On-site genetic analysis for species identification using lab-on-a-chip.

This paper presents a microfluidic device capable of performing genetic analysis on dung samples to identify White Rhinoceros (Ceratotherium simum). The development of a microfluidic device, which can be used in the field, offers a portable and cost-effective solution for DNA analysis and species identification to aid conservation efforts. Optimization of the DNA extraction processes produced equivalent yields compared to conventional kit-based methods within just 5 minutes. The use of a color-changing loop-mediated isothermal amplification reaction for simultaneous detection of the cytochrome B sequence of C. simum enabled positive results to be obtained within as little as 30 minutes. Field testing was performed at Knowsley Safari to demonstrate real-world applicability of the microfluidic device for testing of biological samples.

Gut Microbial Predictors of Type 2 Diabetes Remission Following Bariatric Surgery.

PURPOSE: Distinct anatomical rearrangements of the gastrointestinal tract achieved by various types of bariatric surgery cause changes in nutrient intake and gut microbiota. The contribution of such gut microbiota changes to remission of type 2 diabetes (T2D) remains unclear. AIM: We examined gut microbiota changes following banded Roux-en-Y gastric bypass (RYGB) or sleeve gastrectomy (SG) in a randomised study, in relation to T2D remission. MATERIALS AND METHODS: Whole-metagenome shotgun sequencing was carried out on paired stool samples at pre- and 1-year post-surgery collected from 44 participants with T2D randomised to banded Roux-en-Y gastric bypass (RYGB) or sleeve gastrectomy (SG). Taxonomic composition and predicted functional potential of the gut bacteria were identified using HUMANn2, and annotated using MetaCyc. Five-day dietary records (analysed using FoodWorks v8.0), body weight and diabetes status were recorded at both time points. RESULTS: RYGB participants had higher percentage excess weight loss than SG (p = 0.01), even though dietary intake was similar at 1-year post-surgery. Similar proportions achieved diabetes remission (HbA1c < 48 mmol/mol without medications) after either RYGB (68%) or SG (59%). RYGB resulted in increased abundances of Firmicutes and Proteobacteria, while SG resulted in increased Bacteroidetes. Pre-surgery, an increased abundance of Eubacteriaceae (p = 0.01) and Alistipes putredinis (p = 0.01) was observed in those who went on to remit from T2D post-surgery. Following surgery, Lachnospiraceae (p = 0.04) and Roseburia (p = 0.01) species were more abundant in those who had achieved T2D remission. CONCLUSIONS: Specific stool bacterial taxa may signal likelihood of T2D remission after bariatric surgery which is potentially mediated by increases in Lachnospiraceae and Roseburia.

Fibroblast growth factor 23 levels decline following sleeve gastrectomy.

OBJECTIVE: Levels of fibroblast growth factor 23 (FGF23) have been positively associated with measures of adiposity, cardiovascular disease and mortality. It is unclear whether the relationship of FGF23 with cardiovascular disease and mortality is confounded by obesity. We aimed to determine whether FGF23 concentrations decline following a reduction in adiposity after sleeve gastrectomy (SG). DESIGN: The effect of SG on FGF23 was evaluated in 22 obese adults (59% male) with type 2 diabetes. Fat mass, weight, BMI, plasma intact FGF23, parathyroid hormone (PTH) and leptin were determined at baseline and at 12 months following SG. RESULTS: At baseline, median (IQR) age was 51 (43-54) years, fat mass 47.8 (41.0-59.4) kg, BMI 40.9 (36.9-46.9) kg/m2 and FGF23 66.2 (55.3-82.9) pg/mL. Significant changes in median BMI (-10.8 kg/m2 , 95% CI: -12.9 to -7.2, P < 0.0001), fat mass (-20.0 kg, 95% CI: -26.7 to -12.4, P < 0.0001) and weight (-34.7 kg, 95% CI -40.0 to -23.1, P < 0.0001) were observed after SG. FGF23 (-12.4 pg/mL, 95% CI: -19.5 to 2.0, P = 0.005), PTH (-1.1 pmol/L, 95% CI: -1.7 to 0.2, P = 0.009) and leptin (-1687 pg/mL, 95% CI -4524 to -563, P = 0.01) declined following SG. Change in FGF23 was not significantly associated with change in measures of adiposity, PTH or leptin. CONCLUSIONS: FGF23 concentrations decline in the setting of significant weight loss following SG, implying that increased FGF23 concentrations are a downstream consequence of obesity, which may confound its association with cardiometabolic dysfunction. Mediators of the relationship between adiposity and FGF23 require further elucidation.

Altered gut microbiome after bariatric surgery and its association with metabolic benefits: A systematic review.

Bariatric surgery is currently the recommended therapy for significant weight reduction and remission of type 2 diabetes. Different types of bariatric surgery result in dramatic changes to gut bacteria but the contribution of such changes to the metabolic benefits achieved is still unclear. This systematic review of 14 clinical studies, incorporating 222 participants (146 patients with Roux-en-Y gastric bypass, 25 with sleeve gastrectomy, 30 with biliointestinal bypass, 7 with vertical banded gastroplasty, and 14 with an adjustable gastric band) reveals generally increased microbial diversity and gene richness after surgery. Major species-level changes include a decrease in the relative abundance of Faecalibacterium prausnitzii and increase in Escherichia coli. Decreases in the relative abundance of Firmicutes after sleeve gastrectomy and increases in Bacteroidetes and Proteobacteria after Roux-en-Y gastric bypass were seen. Microbial changes after surgery are discussed in the context of potential confounding effects of baseline diet, medications, and type 2 diabetes, with recommendations to consider these factors in future studies, to identify potentially causal associations with observed metabolic benefits.

Genome editing of Clostridium autoethanogenum using CRISPR/Cas9.

BACKGROUND: Impactful greenhouse gas emissions abatement can now be achieved through gas fermentation using acetogenic microbes for the production of low-carbon fuels and chemicals. However, compared to traditional hosts like Escherichia coli or yeast, only basic genetic tools exist for gas-fermenting acetogens. To advance the process, a robust genetic engineering platform for acetogens is essential. RESULTS: In this study, we report scarless genome editing of an industrially used model acetogen, Clostridium autoethanogenum, using the CRISPR/Cas9 system. Initial efforts to retrofit the CRISPR/Cas9 system for C. autoethanogenum resulted in poor efficiency likely due to uncontrolled expression of Cas9. To address this, we constructed and screened a small library of tetracycline-inducible promoters that can also be used to fine-tune gene expression. With a new inducible promoter, the efficiency of CRISPR/Cas9-mediated desired gene deletion in C. autoethanogenum was improved to over 50 %, making it a viable tool for engineering C. autoethanogenum. CONCLUSIONS: Addition of both an inducible promoter library and a scarless genome editing tool is an important expansion to the genetic tool box of industrial C. autoethanogenum strain.

The feasibility of the interferon gamma release assay and predictors of discordance with the tuberculin skin test for the diagnosis of latent tuberculosis infection in a remote Aboriginal community.

BACKGROUND: The tuberculin skin test (TST) is the standard test used to screen for latent TB infection (LTBI) in the northern Canadian territory of Nunavut. Interferon gamma release assays (IGRA) are T cell blood-based assays to diagnose LTBI. The Bacillus Calmette-Guerin (BCG) vaccine is part of the routine immunization schedule in Nunavut. The objective of this study was to test the feasibility, and predictors of discordance between the Tuberculin Skin Test (TST) and the IGRA assay in a medically under-serviced remote arctic Aboriginal population. METHODS: Both the TST and QuantiFERON-TB Gold (Qiagen group) IGRA tests were offered to people in their homes as part of a public health campaign aimed at high TB risk residential areas in Iqaluit, Nunavut, Canada. Feasibility was measured by the capacity of the staff to do the test successfully as measured by the proportion of results obtained. RESULTS: In this population of predominantly young Inuit who were mostly BCG vaccinated, the use of IGRA for the diagnosis of LTBI was feasible. IGRA testing resulted in more available test results reaching patients (95.6% vs 90.9% p = 0.02) but took longer (median 8 days (IGRA) vs 2 days (TST), p value < 0.0001). 44/256 participants (17.2%) had discordant results. Multivariable regression analysis suggested that discordant results were most likely to have received multiple BCG vaccinations (RR 20.03, 95% CI, 3.94-101.82)), followed by BCG given post infancy (RR 8.13, 95% CI, 2.54-26.03)) and then to a lesser degree when BCG was given in infancy (RR 6.43, 95% CI, 1.72-24.85). INTERPRETATION: IGRA is feasible in Iqaluit, Nunavut, a remote Arctic community. IGRA testing results in more test results available to patients compared to TST. This test could result in fewer patients requiring latent TB treatment among those previously vaccinated with BCG in a region with limited public health human resources.

Taima (stop) TB: the impact of a multifaceted TB awareness and door-to-door campaign in residential areas of high risk for TB in Iqaluit, Nunavut.

BACKGROUND: The incidence rate of active tuberculosis (TB) disease in the Canadian Territory of Nunavut has shown a rising trend over the past 10 years. In 2010 it was 60 times greater than the national incidence rate. The objective of the Taima (translates to "stop" in Inuktitut) TB study was to implement and evaluate a public health campaign to enhance existing TB prevention efforts in Nunavut. METHODS: A TB awareness campaign followed by a door-to-door screening campaign was carried out in Iqaluit, Nunavut. The aim of the campaign was to raise awareness about TB, and to provide in-home screening and treatment for people living in residential areas at high risk for TB. Screening was based on geographic location rather than on individual risk factors. RESULTS: During the general awareness campaign an increase in the number of people who requested TB testing at the local public health clinic was observed. However, this increase was not sustained following cessation of the awareness campaign. Targeted TB screening in high risk residential areas in Iqaluit resulted in 224 individuals having TSTs read, and detection of 42 previously unidentified cases of latent TB, (overall yield of 18.8% or number needed to screen = 5.3). These cases of latent TB infection (LTBI) were extra cases that had not been picked up by traditional screening practices (34% relative increase within the community). This resulted in a 33% relative increase in the completion of LTBI treatment within the community. The program directly and indirectly identified 5/17 new cases of active TB disease in Iqaluit during the study period (29.5% of all incident cases). CONCLUSIONS: While contact tracing investigations remain a cornerstone of TB prevention, additional awareness, screening, and treatment programs like Taima TB may contribute to the successful control of TB in Aboriginal communities.