Understanding the Mechanism and Extent of vlsE Recombination in Borrelia burgdorferi using Next-Generation Sequencing

B. burgdorferi, the causative agent of Lyme disease, have an elaborate antigenic variation system that involves varying the sequence of vlsE. Previous studies have shown that vlsE antigenic variation occurs continuously inside mammalian hosts. Variation has not been shown previously to occur in in vitro or in ticks. We hypothesized that the induction of vlsE recombination requires contact with dense arrays of host tissue cells and/or ECM components. To test this hypothesis, two methods, quantitative PCR and high-throughput sequencing were used determine the extent and nature of vlsE recombination within mouse tissues and in vitro model systems. Using these approaches, we were able to detect vlsE variants in axenic cultures of B. burgdorferi as well as co-cultures with mouse skin and heart tissues; these results were compared with those from mice infected for 7 days. Analysis of PacBio single molecule real-time (SMRT) sequencing indicated the presence of 0.84% to 1.18% variants in pure in vitro cultures and 0.79% to 1.22% in tissue explants, as compared 36% to 57% for organisms from mouse bladder tissue 7 days post inoculation. Statistical evaluation of the variants showed that the rate of recombination in tissue explants was not significantly different from the rate of recombination in in vitro cultures. Thus, tissue explant co-cultures do not seem to promote a higher recombination rate than in in vitro axenic culture. Moreover, high-throughput PacBio sequencing was found to be an effective means of analyzing single molecule sequencing variation in the robust vlsE antigenic variation system

Effect of oral contraceptives on tear film in reproductive age group women

Background: According to WHO 2009, 60-70% women use one of the method of contraception. 8.8% to 15.4% women use oral contraceptives. Objective of present study was to investigate the effect of oral contraceptives on androgen profile and tear film parameters in females within child bearing age.Methods: Present study involve 100 healthy women between 20-45 years, who presented in family planning clinic. Two groups were made according to the use of oral contraceptive pills. Study group consist of 50 women who were on OCPs (Oral contraception pills) and 50 as control group were not taking any hormonal contraceptives. Serum testosterone and DHEA levels of all subjects was done by Enzyme Immuno Assay on blood samples which were collected during 3th to 7th day of menstrual cycle. Quantitative test for tear secretion was done by Schirmer’s test. Stability of tear film was measured by Tear Breakup time (TBUT). Statistical analysis was done to determine the correlation between use of OCPs and androgen profile and tear film parameters.Results: Present results shows decreased androgen levels in women taking oral contraceptives as compared with age matched women who were not taking oral contraceptives. Tear secretion was significantly reduced in study group as indicated by decreased Schirmer’s test values in study group as compared to control group, the tear film stability was also significantly decreased in women taking oral contraceptives.Conclusions: Present study suggest that androgen profile decrease in women taking oral contraceptives. These results support that use of oral contraceptives may be an important etiological factor in pathogenesis of dry eye disease reproductive age group women

Effect of oral contraceptives on tear film in reproductive age group women

Background: According to WHO 2009, 60-70% women use one of the method of contraception. 8.8% to 15.4% women use oral contraceptives. Objective of present study was to investigate the effect of oral contraceptives on androgen profile and tear film parameters in females within child bearing age.Methods: Present study involve 100 healthy women between 20-45 years, who presented in family planning clinic. Two groups were made according to the use of oral contraceptive pills. Study group consist of 50 women who were on OCPs (Oral contraception pills) and 50 as control group were not taking any hormonal contraceptives. Serum testosterone and DHEA levels of all subjects was done by Enzyme Immuno Assay on blood samples which were collected during 3th to 7th day of menstrual cycle. Quantitative test for tear secretion was done by Schirmer’s test. Stability of tear film was measured by Tear Breakup time (TBUT). Statistical analysis was done to determine the correlation between use of OCPs and androgen profile and tear film parameters.Results: Present results shows decreased androgen levels in women taking oral contraceptives as compared with age matched women who were not taking oral contraceptives. Tear secretion was significantly reduced in study group as indicated by decreased Schirmer’s test values in study group as compared to control group, the tear film stability was also significantly decreased in women taking oral contraceptives.Conclusions: Present study suggest that androgen profile decrease in women taking oral contraceptives. These results support that use of oral contraceptives may be an important etiological factor in pathogenesis of dry eye disease reproductive age group women

Direct detection of Mycobacterium tuberculosis rifampin resistance in bio-safe stained sputum smears.

Direct smear microscopy of sputum forms the mainstay of TB diagnosis in resource-limited settings. Stained sputum smear slides can serve as a ready-made resource to transport sputum for molecular drug susceptibility testing. However, bio-safety is a major concern during transport of sputum/stained slides and for laboratory workers engaged in processing Mycobacterium tuberculosis infected sputum specimens. In this study, a bio-safe USP (Universal Sample Processing) concentration-based sputum processing method (Bio-safe method) was assessed on 87 M. tuberculosis culture positive sputum samples. Samples were processed for Ziehl-Neelsen (ZN) smear, liquid culture and DNA isolation. DNA isolated directly from sputum was subjected to an IS6110 PCR assay. Both sputum DNA and DNA extracted from bio-safe ZN concentrated smear slides were subjected to rpoB PCR and simultaneously assessed by DNA sequencing for determining rifampin (RIF) resistance. All sputum samples were rendered sterile by Bio-safe method. Bio-safe smears exhibited a 5% increment in positivity over direct smear with a 14% increment in smear grade status. All samples were positive for IS6110 and rpoB PCR. Thirty four percent samples were RIF resistant by rpoB PCR product sequencing. A 100% concordance (κ value = 1) was obtained between sequencing results derived from bio-safe smear slides and bio-safe sputum. This study demonstrates that Bio-safe method can address safety issues associated with sputum processing, provide an efficient alternative to sample transport in the form of bio-safe stained concentrated smear slides and can also provide information on drug (RIF) resistance by direct DNA sequencing

Butyrate-producing human gut symbiont, Clostridium butyricum, and its role in health and disease

Clostridium butyricum is a butyrate-producing human gut symbiont that has been safely used as a probiotic for decades. C. butyricum strains have been investigated for potential protective or ameliorative effects in a wide range of human diseases, including gut-acquired infection, intestinal injury, irritable bowel syndrome, inflammatory bowel disease, neurodegenerative disease, metabolic disease, and colorectal cancer. In this review we summarize the studies on C. butyricum supplementation with special attention to proposed mechanisms for the associated health benefits and the supporting experimental evidence. These mechanisms center on molecular signals (especially butyrate) as well as immunological signals in the digestive system that cascade well beyond the gut to the liver, adipose tissue, brain, and more. The safety of probiotic C. butyricum strains appears well-established. We identify areas where additional human randomized controlled trials would provide valuable further data related to the strains’ utility as an intervention

Workflow of the study showing timeline for sample collection and processing in this study.

<p>*Only <i>M</i>. <i>tuberculosis</i> culture positive samples were retrospectively selected for the current study.</p

Smear status of culture positive sputum samples.

<p>Smear status of culture positive sputum samples.</p

Direct, USP and bio-safe USP smear.

<p>USP and bio-safe USP smears show a clear background as compared to direct smear.</p

Direct detection of <i>rpoB</i> mutations in bio-safe sputum samples (n = 87) by DNA sequencing.

<p>Direct detection of <i>rpoB</i> mutations in bio-safe sputum samples (n = 87) by DNA sequencing.</p