Simultaneous single-structure and bundle representation of protein NMR structures in torsion angle space

A method is introduced to represent an ensemble of conformers of a protein by a single structure in torsion angle space that lies closest to the averaged Cartesian coordinates while maintaining perfect covalent geometry and on average equal steric quality and an equally good fit to the experimental (e.g. NMR) data as the individual conformers of the ensemble. The single representative ‘regmean structure’ is obtained by simulated annealing in torsion angle space with the program CYANA using as input data the experimental restraints, restraints for the atom positions relative to the average Cartesian coordinates, and restraints for the torsion angles relative to the corresponding principal cluster average values of the ensemble. The method was applied to 11 proteins for which NMR structure ensembles are available, and compared to alternative, commonly used simple approaches for selecting a single representative structure, e.g. the structure from the ensemble that best fulfills the experimental and steric restraints, or the structure from the ensemble that has the lowest RMSD value to the average Cartesian coordinates. In all cases our method found a structure in torsion angle space that is significantly closer to the mean coordinates than the alternatives while maintaining the same quality as individual conformers. The method is thus suitable to generate representative single structure representations of protein structure ensembles in torsion angle space. Since in the case of NMR structure calculations with CYANA the single structure is calculated in the same way as the individual conformers except that weak positional and torsion angle restraints are added, we propose to represent new NMR structures by a ‘regmean bundle’ consisting of the single representative structure as the first conformer and all but one original individual conformers (the original conformer with the highest target function value is discarded in order to keep the number of conformers in the bundle constant). In this way, analyses that require a single structure can be carried out in the most meaningful way using the first model, while at the same time the additional information contained in the ensemble remains available.     

Quinolone resistance in absence of selective pressure: the experience of a very remote community in the Amazon forest

Background

Quinolones are potent broad-spectrum bactericidal agents increasingly employed also in resource-limited countries. Resistance to quinolones is an increasing problem, known to be strongly associated with quinolone exposure. We report on the emergence of quinolone resistance in a very remote community in the Amazon forest, where quinolones have never been used and quinolone resistance was absent in 2002.

Methods

The community exhibited a considerable level of geographical isolation, limited contact with the exterior and minimal antibiotic use (not including quinolones). In December 2009, fecal carriage of antibiotic resistant Escherichia coli was investigated in 120 of the 140 inhabitants, and in 48 animals reared in the community. All fluoroquinolone-resistant isolates were genotyped and characterized for the mechanisms of plasmid- and chromosomal-mediated quinolone resistance.

Principal Findings

Despite the characteristics of the community remained substantially unchanged during the period 2002–2009, carriage of quinolone-resistant E. coli was found to be common in 2009 both in humans (45% nalidixic acid, 14% ciprofloxacin) and animals (54% nalidixic acid, 23% ciprofloxacin). Ciprofloxacin-resistant isolates of human and animal origin showed multidrug resistance phenotypes, a high level of genetic heterogeneity, and a combination of GyrA (Ser83Leu and Asp87Asn) and ParC (Ser80Ile) substitutions commonly observed in fluoroquinolone-resistant clinical isolates of E. coli.

Conclusions

Remoteness and absence of antibiotic selective pressure did not protect the community from the remarkable emergence of quinolone resistance in E. coli. Introduction of the resistant strains from antibiotic-exposed settings is the most likely source, while persistence and dissemination in the absence of quinolone exposure is likely mostly related with poor sanitation. Interventions aimed at reducing the spreading of resistant isolates (by improving sanitation and water/food safety) are urgently needed to preserve the efficacy of quinolones in resource-limited countries, as control strategies based only on antibiotic restriction policies are unlikely to succeed in those settings.     

Migratory Status Is Not Related to the Susceptibility to HPAIV H5N1 in an Insectivorous Passerine Species

Migratory birds have evolved elaborate physiological adaptations to travelling, the implications for their susceptibility to avian influenza are however unknown. Three groups of stonechats (Saxicola torquata) from (I) strongly migrating, (II) weakly migrating and (III) non-migrating populations were experimentally infected with HPAIV H5N1. The different bird groups of this insectivorous passerine species were infected in autumn, when the migrating populations clearly exhibit migratory restlessness. Following infection, all animals succumbed to the disease from 3 through 7 days post inoculation. Viral shedding, antigen distribution in tissues, and survival time did not differ between the three populations. However, notably, endothelial tropism of the HPAIV infection was exclusively seen in the group of resident birds. In conclusion, our data document for the first time the high susceptibility of an insectivorous passerine species to H5N1 infection, and the epidemiological role of these passerine birds is probably limited due to their high sensitivity to HPAIV H5N1 infection. Despite pronounced inherited differences in migratory status, the groups were generally indistinguishable in their susceptibility, survival time, clinical symptoms and viral shedding. Nevertheless, the migratory status partly influenced pathogenesis in the way of viral tropism.     

High haplotype diversity with fine-scale structure in a recently established population of an endangered orchid

New populations of endangered plants may establish outside of protected areas if a suitable habitat becomes available. However, it is unclear whether such populations are genetically uniform, as a result of a founder effect, or whether they contain genetic variation resulting from continuous gene flow from source populations, and subsequent recruitment after establishment. We addressed this by examining haplotype variation in the endangered orchid, Anacamptis robusta, which has formed a new population outside of its protected area within the past 20 years. To assess population growth, the number of A. robusta flowering plants were counted every year for 22 years in both the new population and populations within the reserve. Haplotype diversity and fine-scale structure were examined with spatial autocorrelation analysis, both in the new population and representative populations from the protected area. The number of flowering individuals in the new population increased from 9 to 2,277 between 2003 and 2012, whereas within the reserve flowering population sizes varied between years. Seventeen of 23 haplotypes detected for A. robusta were detected in the new population, with seven of these occurring more frequently in the new population than reserve populations. In the new population, there was strong fine-scale spatial structure of haplotypes, similar to patterns found in large populations from the protected area, suggesting multiple colonization events and subsequent local recruitment. This highlights that ongoing demographic and genetic monitoring of plant populations is vital to improve our understanding of population colonization and the conservation of narrow endemics.     

Highly Pathogenic Avian Influenza Virus Infection of Mallards with Homo- and Heterosubtypic Immunity Induced by Low Pathogenic Avian Influenza Viruses

The potential role of wild birds as carriers of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 is still a matter of debate. Consecutive or simultaneous infections with different subtypes of influenza viruses of low pathogenicity (LPAIV) are very common in wild duck populations. To better understand the epidemiology and pathogenesis of HPAIV H5N1 infections in natural ecosystems, we investigated the influence of prior infection of mallards with homo- (H5N2) and heterosubtypic (H4N6) LPAIV on exposure to HPAIV H5N1. In mallards with homosubtypic immunity induced by LPAIV infection, clinical disease was absent and shedding of HPAIV from respiratory and intestinal tracts was grossly reduced compared to the heterosubtypic and control groups (mean GEC/100 µl at 3 dpi: 3.0×10² vs. 2.3×10⁴ vs. 8.7×10⁴; p<0.05). Heterosubtypic immunity induced by an H4N6 infection mediated a similar but less pronounced effect. We conclude that the epidemiology of HPAIV H5N1 in mallards and probably other aquatic wild bird species is massively influenced by interfering immunity induced by prior homo- and heterosubtypic LPAIV infections.     

Lactase persistence in Tunisia as a result of admixture with other Mediterranean populations

Background

The ability to digest lactose after weaning, namely, lactase persistence (LP), is encoded by polymorphisms in the MCM6 gene and varies widely in frequency among different human populations. Although, evolution of LP-related genetic variants was investigated in many groups of Sub-Saharan African, Middle Eastern, and European ancestry, only few studies have focused on populations from North Africa and no data are especially available from the Tunisian one. For this reason, there is an urgent need to investigate the frequency patterns at these loci in Tunisia since this adaptive trait is implicated in health.

Methods

Forty SNPs covering the LCT/MCM6 genes and including the two functional variants ??13,910 C > T and ??22,018 G > A were genotyped in 117 Tunisian individuals using the Sequenom Mass Array technology. The observed nucleotide and haplotype patterns of variation were then compared with those of several African, European, and Mediterranean human groups for which comparable data were publicly available. Admixture analysis on a 5 Mb genomic region surrounding the LCT/MCM6 loci was also performed by extracting genotypes from a previously generated genome-wide dataset in order to deepen the reconstruction of the evolutionary history of these loci.

Results

We found that lactase non-persistence (LNP)-related alleles and haplotypes were predominantly present in the examined population. A clear differentiation between Tunisian, African, and North European/North Italian samples was found, while the Tunisian population showed more genetic affinity to Central and South Italian groups.

Conclusions

Our study provided a first report of LP-associated alleles and haplotypes in the Tunisian population. We highlighted a gradient followed by LP diffusion from Europe to North Africa. Based on the rich historic background of Tunisia, we suggest that this adaptive trait was introduced in that geographic region by a relatively recent gene flow.

     

Selected markers of proliferation and apoptosis in the parathyroid lesions: a spatial visualization and quantification

The aim of the paper was to apply a method for quantitative assessment of proliferation and apoptosis markers, based on their 3D visualization, in cases of parathyroid adenoma and hyperplasia. Material was obtained from 49 patients (32 females and 17 males) with primary hyperparahyroidism. Quantitative immunohistochemistry studies of Ki-67, proliferating cell nuclear antigen (PCNA) and bcl-2 were performed on digital microscopy images with the use of 3D visualization. The use of spatial visualization method allowed us to perform objective quantitative assessment of the studied immunohistochemical markers. The average cell nuclear fraction of Ki67+ was 1.8% in hyperplasia and 1.9% in adenoma cases while 3.5% in the controls. The highest expression of PCNA was found in parathyroid hyperplasia (22.9%) and significantly decreased in adenoma (12.5%) and in the control group (16.8%). The lower expression of bcl-2 in hyperplasia cases (mean area fraction of 0.172 per 1 μm², in contrast to 0.643 in adenomas and 0.648 in control) suggested that principal cells can be ready for apoptosis and may confirm the important role of bcl-2 protein in etiopathogenesis of hyperplasia of the parathyroid gland while PCNA might be a useful marker for differentiating adenoma from early hyperplasia in primary hyperparahyroidism cases.     

The fluorescence studies of the sol-gel transition by styrylpyridine derivative

The gelation process of tetraethylorthosilanes in acid environment was monitored with the trans-4-(p-N,N-dimethylaminostyryl)-N-vinylbenzylopyridinium chloride (vbDMASP) fluorescent probe. The fluorescence steady-state and anisotropy measurements of material during sol-gel transition are reported. The results are compared with fluorescence studies of the probe in a modeled viscous system of water-glycerol mixtures. A strong increase of anisotropy, from 0.1 to 0.9, with gelation time as well with wavelength, was observed. Although the increase of anisotropy with wavelength is due to specificity of the compounds exhibiting charge transfer properties, the increase of the anisotropy with gelation time is due to an increase of microviscosity. On this basis, suitability of the applied fluorophore in recording of viscosity changes during sol-gel transition is discussed. The molecular structure of vbDMASP in the excited states in dependence on environmental polarity was optimized using the HyperChem and Amsol program. The dynamics of torsional angle C35-C34-N31-C28 of the multichromophore dye in correlation with micropolarity and microviscosity of the network formation during the sol-gel transition is discussed.     

Foraminiferal assemblages of the Bric della Muda laminites (Nizza Monferrato, Piedmont): Proxies of cyclic palaeoenvironmental changes in the early Messinian of Northwestern Italy

Foraminiferal assemblages in laminites cropping out at the Bric (i.e. Hill) della Muda (Nizza Monferrato, Piedmont, Northwestern Italy) document deep marine conditions during the early Messinian, spanning a time-interval approximating, or slightly wider than the stratigraphic range of Globorotalia nicolae (6.82-6.72 Ma). Fluctuations in abundance of planktonic warm-water oligotrophic taxa and cold-water eutrophic species show a cyclical pattern, very comparable to variations measured in cyclically-bedded formations of the pre-evaporitic Messinian in the Mediterranean area. A subtropical climate with cyclic warmer and cooler episodes, characterized by surface water stratification and vertical mixing in the water column, respectively, is also documented. A depositional depth close to or larger than 1000 m is inferred on the basis of planktonic assemblages yielding also the deep planktonic Hastigerinella digitata and of very high P/(P + B) ratios. Anoxic or strongly dysoxic bottom conditions are indicated by the absence or rareness of benthic foraminifers, which are represented by taxa tolerant of low-oxygen or high organic content (Bolivina pseudoplicata, Chilostomella oolina, Globobulimina affinis). A slight decrease in P/(P + B) ratios, and the presence of rare shallow-water forms in the uppermost layers suggest a mild dysoxia and a probable shallowing, when compared with coeval events in Northern Apennines and Southern Greece sections.