Mycobacterial proteome extraction: comparison of disruption methods

Mycobacterium avium subsp. paratuberculosis has long been recognized as the causative agent of Johne's disease, a chronic inflammatory intestinal disease of sheep, cattle and other ruminants. Mycobacterial cells are extremely hardy, and proteomic analyses require the use of harsh conditions to effect their disruption. We compared the effectiveness of bead beating and sonication as cell lysis methods for the extraction of the proteomes of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis. Broad and narrow range two-dimensional gel electrophoresis was used to compare the numbers of silver stained protein spots that were observed in mycobacterial lysates. Despite differences in the yield of total protein from either species, and at different ages, the two methods appeared to give similar representations of the mycobacterial proteomes analyzed. Bead beating therefore represents a rapid and effective method of extracting the proteomes of mycobacterial species without the risks associated with an open tube sonication procedure.     

Biochemical and ultrastructural study of two familial cases of Winchester syndrome

Two new familial cases of Winchester syndrome with the characteristic features allowed to be more explicit on a few data of this syndrome. As reported in a previous paper an abnormal oligosaccharide was detected in urine of patients but the pathological significance of this oligosaccharide must be discussed and its finding in patients with Winchester syndrome does not lead to further elucidation of the aetiology of this condition. Cultured fibroblasts were obtained from a skin biopsy performed in thickened area. These cells had a normal level of the hydrolases studied whereas they showed ultrastructural abnormalities with numerous secondary lysosomes and pseudomyelinic figures.     

Complex links between dietary lipids, endogenous endotoxins and metabolic inflammation

Metabolic diseases such as obesity are characterized by a subclinical inflammatory state that contributes to the development of insulin resistance and atherosclerosis. Recent reports also indicate that (i) there are alterations of the intestinal microbiota in metabolic diseases and (ii) absorption of endogenous endotoxins (namely lipopolysaccharides, LPS) can occur, particularly during the digestion of lipids. The aim of the present review is to highlight recently gained knowledge regarding the links between high fat diets, lipid digestion, intestinal microbiota and metabolic endotoxemia & inflammation.     

A model actin comet tail disassembling by severing

We use a numerical simulation to model an actin comet tail as it grows from the surface of a small object (a bead) and disassembles by severing. We explore the dependence of macroscopic properties such as the local tail radius and tail length on several controllable properties, namely the bead diameter, the bead velocity, the severing rate per unit length, and the actin gel mesh size. The model predicts an F-actin density with an initial exponential decay followed by an abrupt decay at the edge of the tail, and predicts that the comet tail diameter is constant along the length of the tail. The simulation results are used to fit a formula relating the comet tail length to the control parameters, and it is proposed that this formula offers a means to extract quantitative information on the actin gel mesh size and severing kinetics from simple macroscopic measurements.     

More than 100,000 detectable peptide species elute in single shotgun proteomics runs but the majority is inaccessible to data-dependent LC-MS/MS

Shotgun proteomics entails the identification of as many peptides as possible from complex mixtures. Here we investigate how many peptides are detectable by high resolution MS in standard LC runs of cell lysate and how many of them are accessible to data-dependent MS/MS. Isotope clusters were determined by MaxQuant and stringently filtered for charge states and retention times typical of peptides. This resulted in more than 100,000 likely peptide features, of which only about 16% had been targeted for MS/MS. Three instrumental attributes determine the proportion of additional peptides that can be identified: sequencing speed, sensitivity, and precursor ion isolation. In our data, an MS/MS scan rate of 25/s would be necessary to target all peptide features, but this drops to less than 17/s for reasonably abundant peptides. Sensitivity is a greater challenge, with many peptide features requiring long MS/MS injection times (>250 ms). The greatest limitation, however, is the generally low proportion of the target peptide ion intensity in the MS/MS selection window (the "precursor ion fraction" or PIF). Median PIF is only 0.14, making the peptides difficult to identify by standard MS/MS methods. Our results aid in developing strategies to further increase coverage in shotgun proteomics.     

Successful carnivore identification with faecal DNA across a fragmented Amazonian landscape

The use of scat surveys to obtain DNA has been well documented in temperate areas, where DNA preservation may be more effective than in tropical forests. Samples obtained in the tropics are often exposed to high humidity, warm temperatures, frequent rain and intense sunlight, all of which can rapidly degrade DNA. Despite these potential problems, we demonstrate successful mtDNA amplification and sequencing for faeces of carnivores collected in tropical conditions and quantify how sample condition and environmental variables influence the success of PCR amplification and species identification. Additionally, the feasibility of genotyping nuclear microsatellites from jaguar (Panthera onca) faeces was investigated. From October 2007 to December 2008, 93 faecal samples were collected in the southern Brazilian Amazon. A total of eight carnivore species was successfully identified from 71% of all samples obtained. Information theoretic analysis revealed that the number of PCR attempts before a successful sequence was an important negative predictor across all three responses (success of species identification, success of species identification from the first sequence and PCR amplification success), whereas the relative importance of the other three predictors (sample condition, season and distance from forest edge) varied between the three responses. Nuclear microsatellite amplification from jaguar faeces had lower success rates (15-44%) compared with those of the mtDNA marker. Our results show that DNA obtained from faecal samples works efficiently for carnivore species identification in the Amazon forest and also shows potential for nuclear DNA analysis, thus providing a valuable tool for genetic, ecological and conservation studies.     

Isolation by elevation: genetic structure at neutral and putatively non-neutral loci in a dominant tree of subtropical forests, Castanopsis eyrei

Background

The distribution of genetic diversity among plant populations growing along elevational gradients can be affected by neutral as well as selective processes. Molecular markers used to study these patterns usually target neutral processes only, but may also be affected by selection. In this study, the effects of elevation and successional stage on genetic diversity of a dominant tree species were investigated controlling for neutrality of the microsatellite loci used.

Methodology/Principal Findings

Diversity and differentiation among 24 populations of Castanopsis eyrei from different elevations (251–920 m) and successional stages were analysed by eight microsatellite loci. We found that one of the loci (Ccu97H18) strongly deviated from a neutral model of differentiation among populations due to either divergent selection or hitchhiking with an unknown selected locus. The analysis showed that C. eyrei populations had a high level of genetic diversity within populations (A_R = 7.6, H_E = 0.82). Genetic variation increased with elevation for both the putatively selected locus Ccu97H18 and the neutral loci. At locus Ccu97H18 one allele was dominant at low elevations, which was replaced at higher elevations by an increasing number of other alleles. The level of genetic differentiation at neutral loci was similar to that of other Fagaceae species (F_ST = 0.032,  = 0.15). Population differentiation followed a model of isolation by distance but additionally, strongly significant isolation by elevation was found, both for neutral loci and the putatively selected locus.

Conclusions/Significance

The results indicate higher gene flow among similar elevational levels than across different elevational levels and suggest a selective influence of elevation on the distribution of genetic diversity in C. eyrei. The study underlines the importance to check the selective neutrality of marker loci in analyses of population structure.     

Strong genetic differentiation between Gymnadenia conopsea and G. densiflora despite morphological similarity

The fragrant orchid Gymnadenia conopsea s.l. is a controversial taxon with two commonly distinguished species, G. conopsea s.str. and G. densiflora. Despite morphological similarity, differentiation between the taxa has been reported for several characters; however, character variation within taxa has obviated a clear consensus. We assessed ITS sequences, microsatellite variation and chromosome numbers on the European scale (1,420 samples) and conducted morphological analyses for 626 samples from Germany. ITS analysis revealed a 2% nucleotide divergence between the taxa, similar to the divergence between other Gymnadenia species. The ITS sequences of G. densiflora form a well-supported monophyletic group sharing a most recent common ancestor with G. nigra and G. austriaca. Thus, G. conopsea and G. densiflora are not sister species, and a species rank is supported for G. densiflora (Wahlenb.) Dietrich and G. conopsea (L.) R.Br. s.str. This was confirmed by the microsatellite analysis, which revealed a strong genetic differentiation between the taxa because of largely non-overlapping sets of alleles. Chromosome numbers showed that G. conopsea was either diploid or tetraploid, whereas G. densiflora was diploid throughout. Morphologically, the taxa differed significantly in the mean value of a number of diagnostic characters. However, a discriminant analysis showed that the morphological variability is substantial, and on the individual level an unequivocal assignment is not possible as 96% of G. conopsea, but only 77% of G. densiflora could be assigned correctly. Further studies are needed on character variation within and among species and ploidy levels to allow for a better identification of the genetically differentiated but morphologically similar taxa.