Biology subjects

Mukhopadhyay, S.

Publications and source records attributed to Mukhopadhyay, S..

88 records · Page 5Linked to original sources

bioRxiv2020

No evidence that androgen regulation of pulmonary TMPRSS2 explains sex-discordant COVID-19 outcomes

The sex discordance in COVID-19 outcomes has been widely recognized, with males generally faring worse than females and a potential link to sex steroids. A plausible mechanism is androgen-induced expression of TMPRSS2 and/or ACE2 in pulmonary tissues that may increase susceptibility or severity in males. This hypothesis is the subject of several clinical trials of anti-androgen therapies around the world. Here, we investigated the sex-associated TMPRSS2 and ACE2 expression in human and mouse lungs and interrogated the possibility of pharmacologic modification of their expression with anti-androgens. We found no evidence for increased TMPRSS2 expression in the lungs of males compared to females in humans or mice. Furthermore, in male mice, treatment with the androgen receptor antagonist enzalutamide did not decrease pulmonary TMPRSS2. On the other hand, ACE2 and AR expression was sexually dimorphic and higher in males than females. ACE2 was moderately suppressible with enzalutamide therapy. Our work suggests that sex differences in COVID-19 outcomes attributable to viral entry are independent of TMPRSS2. Modest changes in ACE2 could account for some of the sex discordance.

physiology↗

bioRxiv2020

Chronic chemogenetic activation of forebrain excitatory neurons in postnatal life evokes long-lasting changes in mood-related behavior

Early adversity is a key risk factor for the development of adult psychopathology, including anxiety, depression and schizophrenia. Rodent models of early adversity program persistent behavioral, molecular, metabolic, and neurophysiological changes. Perturbed signaling via forebrain Gq-coupled neurotransmitter receptors is a common feature across multiple models of early adversity. We addressed whether enhanced Gq-mediated signaling in forebrain excitatory neurons during postnatal life can evoke long-lasting mood-related behavioral changes. Excitatory hM3Dq DREADD-mediated chemogenetic activation of CamKII-positive forebrain excitatory neurons during postnatal life (P2-14) increased anxiety- and despair-like behavior, and evoked sensorimotor gating deficits in adulthood. In contrast, chronic chemogenetic hM3Dq DREADD activation of forebrain excitatory neurons in the juvenile or adult window did not evoke any mood-related behavioral alterations, highlighting the criticality of the postnatal temporal window. The enhanced anxiety-, despair- and schizophrenia-like behavioral changes evoked by chronic chemogenetic activation of forebrain excitatory neurons in postnatal life, was accompanied by an increased cortical and hippocampal metabolic rate of glutamatergic and GABAergic neurons in adulthood. Furthermore, animals with a history of postnatal hM3Dq activation exhibited a decline in the expression of activity-dependent and plasticity-associated markers within the hippocampus, along with perturbed hippocampal excitatory and inhibitory currents in adulthood. These results indicate that Gq signaling mediated activation of forebrain excitatory neurons during the critical postnatal window is sufficient to program altered mood-related behavior, as well as metabolic and neurophysiological changes in forebrain glutamate and GABA systems, recapitulating specific aspects of the consequences of early adversity.

neuroscience↗

bioRxiv2020

A synthetic biochemical device for sensing microgravity

Biological solutions to human space travel must consider microgravity as an important component, which is unknown by the biochemical worlds on the Earth. Thus, one of the fundamental challenges of space biotechnology is to create engineered biochemical systems to integrate microgravity as a signal within molecular and cellular processes. Here we created the first molecular or biochemical microgravity sensor by creating a synthetic-small-regulatory-RNA based molecular network in E.coli, which sensed microgravity and responded by altering the expression of a target protein. We demonstrated that the design was universal, could work potentially with any promoter and against any target gene. This device was applied to target cell division process and rescue the deformed cell shape by applying microgravity. The work showed for the first time, a way to integrate microgravity as physical signals within biochemical process of a living cell in a human designed way and thus, opens a new direction in space biotechnology, space chemistry and space technology.

synthetic biology↗

bioRxiv2020

Virion-associated spermidine transmits with Rift Valley fever virus particles to maintain infectivity

Viruses require host cell metabolites to productively infect, and the mechanisms by which viruses usurp these molecules is diverse. One group of cellular metabolites important in virus infection is the polyamines, small positively-charged molecules involved in cell cycle, translation, and nucleic acid synthesis, among other cellular functions. Polyamines also support replication of diverse viruses, and they are important for processes such as transcription, translation, and viral protein enzymatic activity. Rift Valley fever virus (RVFV) is a negative-sense RNA virus that requires polyamines to produce infectious particles. In polyamine depleted conditions, noninfectious particles are produced that interfere with virus replication and stimulate immune signaling. Here, we find that RVFV relies on virion-associated polyamines to maintain infectivity. We show that RVFV replication is facilitated by any of the three biogenic polyamines; however, we specifically find spermidine associated with purified virions. Using a panel of polyamine homologs, we observe that virions can also associate with (R)-3-methylspermidine and norspermidine, though not with other less homologous molecules. Using polyamine reporter cells, we demonstrate that virion-associated polyamines transmit from one infected cell to another. Finally, we find that virions devoid of polyamines are unstable and cannot be supplemented with exogenous polyamines to regain stability or infectivity. These data highlight a unique role for polyamines, and spermidine in particular, in maintaining virus infectivity, a function not previously appreciated. Further, these studies are the first to identify polyamines associated with RVFV virions. Targeting polyamines represents a promising antiviral strategy, and this work highlights a new mechanism by which we can inhibit virus replication through FDA-approved polyamine depleting pharmaceuticals.

microbiology↗

bioRxiv2019

TF protein of Sindbis virus antagonizes host type I interferon responses in a palmitoylation-dependent manner

Sindbis virus (SINV) produces the small membrane protein TF from the 6K gene via a (-1) programmed ribosomal frameshifting. While several groups have shown that TF-deficient virus exhibits reduced virulence, mechanism(s) by which this occurs remain unknown. Here, we demonstrate a role for TF in antagonizing the host interferon response. Using wild-type and type 1 interferon receptor-deficient mice and primary cells derived from these animals, we show that TF controls the induction of the host interferon responses at early times during infection. Loss of TF production leads to elevated interferon and a concurrent reduction in viral loads with a loss of pathogenicity. Palmitoylation of TF has been shown to be important for particle assembly and morphology. We find that palmitoylation of TF also contributes to the ability of TF to antagonize host interferon responses as dysregulated palmitoylation of TF reduces virulence in a manner similar to loss of TF.

microbiology↗

bioRxiv2019

Cotranslational Folding Stimulates Programmed Ribosomal Frameshifting in the Alphavirus Structural Polyprotein

Viruses maximize their genetic coding capacity through a variety of biochemical mechanisms including programmed ribosomal frameshifting (PRF), which facilitates the production of multiple proteins from a single transcript. PRF is typically stimulated by structural elements within the mRNA that generate mechanical tension between the transcript and ribosome. However, in this work we show that the forces generated by the cotranslational folding of the nascent polypeptide chain can also enhance PRF. Using an array of biochemical, cellular, and computational techniques, we first demonstrate that the Sindbis virus structural polyprotein forms two competing topological isomers during biosynthesis at the ribosome-translocon complex. We then show that the formation of one of these topological isomers is linked to PRF. Coarse-grained molecular dynamic simulations reveal that the translocon-mediated membrane integration of a transmembrane domain upstream from the ribosomal slip-site generates a force on the nascent polypeptide chain that scales with observed frameshifting. Together, our results demonstrate that cotranslational folding of this protein generates a tension that stimulates PRF. To our knowledge, this constitutes the first example in which the conformational state of the nascent chain has been linked to PRF. These findings raise the possibility that, in addition to RNA-mediated translational recoding, a variety of cotranslational folding and/ or binding events may also stimulate PRF.

biochemistry↗

bioRxiv2019

Identification of Chikungunya virus nucleocapsid core assembly modulators

AbstractThe alphavirus Chikungunya virus is transmitted to humans via infected mosquitos. Most infected humans experience symptoms which can range from short-term fatigue and fever to debilitating arthritis that can last for months or years. Some patients relapse and experience symptoms months or years after the initial bout of disease. The capsid protein of Chikungunya virus forms a shell around the viral RNA genome; this structure is called the nucleocapsid core. The core protects the genome during virus transmission and with the correct environmental trigger, this proteinaceous shell dissociates and releases the viral genome to initiate infection. We hypothesized that targeting compounds to interfere with the nucleocapsid cores function would constrain virus spread either by inhibiting the release of viral genomes during entry or by reducing the number of infectious virus particles assembled. We implemented a high throughput, in vitro, FRET-based assay to monitor nucleic acid packaging by purified Chikungunya capsid protein as a proxy for nucleocapsid core assembly and disassembly. We screened 10,000 compounds and found 45 that substantially modulated the assembly of core-like particles. A subset of compounds was selected to study their effects in virus-infected vertebrate cells. Our results show that four compounds inhibit infectious virus production by at least 90% in a dose-dependent manner. The most promising inhibitor was tested and found to reduce the amount of nucleocapsid cores inside the cell during Chikungunya virus infection. These compounds could be the foundation for anti-viral therapeutics.\n\nHighlightsO_LIA FRET-based assay to detect nucleic acid packaging by Chikungunya virus capsid protein\nC_LIO_LIIdentification of small molecules that modulate core-like particle assembly\nC_LIO_LIA subset of compounds that interfere with in vitro assembly also inhibit Chikungunya virus production in cell culture\nC_LIO_LIIdentification of antiviral molecules that may not be identified by assays using reporter viruses\nC_LIO_LIPotential starting compounds for developing direct-acting antivirals\nC_LI

microbiology↗

bioRxiv2019

Immunosuppression, rather than inflammation, is a salientfeature of sepsis in an Indian cohort

1Sepsis remains a lethal ailment with imprecise treatment and ill-understood biology. A clinical transcriptomic analysis of sepsis patients was performed for the first time in India and revealed large-scale change in blood gene expression in patients of severe sepsis and septic shock admitted to ICU. Three biological processes were quantified using scores derived from the corresponding transcriptional modules. Comparison of the module scores revealed that genes associated with immune response were more suppressed compared to the inflammation-associated genes. These findings will have great implication in the treatment and prognosis of severe sepsis/septic shock if it can be translated into a bedside tool.

genomics↗

bioRxiv2019

Some common pathways perturbed in septic shock and cancer

BackgroundSepsis and cancer are both leading causes of death, and occurrence of any one, increases the likelihood of the other. While cancer patients are susceptible to sepsis, survivors of sepsis are also susceptible to develop certain cancers. This mutual dependence for susceptibility suggests shared biology between the two disease categories. Earlier analysis had revealed cancer-related pathway to be up-regulated in Septic Shock (SS), an advanced stage of sepsis. This has motivated a more comprehensive comparison of the transcriptomes of SS and cancer. MethodsGene Set Enrichment Analysis was performed to detect the pathways enriched in SS and cancer. Thereafter, hierarchical clustering was applied to identify relative segregation of 17 cancer types in to two groups vis-a-vis SS. Biological significance of the selected pathways was explored by network analysis. Clinical significance of the pathways was tested by survival analysis. A robust classifier of cancer groups was developed based on machine learning. ResultsA total of 66 pathways were observed to be enriched in both SS and cancer. However, clustering segregated cancer types into two categories based on the direction of transcriptomic change. In general, there was up-regulation in SS and one group of cancer (termed Sepsis-Like Cancers, or SLC), but not in other cancers. SLC group mainly consisted of malignancies of the gastrointestinal tract (head and neck, oesophagus, stomach, liver and biliary system) often associated with infection. Machine learning classifier successfully segregated the two cancer groups with high accuracy (> 98%). Additionally, pathway up-regulation was observed to be associated with survival in the SLC group of cancers. ConclusionTranscriptome-based systems biology approach segregates cancer into two groups (SLC and CA) based on similarity with SS. Host response to infection plays a key role in pathogenesis of SS and SLC. However, we hypothesize that some component of the host response is protective in both SS and SLC.

genomics↗

bioRxiv2019

Liquid-Liquid Phase Separation is Driven by Large-Scale Conformational Unwinding and Fluctuations of Intrinsically Disordered Protein Molecules

Liquid-liquid phase separation occurs via a multitude of transient, non-covalent, intermolecular interactions resulting in phase transition of intrinsically disordered proteins/regions (IDPs/IDRs) and other biopolymers into mesoscopic, dynamic, non-stoichiometric, supramolecular condensates. IDPs resemble associative polymers possessing stereospecific \"stickers\" and flexible \"spacers\" that govern the transient chain-chain interactions and fluidity in phase-separated liquid droplets. However, the fundamental molecular origin of phase separation remains elusive. Here we present a unique case to demonstrate that unusual conformational expansion events coupled with solvation and fluctuations drive phase separation of tau, an IDP associated with Alzheimers disease. Using intramolecular excimer emission as a powerful proximity readout, we show the unraveling of polypeptide chains within the protein-rich interior environment that can promote critical interchain contacts. Using highly-sensitive picosecond time-resolved fluorescence depolarization measurements, we directly capture rapid large-amplitude torsional fluctuations in the extended chains that can control the relay of making-and-breaking of noncovalent intermolecular contacts maintaining the internal fluidity. Our observations, together with the existing polymer theories, suggest that such an orchestra of concerted molecular shapeshifting events involving chain expansion, solvation, and fluctuations can provide additional favorable free energies to overcome the entropy of mixing term during phase separation. The interplay of these key molecular parameters can also be of prime importance in modulating the mesoscale material property of liquid-like condensates and their maturation of into pathological gel-like and solid-like aggregates.

biophysics↗

bioRxiv2019

Salmonella Typhi survives in human macrophages by neutralizing the RAB32/BLOC-3 host-defence pathway

Macrophages provide a first line of defence against microorganisms, and while some mechanisms to kill pathogens such as the oxidative burst are well described, others are still undefined or unknown. Here we report that the Rab32 GTPase and its guanine nucleotide exchange factor BLOC-3 are central components of a trafficking pathway that controls both bacterial and fungal intracellular pathogens. This broad host-defence mechanism is active in both human and murine macrophages and is independent of well known antimicrobial mechanisms such as the NADPH-dependent oxidative burst, production of nitric oxide and antimicrobial peptides. To survive in human macrophages, Salmonella Typhi actively counteracts the Rab32/BLOC-3 pathway through its Salmonella pathogenicity island-1-encoded type III secretion system. These findings demonstrate that the Rab32/BLOC-3 pathway is a novel and universal host-defence pathway and protects mammalian species from a wide range of intracellular pathogens.

microbiology↗

bioRxiv2019

Mammogram Segmentation using Multi-atlas Deformable Registration

Accurate breast region segmentation is an important step in various automated algorithms involving detection of lesions like masses and microcalcifications, and efficient telemammography. Existing segmentation algorithms underperform due to variations in image quality and shape of the breast region. In this paper, we propose to segment breast region by combining data-driven clustering with deformable image registration. In the first phase of the approach, we identify atlas images from a dataset of mammograms using data-driven clustering. Then, we segment these atlas images and use in the next phase of the algorithm. The second phase is atlas-based registration. For a candidate image, we find the most similar atlas image from the set of atlases identified in phase one. We deform the selected atlas image to match the given test image using the Demon's registration algorithm. Then, the segmentation mask of the deformed atlas is transferred to the mammogram in consideration. Finally, we refine the segmentation mask with some morphological operations in order to obtain accurate breast region boundary. We evaluated the performance of our method using ground-truth segmentation masks verified by an expert radiologist. We compared the proposed method with three existing state-of-the-art algorithms for breast region segmentation and the proposed approach outperformed all three in most of the cases.

bioinformatics↗

bioRxiv2018

Domains of the TF protein important in regulating its own palmitoylation

Sindbis virus particles contain the viral proteins capsid, E1 and E2, and low levels of a small membrane protein called TF. TF is produced during a (-1) programmed ribosomal frameshifting event during the translation of the structural polyprotein. TF from Sindbis virus-infected cells is present in two palmitoylated states, basal and maximal; unpalmitoylated TF is not detectable. Mutagenesis studies demonstrated that without palmitoylation, TF is not incorporated into released virions, suggesting palmitoylation of TF is a regulated step in virus assembly. In this work, we identified Domains within the TF protein that regulate its palmitoylation state. Mutations and insertions in Domain III, a region proposed to be in the cytoplasmic loop of TF, increase levels of unpalmitoylated TF found during an infection and even allow incorporation of unpalmitoylated TF into virions. Mutations in Domain IV, the TF unique region, are likely to impact the balance between basal and maximal palmitoylation.

molecular biology↗

bioRxiv2018

The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K

Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase and ion channel activity, or specific chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident calcium-regulated lipid scramblase. Our crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional structures solved by cryo-EM reveal extensive conformational changes extending from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity. Our results suggest mechanisms by which missense variants of TMEM16K could cause SCAR10 ataxia, providing new hypotheses to explore for therapy.

biochemistry↗

bioRxiv2018

Structures of DPAGT1 explain glycosylation disease mechanisms and advance TB antibiotic design

Protein glycosylation is a widespread post-translational modification. The first committed step to the lipid-linked glycan used for this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and congenital disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use as antibiotics. However, little is known about the mechanism or the effects of disease-associated mutations in this essential enzyme. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (and thus cause disease) and allow design of non-toxic lipid-altered tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo thereby providing a promising new class of antimicrobial drug.\n\nHighlightsO_LIStructures of DPAGT1 with UDP-GlcNAc and tunicamycin reveal mechanisms of catalysis\nC_LIO_LIDPAGT1 mutants in patients with glycosylation disorders modulate DPAGT1 activity\nC_LIO_LIStructures, kinetics and biosynthesis reveal role of lipid in tunicamycin\nC_LIO_LILipid-altered, tunicamycin analogues give non-toxic antibiotics against TB\nC_LI

biochemistry↗

bioRxiv2017

Genetic effects on chromatin accessibility foreshadow gene expression changes in macrophage immune response

Noncoding regulatory variants play an important role in the genetics of complex traits. Although quantitative trait locus (QTL) mapping is a powerful approach to identify these variants, many genetic effects may remain unobserved when cells are sampled in only one of a large number of possible environments. Using a novel induced pluripotent stem cell-derived system, we mapped QTLs regulating chromatin accessibility and gene expression in macrophages in four conditions mimicking the interplay between interferon-gamma response and Salmonella infection. We found that approximately 50% of condition-specific effects on gene expression altered chromatin accessibility prior to stimulation. Furthermore, 6% of the chromatin accessibility QTLs regulated multiple neighbouring regions and these interactions were modulated by stimulation, occasionally producing condition-specific changes in gene expression. Profiling additional states also doubled the number of expression QTLs that could be confidently colocalised with disease associations. Thus, a substantial fraction of disease-associated variants may affect primed regulatory elements in naive cells.

genomics↗