Biology subjects

Ghosh, S.

Publications and source records attributed to Ghosh, S..

At least 343 records · Page 19Linked to original sources

bioRxiv2020

Whole Genome Sequences of Aedes aegypti (Linn.) Field Isolates from Southern India

Aedes spp. mosquitoes are a major health concern as they transmit several viral pathogens resulting in millions of deaths annually around the world. This is compounded by the emergence of insecticide-resistant strains and global warming, which could expose more than half of the world's population to Aedes-borne diseases in the future. Therefore, a comprehensive understanding of vector biology and the genomic basis of phenotypes such as insecticide resistance in natural populations are of paramount importance. Here, we sequenced the genome of Aedes aegypti mosquitos sampled from dengue-endemic areas and investigated the genetic variations between the previously reported laboratory-reared strain and our field isolates. The mosquito genomic DNA was used for paired-end sequencing using the Illumina platform. The reads were used for template-based assembly and mapped to the Aedes aegypti reference genome. Stringent parameters and multiple variant calling methods were used to identify unique single nucleotide variants (SNVs) and insertions-deletions (indels) and mapped to the Aedes chromosomes to create a draft consensus genome. Gene Ontology analyses was performed on the variant-enriched genes while two gene families involved in insecticide resistance were used for comparative sequence and phylogenetic analyses. Comparative sequence variant analyses showed that the majority of the high-quality variants in our samples mapped to non-coding regions of the genome, while gene ontology analyses of genic variants revealed enrichment of terms relevant to drug binding and insecticide resistance. Importantly, one mutation implicated in pyrethroid resistance was found in one Aedes sample. This is the first report of genome sequences of A. aegypti field isolates from India which reveals variants specific to the wild population. This is a useful resource which will facilitate development of robust integrated vector control strategies for management of Aedes-borne diseases through genetic manipulation of local mosquito populations.

genomics↗

bioRxiv2020

Type III interferons disrupt the lung epithelial barrier upon viral recognition.

Lower respiratory tract infections are a leading cause of mortality driven by infectious agents. RNA viruses such as influenza virus, respiratory syncytial virus and the new pandemic coronavirus SARS-CoV-2 can be highly pathogenic. Clinical and experimental evidence indicate that most severe and lethal cases do not depend on the viral burden and are, instead, characterized by an aberrant immune response. In this work we assessed how the innate immune response contributes to the pathogenesis of RNA virus infections. We demonstrate that type III interferons produced by dendritic cells in the lung in response to viral recognition cause barrier damage and compromise the host tissue tolerance. In particular, type III interferons inhibit tissue repair and lung epithelial cell proliferation, causing susceptibility to lethal bacterial superinfections. Overall, our data give a strong mandate to rethink the pathophysiological roles of this group of interferons and their possible use in the clinical practice against endemic as well as emerging viral infections.

immunology↗

bioRxiv2020

Integrator terminates promoter-proximal Pol II to generate C. elegans piRNA precursors

Piwi-interacting RNAs (piRNAs) play key roles in germline development and genome defence in metazoans. In C. elegans, piRNAs are transcribed from >15000 discrete genomic loci by RNA polymerase II, resulting in 28 nt short-capped piRNA precursors. Here we investigate transcription termination at piRNA loci. We show that the Integrator complex, which terminates snRNA transcription, is recruited to piRNA loci. We show that the catalytic activity of Integrator cleaves nascent capped piRNA precursors associated with promoter-proximal Pol II, resulting in termination of transcription. Loss of Integrator activity, however, does not result in transcriptional readthrough at the majority of piRNA loci. Our results draw new parallels between snRNA and piRNA biogenesis in nematodes, and provide evidence of a role for the Integrator complex as a terminator of promoter-proximal RNA polymerase II. Highlights- Integrator localises to sites of piRNA biogenesis in nematodes - Integrator cleaves nascent RNAs associated with promoter-proximal Pol II at piRNA loci to release short capped piRNA precursors from chromatin - Repression of Pol II elongation at the majority of piRNA loci is independent of Integrator

molecular biology↗

bioRxiv2020

Ribosome profiling reveals ribosome stalling on tryptophan codons upon oxidative stress in fission yeast

Modulation of translation is an essential response to stress conditions. We have investigated the translational programmes launched by the fission yeast Schizosaccharomyces pombe subject to five environmental stresses: oxidative stress, heavy metal, heat shock, osmotic shock and DNA damage. We also explored the contribution of two major defence pathways to these programmes: The Integrated Stress Response, which directly regulates translation initiation, and the stress-response MAPK pathway. To obtain a genome-wide and high-resolution view of this phenomenon, we performed ribosome profiling of control cells and of cells subject to each of the five stresses mentioned above, both in wild type background and in cells in which the Integrated Stress Response or the MAPK pathway were inactivated. Translational changes were partially dependent on the integrity of both signalling pathways. Interestingly, we found that the transcription factor Fil1, a functional homologue of the Gcn4 and Atf4 proteins (from budding yeast and mammals, respectively), was highly upregulated in most stresses. Consistent with this result, Fil1 was required for the normal response to most stresses. A large group of mRNAs were translationally downregulated, including many required for ribosome biogenesis. Overall, our data suggest that severe stresses lead to the implementation of a universal translational response, which includes energy-saving measures (reduction of ribosome production) and induction of a Fil1-mediated transcriptional programme. Surprisingly, ribosomes stalled on tryptophan codons specifically upon oxidative stress, a phenomenon that is likely caused by a decrease in charged tRNA-Tryptophan. Tryptophan stalling led to a mild translation elongation reduction and contributed to the inhibition of initiation by the Integrated Stress Response. Taken together, our results show that different stresses elicit common and specific translational responses, revealing a special and so far unknown role in Tryptophan-tRNA availability.

molecular biology↗

bioRxiv2020

Corticostriatal Plasticity Established by Initial Learning Persists After Behavioral Reversal

The neural mechanisms that allow animals to adapt their previously learned associations in response to changes in the environment remain poorly understood. To probe the synaptic mechanisms that mediate such adaptive behavior, we trained mice on an auditory-motor reversal task, and tracked changes in the strength of corticostriatal synapses associated with the formation of learned associations. Using a ChR2-based electrophysiological assay in acute striatal slices, we measured the strength of these synapses after animals learned to pair auditory stimuli with specific actions. Here we report that the pattern of synaptic strength initially established by learning remains unchanged even when the task contingencies are reversed. Our results suggest that synaptic changes associated with the initial acquisition of this task are not erased or over-written, and that behavioral reversal of learned associations may recruit a separate neural circuit.

neuroscience↗

bioRxiv2020

Image-based elastography of heterochromatin and euchromatin domains in the deforming cell nucleus

Chromatin of the eukaryotic cell nucleus comprises of microscopically dense heterochromatin and loosely packed euchromatin domains, each with distinct transcriptional ability and roles in cellular mechanotransduction. While recent methods have been developed to characterize the nucleus, measurement of intranuclear mechanics remains largely unknown. Here, we describe the development of nuclear elastography, which combines microscopic imaging and computational modeling to quantify the relative elasticity of the heterochromatin and euchromatin domains. Using contracting murine embryonic cardiomyocytes, nuclear elastography reveals that the heterochromatin is almost four times stiffer than the euchromatin at peak deformation. The relative elasticity between the two domains changes rapidly during the active deformation of the cardiomyocyte in the normal physiological condition but progresses more slowly in cells cultured in a mechanically stiff environment, although the relative stiffness at peak deformation does not change. Further, we found that the disruption of the LINC complex in cardiomyocytes compromises the intranuclear elasticity distribution resulting in elastically similar heterochromatin and euchromatin. These results provide insight into the elastography dynamics of heterochromatin and euchromatin domains, and provide a non-invasive framework to further investigate the mechanobiological function of subcellular and subnuclear domains limited only by the spatiotemporal resolution of the image acquisition method.

cell biology↗

bioRxiv2020

COP9 signalosome is an essential and druggable parasite target that regulates protein degradation

Understanding how the protozoan protein degradation pathway is regulated could uncover new parasite biology for drug discovery. We found the COP9 signalosome (CSN) conserved in multiple pathogens such as Leishmania, Trypanosoma, Toxoplasma, and used the severe diarrhea-causing Entamoeba histolytica to study its function in medically significant protozoa. We show that CSN is an essential upstream regulator of parasite protein degradation. Genetic disruption of E. histolytica CSN by two distinct approaches inhibited cell proliferation and viability. Both CSN5 knockdown and dominant negative mutation trapped cullin in a neddylated state, disrupting UPS activity and protein degradation. In addition, zinc ditiocarb (ZnDTC), a main metabolite of the inexpensive FDA-approved alcohol-abuse drug disulfiram, was active against parasites acting in a COP9-dependent manner. ZnDTC, given as disulfiram-zinc, had oral efficacy in clearing parasites in vivo. Our findings provide insights into the regulation of parasite protein degradation, and supports the significant therapeutic potential of COP9 inhibition. Summary sentenceParasite-encoded COP9 signalosome is an essential upstream regulator of ubiquitin-proteasome mediated protein degradation, and shows significant potential as a therapeutic target.

microbiology↗

bioRxiv2020

A Novel Small Animal Model of Norovirus Diarrhea

Human noroviruses are the leading cause of severe childhood diarrhea worldwide yet we know very little about their pathogenic mechanisms. Murine noroviruses cause diarrhea in interferon-deficient adult mice but these hosts also develop systemic pathology and lethality, reducing confidence in the translatability of findings to human norovirus disease. Herein we report that a murine norovirus causes self-resolving diarrhea in the absence of systemic disease in wild-type neonatal mice, thus mirroring the key features of human norovirus disease and representing a robust norovirus small animal disease model. Intriguingly, lymphocytes are critical for controlling acute norovirus replication while simultaneously contributing to disease severity, likely reflecting their dual role as targets of viral infection and key components of the host response.

microbiology↗

bioRxiv2020

Kinetics of charged polymer collapse: effects of additional salt

Extensive molecular dynamics simulations, using simple charged polymer models, have been employed to probe the kinetics and dynamics of early-stage collapse of charged polymers and the effect of additional monovalent salt on such kinetics. The exponents characterizing the coarsening dynamics during such early-collapse stage via finite size scaling for the case of charged polymers are found to be different from the neutral polymers, suggesting that the collapse kinetics of charged polymers are inherently different from that neutral polymers. The kinetics of coarsening of the clusters along the collapsed trajectory also depends significantly on the counterion valency and for higher valency counterions, multiple regimes are observed and unlike the neutral polymer case, the collapse kinetics are a function of charge density along the charged polymer. Inclusion of additional salt affects the kinetics and conformational landscape along the collapse trajectory. Addition of salt increases the value of critical charge density required to initiate collapse for all the counterion valencies, though the effect is more pronounced for monovalent counterion systems. The addition of salt significantly affects the collapse trajectory in the presence of trivalent counterions via promotion of transient long-distance loop structures inducing a parallel and hierarchical local collapsed conformation leading to faster global collapsed states. This may play a role in understanding the fast folding rates of biopolymers such as proteins and RNA from extended state to a collapsed state in the presence of multivalent counterions before reorganizing into a native fold.

biophysics↗

bioRxiv2020

Redundant function of Ets1 and Ets2 in regulating M-phase progression in post-natal angiogenesis

Angiogenesis is a highly orchestrated process involving complex crosstalk between several endothelial cell processes including cell cycle, cell survival and migration. Transcription factors ETS1 and ETS2 are required for EC functions necessary for embryonic angiogenesis. Here, by utilizing endothelial cell-specific deletion of Ets1 and Ets2 on post-natal angiogenesis we demonstrate that ETS1 and ETS2 have a redundant regulatory role in controlling the expression of key G2/M regulators and anti-apoptotic gene modules by recruiting the transcriptional activator CBP/p300 to the enhancers of these genes. Further, cultured aortic ECs lacking both ETS1 and ETS2 demonstrated G2/M phase arrest and increased apoptosis. In vivo, EC infiltration and invasion was attenuated when matrigel admixed with mouse mammary tumor cells was injected into adult mice with EC-specific ablation of Ets1 and Ets2. These results demonstrate that deletion of Ets1 and Ets2 in endothelial cells inhibits angiogenesis by altering cell cycle progression and decreasing cell survival.

genomics↗

bioRxiv2020

A gatekeeping role of ESR2 to maintain the primordial follicle reserve

Over the entire reproductive lifespan in mammals, a fixed number of primordial follicles serve as the source of mature oocytes. Uncontrolled and excessive activation of primordial follicles can lead to depletion of the ovarian reserve. We observed that disruption of ESR2-signaling results in increased activation of primordial follicles in Esr2-null (Esr2-/-) rats. However, follicle assembly was unaffected, and the total number of follicles remained comparable between neonatal wildtype and Esr2-/- ovaries. While the activated follicle counts were increased in Esr2-/- ovary, the number of primordial follicles were markedly decreased. Excessive recruitment of primordial follicles led to premature ovarian senescence in Esr2-/- rats and was associated with reduced levels of serum AMH and estradiol. Disruption of ESR2-signaling through administration of a selective antagonist (PHTPP) increased the number of activated follicles in wildtype rats, whereas a selective agonist (DPN) decreased follicle activation. In contrast, primordial follicle activation was not increased in the absence of ESR1 indicating that the regulation of primordial follicle activation is ESR2-specific. Follicle activation was also increased in Esr2-mutants lacking the DNA-binding domain, suggesting a role for the canonical transcriptional activation function. Both primordial and activated follicles express ESR2 suggesting a direct regulatory role for ESR2 within these follicles. We also detected that loss of ESR2 augmented the activation of AKT, ERK and mTOR pathways. Our results indicate that the lack of ESR2 upregulated both granulosa and oocyte factors, which can facilitate AKT and mTOR activation in Esr2-/- ovaries leading to increased activation of primordial follicles.

developmental biology↗

bioRxiv2020

Loss of mitochondrial localization of human FANCG causes defective FANCJ helicase

Fanconi anemia (FA) is a unique DNA damage repair pathway. Almost twenty-two genes have been identified which are associated with the FA pathway. Defect in any of those genes causes genomic instability, and the patients bear the mutation become susceptible to cancer. In our earlier work, we have identified that Fanconi anemia protein G (FANCG) protects the mitochondria from oxidative stress. In this report, we have identified eight patients having mutation (C.65G>C; p.Arg22Pro) in the N-terminal of FANCG. The mutant protein hFANCGR22P is able to repair the DNA and able to retain the monoubiquitination of FANCD2 in FANCGR22P/FGR22P cell. However, it lost mitochondrial localization and failed to protect mitochondria from oxidative stress. Mitochondrial instability in the FANCGR22P cell causes the transcriptional down-regulation of mitochondrial iron-sulphur cluster biogenesis protein Frataxin (FXN) and resulting iron deficiency of FA protein FANCJ, an iron-sulphur containing helicase involved in DNA repair.

cancer biology↗

bioRxiv2020

ECSIT prevents Alzheimer's disease pathology by regulating neuronal mitochondrial ROS and mitophagy

Altered mitochondrial fitness is a potential triggering factor in Alzheimers disease (AD). Mitochondrial quality control pathways are dysfunctional and mitochondrially-derived reactive oxygen species (mROS) levels are increased in AD patient brains. However, the pathways responsible for dysregulated mROS accumulation have remained relatively unclear. In this study, we demonstrate that levels of ECSIT, a mitochondrial oxidative phosphorylation (OxPhos) complex I (CI)-associated protein, are reduced in AD-affected brains. Neuronal ECSIT downregulation increased mROS generation and impaired mitophagy of defective mitochondria. Consequently, decreasing neuronal ECSIT caused AD-like changes, including memory loss and neuropathology. In contrast, augmented neuronal expression of ECSIT protected against the development of an AD-like phenotype. Decreased levels of ECSIT in AD patient brains therefore likely contribute to oxidative stress, neuroinflammation and AD pathogenesis.

neuroscience↗

bioRxiv2020

A novel cross talk of AtRAV1, an ethylene responsive transcription factor with MAP kinases imparts broad spectrum disease resistance in plants

Plant diseases pose a serious threat to sustainable agriculture as controlling them in eco-friendly manner remains a challenge. In this study, we establish RAV1 as a master transcriptional regulator of defense genes in model plant Arabidopsis. The overexpression of AtRAV1 provided disease resistance against necrotrophic fungal pathogen (Rhizoctonia solani) infection in A. thaliana. The transgenic lines exhibited enhanced expression of several defense genes including mitogen associated protein kinases (MAPKs) and the amplitude of their expression was further enhanced upon pathogen infection. Conversely, the atrav1 mutant plants were unable to induce the expression of these defense genes and were highly susceptible to infection. Our data suggests that upon pathogen attack, AtRAV1 transcriptionally upregulate the expression of MAPKs (AtMPK3, AtMPK4 and AtMPK6) and AtMPK3 and AtMPK6 are essential for AtRAV1 mediated disease resistance. Further, we demonstrate that AtRAV1 is a phosphorylation target of AtMPK3 (but not AtMPK6) and the phospho-defective variants of AtRAV1 are unable to induce disease resistance in A. thaliana. Considering the presence of AtRAV1 orthologs in diverse plant species, we propose that they can be gainfully deployed to control economically important diseases. In deed we observe that overexpression of tomato ortholog of AtRAV1 (SlRAV1) provides broad spectrum disease resistance against bacterial (Ralstonia solanacearum), fungal (R. solani) and viral (Tomato leaf curl virus) infections in tomato.

plant biology↗

bioRxiv2020

Improved Supra-Organization Is A Conformational And Functional Adaptation Of Respiratory Complexes

Multiple surveillance mechanisms accelerate proteasome mediated degradation of misfolded proteins to prevent protein aggregation inside and outside mitochondria. But how cells safeguard mitochondrial function despite increased protein aggregation during proteasome inactivation? Here, using two-dimensional complexome profiling, we extensively characterize the dynamic states of respiratory complexes (RCs) in proteasome-inhibited cells. We report that RC-subunits are increasingly integrated into supra-organizations to optimize catalytic activity simultaneous to their aggregation inside mitochondria. Complex-II (CII) and CV are incorporated into oligomers. CI, CIII, and CIV subcomplexes are associated into holocomplexes followed by integration into supercomplexes. Time-course experiments reveal that the core (CI+CIII2) stoichiometry of supercomplex (I+III2+IV) is preserved during early-stress while CIV composition varies. Simultaneously, increased CI-activity suggests conformational optimization of supercomplexes for better function. Re-establishment of steady-state stoichiometry and relative increase in supercomplex-quantity consolidates functional adaptation during prolonged proteasome-inhibition. Together, we name this pre-emptive adaptive mechanism as improved Supra-organization of Respiratory Complexes (iSRC). We find that iSRC is active in multiple protein-unfolding stresses, in multiple cell-types that differ in proteostatic and metabolic demands, and reversible upon stress-withdrawal.

biochemistry↗

bioRxiv2019

α-synuclein aggregates induce c-Abl activation and neurodegeneration by a redox stress mechanism

ObjectiveRedox stress, c-Abl activation, and -synuclein aggregates each independently contribute to neurodegeneration in Parkinsons disease. Interactions between these factors may underlie convergent and feed-forward mechanisms of disease progression. Methods-synuclein aggregate formation was induced in neuronal cultures and mouse substantia nigra by exposure to pre-formed human -synuclein fibrils or by AAV-mediated over-expression of -synuclein. Aggregate formation, c-Abl activation, redox stress, and neurodegeneration were evaluated by immunohistochemistry and Western blots, and mouse motor function was evaluated using the rota-rod and pole tests. To suppress redox stress, cultures and mice were treated with N-acetyl cysteine, a thiol repletion agent that supports neuronal glutathione metabolism. ResultsIn primary neuron cultures, the formation of -synuclein aggregates led to redox stress and c-Abl activation. Redox stress alone, in the absence of -synuclein aggregates, was also sufficient to induced c-Abl activation. N-acetyl cysteine suppressed redox stress, and likewise suppressed both c-Abl activation and -synuclein aggregation. A similar pattern was observed in the two mouse models of Parkinsons disease. In both models, -synuclein aggregates in the substantia nigra were accompanied by redox stress, c-Abl activation, dopaminergic neurodegeneration and motor impairment, all of which were attenuated in mice treated with oral N-acetyl cysteine. InterpretationThese results indicate that -synuclein aggregates induce c-Abl activation by a redox stress mechanism. c-Abl in turn promotes -synuclein aggregation, and this potentially feed-forward process can be blocked by N-acetyl cysteine. The findings thus add mechanistic support for N-acetyl cysteine as a therapeutic for Parkinsons disease.

neuroscience↗

bioRxiv2019

Conversion of a defensive toxin-antitoxin system into an offensive T6SS effector in Burkholderia

Bacteria use various kinds of toxins to either inhibit the growth of co-habiting bacteria or when needed control their own growth. Here we report that Burkholderia and certain other bacteria have altered the potential defensive function of Tox-REase-5 domain containing toxins into offensive function. The Burkholderia gladioli strain NGJ1 encodes such toxins as type VI secretion system (T6SS) effectors (Tse) and potentially deploys them to kill co-habiting rice endophytic bacteria. Notably, the immunity (Tsi) proteins associated with Tse effectors demonstrate functional similarity with the antitoxin of type II toxin-antitoxin (TA) system. Genome analysis of diverse bacteria revealed that various Tse orthologs are either encoded as TA or T6SS effectors. In addition, potential evolutionary events associated with conversion of TA into T6SS effectors have been delineated. Our results indicate that the transposition of IS3 elements has led to the operonic fusion of certain T6SS related genes with TA genes resulting in their conversion into T6SS effectors. Such a genetic change has enabled bacteria to utilize novel toxins to precisely target co-habiting bacteria.

microbiology↗

bioRxiv2019

The Interaction of Viperin with Hepatitis C Virus Non-Structural Protein 5A Inhibits the Catalytic Activity of Viperin

The radical SAM enzyme viperin exerts a wide range of antiviral effects through both the synthesis of the antiviral nucleotide 3-deoxy-3, 4-didehydro-CTP (ddhCTP) and through its interactions with various cellular and viral proteins. Here we investigate the interaction of viperin with hepatitis C virus non-structural protein 5A (NS5A) and the host sterol regulatory protein, vesicle-associated membrane protein A (VAP-33). NS5A and VAP-33 form part of the viral replication complex that is essential for copying the RNA genome of the virus. Using transfected enzymes in HEK293T cells, we show that viperin binds to both NS5A and VAP-33 independently and that this interaction is dependent on all three proteins being localized to the ER membrane. Co-expression of viperin with VAP-33 and NS5A was found to reduce NS5A levels, most likely by increasing the rate of proteasomal degradation. However, co-expression of viperin with VAP-33 and NS5A also reduces the specific activity of viperin by ~ 3-fold. This observation suggests that NS5A may have evolved to bind viperin as a strategy to reduce ddhCTP synthesis, thereby reducing possibility of the replication complex introducing this chain-terminating nucleotide during genome synthesis.

biochemistry↗