Biology subjects

Mukherjee, M.

Publications and source records attributed to Mukherjee, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

The genome and population genomics of allopolyploid Coffea arabica reveal the diversification history of modern coffee cultivars

Coffea arabica, an allotetraploid hybrid of C. eugenioides and C. canephora, is the source of approximately 60% of coffee products worldwide, and its cultivated accessions have undergone several population bottlenecks. We present chromosome-level assemblies of a di-haploid C. arabica accession and modern representatives of its diploid progenitors, C. eugenioides and C. canephora. The three species exhibit largely conserved genome structures between diploid parents and descendant subgenomes, with no obvious global subgenome dominance. We find evidence for a founding polyploidy event 350,000-610,000 years ago, followed by several pre-domestication bottlenecks, resulting in narrow genetic variation. A split between wild accessions and cultivar progenitors occurred [~]30.5 kya, followed by a period of migration between the two populations. Analysis of modern varieties, including lines historically introgressed with C. canephora, highlights their breeding histories and loci that may contribute to pathogen resistance, laying the groundwork for future genomics-based breeding of C. arabica.

genomics↗

bioRxiv2023

Mild ER Stress Impedes Regulated Secretion By Governing Key Exocytotic and granulogenic Molecular Switches

Dense core vesicles (DCVs) and synaptic vesicles (SVs) are specialised secretory vesicles (SSVs) in neurons/neuroendocrine cells harbouring cargo whose abnormal release is associated with pathophysiology. Endoplasmic Reticulum (ER) stress and inter-organellar communication are also associated with disease biology. In pursuit of investigating the cell physiological consequences arising from the crosstalk of a stressed ER and DCVs, ER stress was modelled in PC12 neuroendocrine cells using Thapsigargin (Tg). DCV exocytosis was severely compromised in ER-stressed PC12 cells, reversed by Docosahexaenoic acid (DHA). Experiments with Tunicamycin(Tm), an independent ER stressor, yielded similar results. Concurrently, ER stress caused impaired DCV exocytosis also in INS-1 cells. Molecular analysis revealed blunted SNAP25 expression, potentially attributed to augmented levels of ATF4 (a well-known CREB inhibitor) and its transcriptional regulator CREB (also known to regulate key granulogenic players Chromogranin A, Secretogranin II). Our studies revealed severe defects in DCV exocytosis in ER-stressed cells for the first time, mediated by reduced levels of key exocytotic and granulogenic switches regulated via the CREB/ATF4/eIF2 axis.

cell biology↗

bioRxiv2023

Biotic Assessment of Crowdsourced Data Defines Four Ecoregions in Thar: A Novel Approach for Citizen Engagement in Conservation

Distinct ecosystems have representative biota that can allow delineation of ecoregions. Despite being one of the most populated desert ecosystems in the world with extreme climatic conditions, Thar has been classified as a single ecoregion by WWF. The present study aimed to test this on the basis of biotic assessment using a citizen science-based crowdsourced data from ebird (492 species of birds through 50,000 checklists of 4000 birders). Unsupervised clustering (k=4) and mathematical validation using cosine similarity revealed the presence of four distinct ecoregionsEastern Thar (ET), Western Thar (WT), Transitional Zone (TZ) and Cultivated Zone (CZ) in Thar. Most strikingly, these ecoregions when overlaid on geographical regions of Thar show that the CZ ecoregion was split between three distant and distinct geographic regions. Further spatial diversity estimates show CZ had the least diversity (273) and highest {beta} diversity (1.8) indicating the least similarity between the districts that comprised this ecoregion. The results suggest CZ as an evolving ecoregion or a consequence of habitat fragmentation due to anthropogenic effects. The presence of least number of exclusive species in CZ (0.2%) and TZ (0.8%) including the near-threatened species like Laticilla burnesii (Rufous-vented grass babbler) and Pelecanus philippensis (Spot-billed pelican) and an endangered species Athene blewitti (Forest Owlet) highlights the need for restoration of these threatened biota and ecoregions. This study for the first time proposes crowd-sourced bird data as an important biota-based tool to understand (a) established ecoregions in an ecosystem, (b) anthropogenic effects of agri-farmlands and (c) relation between geographic regions and biota.

ecology↗

bioRxiv2023

Right Ventricular Sarcomere Contractile Depression and the Role of Thick Filament Activation in Human Heart Failure with Pulmonary Hypertension

RationaleRight ventricular (RV) contractile dysfunction commonly occurs and worsens outcomes in heart failure patients with reduced ejection fraction and pulmonary hypertension (HFrEF-PH). However, such dysfunction often goes undetected by standard clinical RV indices, raising concerns that they may not reflect aspects of underlying myocyte dysfunction. ObjectiveTo determine components of myocyte contractile depression in HFrEF-PH, identify those reflected by clinical RV indices, and elucidate their underlying biophysical mechanisms. Methods and ResultsResting, calcium- and load-dependent mechanics were measured in permeabilized RV cardiomyocytes isolated from explanted hearts from 23 HFrEF-PH patients undergoing cardiac transplantation and 9 organ-donor controls. Unsupervised machine learning using myocyte mechanical data with the highest variance yielded two HFrEF-PH subgroups that in turn mapped to patients with depressed (RVd) or compensated (RVc) clinical RV function. This correspondence was driven by reduced calcium-activated isometric tension in RVd, while surprisingly, many other major myocyte contractile measures including peak power, maximum unloaded shortening velocity, and myocyte active stiffness were similarly depressed in both groups. Similar results were obtained when subgroups were first defined by clinical indices, and then myocyte mechanical properties in each group compared. To test the role of thick-filament defects, myofibrillar structure was assessed by X-ray diffraction of muscle fibers. This revealed more myosin heads associated with the thick filament backbone in RVd but not RVc, as compared to controls. This corresponded to reduced myosin ATP turnover in RVd myocytes, indicating less myosin in a cross-bridge ready disordered-relaxed (DRX) state. Altering DRX proportion (%DRX) affected peak calcium-activated tension in the patient groups differently, depending on their basal %DRX, highlighting potential roles for precision-guided therapeutics. Lastly, increasing myocyte preload (sarcomere length) increased %DRX 1.5-fold in controls but only 1.2-fold in both HFrEF-PH groups, revealing a novel mechanism for reduced myocyte active stiffness and by extension Frank-Starling reserve in human HF. ConclusionsWhile there are multiple RV myocyte contractile deficits In HFrEF-PH, clinical indices primarily detect reduced isometric calcium-stimulated force related to deficits in basal and recruitable %DRX myosin. Our results support use of therapies to increase %DRX and enhance length-dependent recruitment of DRX myosin heads in such patients.

biophysics↗

bioRxiv2022

The Alternate Ligand Jagged Enhances the Robustness of Notch Signaling Patterns

The Notch pathway, an example of juxtacrine signaling, is an evolutionary conserved cell-cell communication mechanism. It governs emergent spatiotemporal patterning in tissues during development, wound healing and tumorigenesis. Communication occurs when Notch receptors of one cell bind to either of its ligands, Delta/Jagged of neighboring cell. In general, Delta-mediated signaling drives neighboring cells to have an opposite fate (lateral inhibition) whereas Jagged-mediated signaling drives cells to maintain similar fates (lateral induction). Here, By deriving and solving a reduced set of 12 coupled ordinary differential equations for Notch-Delta-Jagged system on a hexagonal grid of cells, we determine the allowed states across different parameter sets. We also show that Jagged (at low dose) acts synergistically with Delta to enable more robust pattern formation, despite of its lateral induction property; this effect is due to competition with Delta over binding with Notch, as experimentally observed in the case of chick inner ear development. Finally, we show that how Jagged can help to expand the bistable (both Uniform and Hexagon phases are stable) region, where a local perturbation can spread over time in a ordered manner to create a biologically relevant, defect-free lateral inhibition pattern.

biophysics↗

bioRxiv2022

Infiltration of Tumor Spheroids by Activated Immune Cells

Recent years have seen a tremendous growth of interest in understanding the role that the adaptive immune system could play in interdicting tumor progression. In this context, it has been shown that the density of adaptive immune cells inside a solid tumor serves as a favorable prognostic marker across different types of cancer. The exact mechanisms underlying the degree of immune cell infiltration is largely unknown. Here, we quantify the temporal dynamics of the density profile of activated immune cells around a solid tumor spheroid. We propose a computational model incorporating immune cells with active, persistent movement and a proliferation rate that depends on the presence of cancer cells, and show that the model able to reproduce quantitatively the experimentally measured infiltration profile. Studying the density distribution of immune cells inside a solid tumor can help us better understand immune trafficking in the tumor micro-environment, hopefully leading towards novel immunotherapeutic strategies.

cancer biology↗

bioRxiv2022

A Well-Characterized Polycistronic-Like Gene Expression System in Yeast

Efficient expression of multiple genes is critical to yeast metabolic engineering because of the increasing complexity of engineered pathways. A yeast polycistronic expression system is of particular interest because one promoter can drive the expression of multiple genes. Polycistronic expression thus requires less genetic material and minimizes undesirable recombination due to repeated use of the same promoter. It also decreases the number of DNA parts necessary for cloning a multi-gene construct. 2A viral peptides enable the co-translation of multiple proteins from one mRNA by ribosomal skipping. However, the wide adaptation of this strategy for polycistronic-like gene expression in yeast awaits in-depth characterizations. Additionally, a one-step assembly of such a polycistronic-like system is highly desirable. To this end, we have developed a MoClo compatible 2A peptide-based polycistronic-like system capable of expressing multiple genes from a single promoter in yeast. Characterizing the bi-, tri, and quad-cistronic expression of fluorescent proteins showed high cleavage efficiencies of three 2A peptides. The expression level of each protein decreases as it moves away from the promoter. Additionally, the impact of a C-terminal 2A peptide on protein function is dependent on the protein sequence. Applying the polycistronic-like system for geraniol production resulted in similar or higher titers compared to a conventional monocistronic construct. In summary, this highly-characterized polycistronic-like gene expression system is another tool to facilitate multi-gene expression in yeast.

bioengineering↗

bioRxiv2021

Three-dimensional Cancer Cell Migration Directed by Dual Mechanochemical Guidance

Directed cell migration guided by external cues plays a central role in many physiological and pathophysiological processes. The microenvironment of cells often simultaneously contains various cues and the motility response of cells to multiplexed guidance is poorly understood. Here we combine experiments and mathematical models to study the three-dimensional migration of breast cancer cells in the presence of both contact guidance and a chemoattractant gradient. We find that the chemotaxis of cells is complicated by the presence of contact guidance as the microstructure of extracellular matrix (ECM) vary spatially. In the presence of dual guidance, the impact of ECM alignment is determined externally by the coherence of ECM fibers, and internally by cell mechanosensing Rho/Rock pathways. When contact guidance is parallel to the chemical gradient, coherent ECM fibers significantly increase the efficiency of chemotaxis. When contact guidance is perpendicular to the chemical gradient, cells exploit the ECM disorder to locate paths for chemotaxis. Our results underscores the importance of fully characterizing the cancer cell microenvironment in order to better understand invasion and metastasis.

biophysics↗

bioRxiv2021

Debunking the "junk": Unraveling the role of lncRNA-miRNA-mRNA networks in fetal hemoglobin regulation

Fetal hemoglobin (HbF) induction is considered to be a promising therapeutic strategy to ameliorate the clinical severity of {beta}-hemoglobin disorders, and has gained a significant amount of attention in recent times. Despite the enormous efforts towards the pharmacological intervention of HbF reactivation, progress has been stymied due to limited understanding of {gamma}-globin gene regulation. In this study, we intended to investigate the implications of lncRNA-associated competing endogenous RNA (ceRNA) interactions in HbF regulation. Probe repurposing strategies for extraction of lncRNA signatures and subsequent in silico analysis on publicly available datasets (GSE13284, GSE71935 and GSE7874) enabled us to identify 46 differentially expressed lncRNAs (DElncRNAs). Further, an optimum set of 11 lncRNAs that could distinguish between high HbF and normal conditions were predicted from these DElncRNAs using supervised machine learning and a stepwise selection model. The candidate lncRNAs were then linked with differentially expressed miRNAs and mRNAs to identify lncRNA-miRNA-mRNA ceRNA networks. The network revealed that 2 lncRNAs (UCA1 and ZEB1-AS1) and 4 miRNAs (hsa-miR-19b-3p,hsa-miR-3646,hsa-miR-937 and hsa-miR-548j) sequentially mediate cross-talk among different signaling pathways which provide novel insights into the lncRNA-mediated regulatory mechanisms, and thus lay the foundation of future studies to identify lncRNA-mediated therapeutic targets for HbF reactivation.

bioinformatics↗

bioRxiv2021

Cluster Size Distribution of Cells Disseminating from a Primary Tumor

The first stage of the metastatic cascade often involves motile cells emerging from a primary tumor either as single cells or as clusters. These cells enter the circulation, transit to other parts of the body and finally are responsible for growth of secondary tumors in distant organs. The mode of dissemination is believed to depend on the EMT nature (epithelial, hybrid or mesenchymal) of the cells. Here, we calculate the cluster size distribution of these migrating cells, using a mechanistic computational model, in presence of different degree of EMT-ness of the cells; EMT is treated as given rise to changes in their active motile forces () and cell-medium surface tension ({Gamma}). We find that, for ( > min, {Gamma} > 1), when the cells are hybrid in nature, the mean cluster size, [Formula], where min increases with increase in {Gamma}. For {Gamma} [≤] 0, [Formula], the cells behave as completely mesenchymal. In presence of spectrum of hybrid states with different degree of EMT-ness (motility) in primary tumor, the cells which are relatively more mesenchymal (higher ) in nature, form larger clusters, whereas the smaller clusters are relatively more epithelial (lower ). Moreover, the heterogeneity in is comparatively higher for smaller clusters with respect to that for larger clusters. We also observe that more extended cell shapes promote the formation of smaller clusters. Overall, this study establishes a framework which connects the nature and size of migrating clusters disseminating from a primary tumor with the phenotypic composition of the tumor, and can lead to the better understanding of metastasis. Author summaryIn the process of metastasis, tumor cells disseminate from the primary tumor either as single cells or multicellular clusters. These clusters are potential contributor to the initiation of secondary tumor in distant organs. Our computational model captures the size distribution of migrating clusters depending on the adhesion and motility of the cells (which determine the degree of their EMT nature). Furthermore, we investigate the effect of heterogeneity of cell types in the primary tumor on the resultant heterogeneity of cell types in clusters of different sizes. We believe that the understanding the formation and nature of these clusters, dangerous actors in the deadly aspect of cancer progression, will be useful for improving prognostic methods and eventually better treatments.

cancer biology↗

bioRxiv2020

Global cataloguing of variations in untranslated regions of viral genome and prediction of key host RNA binding protein-microRNA interactions modulating genome stability in SARS-CoV2

BackgroundThe world is going through the critical phase of COVID-19 pandemic, caused by human coronavirus, SARS-CoV2. Worldwide concerted effort to identify viral genomic changes across different sub-types has identified several strong changes in the coding region. However, there have not been many studies focusing on the variations in the 5 and 3 untranslated regions and their consequences. Considering the possible importance of these regions in host mediated regulation of viral RNA genome, we wanted to explore the phenomenon. MethodsTo have an idea of the global changes in 5 and 3-UTR sequences, we downloaded 8595 complete and high-coverage SARS-CoV2 genome sequence information from human host in FASTA format from Global Initiative on Sharing All Influenza Data (GISAID) from 15 different geographical regions. Next, we aligned them using Clustal Omega software and investigated the UTR variants. We also looked at the putative host RNA binding protein (RBP) and microRNA binding sites in these regions by RBPmap and RNA22 v2 respectively. Expression status of selected RBPs and microRNAs were checked in lungs tissue. ResultsWe identified 28 unique variants in SARS-CoV2 UTR region based on a minimum variant percentage cut-off of 0.5. Along with 241C>T change the important 5-UTR change identified was 187A>G, while 29734G>C, 29742G>A/T and 29774C>T were the most familiar variants of 3UTR among most of the continents. Furthermore, we found that despite of the variations in the UTR regions, binding of host RBP to them remains mostly unaltered, which further influenced the functioning of specific miRNAs. ConclusionOur results, shows for the first time in SARS-Cov2 infection, a possible cross-talk between host RBPs-miRNAs and viral UTR variants, which ultimately could explain the mechanism of escaping host RNA decay machinery by the virus. The knowledge might be helpful in developing anti-viral compounds in future.

genomics↗

bioRxiv2020

Unifying the Ambivalent Mechanisms of Protein-Stabilising Osmolytes

The mechanism of protein stabilization by zwiterionic osmolytes has remained a long-standing puzzle. While the prevalent mechanistic hypothesis suggests an osmo-phobic model in which osmolytes are assumed to stabilize proteins by preferentially excluding themselves from the protein surface, emerging evidences of preferential binding of popular osmolyte trimethyl amine N-oxide (TMAO) with hydrophobic macromolecules contradict this view. Here we address these contrasting perspectives by investigating the folding mechanism of a set of mini proteins in aqueous solutions of two different osmolytes glycine and TMAO, via free energy simulations. Our results demonstrate that, while both osmolytes are found to stabilize the folded conformation of the mini proteins, their mechanism of actions are mutually diverse: Specifically, glycine always depletes from the surface of all mini proteins, thereby conforming to the osmophobic model; but TMAO is found to display ambivalent signatures of protein-specific preferential binding and exclusion to/from the protein surface. At molecular level, the presence of an extended hydrophobic patch in protein topology is found to be recurrent motif in proteins leading to favorable binding with TMAO. Finally, an analysis based upon the preferential interaction theory and folding free energetics reveals that irrespective of preferential binding vs exclusion of osmolytes, it is the relative preferential depletion of osmolytes on transition from folded to unfolded conformation of proteins, which drives the overall conformational equilibrium towards the folded state in presence of osmolytes. Taken together, moving beyond the model system and hypothesis, this work brings out ambivalent mechanism of osmolytes on proteins and provides an unifying justification.

biophysics↗

bioRxiv2020

Identification of QTLs for high grain yield and component traits in New Plant Types of rice

A panel of 60 genotypes consisting of New Plant Types (NPTs) along with indica, tropical and temperate japonica genotypes were phenotypically evaluated for four seasons in irrigated situation for grain yield per se and component traits. Twenty NPT genotypes were found to be promising with an average grain yield of 5.45 to 8.8 t/ha. A total of 85 SSR markers were used in the study to identify QTLs associated with grain yield per se and related traits. Sixty-six (77.65%) markers were found to be polymorphic. The PIC values varied from 0.516 to 0.92 with an average of 0.704. A moderate level of genetic diversity (0.39) was detected among genotypes. Variation to the tune of 8% within genotypes, 68% among the genotypes within the population and 24% among the populations were observed (AMOVA). The association analysis using GLM and MLM models led to the identification of 30 and 10 SSR markers were associated with 70 and 16 QTLs, respectively. Thirty novel QTLs linked with 16 SSRs were identified to be associated with eleven traits, namely, tiller number (qTL-6.1, qTL-11.1, qTL-4.1), panicle length (qPL-1.1, qPL-5.1, qPL-7.1, qPL-8.1), flag leaf length (qFLL-8.1, qFLL-9.1), flag leaf width (qFLW-6.2, qFLW-5.1, qFLW-8.1, qFLW-7.1), total no. of grains (qTG-2.2, qTG-a7.1), thousand-grain weight (qTGW-a1.1, qTGW-a9.2, qTGW-5.1, qTGW-8.1), fertile grains (qFG-7.1), seed length-breadth ratio (qSlb-3.1), plant height (qPHT-6.1, qPHT-9.1), days to 50% flowering (qFD-1.1) and grain yield per se (qYLD-5.1, qYLD-6.1a, qYLD-11.1). This information could be useful for identification of highly potential parents for development of transgressive segregants. Moreover, super rice genotypes could be developed through pyramiding of these QTLS for important yield traits for prospective increment in yield potentiality and breaking yield ceiling.

plant biology↗

bioRxiv2019

Human NK cell deficiency as a result of biallelic mutations in MCM10

Human natural killer cell deficiency (NKD) arises from inborn errors of immunity that lead to impaired NK cell development, function or both. Through the understanding of the biological perturbations in individuals with NKD, requirements for the generation of terminally mature functional innate effector cells can be elucidated. Here we report a novel cause of NKD resulting from compound heterozygous mutations in MCM10 that impaired NK cell maturation in a child with fatal susceptibility to CMV. MCM10 has not been previously associated with monogenic disease and plays a critical role in the activation and function of the eukaryotic DNA replisome. By modeling MCM10 deficiency in human NK cell lines and primary NK cell precursors, we demonstrate that MCM10 is required for NK cell terminal maturation and acquisition of immunological system function.

immunology↗

bioRxiv2019

Foxi1 inactivation rescues loss of principal cell fate selection in Hes1-deficient kidneys but does not ensure maintenance of principal cell gene expression.

The distal nephron and collecting duct segments of the mammalian kidney consist of intercalated cell types intermingled among principal cell types. Notch signaling ensures that a sufficient number of cells select a principal instead of an intercalated cell fate. However, the precise mechanisms by which Notch signaling patterns the distal nephron and collecting duct cell fates is unknown. Here we observed that Hes1, a direct target of Notch signaling pathway, is required within the mouse developing collecting ducts for repression of Foxi1 expression, an essential intercalated cell specific transcription factor. Interestingly, inactivation of Foxi1 in Hes1-deficient collecting ducts rescues the deficiency in principal cell fate selection, overall urine concentrating deficiency, and reduces the occurrence of hydronephrosis. However, Foxi1 inactivation does not rescue the reduction in expression of all principal cell genes in the Hes1-deficient kidney collecting duct cells that select the principal cell fate. Additionally, suppression of Notch/Hes1 signaling in mature principal cells reduces principal cell gene expression without activating Foxi1. We conclude that Hes1 is a Notch signaling target that is essential for normal patterning of the collecting ducts with intermingled cell types by repressing Foxi1, and for maintenance of principal cell gene expression independent of repressing Foxi1.

developmental biology↗

bioRxiv2019

Notch signaling regulates Akap12 expression and primary cilia length during renal tubule morphogenesis.

Alagille syndrome patients present with loss of function mutations in either JAG1 or NOTCH2. About 40-50% of patients have kidney abnormalities, with multi-cystic, dysplastic kidneys being one of the more frequent kidney defects. Additionally, gain-of-function mutations in NOTCH2 are associated with cystic kidneys in Hajdu-Cheney syndrome patients. Conditional inactivation of Notch1, Notch2, or RBPJ within the nephrogenic lineage impairs nephrogenesis and produces proximal tubule cysts in mice. How perturbations in Notch signaling cause renal tubular cysts remains unclear. Here we have determined that inhibition of Notch signaling in the kidney increases Akap12 expression. Ectopic expression of Akap12 in renal epithelia results in abnormally long primary cilia similar to those observed in Notch-signal-deficiency. Both loss of Notch signaling and elevated Akap12 expression disrupt the ability of renal epithelial cells to form spherical structures with a single lumen when grown embedded in matrix. We conclude that Notch signaling regulates Akap12 expression to ensure normal primary cilia length and renal epithelial morphogenesis, and suggest that diseases associated with defective Notch signaling, such as Alagille syndrome, maybe mechanistically related to ciliopathies.\n\nTranslational StatementThe current study examines how a reduction in Notch signaling results in abnormal renal tubule formation, as occurs in Alagille Syndrome patients with mutations in JAG1 or NOTCH2. The finding that reduced Notch signaling results in abnormally long cilia is suggestive that some of the clinical manifestations in Alagille Syndrome, such as small cystic kidneys, may originate due to defective cilia function. Linking Notch to primary cilia also opens up the possibility that coinheritance of mutations in ciliopathy genes along with a mutation in JAG1 or NOTCH2 may enhance the severity of the clinical phenotypes such as cystic kidney disease and may explain the variable occurrence and onset of kidney disease among Alagille Syndrome patients.

cell biology↗

bioRxiv2019

Unraveling the substrate preference of an uncharacterized phylogenetic subgroup in Formate/Nitrite Transporter (FNT) family: Computational studies of anion transport in Escherichia coli FNT homolog

Formate/Nitrite Transporters (FNTs) selectively transport monovalent anions and are found in prokaryotes and lowers eukaryotes. They play significant role in bacterial growth and act against the defense mechanism of infected host. Since FNTs dont occur in higher animals, they are attractive drug targets for many bacterial diseases. Phylogenetic analysis revealed that they can be classified into eight subgroups and two of which belong to the uncharacterized YfdC- and YfdC-{beta} groups. Experimentally determined structures of FNTs belonging to different phylogenetic groups adopt the unique aquaporin-like hourglass helical fold. We considered formate channel from Vibrio Cholerae (VcFocA), hydrosulphide channel from Clostridium difficile (CdHSC) and the uncharacterized channel from Escherchia coli (EcYfdC) to investigate the mechanism of transport and selectivity. Using equilibrium molecular dynamics (MD) and umbrella sampling studies, we determined temporal channel radius profiles, permeation events and potential of mean force (PMF) profiles of different substrates with the conserved central histidine residue in protonated or neutral form. Unlike the VcFocA and CdHSC, MD studies showed that the formate substrate was unable to enter the vestibule region of EcYfdC. Absence of a conserved basic residue and presence of acidic residues in the vestibule regions, conserved only in YfdC-, were found to be responsible for high energy barriers for the anions to enter EcYfdC. PMF profiles generated for ammonia and ammonium ion revealed that EcYfdC can transport neutral solutes and could possibly be involved in the transport of cations analogous to the mechanism proposed for ammonium transporters. Although YfdC members belong to the FNT family, our studies convincingly reveal that EcYfdC is not an anion channel. Absence/presence of specific charged residues at particular positions makes EcYfdC selective for neutral or possibly cationic substrates. This adds to the repertoire of membrane proteins that use the same fold but transport substrates with different chemical nature.\n\nAuthor SummaryChannels and transporters are membrane proteins involved in the transport of solutes selectively across the cell membranes. Drugs for many diseases have been developed to inhibit ion channels. Formate/Nitrite Transporters (FNTs) are ion channels selective for monovalent anions and are present in bacteria and lower eukaryotes. Absence of FNTs in humans makes them as attractive drug targets against many pathogenic bacteria. To develop inhibitors for a protein, it is important to understand the mechanism of its function. Selectivity and transport mechanism of FNTs have been investigated for some members. One of the subgroups of FNTs, YfdC-, is uncharacterized. In this study we used computer simulation approach to investigate the molecular mechanism of selectivity and transport of three FNTs including one from YfdC- group from Escherichia coli. Our studies show that E. coli YfdC is not an anion channel although it belongs to FNT family. We hypothesize that the YfdC- members could be involved in the transport of neutral or possibly cationic substrates. This is further supported by the conservation of specific acidic residues found only in YfdC- in the vestibule regions. This finding has major implications in developing blockers for FNT members belonging to YfdC- group.

biophysics↗

bioRxiv2019

Bacillus clarus sp. nov. is a new Bacillus cereus group species isolated from soil

Bacillus cereus group or B. cereus sensu lato (s.l.), is comprised of Gram-positive spore-forming, rod-like bacteria that are widespread in natural environments. Although the species in this group are known to be highly related in terms of phenotypic characteristics, they display different levels of pathogenicity. Biochemical assays are therefore considered to be insufficient for accurate taxonomic classification of B. cereus group species. To facilitate accurate taxonomic classification and associated prediction of pathogenic potential, we have conducted comparative genomic analyses of publicly available genome assemblies of B. cereus group isolates. Through that, we found that an isolate previously known as B. mycoides ATCC 21929 was sufficiently distant from valid and effective type strains to be considered a putative new species. We have conducted biochemical and bioinformatic characterization of strain ATCC 21929 that had been isolated from soil in Papua New Guinea. Strain ATCC 21929 most closely resembles B. paramycoides NH24A2T, producing ANIb and DDH values of 86.70% and 34.1%, respectively. Phenotypically, isolate ATCC 21929 does not possess cytochrome c oxidase activity, and is able to grow at a range of temperatures 15{degrees}C - 43{degrees}C and at a range of pH 6 - 9. With regards to fatty acid composition, this isolate has iso-C17:0 in highest abundance. We propose the strain ATCC 21929T (=PS00077AT = PS00077BT = PSU-0922T = BHPT) as a new species named Bacillus clarus sp. nov. to facilitate accurate taxonomic classification of B. cereus group isolates.

microbiology↗