Comparison of Storage Conditions for Human Vaginal Microbiome Studies

BACKGROUND: The effect of storage conditions on the microbiome and metabolite composition of human biological samples has not been thoroughly investigated as a potential source of bias. We evaluated the effect of two common storage conditions used in clinical trials on the bacterial and metabolite composition of the vaginal microbiota using pyrosequencing of barcoded 16S rRNA gene sequencing and (1)H-NMR analyses. METHODOLOGY/PRINCIPAL FINDINGS: Eight women were enrolled and four mid-vaginal swabs were collected by a physician from each woman. The samples were either processed immediately, stored at -80°C for 4 weeks or at -20°C for 1 week followed by transfer to -80°C for another 4 weeks prior to analysis. Statistical methods, including Kolmogorovo-Smirnov and Wilcoxon tests, were performed to evaluate the differences in vaginal bacterial community composition and metabolites between samples stored under different conditions. The results showed that there were no significant differences between samples processed immediately after collection or stored for varying durations. (1)H-NMR analysis of the small molecule metabolites in vaginal secretions indicated that high levels of lactic acid were associated with Lactobacillus-dominated communities. Relative abundance of lactic acid did not appear to correlate with relative abundance of individual Lactobacillus sp. in this limited sample, although lower levels of lactic acid were observed when L. gasseri was dominant, indicating differences in metabolic output of seemingly similar communities. CONCLUSIONS/SIGNIFICANCE: These findings benefit large-scale, field-based microbiome and metabolomic studies of the vaginal microbiota

Highly Precise and Developmentally Programmed Genome Assembly in Paramecium Requires Ligase IV–Dependent End Joining

During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5′ overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi–mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5′-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3′ ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a “cut-and-close” mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms involved in genome dynamics

Longitudinal Study of the Dynamics of Vaginal Microflora during Two Consecutive Menstrual Cycles

Although the vaginal microflora (VMF) has been well studied, information on the fluctuation of the different bacterial species throughout the menstrual cycle and the information on events preceding the presence of disturbed VMF is still very limited. Documenting the dynamics of the VMF during the menstrual cycle might provide better insights. In this study, we assessed the presence of different Lactobacillus species in relation to the BV associated species during the menstrual cycle, assessed the influence of the menstrual cycle on the different categories of vaginal microflora and assessed possible causes, such as menstruation and sexual intercourse, of VMF disturbance. To our knowledge, this is the first longitudinal study in which swabs and Gram stains were available for each day of two consecutive menstrual cycles, whereby 8 grades of VMF were distinguished by Gram stain analysis, and whereby the swabs were cultured every 7(th) day and identification of the bacterial isolates was carried out with a molecular technique.status: publishe

Actin-interacting and flagellar proteins in Leishmania spp.: Bioinformatics predictions to functional assignments in phagosome formation

Several motile processes are responsible for the movement of proteins into and within the flagellar membrane, but little is known about the process by which specific proteins (either actin-associated or not) are targeted to protozoan flagellar membranes. Actin is a major cytoskeleton protein, while polymerization and depolymerization of parasite actin and actin-interacting proteins (AIPs) during both processes of motility and host cell entry might be key events for successful infection. For a better understanding the eukaryotic flagellar dynamics, we have surveyed genomes, transcriptomes and proteomes of pathogenic Leishmania spp. to identify pertinent genes/proteins and to build in silico models to properly address their putative roles in trypanosomatid virulence. In a search for AIPs involved in flagellar activities, we applied computational biology and proteomic tools to infer from the biological meaning of coronins and Arp2/3, two important elements in phagosome formation after parasite phagocytosis by macrophages. Results presented here provide the first report of Leishmania coronin and Arp2/3 as flagellar proteins that also might be involved in phagosome formation through actin polymerization within the flagellar environment. This is an issue worthy of further in vitro examination that remains now as a direct, positive bioinformatics-derived inference to be presented