The Mycobacterium tuberculosis KatG gene : identification of a novel function and analysis of the regulation of expression

A clone containing the terminal third of the Mycobacterium tuberculosis katG gene was previously shown to confer resistance to ethyl methane sulfonate on DNA repair-deficient Escherichia coli cells. The first aim of this study, therefore, was to examine the role played by the M tuberculosis katG gene in DNA repair. The strategy used was overexpression of different regions of the gene in DNA repair-deficient mutants of E. coli, and examination of the sensitivities of the transformants to DNA damaging agents. Overexpression of the gene resulted in an increase in the survival of recA mutants exposed to ultraviolet (UV) light irradiation (254 run) and hydrogen peroxide, and uvr mutants exposed to mitomycin C. Both the 5' and 3' regions of the M tuberculosis KatG protein conferred the above effects, and this was independent of the catalase or peroxidase activity of the enzyme. The results suggest that the M tuberculosis katG gene may encode a novel function related to the repair of DNA damage, and this may have implications for the survival of M tuberculosis in the presence of DNA damaging agents, for example, in the macrophage. UV sensitivity tests on M intracellulare and M tuberculosis strains mutant in katG revealed that the katG gene product does not play a demonstrable role in the survival of repair-competent mycobacterial cells after exposure to UV irradiation. The second aim of this study was to examine the regulation of expression of the M tuberculosis katG gene. An E. coli-mycobacterial shuttle vector, pJCluc, containing the luciferase reporter gene, was constructed and used to examine the katG promoter sequences. The region required for optimal expression in M. smegmatis was localized to a 559 hp fragment immediately upstream of the gene. Two transcription start sites were mapped and putative -10 and -35 promoter sequences identified. It was demonstrated that expression from the promoter peaks during late exponential phase, and declines during stationary phase, and that the promoter is induced by ascorbic acid, and is repressed by oxygen limitation and growth at elevated temperatures. An upstream element that increased expression from the M. tuberculosis katG and the M. paratuberculosis PAN promoters was identified, and shown to bind to one or more M smegma/is proteins. Similar results were obtained in M bovis BCG. Understanding the regulation of gene expression in mycobacteria is essential for determining the processes that govern interaction with the host. This study provides information on both the mycobacterial transcription signals and gene regulatory mechanisms

The Effect of Electronic Cigarette User Modifications and E-liquid Adulteration on the Particle Size Profile of an Aerosolized Product

Electronic cigarettes (e-cigarettes) are an alternate nicotine delivery system that generate a condensation aerosol to be inhaled by the user. The size of the droplets formed in the aerosol can vary and contributes to drug deposition and ultimate bioavailability in the lung. The growing popularity of e-cigarette products has caused an increase in internet sources promoting the use of drugs other than nicotine (DOTNs) in e-cigarettes. The purpose of this study was to determine the effect of various e-cigarette and e-liquid modifications, such as coil resistance, battery voltage, and glycol and drug formulation, on the aerosol particle size. E-liquids containing 12 mg/mL nicotine prepared in glycol compositions of 100% propylene glycol (PG), 100% vegetable glycerin (VG), or 50:50 PG:VG were aerosolized at three voltages and three coil resistances. Methamphetamine and methadone e-liquids were prepared at 60 mg/mL in 50:50 PG:VG and all e-liquids were aerosolized onto a 10 stage Micro-Orifice Uniform Deposit Impactor. Glycol deposition correlated with drug deposition, and the majority of particles centered between 0.172–0.5 μm in diameter, representing pulmonary deposition. The 100% PG e-liquid produced the largest aerosol particles and the 100% VG and 50:50 PG:VG e-liquids produced ultra-fine particles \u3c0.3 μm. The presence of ultrafine particles indicates that drugs can be aerosolized and reach the pulmonary alveolar regions, highlighting a potential for abuse and risk of overdose with DOTNs aerosolized in an e-cigarette system

Behavioral and neural responses to social rejection: Individual differences in developmental trajectories across childhood and adolescence

Dealing with social rejection is challenging, especially during childhood when behavioral and neural responses to social rejection are still developing. In the current longitudinal study, we used a Bayesian multilevel growth curve model to describe individual differences in the development of behavioral and neural responses to social rejection in a large sample (n > 500). We found a peak in aggression following negative feedback (compared to neutral feedback) during late childhood, as well as individual differences during this developmental phase, possibly suggesting a sensitive window for dealing with social rejection across late childhood. Moreover, we found evidence for individual differences in the linear development of neural responses to social rejection in our three brain regions of interest: The anterior insula, the medial prefrontal cortex, and the dorsolateral prefrontal cortex. In addition to providing insights in the individual trajectories of dealing with social rejection during childhood, this study also makes a meaningful methodological contribution: Our statistical analysis strategy (and online supplementary information) can be used as an example on how to take into account the many complexities of developmental neuroimaging datasets, while still enabling researchers to answer interesting questions about individual-level relationships

The effects of dapagliflozin on urinary metabolites in patients with type 2 diabetes

AIMS: Previously, a panel of 13 urinary metabolites linked to mitochondrial metabolism was found to be significantly reduced in patients with diabetic kidney disease and eGFR>60 ml/min/1.73m2 . The beneficial effects of SGLT-2 inhibition on cardio-renal outcomes are hypothesized in part due to improved work efficiency at the mitochondrial level. We therefore assessed the effects of the SGLT-2 inhibitor dapagliflozin, on this pre-specified panel of 13 urinary metabolites linked to mitochondrial metabolism in patients with type 2 diabetes and elevated albuminuria. MATERIALS AND METHODS: Urine and plasma samples were used from a double-blind, randomized, placebo controlled crossover trial in 31 patients with type 2 diabetes, albumin:creatinine ratio >100 mg/g, and on a stable dose of an Angiotensin Converting Enzyme inhibitor (ACEi) or angiotensin receptor blocker (ARB). Dapagliflozin or placebo treatment periods each lasted for 6 weeks, with 6 weeks wash-out in between. Urinary and plasma metabolites were quantified by gas-chromatography mass spectrometry, corrected for creatinine, and then combined into a single-valued urinary metabolite index. Fractional excretion of the metabolites was calculated. RESULTS: All 13 urinary metabolites were detectable. After 6 weeks of dapagliflozin therapy, nine of the 13 metabolites were significantly increased from baseline. The urinary metabolite index increased by 42% (95%CI: 8.5 - 85.6, p=0.01) with placebo compared to 121% (69 - 189, p<0.001) with dapaglifozin. Accordingly, the placebo-adjusted effect was 56% (11 - 118, p=0.012). In plasma, seven of the 13 metabolites were detectable, and none were modified by dapagliflozin. CONCLUSIONS: Dapagliflozin significantly increased a panel of urinary metabolites previously linked to mitochondrial metabolism. These data support the hypothesis that SGLT-2 inhibitors may improve mitochondrial function, and improvements in mitochondrial function may be a mechanism for kidney protection. Future studies of longer treatment duration and clinical outcomes are needed to confirm the clinical impact of these findings. This article is protected by copyright. All rights reserved

Peer review as alearning tool

In recent years it has become quite common to use a routine research practice within our classrooms: that of peer review. Involving students in peer review is often regarded as an effective way to help students improve the quality of their assignment work before it is submitted. The peer review process offers many other benefits to students: an opportunity to reflect on their work in the light of the work of others; to observe the quality and scope of other students' work; to critically evaluate a piece of work and construct appropriate and constructive feedback; to develop critical thinking, higher-order cognitive, negotiation, and diplomacy skills. However, one benefit less often acknowledged is the impact that the peer review process can have on students' learning. In this presentation we will report on two case studies that illustrate how peer review improved the conceptual understanding of students. One study was a formal research study that assessed the effect of peer review on the performance of third year zoology students. The other less formal study describes students' self-reported conceptual gains in a first year physics subject

Determining ancestry proportions in complex admixture scenarios in South Africa using a novel proxy ancestry selection method

Admixed populations can make an important contribution to the discovery of disease susceptibility genes if the parental populations exhibit substantial variation in susceptibility. Admixture mapping has been used successfully, but is not designed to cope with populations that have more than two or three ancestral populations. The inference of admixture proportions and local ancestry and the imputation of missing genotypes in admixed populations are crucial in both understanding variation in disease and identifying novel disease loci. These inferences make use of reference populations, and accuracy depends on the choice of ancestral populations. Using an insufficient or inaccurate ancestral panel can result in erroneously inferred ancestry and affect the detection power of GWAS and meta-analysis when using imputation. Current algorithms are inadequate for multi-way admixed populations. To address these challenges we developed PROXYANC, an approach to select the best proxy ancestral populations. From the simulation of a multi-way admixed population we demonstrate the capability and accuracy of PROXYANC and illustrate the importance of the choice of ancestry in both estimating admixture proportions and imputing missing genotypes

Baseline urinary metabolites predict albuminuria response to spironolactone in type 2 diabetes

The mineralocorticoid receptor antagonist spironolactone significantly reduces albuminuria in subjects with diabetic kidney disease, albeit with a large variability between individuals. Identifying novel biomarkers that predict response to therapy may help to tailor spironolactone therapy. We aimed to identify a set of metabolites for prediction of albuminuria response to spironolactone in subjects with type 2 diabetes. Systems biology molecular process analysis was performed a priori to identify metabolites linked to molecular disease processes and drug mechanism of action. Individual subject data and urine samples were used from 2 randomized placebo controlled double blind clinical trials (NCT01062763, NCT00381134). A urinary metabolite score was developed to predict albuminuria response to spironolactone therapy using penalized ridge regression with leave-one-out cross validation. Bioinformatic analysis identified a set of 18 metabolites linked to a diabetic kidney disease molecular model and potentially affected by spironolactone mechanism of action. Spironolactone reduced UACR relative to placebo by median -42% (25th to 75% percentile -65 to 6) and -29% (25th to 75% percentile -37 to -1) in the test and replication cohorts, respectively. In the test cohort, UACR reduction was higher in the lowest tertile of the baseline urinary metabolite score compared with middle and upper tertiles -58% (25th to 75% percentile -78 to 33), -28% (25th to 75% percentile -46 to 8), -40% (25th to 75% percentile -52% to 31), respectively, P= 0.001 for trend). In the replication cohort, UACR reduction was -54% (25th to 75% percentile -65 to -50), -41 (25th to 75% percentile -46% to 30), and -17% (25th to 75% percentile -36 to 5), respectively, P= 0.010 for trend). We identified a set of 18 urinary metabolites through systems biology to predict albuminuria response to spironolactone in type 2 diabetes. These data suggest that urinary metabolites may be used as a tool to tailor optimal therapy and move in the direction of personalized medicine

High throughput surface plasmon resonance imaging method for clinical detection of presence and strength of binding of IgM, IgG and IgA antibodies against SARS-CoV-2 during CoViD-19 infection

Surface Plasmon Resonance imaging (SPRi) was used to determine the presence and strength of binding of IgG, IgM and IgA against the Receptor Binding Domain (RBD) of SARS-CoV-2 in sera of 102 CoViD-19 and non-CoViD-19 patients. The SPRi assay simultaneously measures the antibody isotype levels and the strength of binding to the RBD of ultimate 384 patient samples in one run. It turns out that during the course of the disease, the IgG levels and strength of binding increased while generally the IgM and IgA levels go down. Recovered patients all show high strength of binding of the IgG type to the RBD protein. The anti-RBD immunoglobulins SPRi assay provides additional insights in the immune status of patients recovering from CoViD-19. This new high throughput method can be applied for the assessment of the quality of the immune reaction of healthy individuals to SARS-CoV-2 and its mutants in vaccination programs.•Surface Plasmon Resonance imaging is an unprecedented technology for high throughput screening of antibody profiling of CoViD19 patients.•Fingerprinting of isotypes IgM, IgG and IgA can be performed for 384 patients in one run.•An affinity maturation effect was shown for patients recovering from CoViD19

The development and application of bioinformatics core competencies to improve bioinformatics training and education

Bioinformatics is recognized as part of the essential knowledge base of numerous career paths in biomedical research and healthcare. However, there is little agreement in the field over what that knowledge entails or how best to provide it. These disagreements are compounded by the wide range of populations in need of bioinformatics training, with divergent prior backgrounds and intended application areas. The Curriculum Task Force of the International Society of Computational Biology (ISCB) Education Committee has sought to provide a framework for training needs and curricula in terms of a set of bioinformatics core competencies that cut across many user personas and training programs. The initial competencies developed based on surveys of employers and training programs have since been refined through a multiyear process of community engagement. This report describes the current status of the competencies and presents a series of use cases illustrating how they are being applied in diverse training contexts. These use cases are intended to demonstrate how others can make use of the competencies and engage in the process of their continuing refinement and application. The report concludes with a consideration of remaining challenges and future plans