Biology subjects

Banerjee, R.

Publications and source records attributed to Banerjee, R..

80 records · Page 5Linked to original sources

bioRxiv2020

Isolation, identification and functional characterization of cultivable bacteria from Arabian Sea and Bay of Bengal water samples reveals high diversity

The oxygen minimum zone of the Arabian Sea (AS) and Bay of Bengal (BOB) is rich in organic matter and is an unusual niche. Bacteria present in the oceanic water play an important role in ecology since they are responsible for decomposing, mineralizing of organic matter and in elemental cycling like nitrogen, sulfur, phosphate. This study focuses on culturing bacteria from oxygen minimum zones (OMZ) and non-OMZ regions and their phylogenetic as well as the functional characterization. Genotypic characterization of the isolates using amplified rDNA based 16SrRNA sequencing grouped them into various phylogenetic groups such as alpha-proteobacteria, gamma-proteobacteria and unaffiliated bacteria. The cultivable bacterial assemblages encountered belonged to the genus Halomonas, Marinobacter, Idiomarina, Pshyctobacter and Pseudoalteromonas. Among the enzymatic activities, carbohydrate utilization activity was most predominant (100%) and microorganisms possessed amylase, cellulase, xylanase and chitinase. A large proportion of these bacteria (60%) were observed to be hydrocarbon consuming and many were resistant to ampicillin, chloramphenicol, kanamycin and streptomycin. The high diversity and high percentage of extracellular hydrolytic enzyme activities along with hydrocarbon degradation activity of the culturable bacteria reflects their important ecological role in oceanic biogeochemical cycling. Further assessment confirmed the presence of nitrogen reduction capability in these cultivable bacteria which highlights their importance in oceanic geochemical cycling.

microbiology↗

bioRxiv2020

Dismantling and rebuilding the trisulfide cofactor demonstrates its essential role in human sulfide quinone oxidoreductase

Sulfide quinone oxidoreductase (SQR) catalyzes the first step in sulfide clearance, coupling H2S oxidation to coenzyme Q reduction. Recent structures of human SQR revealed a sulfur atom bridging the SQR active site cysteines in a trisulfide configuration. Here, we assessed the importance of this cofactor using kinetic, crystallographic and computational modeling approaches. Cyanolysis of SQR proceeds via formation of an intense charge transfer complex that subsequently decays to eliminate thiocyanate. Cyanolysis leads to reversible loss of SQR activity, which is restored in the presence of sulfide. We captured a crystallographic intermediate in SQR that provides clues as to how the oxidized state of the cysteines is preserved. Computational modeling and MD simulations revealed an ~105-fold rate enhancement for nucleophilic addition of sulfide into the trisulfide versus a disulfide cofactor. The cysteine trisulfide in SQR is thus critical for activity and provides a significant catalytic advantage over a cysteine disulfide.

biochemistry↗

bioRxiv2020

Structural insights into loss of function of a pore forming toxin and its role in pneumococcal adaptation to an intracellular lifestyle

The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel inter-molecular cation-{pi} interaction, governing formation of the transmembrane {beta}-hairpins (TMH) in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal carriage, with loss of pore forming ability of Ply facilitating a benign association of SPN in an alternative, intracellular host niche. AUTHOR SUMMARYStreptococcus pneumoniae, the main causative agent of pneumonia, triggers inflammation and tissue damage by expressing a pore-forming toxin, pneumolysin (Ply). Ply-induced inflammation drives pneumococcal transmission from nasopharynx (its primary reservoir), but also contributes to host mortality, limiting its occupiable habitats. Here, we uncovered the structural basis for loss of pore-forming activity of a Ply variant, present in Serotype 1 ST306, and observed that this enabled adoption of an intracellular lifestyle, attenuating inflammatory responses and prolonging host tolerance of pneumococcus in the lower airways. This commensal-like lifestyle, resembling that of members of the mitis group of Streptococci, might have evolved within ST306 by loss of function ply mutations, compensating for limited nasopharyngeal carriage capacity by facilitating adaptation to an alternate niche.

microbiology↗

bioRxiv2020

The hepatic compensatory response to elevated systemic sulfide impairs medium chain fat oxidation and promotes diabetes

Impaired hepatic glucose and lipid metabolism are hallmarks of type-2 diabetes. Increased sulfide production from cysteine, or sulfide-donor compounds, may beneficially regulate hepatic metabolism. Disposal of sulfide through the sulfide oxidation pathway (SOP) is critical for maintaining sulfide within a safe physiological range. We show that mice lacking the liver-enriched mitochondrial SOP enzyme thiosulfate sulfur-transferase (Tst-/- mice) exhibit high circulating sulfide, increased gluconeogenesis, hypertriglyceridemia and fatty liver, despite whole-body insulin-sensitisation. Unexpectedly, hepatic sulfide levels were normal in Tst-/- mice, a result of homeostatic induction of mitochondrial sulfide disposal and glutathione excretion associated with net suppression of protein persulfidation and nuclear respiratory factor-2 target proteins. Proteomic and persulfidomic profiling converged on gluconeogenesis and hepatic lipid metabolism and revealed a selective deficit in medium-chain fatty acid oxidation in Tst-/- mice. We reveal a critical role for TST in hepatic metabolism that raises implications for sulfide-donor strategies in the context of liver function and metabolic disease.

physiology↗

bioRxiv2019

A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye, and inhibitor-resistant high-fidelity DNA polymerase

Deoxyribonucleotide triphosphates (dNTPs) are vital for the biosynthesis and repair of DNA. Their cellular concentration peaks during the S phase of the cell cycle. In non-proliferating cells dNTP concentrations are low, making their reliable quantification from tissue samples of heterogeneous cellular composition challenging. Partly because of this, the current knowledge related to regulation of and disturbances in cellular dNTP concentrations derive from cell culture experiments with little corroboration at the tissue or organismal level. Here, we fill the methodological gap by presenting a simple non-radioactive microplate assay for the quantification of dNTPs with a minimum requirement of 10 to 30 mg of biopsy material. In contrast to published assays, this assay is based on long (~200 nucleotides) synthetic single-stranded DNA templates, an inhibitor-resistant high-fidelity DNA polymerase, and the double-stranded-DNA-binding EvaGreen dye. The assay quantifies reliably as little as 100 fmol of each of the four dNTPs. Importantly, the assay allowed measurement of minute dNTP concentrations in mouse liver, heart, and skeletal muscle.

molecular biology↗

bioRxiv2019

GSMN-ML- a genome scale metabolic network reconstruction of the obligate human pathogen Mycobacterium leprae

Leprosy, caused by Mycobacterium leprae, has plagued humanity for thousands of years and continues to cause morbidity, disability and stigmatization in two to three million people today. Although effective treatment is available, the disease incidence has remained approximately constant for decades so new approaches, such as vaccine or new drugs, are urgently needed for control. Research is however hampered by the pathogens obligate intracellular lifestyle and the fact that it has never been grown in vitro. Consequently, despite the availability of its complete genome sequence, fundamental questions regarding the biology of the pathogen, such as its metabolism, remain largely unexplored. In order to explore the metabolism of the leprosy bacillus with a long-term aim of developing a medium to grow the pathogen in vitro, we reconstructed an in silico genome scale metabolic model of the bacillus, GSMN-ML. The model was used to explore the growth and biomass production capabilities of the pathogen with a range of nutrient sources, such as amino acids, glucose, glycerol and metabolic intermediates. We also used the model to analyze RNA-seq data from M. leprae grown in mouse foot pads, and performed Differential Producibility Analysis (DPA) to identify metabolic pathways that appear to be active during intracellular growth of the pathogen, which included pathways for central carbon metabolism, co-factor, lipids, amino acids, nucleotides and cell wall synthesis. The GSMN-ML model is thereby a useful in silico tool that can be used to explore the metabolism of the leprosy bacillus, analyze functional genomic experimental data, generate predictions of nutrients required for growth of the bacillus in vitro and identify novel drug targets.\n\nAuthor SummaryMycobacterium leprae, the obligate human pathogen is uncultivable in axenic growth medium, and this hinders research on this pathogen, and the pathogenesis of leprosy. The development of novel therapeutics relies on the understanding of growth, survival and metabolism of this bacterium in the host, the knowledge of which is currently very limited. Here we reconstructed a metabolic network of M. leprae-GSMN-ML, a powerful in silico tool to study growth and metabolism of the leprosy bacillus. We demonstrate the application of GSMN-ML to identify the metabolic pathways, and metabolite classes that M. leprae utilizes during intracellular growth.

microbiology↗

bioRxiv2019

Functional linkage of gene fusions to cancer cell fitness assessed by pharmacological and CRISPR/Cas9 screening

Many gene fusions have been reported in tumours and for most their role remains unknown. As fusions can be used clinically for diagnostic and prognostic purposes, and are targets for treatment, it is crucial to assess their functional implications in cancer. To investigate the role of fusions in tumor cell fitness, we developed a systematic analysis utilising RNA-sequencing data from 1,011 human cancer cell lines to functionally link 8,354 gene fusion events with genomic data, sensitivity to >350 anti-cancer drugs and CRISPR-Cas9 loss-of-fitness information. Established clinically-relevant fusions were readily identified. Overall, functional fusions were rare, including those involving cancer driver genes, suggesting that many fusions are dispensable for tumor cell fitness. Novel therapeutically actionable fusions involving RAF1, BRD4 and ROS1 were verified in new histologies. In addition, recurrent YAP1-MAML2 fusions were identified as activators of Hippo-pathway signaling in multiple cancer types, supporting therapeutic targeting of Hippo signalling. Our approach discriminates functional fusions, identifying new drivers of carcinogenesis and fusions that could have important clinical implications. SignificanceWe identify fusions as new potential candidates for drug repurposing and drivers of carcinogenesis. These results support histology agnostic marker-driven precision cancer medicine. Most fusions are not functional with implications for interpreting cancer fusions reported from clinical sequencing studies.

cancer biology↗

bioRxiv2018

Non-invasive Assessment of Liver Disease in Rats Using Multiparametric Magnetic Resonance Imaging: A Feasibility Study

Background & AimsNon-invasive quantitation of chronic liver disease using multiparametric MRI has the potential to refine clinical care pathways, trial design and preclinical drug development. The aim of the study was to evaluate the use of multiparametric liver MRI in experimental rat models of chronic liver disease. Methods: Liver injury was induced in male wild-type Sprague-Dawley rats using 4 or 12 weeks carbon tetrachloride (CCl4) intoxication and 4 or 8 weeks methionine and choline deficient (MCD) diet. Liver MRI was performed using a 7.0 Tesla small animal scanner at baseline and specified timepoints after liver injury. Multiparametric liver MRI parameters (T1 mapping, T2* mapping and proton density fat fraction (PDFF)) were correlated with gold standard histopathological measures, assessed by a blinded expert adjudicator. One-way analysis of variance with Tukeys post-hoc test was used to assess differences between groups and Spearmans rank-order correlation to examine associations.\n\nResultsMean hepatic T1 increased significantly in rats treated with CCl4 for 12 weeks compared to controls (1122{+/-}78 ms vs. 959{+/-}114 ms; d=162.7, 95% CI (11.92, 313.4), P=0.038) and correlated strongly with histological collagen content (rs=0.717, P=0.037). In MCD diet-treated rats, hepatic PDFF correlated strongly with histological fat content (rs=0.819, P<0.0001), steatosis grade (rs=0.850, P<0.0001) and steatohepatitis score (rs=0.818, P<0.0001). Although there was minimal histological iron, progressive fat accumulation in MCD diet-treated liver significantly shortened T2*.\n\nConclusionsIn preclinical models, quantitative MRI markers correlated with histopathological assessments, especially for fatty liver disease. Validation in longitudinal studies is required.\n\nKey pointsO_LINon-invasive quantitative measures of chronic liver injury are needed for stratification and monitoring of disease in clinical practice and in drug development\nC_LIO_LIWe adapted and applied a clinically-relevant non-contrast multiparametric MRI protocol in well-established preclinical liver disease models, for simultaneous quantitation of hepatic fat, fibro-inflammatory injury and iron\nC_LIO_LIMultiparametric liver MRI was feasible at 7.0 Tesla in experimental rat models and imaging parameters correlated with gold standard histopathological assessments, especially characteristics of fatty liver disease\nC_LIO_LIFurther development of multiparametric liver MRI could refine drug development strategies and impact upon trial design and clinical care pathways\nC_LI

pathology↗