, LbpB, and HpuA. A number of housekeeping genes were represented among those with high πN and πS, including shikimate 5-dehydrogenase (aroE), one of the seven genes used for MLST. The late strain type is a single locus variant of ST-23; single locus variants are considered to have arisen by horizontal gene transfer if the variant allele exhibits extensive differences from the allele found in the parent sequence type. Therefore, the πN and πS values for shikimate 5-dehydrogenase can be used as a marker for horizontal gene transfer. Genes in the early and late genomes whose πN and πS are similar to that of aroE have likely been involved in horizontal gene transfer.The majority of ORFs in the two genomes had few or no mutations, leading to an overall low level of πN and πS, loci which differed between the early and late strain type genomes exhibited a surplus of πS over πN. This phenomenon has been documented in methicillin-resistant Staphylococcus aureus and Clostridium difficileIn addition to the overall low values of πN. meningitidis and many others can be predicted to be immunogenic based upon their structure or their function. Our comparison of whole genomes found a preponderance of antigens unchanged but also found differences in a number of loci that contribute to the antigenic profile and these differences may have been instrumental in disease emergence. Genes exhibiting differences include antigenic outer membrane proteins, genes involved in pilus structure, function and glycosylation, and genes involved in iron acquisition and uptake. Differences in the genes encoding the antigens PorA, PorB, and FetA are more extensive than previously described (Tfp) are necessary for adhesion of bacterial cells to human mucosal cells and are major antigens pilH/I/J loci differed between early and late strains but most strains within the population tested contained the late strain type alleles. These loci may not have been involved in emergence of the late strain type. Alternatively, the late strain type alleles may have been acquired early in the genetic transformation of the early to late strain type. The presence of the early strain type alleles in isolates obtained in three different years  may imply persistence of a sub-population possessing those alleles or multiple instances of horizontal transfer. Pilin glycosylation is believed to be an important mechanism for decreasing immunogenicity of the pilin subunits by masking exposed areas of the protein. In N. meningitidis, the gene encoding pilin glycosylation protein B (PglB) has been documented to exist in 2 forms, pglB and pglB2pglB, while the late strain type contains pglB2. Based upon the number and extent of differences in genes affecting the structure and function of the pilus, it can be theorized that these differences contributed to the change in disease epidemiology.Given that both strain types caused similar rates of disease in the same population during different periods of the same decade, we did not expect major differences in virulence between them. Accordingly, our principal hypothesis was that emergence of the late strain type was primarily due to antigenic changes that allowed escape from population immunity. A number of proteins are known to be immunogenic in N. meningitidis in the human host and iron is sequestered in a variety of forms, including lactoferrin, transferrin, and hemoglobin/haptoglobin. N. meningitidis has evolved mechanisms for obtaining iron from these sources using several two component systems. These systems are antigenic, as they require surface exposure to bind their substrates. In addition, they are highly variable, with different alleles found in otherwise similar strains during epidemics, invoking the concept of a “genocloud”, a combination of a dominant strain and its close relatives lbpB and the early strain type isolates contained multiple variants of tbpB, which did not match either the early or late allele. These results may indicate selection for recombinants at these loci in a population of isolates against which human immunity has developed.Iron acquisition and uptake are necessary for survival of fHbp), neisserial heparin binding antigen (nhbA) and nadA have been identified as encoding possible targets for vaccines for prevention of serogroup B meningococcal disease as well as that caused by other serogroups nadA was absent in the early and late strain type genomes, but fHbp and nhbA were highly conserved. This study found 46 other genes encoding predicted lipoproteins that were identical in amino acid sequence between the early and late strain types and an additional 20 with greater than 99% amino acid identity. The majority of the identical lipoproteins were also highly conserved across 20 sequenced genomes, suggesting that some of them may be appropriate for investigation as vaccine targets. Conservation of antigens may indicate that structural constraints based upon function limit variation in those proteins. Conserved antigens are superior vaccine candidates, since highly variable vaccine targets complicate vaccine formulation, requiring the inclusion of many variants. However, highly conserved antigens may be a disadvantage to the bacteria, since their conservation predicts that any immunity they engender will be broad and will therefore limit spread of many strains.We also identified genes encoding known and predicted lipoproteins that were highly similar or identical in the two genomes. Factor H binding protein  to 100 (top) percent in each row.(TIF)Click here for additional data file.Figure S2Alignment of predicted amino acid sequence of early strain type (upper sequence) and late strain type (lower sequence) PorB (NMY220_1828 and NMY233_1807) showing non-conserved amino acids. Boxes outline loops V (*) and VII (+), which were previously reported.(TIF)Click here for additional data file.Figure S3Alignment of predicted protein lactoferrin binding protein B (LbpB) showing non-conserved amino acids. Upper sequence early clone (NM220), lower sequence late strain type (NM233).(TIF)Click here for additional data file.Figure S4Alignment of predicted protein hemoglobin-haptoglobin utilization protein A (hpuA) showing non-conserved amino acids. Upper sequence early strain type (NM220), lower sequence late strain type (NM233).(TIF)Click here for additional data file.Figure S5Alignment of predicted protein PilC1, showing non-conserved amino acids. Upper sequence early strain type (NM220), lower sequence late strain type (NM233).(TIF)Click here for additional data file.Table S1Available sequenced genomes.(DOCX)Click here for additional data file.Table S2Lipoproteins exhibiting identical nucleotide and amino acid sequence in early and late clones and their amino acid identity across sequenced genomes.(DOCX)Click here for additional data file.Table S3Early clone (NM220) and late clone (NM233) differences in predicted outer membrane proteins, lipoproteins, proteins involved in pilus biogenesis and non-lipoproteins involved in iron acquisition and uptake.(DOCX)Click here for additional data file.Table S4Proteins encoded by genes found in only one of the clones.(DOCX)Click here for additional data file.


Drosophila melanogaster to be used to better understand these myopathies and suggest novel therapeutic strategies. Integrin-mediated adhesion is required to support muscle structure and function, and expression of Integrin adhesive complex (IAC) proteins is modulated to adapt to varying levels of mechanical stress within muscle. Mutations in flapwing (flw), a catalytic subunit of myosin phosphatase, result in non-muscle myosin hyperphosphorylation, as well as muscle hypercontraction, defects in size, motility, muscle attachment, and subsequent larval and pupal lethality. We find that moderately elevated expression of the IAC protein PINCH significantly rescues flw phenotypes. Rescue requires PINCH be bound to its partners, Integrin-linked kinase and Ras suppressor 1. Rescue is not achieved through dephosphorylation of non-muscle myosin, suggesting a mechanism in which elevated PINCH expression strengthens integrin adhesion. In support of this, elevated expression of PINCH rescues an independent muscle hypercontraction mutant in muscle myosin heavy chain, Samba1Mhc. By testing a panel of IAC proteins, we show specificity for PINCH expression in the rescue of hypercontraction mutants. These data are consistent with a model in which PINCH is present in limiting quantities within IACs, with increasing PINCH expression reinforcing existing adhesions or allowing for the de novo assembly of new adhesion complexes. Moreover, in myopathies that exhibit hypercontraction, strategic PINCH expression may have therapeutic potential in preserving muscle structure and function.A variety of human diseases arise from mutations that alter muscle contraction. Evolutionary conservation allows genetic studies in  A wide variety of diseases of the muscle are caused by mutations that alter either the actin and myosin contractile machinery or its regulation. One class of mutations of interest results in hypercontraction of the muscle—actin and myosin fibers contract, but cannot efficiently relax. We have used the fruit fly as a model to study these mutations because of the striking similarity of fly and human muscle and because of the many genetic techniques that are available in the fly. Using a genetic approach we identified a protein, PINCH, whose increased expression can rescue the defects observed in hypercontraction mutants. PINCH is a component of integrin adhesion complexes, responsible for anchoring cells in their environment. This suggests that strengthening the anchorage of muscles via PINCH may be an effective strategy to prevent or reduce the muscle damage that occurs in diseases of muscle hypercontraction. Drosophila melanogaster have made it an attractive system in which to characterize these genes and mutations Drosophila exhibits myopathy due to muscle hypercontraction, leading to a range of phenotypes from flightless adults to early lethality Myosin heavy chain (Mhc) can decrease overall actomyosin force, which is sufficient to suppress hypercontraction defects SambaMhc hypercontraction mutants suggested an extensive actin cytoskeletal remodeling response, as well as upregulation of non-muscle myosin phosphatase expression to promote relaxation of the actomyosin cytoskeleton C. elegans muscles developed in the microgravity of spaceflight http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE36358) also reveals increased UNC-97 mRNA levels under conditions of hypergravity. This supports the idea that modulating gene expression of IAC components may be an evolutionarily conserved mechanism to adapt to widely divergent levels of mechanical stress to maintain muscle integrity.Numerous human diseases, including muscular dystrophies spaghetti squash (sqh) in Drosophilaflw exhibit hyperphosphorylation of non-muscle myosin. Notably, though flw does not genetically interact with the muscle version of MRLC (Mlc2) flw mutants is hypercontraction of myofibrillar myosin, eventually resulting in detachment of striated muscles and subsequent lethality at both larval and pupal stages Flapwing (Flw) is a serine/threonine Protein Phosphatase 1β (PP1β) that acts broadly upon many phosphorylated protein substrates, but it is required solely for its activity in dephosphorylating non-muscle myosin regulatory light chain (MRLC), encoded by flw muscle hypercontraction phenotype could be rescued by strengthening the anchoring of muscles at integrin-based adhesion sites to better withstand the force of hypercontraction. We show here that moderately elevated expression of the IAC protein PINCH alleviates the phenotypes of flw mutants. PINCH is an integrin-associated LIM domain adaptor protein encoded by the steamer duck (stck) gene in Drosophila. PINCH is part of an evolutionarily conserved, high affinity protein complex comprised of Ras Suppressor 1 (RSU1), Integrin-linked kinase (ILK) and Parvin that is targeted to the cell membrane at sites of integrin adhesion Drosophila, null mutants in PINCH, ILK, and Parvin each die late in embryogenesis as a consequence of failure of integrin-based muscle attachment sites within the segmental musculature flw mutant.We hypothesized that the flw mutants. PINCH does not appear to alleviate hypercontraction of the myofibrils, as muscle attachment is better maintained even in the presence of muscle hypercontraction. We also eliminate phospho-regulation as a plausible mechanism, as elevated PINCH expression does not alter the phosphorylation status of the only essential Flapwing substrate, MRLC/Sqh. We show that moderately elevated expression of PINCH partially rescues another hypercontraction mutant, the 1Samba allele of Mhc. Furthermore, we show that among a panel of IAC proteins tested, elevated expression of Talin also affords marginal rescue of 1Samba hypercontraction defects. However, transgenic Talin expression is unable to afford rescue of flw larval lethality, underscoring the specificity of PINCH expression in strengthening adhesion under multiple conditions of hypercontraction. These studies have broad implications for understanding and treating a variety of myopathies and muscle degenerative diseases.We demonstrate that structural stabilization of integrin-based adhesions by PINCH is likely to be a mechanism for the rescue of Drosophila proteins also interact. Our analyses included two recessive alleles of the PP1β family member, 6flapwing. flw is a point mutant with decreased substrate affinity, and 7flw contains a lacZ enhancer trap inserted in the flw 5′ UTR which reduces the level of Flw expression flw alleles. We observed that expression of transgenic wild type PINCH-Flag significantly increased the frequency of hemizygous flw adult male escapers, and restored their fertility  and the integrin-associated protein PINCH was described ertility . These cflw mutants, we assessed the viability across development at 25°C of male hemizygotes selected from balanced stocks. Compared to a wild type 1118w control, we found significant larval lethality—approximately 70% of 7flw larvae failed to successfully pupariate, and of the 30% that do form pupae, essentially all arrest development prior to adult eclosion  the number of adult escapers at 18°C, 2) the developmental profile at 25°C, and 3) the rescue of larval lethality by expression of transgenic PINCH, we focus on 7flw in the remaining experiments.To more carefully characterize the spectrum of defects in eclosion . Expresseclosion . However7 mutant . At 25°C7 mutant , pupal l7 mutant , which mpression . Becauseflw lethality. Decreasing PINCH dosage using a heterozygous 17stck mutation in the 7flw background was challenging due to a high rate of balancer breakdown in the required crosses. However, the limited number of 7; stck17/+flw animals that were isolated exhibited complete larval lethality (n = 44)  . These dflw larval lethality is through reversing protein hyper-phosphorylation that results from flw loss-of-function. Because the sole essential activity of Flapwing is the dephosphorylation of non-muscle myosin regulatory light chain (MRLC)/Sqh 7flw lysates as compared to wild type 1118w controls , indicating either that Akt, JNK, and ERK are not actively signaling in these samples, are not substrates of Flapwing, or that other phosphatases compensate to appropriately dephosphorylate Akt, JNK and ERK in the flw mutant. While it is formally possible that PINCH could contribute to the phospho-regulation of non-essential Flapwing targets, it seems unlikely that phospho-regulation is a major contributor to the suppression of flw lethality observed upon elevated PINCH expression.We also examined the phosphorylation states of several other phospho-proteins that have been connected to PINCH: Akt flw larval lethality, we employed several mutants of PINCH that disrupt binding of its known partners, Integrin-linked kinase (ILK) and Ras Suppressor 1 (RSU1)  . One of of PINCH  [30], how, and ZCL0663Zasp66 were obtained from the Bloomington Stock Center. Samba1Mhc17stck and 18stckQ38AFlag w; PINFL2 and both 6/FM7iflw and 7/FM7iflw were set at 18°C and progeny counted. The percent escape was calculated as 100×[flw males/]. Escaper male hemizygotes were tested for fertility by crossing to 1118w virgin females.Crosses between FM7i balanced, flw hemizygous males were selected by sorting against the GFP expressed from the FM7i balancer chromosome. A cross between 7/FM7iflw and 17/TM3, twi>GFPFM7i/Y; stck was used to generate 7; stck17/+flw animals, by selecting against the GFP expressed on both balancer chromosomes. On subsequent days, the number of viable animals at each stage of development was counted and live animals were moved to a fresh plate. Putative larval stages were determined by size. In 1118w control was included to determine baseline lethality arising from experimental manipulation of the samples. Graphical analyses were done using GraphPad Prism.Stocks of the indicated genotypes were placed in cages and allowed to lay for approximately 6 hours on yeasted grape agar plates at 25°C. Except where noted, >80 animals of the appropriate genotype were selected from stocks as early L1s (24–30 hours) and placed on fresh grape agar plates. For stocks that are For each analysis that involves a PINCH transgene, one of at least two independent insertion lines that were analyzed is shown, to confirm that the results were not dependent upon the locus of the transgene insertion.Four-day-old L3 larvae were homogenized in RIPA buffer with phosphatase inhibitors and normalized by protein content (BioRad DC protein assay). Antibodies employed were anti-PINCH In prior experiments that mapped the site of RSU1 interaction to LIM5 of PINCH, we constructed a LIM5 bait and RSU1 prey that activated an ADE2 reporter in a yeast two-hybrid system wt-His construct wtHis and pMT-PINCHD303VHis were stably transfected into S2 cells using standard methods and expression induced by the addition of CuSO4. S2 cell lysates were prepared in lysis buffer  plus protease inhibitors, and were incubated with Ni-NTA agarose (Qiagen), clarified by centrifugation, washed with lysis buffer, then boiled in 2× Laemmli sample buffer prior to western blotting.PCR mutagenesis of a previously described pMT-PINCHD303VFlag were generated by PCR mutagenesis of a previously described pCasper construct containing genomic PINCHwt-Flag 17/stck18stck). Adult fly lysates were prepared in lysis buffer plus protease inhibitors, clarified by centrifugation and 0.45 µm filtration, and were incubated with anti-Flag M2 agarose (Sigma), washed, and boiled in 2× Laemmli sample buffer for western blotting.Transgenic flies carrying PINCH5′GCACTGGCATGTGGAACATT3′, reverse: 5′ACTAGTCTACCTGTCATCGTC3′), GAPDH .Twelve adult flies per sample were lysed in 350 µl RLT Buffer using Qiashredder columns (Qiagen), and RNA was extracted using RNEasy Mini Kits (Qiagen) according to the manufacturer's recommendations. RT-PCR was conducted with 50 ng RNA per reaction using the Access RT-PCR System (Promega), with the following primers: PINCH-Flag (Forward: rd instar). Prior to size measurements, larvae were heat fixed by rapidly submerging in boiling embryo wash , and bright field images captured on an Olympus MVX10 dissecting microscope. Length measurements of individual larvae were normalized to the mean of a matched wild type 1118w control. To measure larval motility, we adapted an existing protocol Larvae of the given genotypes were sorted at 24–30 hr after egg lay and analyzed when they reached 4 days of age , then the Zasp66-GFP pattern in the Ventral Intersegmental (VIS) muscles was examined using an Olympus MVX10 GFP-dissecting microscope. Any animal in which the medial pair of VIS muscles does not correctly span body segments T1-T3 is scored as detached. Discontinuities in the Zasp-GFP signal are characteristic of breaks/detachments in the VIS muscles. Areas of hypercontraction are characterized by intense Zasp-GFP signal, as the Z-lines are more closely spaced. For each genotype, analyses were conducted in triplicate. Paired t tests were performed to determine statistically significant differences between genotypes.Four-day-old L3s of the given genotypes were generated in a cross with t tests were performed to determine statistically significant differences between genotypes.Assays were done according to a previously described protocol IFM was analyzed as described previously Figure S16flw mutant. A,B) For each genotype shown, graphs depict the percentage of animals of the given developmental stage at the indicated time points. Pupae present at day 11 do not progress to adulthood.Expression of the PINCH-Flag transgene rescues the larval lethality of the (EPS)Click here for additional data file.Figure S2Differences in the levels of PINCH transgene expression are post-transcriptional. RT-PCR from adult fly lysates shows that mRNA levels transcribed from the wild type, Q38A and D303V versions of the PINCH transgene are identical. GAPDH is used as a loading control.(EPS)Click here for additional data file.Figure S3Expression of wild type or PINCH mutant transgenes alone does not significantly affect larval motility. The average percentage of larvae located in each zone at the end of the larval motility assay is plotted for the indicated genotypes.(EPS)Click here for additional data file.Figure S47flw genetic background does not significantly alter viability or motility in four-day-old larvae. A) The graph shows the percentage of 7; Zasp-GFPflw animals of the given developmental stage at the indicated time points. Expression of Zasp-GFP in the 7flw mutant prevents pupariation, but at the time at which VIS muscle integrity is assessed (indicated by arrow), larval viability is not compromised (compare to Expression of Zasp-GFP in the (EPS)Click here for additional data file.Figure S5Expression of wild type or mutant PINCH transgenes does not significantly affect VIS muscle attachment. The graph shows the percent of total animals in which the VIS muscles exhibit detachment. Number of animals examined of each genotype is shown above the bars.(EPS)Click here for additional data file.


Xenopus tadpole tail is a complex appendage, containing epidermis, muscle, nerves, spinal cord, and vasculature, which regenerates after amputation. Understanding the mechanisms of tail regeneration may lead to new insights to promote biomedical regeneration in non-regenerative tissues. Although chromatin remodeling is known to be critical for stem cell pluripotency, its role in complex organ regeneration in vivo remains largely uncharacterized. Here we show that histone deacetylase (HDAC) activity is required for the early stages of tail regeneration. HDAC1 is expressed during the 1st two days of regeneration. Pharmacological blockade of HDACs using Trichostatin A (TSA) increased histone acetylation levels in the amputated tail. Furthermore, treatment with TSA or another HDAC inhibitor, valproic acid, specifically inhibited regeneration. Over-expression of wild-type Mad3, a transcriptional repressor known to associate in a complex with HDACs via Sin3, inhibited regeneration. Similarly, expression of a Mad3 mutant lacking the Sin3-interacting domain that is required for HDAC binding also blocks regeneration, suggesting that HDAC and Mad3 may act together to regulate regeneration. Inhibition of HDAC function resulted in aberrant expression of Notch1 and BMP2, two genes known to be required for tail regeneration. Our results identify a novel early role for HDAC in appendage regeneration and suggest that modulation of histone acetylation is important in regenerative repair of complex appendages.The ability to fully restore damaged or lost organs is present in only a subset of animals. The  Xenopus laevis, have the ability to rapidly regenerate their tails upon amputation Tadpoles of the African clawed frog, Recent studies have identified a set of processes that occur when the tail is lost. Amputation of a tail results in migration of the epidermal cells to cover the wound within 2–3 hours in vivo model well-suited to understand how differentiated cell types can transiently convert to a highly proliferative state that also recapitulates developmental gene expression programs The critical process of tail regeneration requires that cells re-enter the cell cycle and differentiate from a lineage-restricted progenitor cell population into a specific cell type to replace the damaged tissue and reconstitute the missing tissue. Organ rebuilding using the newly generated cells must also be orchestrated in three dimensions in order to properly restore the complex morphology of the intact neuromuscular appendage. Thus, tail regeneration is a tractable The proliferation of differentiated somatic cells upon injury is a process mostly dictated by the epigenetic markers they harbor on regulatory regions of tissue specific genes One important aspect of chromatin remodeling is controlling DNA through histone acetylation. Histones are dynamic components of the transcriptional machinery that can be modified by post-translational modifications such as acetylation, methylation and phosphorylation In particular, the acetylation of the ε-amino group of lysines residues on the histone tail by Histones acetyltranferases (HAT) is tightly correlated to gene transcription during development Xenopus, and zebrafish HDACs are highly conserved enzymes with homologues in yeast, humans, Because HDACs are transcriptional repressors that lack DNA binding domains, their specificity is mediated by direct interaction with transcriptional repressors in large multi-protein complexes containing components such as NuRD, CoREST, or Sin3 proteins in vivo. Previous studies have identified methylation states and histone demethylases as regulatory components of regeneration in Xenopus tadpole limbs Xenopus tail model and used molecular and pharmacological tools to show that HDAC activity is required for regeneration. Inhibition of HDAC function blocks regeneration by altering the histone acetylation state and results in aberrant expression of the downstream genes involved in driving regenerative outgrowth. Furthermore, HDACs likely associate with the transcriptional repressor Mad3 to regulate histone acetylation.As epigenetic regulation of DNA has been shown to be important for modulating stem cell states, such regulation may also play an important role in complex organ regeneration Xenopus HDAC1 in situ hybridization with gene-specific probes showed that HDAC1  was strongly expressed at 24, 48, and 72 hpa (hours post amputation) in the mesenchymal cells of the regeneration bud , a well-known specific and potent chemical inhibitor of both Class I and Class II HDACs Our RNA expression data showed that HDAC1 is present during the events occurring right after tail amputation. Thus, we tested the hypothesis that HDAC activity is required during the early stages of regeneration by determining the temporal requirement for HDAC function. Tadpole tails were amputated and incubated for specific durations with TSA and assayed for their regenerative ability at 7 dpa (days post amputation) . TreatmeClass I HDACs are widely expressed transcriptional repressors that lack DNA binding domains. Thus their specificity is due to direct interaction with transcriptional repressors in large multi-protein complexes harboring NuRD, CoREST, or Sin3 proteins Xenopus development Our previous work showed that Mad3 and the maternal HDAC functionally interact in the establishment of left-right asymmetry during early First, we characterized the potential role of Mad3 in regeneration. The over-expression of Mad3 has been shown to exert a dominant-negative effect on the HDAC1-Mad3 complex The ability of Mad3 to repress gene expression is dependent on its Sin3-Interacting Domain (SID), which enables Mad3 to interact with Sin3 co-factors. Sin3 in turn, recruits HDAC1 multi-protein complexes containing Mad, leading to transcriptional repression Histone Deacetylases (HDAC) act to remove acetyl groups from the lysine amino acid on histones. To better understand the mechanism by which inhibition of HDAC activity blocks regeneration, we examined the effect of the HDAC inhibitor, TSA, on the acetylation state of the tail regenerate. Using an antibody that specifically detects acetylation on Histone H4, we observed a weak signal on control 24 hpa regeneration buds, consistent with our data that a low acetylation level of histone H4 is necessary for regeneration . In contThe acetylation state of histones modulates genes expression. The removal of acetyl groups on histones by HDACs acts to repress transcription whereas the presence of acetyl groups enables transcription Xenopus BMP2 is up-regulated in the regeneration bud , affects regenerative ability. They showed that the Xenopus work used constitutively-active forms of either the BMP receptor Alk3 or the intracellular active Notch domain (NICD) Numerous studies support the hypothesis that HDACs can act as promoters of growth and proliferation Notably, our data show that HDAC function is critical for properly regulating the expression patterns of regenerative genes as an essential component of this process. Importantly, the direct regulation of Notch1 and BMP2 by histone acetylation is unlikely to account for the regenerative failure due to HDAC inhibition. It has been shown that HDAC inhibitor treatment in myeloma cells can modulate the mRNA levels of approximately 2% of expressed genes Xenopus laevis larvae were cultured via approved protocols . Tails at stages (st.) 40–41 Xenopus Mad3 and Mad3ΔSID were generated using the mMessage mMachine kit (Ambion). Embryos at the 4-cell stage were transferred to 0.1X MMR containing 3% Ficoll and synthetic mRNA was injected into each of the 4 blastomeres along with the lineage tracer rhodamine-labeled dextran (Invitrogen). At 30 minutes post-injection, embryos were transferred to plain 0.1X MMR and cultured at 18°C until they reached st. 40–41.For microinjections, capped, synthetic mRNAs http://www.xenbase.org/. Wholemount RNA in situ hybridization was performed according to standard protocols Xenopus Mad3 (Open Biosystems clone ID: 4175511), HDAC1/Rpd3 (Open Biosystems clone ID:4683530), HDAC6 (Open Biosystems clone ID:5542663), Notch1 Xenopus embryos were fixed overnight in MEMFA buffer Sequence data for this paper were retrieved from Xenbase, University of Calgary, Alberta T2N 1N4, Canada; URL: U test for ordinal data with tied ranks, using normal approximation for large sample sizes. Multiple treatments were compared using a Kruskal–Wallis test, with Dunn's Q corrected for tied ranks.To compare tail regeneration experiments, raw data from scoring was used. Comparison of two treatments was analyzed with Mann–Whitney Figure S1Expression of HDAC1 and Mad3 in Xenopus Tail. RNA in situ hybridization to detect gene expression in st. 40 uncut and amputated tail for (A–B) HDAC1, (C–D) Mad3, and (E–F) β-gal. Probe targets are shown to the left of the panels.(TIF)Click here for additional data file.Figure S2Tail Regeneration Assay. Individual animals for each specific treatment were scored as follows: Full: complete regeneration. Good: robust regeneration with minor defects . Weak: poor regeneration (hypomorphic/defective regenerates). None: no regeneration. Shown are representative examples of each regenerate class.(TIF)Click here for additional data file.