Biology subjects

Biswas, A.

Publications and source records attributed to Biswas, A..

106 records · Page 6Linked to original sources

bioRxiv2021

High viral abundance and low diversity are associated with increased CRISPR-Cas prevalence across microbial ecosystems

CRISPR-Cas are adaptive immune systems that protect their hosts against viruses and other parasitic mobile genetic elements. Consequently, selection from viruses and other genetic parasites is often assumed to drive the acquisition and maintenance of these immune systems in nature, but this remains untested. Here, we analyse the abundance of CRISPR arrays in natural environments using metagenomic datasets from 332 terrestrial, aquatic and host-associated ecosystems. For each metagenome we quantified viral abundance and levels of viral community diversity to test whether these variables can explain variation in CRISPR-Cas abundance across ecosystems. We find a strong positive correlation between CRISPR-Cas abundance and viral abundance. In addition, when controlling for differences in viral abundance, we found that the CRISPR-Cas systems are more abundant when viral diversity is low. We also found differences in relative CRISPR-Cas abundance among environments, with environmental classification explaining [~]24% of variation in CRISPR-Cas abundance. However, the correlations with viral abundance and diversity are broadly consistent across diverse natural environments. These results indicate that viral abundance and diversity are major ecological factors that drive the selection and maintenance of CRISPR-Cas in microbial ecosystems. Significance statementNumerous studies demonstrate that CRISPR-Cas immune systems can provide defence against bacteriophage and archaeal viruses, yet little is known about the ecological conditions where CRISPR-Cas immunity is favoured. Moreover, our knowledge is largely confined to laboratory studies and it is unknown if viruses are a key selective driver of CRISPR-Cas in nature. Using metagenomic data from diverse environments we find that both viral abundance and the abundance of CRISPR-Cas immune systems correlate positively across most environments. Furthermore, CRISPR-Cas systems are more prevalent when viral diversity is low. These results extend previous theoretical work by demonstrating that viruses are a key driver of selection of CRISPR-Cas immune systems across many natural ecosystems.

microbiology↗

bioRxiv2021

The role of epistasis in determining the fitness landscape of HIV proteins

The rapid evolution of HIV is constrained by interactions between mutations which affect viral fitness. In this work, we explore the role of epistasis in determining the fitness landscape of HIV for multiple drug target proteins, including Protease, Reverse Transcriptase, and Integrase. Epistatic interactions between residues modulate the mutation patterns involved in drug resistance with unambiguous signatures of epistasis best seen in the comparison of a maximum entropy sequence co-variation (Potts) model predicted and experimental HIV sequence "prevalences" when expressed as higher-order marginals (beyond triplets) of the sequence probability distribution. In contrast, the evidence for epistasis based on experimental measures of fitness such as replicative capacity is weak; the correspondence with Potts model "prevalence"-based predictions is obscured by site conservation and limited precision. Double mutant cycles provide in principle one of the best ways to probe epistatic interactions experimentally without reference to a particular background, and we find they reveal that the most strongly interacting mutations in HIV involve correlated sets of drug-resistance-associated residues, however the analysis is complicated by the small dynamic range of measurements. The use of correlated models for the design of experiments to probe viral fitness can help identify the epistatic interactions involved in mutational escape, and lead to better inhibitor therapies. Author summaryProtein covariation models provide an alternative to experimental measures for estimating the fitness of mutations in proteins from across a variety of organisms. Yet, for viral proteins, it has been shown that models including epistatic couplings between residues, or other machine learning models perform no better or even worse than a simpler independent model devoid of such epistatic couplings in estimating viral fitness measurements such as replicative capacities, providing weak or ambiguous evidence for epistasis. We show that the evidence for long-range epistasis is strong by the analysis of the high-order marginals of the MSA distribution (up to subsequences of length 14), which are accurately captured by a correlated Potts sequence-covariation model but not by an independent model. While double mutant cycles in principle provide well-established biophysical probes for epistatic interactions, we demonstrate that the analysis and comparison between model and experiment is difficult due to the much smaller dynamic range of the measurements, making them more susceptible to noise.

biophysics↗

bioRxiv2021

SARS-CoV-2 Nsp14 activates NF-κB signaling and induces IL-8 upregulation

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to NF-{kappa}B activation and induction of pro-inflammatory cytokines, though the underlying mechanism for this activation is not fully understood. Our results reveal that the SARS-CoV-2 Nsp14 protein contributes to the viral activation of NF-{kappa}B signaling. Nsp14 caused the nuclear translocation of NF-{kappa}B p65. Nsp14 induced the upregulation of IL-6 and IL-8, which also occurred in SARS-CoV-2 infected cells. IL-8 upregulation was further confirmed in lung tissue samples from COVID-19 patients. A previous proteomic screen identified the putative interaction of Nsp14 with host Inosine-5-monophosphate dehydrogenase 2 (IMPDH2) protein, which is known to regulate NF-{kappa}B signaling. We confirmed the Nsp14-IMPDH2 protein interaction and found that IMPDH2 knockdown or chemical inhibition using ribavirin (RIB) and mycophenolic acid (MPA) abolishes Nsp14-mediated NF-{kappa}B activation and cytokine induction. Furthermore, IMDPH2 inhibitors (RIB, MPA) efficiently blocked SARS-CoV-2 infection, indicating that IMDPH2, and possibly NF-{kappa}B signaling, is beneficial to viral replication. Overall, our results identify a novel role of SARS-CoV-2 Nsp14 in causing the activation of NF-{kappa}B.

microbiology↗

bioRxiv2021

FACT subunit SUPT16H associates with BRD4 and contributes to silencing of antiviral interferon signaling

Summary/AbstractFACT (FAcilitates Chromatin Transcription) is a heterodimeric protein complex composed of SUPT16H and SSRP1, and a histone chaperone participating in chromatin remodeling during gene transcription. FACT complex is profoundly regulated, and contributes to both gene activation and suppression. Here we reported that SUPT16H, a subunit of FACT, is acetylated at lysine 674 (K674) of middle domain (MD), which involves TIP60 histone acetyltransferase. Such acetylation of SUPT16H is recognized by bromodomain protein BRD4, which promotes protein stability of SUPT16H. We further demonstrated that SUPT16H-BRD4 associates with histone modification enzymes (EZH2, HDAC1) and affects histone marks (H3K9me3, H3K27me3 and H3ac). BRD4 is known to profoundly regulate interferon (IFN) signaling, while such function of SUPT16H has never been explored. Surprisingly, our results revealed that SUPT16H genetic knockdown via RNAi or pharmacological inhibition by using its inhibitor, curaxin 137 (CBL0137), results in the induction of IFNs and interferon-stimulated genes (ISGs). Through this mechanism, CBL0137 is shown to efficiently inhibit infection of multiple viruses, including Zika, influenza, and SARS-CoV-2. Furthermore, we demonstrated that CBL0137 also causes the remarkable activation of IFN signaling in natural killer (NK) cells, which promotes the NK-mediated killing of virus-infected cells in a co-culture system using human primary NK cells. Overall, our studies unraveled the previously un-appreciated role of FACT complex in regulating IFN signaling in both epithelial and NK cells, and also proposed the novel application of CBL0137 to treat viral infections.

molecular biology↗

bioRxiv2021

A study of Mutation in ATP7B gene and its correlation with clinical phenotype and radiological features in Wilson Disease patients

IntroductionWilson Disease (WD) is an autosomal recessive disease caused by mutations in the ATP7B gene. Clinical manifestations of WD are variable. Identification of prevalent mutations in a given population is necessary to provide mutation-based molecular diagnosis. Previous studies have detected common mutations in this part of the world and our study aimed to correlate genotype with clinical and radiological features. MethodsA descriptive cross-sectional observational study was conducted over a period of two years in a tertiary care hospital and neurology referral unit of Kolkata, India. All WD patients within the study period and meeting the inclusion criteria were included. Demographic data collection, clinical examination and relevant laboratory investigations were done. Magnetic resonance imaging of brain and cognitive assessment by Mini Mental Score Exam (MMSE) were also performed. Blood was collected for genetic analyses. PCR-Sanger sequencing of exons 2,4,6,8,10,14,16,18 of ATP7B gene was done based on previous reports of mutation hotspots of ATP7B gene for WD in Eastern India. Genotype phenotype correlation was attempted using two supervised machine learning methods, viz. logistic regression with an elastic-net penalty and the random forest. ResultsOf 52 WD patients were included in the study, 57.7% were males. The mean age at diagnosis was 13.96 years. Majority (61.8%) of the patients had dystonia on presentation, followed by dysarthria (41.2%), tremor (17.6%) and ataxia (11.8%). The mean MMSE and Frontal Assessment Battery score were 23.74 and 10.63 respectively and both were lower than the normal baseline values.Out of the total cohort of 52 patients,15(28.8%) harbored previously reported common mutations from this part of the country. Of the 15, 12 had the same mutation of c.813C>A(p.cys271Ter).The presence of common mutationswas associated with several distinct clinical phenotypes in the mathematical models but larger sample sizes are needed to corroborate the correlation. ConclusionsWD patients in eastern India have significant genotypic and phenotypic diversity. Further studies with larger samples and screening of remaining exons are warranted.

neuroscience↗

bioRxiv2020

Polyamine biosynthesis and eIF5A hypusination are modulated by the DNA tumor virus KSHV and promote KSHV viral infection

Polyamines are critical metabolites involved in various cellular processes and often dysregulated in cancers. Kaposis sarcoma associated Herpesvirus (KSHV) is a defined oncogenic virus belonging to the sub-family of human gamma-herpesviruses. KSHV infection leads to the profound alteration of host metabolic landscape to favor the development of KSHV-associated malignancies. In our studies, we identified that polyamine biosynthesis and eIF5A hypusination are dynamically regulated by KSHV infection likely through the modulation of key enzymes of these pathways, such as ODC1, and that in return these metabolic pathways are required for both KSHV lytic switch from latency and de novo infection. The further analysis unraveled that translation of critical KSHV latent and lytic proteins (LANA, RTA) depends on eIF5A hypusination. We also demonstrated that KSHV infection can be efficiently and specifically suppressed by using inhibitors targeting either polyamine biosynthesis or eIF5A hypusination. Above all, our results illustrated that the dynamic and profound interaction of a DNA tumor virus (KSHV) with host polyamine biosynthesis and eIF5A hypusination metabolic pathways promote viral propagation and oncogenesis, which serve as new therapeutic targets to treat KSHV-associated malignancies.

microbiology↗

bioRxiv2020

Heterogenous adhesion of follower cells affects tension profile and velocity of leader cells in primary keratocyte collective cell migration

Single cell studies demonstrate membrane tension to be a central regulator of lamellipodia-driven motility bringing in front-coherence. During collective cell migration, however, tension mapping or existence of intracellular tension-gradients and the effect of cell-cell interactions have remained unexplored. In this study of membrane fluctuations and fluctuation-tension of migrating primary keratocyte cell-sheets, we first show that some leader cells are followed by followers which remain de-adhered from the substrate while being attached to other cells and thus appear to be "taking a ride". A subtle yet significant enhanced long-timescale velocity in these leaders indicate increased directionality. Intriguingly, such leaders mostly have front-high tension gradients like single keratocytes, while followers and other leaders usually display front-low membrane tension gradients. The front-high tension gradient and higher membrane tension observed in these leaders, despite the high cell-to-cell variability in membrane tension demonstrate how leader-follower interactions and heterogenous adhesion profiles are key in collective cell migration.

biophysics↗

bioRxiv2020

A universal framework for detecting cis-regulatory diversity in DNA regulatory regions

High-throughput sequencing-based assays measure different biochemical activities pertaining to gene regulation, genome-wide. These activities include protein-DNA binding, enhancer-activity, open chromatin, and more. A major goal is to understand underlying sequence components, or motifs, that can explain the measured activity. It is usually not one motif, but a combination of motifs bound by cooperatively acting proteins that confers activity to such regions. Furthermore, although having a single type of activity, the regions can still be diverse, governed by different combinations of proteins/motifs. Current approaches do not take into account this issue of combinatorial diversity. We present a new statistical framework O_SCPLOWCISC_SCPLOWDO_SCPLOWIVERSITYC_SCPLOW, which models regions as diverse modules characterized by combinations of motifs, while simultaneously learning the motifs themselves. We show that ChIP-seq data for the CTCF protein in fly contains diverse sequence structures, with most direct CTCF-binding sites situated far from promoters, giving insights into its co-factors and potential role in looping. Human CTCF-bound regions, on the other hand, have a different architecture. Because O_SCPLOWCISC_SCPLOWDO_SCPLOWIVERSITYC_SCPLOW does not rely on knowledge of motifs, modules, cell-type, or organism, it is general enough to be applied to regions reported by most high-throughput assays. Indeed, enhancer predictions resulting from different assays--GRO-cap, STARR-seq, and those measuring chromatin structure--show distinct modules and combinations of TF binding sites, some specific to the assay. No module occurs universally in all enhancer-assays. Finally, analysis of accessible chromatin suggests that regions open in one cell-state encode information about future states, with certain modules staying open and others closing down later. The code is freely available at https://github.com/NarlikarLab/cisDIVERSITY.

genomics↗

bioRxiv2020

Inhibition of Polo-like kinase 1 (PLK1) to facilitate the reactivation and elimination of latent gamma-herpesviruses.

Both Kaposis sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) establish the persistent, life-long infection primarily at the latent status, and contribute to certain types of tumors, including B cell lymphomas, especially in immuno-compromised individuals, such as people living with HIV (PLWH). Lytic reactivation of these viruses can be employed to kill tumor cells harboring latently infected viral episomes, through the viral cytopathic effects and the subsequent antiviral immune responses. In this study, we identified that polo-like kinase 1 (PLK1) is induced by KSHV de novo infection as well as lytic switch from KSHV latency. We further demonstrated that PLK1 depletion or inhibition facilitates KSHV reactivation and promotes cell death of KSHV-reactivated lymphoma cells. Mechanistically, PLK1 regulates Myc that is critical to both maintenance of KSHV latency and support of cell survival, and preferentially affects the level of H3K27me3 inactive mark both globally and at certain loci of KSHV viral episomes. Lastly, we recognized that PLK1 inhibition synergizes with STAT3 inhibition to efficiently induce KSHV reactivation. Furthermore, we also confirmed that PLK1 depletion or inhibition yields the similar effect on EBV reactivation and cell death of EBV-reactivated lymphoma cells. Additionally, we noticed the PLK1 in B cells is elevated in the context of HIV infection and by HIV Nef protein, which would favor KSHV/EBV latency. Above all, our findings illustrated that PLK1 is a novel host target that can be inhibited to benefit the viral oncolysis to eliminate KSHV/EBV-infected lymphoma cells, particularly for PLWH.

cancer biology↗

bioRxiv2020

Interruption in POSTN-ITGAV axis affects lympho-hematopoiesis in mouse spleen

Outside-in integrin signaling regulates cell fate decisions in a variety of cell types, including hematopoietic stem cells (HSCs). Our earlier published studies showed that interruption of Periostin (POSTN) and Integrin-v (ITGAV) interaction induces faster proliferation in HSCs with developmental stage dependent functional effects. Here, we examined the role of POSTN-ITGAV axis in lympho-hematopoietic activity in spleen that hosts rare population of HSCs, the functional regulation of which is not clearly known. Vav-iCre mediated deletion of Itgav in hematopoietic system led to higher proliferation rates, resulting in increased frequency of primitive HSCs in adult spleen. However, in vitro CFU-C assays demonstrated a poorer differentiation potential following Itgav deletion. This also led to a decrease in the white pulp area with a significant decline in the B-cell numbers. Systemic deletion of its ligand, POSTN, phenocopied the effects noted in Vav-Itgav-/- mice. Histological examination of Postn deficient spleen also showed increase in the spleen trabecular areas. Surprisingly, these were the myofibroblasts of the trabecular and capsular areas that expressed high levels of POSTN within the spleen tissue. In addition, vascular smooth muscle cells also expressed POSTN. Through CFU-S12 assays, we showed that hematopoietic support potential of stroma in Postn deficient splenic hematopoietic niche was defective. Overall, we demonstrate that POSTN-ITGAV interaction plays important role in spleen lympho-hematopoiesis.

immunology↗

bioRxiv2019

Transient CRISPR immunity leads to coexistence with phages

Phages play a major role in shaping the composition, evolution and function of bacterial communities. While bacteria and phages coexist in many natural environments, their coexistence is often short-lived in the lab due to the evolution of phage resistance. However, fitness costs associated with resistance and mutational loss of resistance alleles may limit the durability of acquired resistances, potentially allowing phages to re-invade the population. Here, we explore this idea in the context of bacteria that evolve CRISPR-based immunity against their phages. Consistent with previous studies, we found that the bacterium Pseudomonas aeruginosa PA14 evolved high levels of CRISPR-based immunity and low levels of surface-based resistance following infection with phage DMS3vir, which led to rapid phage extinction. However, when these pre-immunized bacterial populations were subsequently challenged with the same phage, they failed to clear the infection and instead stably coexisted with the phage. Analysis of bacterial genotypes and phenotypes over time explained why CRISPR-Cas immunity provides only a transient advantage: in the absence of phage (i.e. following the initial phage extinction) formerly CRISPR-immune bacteria regain sensitivity due to evolutionary loss of spacers, whereas in the presence of phage (i.e. upon reinfection) selection favours surface-based resistance over CRISPR immunity. The latter results from an infection-induced fitness cost of CRISPR-immunity that is due to phage gene expression prior to target DNA cleavage by the immune system. Together, these results show that CRISPR-Cas immune systems provide only a transient benefit to bacteria upon phage infection and help to explain why bacteria and phages can coexist in natural environments even when bacteria carry CRISPR-Cas adaptive immune systems that allow for rapid acquisition of immunity against phages.

microbiology↗

bioRxiv2019

The plasma Factor XIII heterotetrameric complex structure: unexpected unequal pairing within a symmetric complex

Factor XIII (FXIII) is a predominant determinant of clot stability, strength, and composition. Plasma FXIII circulates as a pro-transglutaminase with 2 catalytic A subunits and 2 carrier-protective B subunits in a heterotetramer (FXIII-A2B2). FXIII-A2 and -B2 subunits are synthesized separately and then assembled in plasma. Following proteolytic activation by thrombin and calcium-mediated dissociation of the B-subunits, activated FXIII (FXIIIa) covalently cross-links fibrin, promoting clot stability. The zymogen and active states of the FXIII-A subunits have been structurally characterized; however, the structure of FXIII-B subunits and the FXIII-A2B2 complex have remained elusive. Using integrative hybrid approaches including atomic force microscopy, cross-linking mass spectrometry, and computational approaches, we have constructed the first all-atom model of the FXIII-A2B2 complex. We also used molecular dynamic simulations in combination with isothermal titration calorimetry to characterize FXIII-A2B2 assembly, activation, and dissociation. Our data reveal unequal pairing of individual subunit monomers in an otherwise symmetric complex, and suggest this unusual structure is critical for both assembly and activation of this complex. Our findings enhance understanding of mechanisms associating FXIII-A2B2 mutations with disease and have important implications for the rational design of molecules to alter FXIII assembly and/or activity to reduce bleeding and thrombotic complications.

molecular biology↗

bioRxiv2019

Exploitation of the cooperative behaviours of anti-CRISPR phages

Many bacteria encode CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR-associated) adaptive immune systems to protect themselves against their viruses (phages)1. To overcome resistance, phages have evolved anti-CRISPR proteins (Acr), which naturally vary in their potency to suppress the host immune system and avoid phage extinction2,3,4,5. However, these Acr-phages need to cooperate in order to overcome CRISPR-based resistance4,5: while many initial infections by Acr-phages are unsuccessful, they nonetheless lead to the production of Acr proteins, which generate immunosuppressed cells that can be successfully exploited by other Acr-phages in the population4,5. Here we test the prediction that phages lacking acr genes (Acr-negative phages) may exploit this cooperative behaviour6. We demonstrate that Acr-negative phages can indeed benefit from the presence of Acr-positive phages during pairwise competitions, but the extent of this exploitation depends on the potency of the Acr protein. Specifically, \"strong\" Acr proteins are more exploitable and benefit both phage types, whereas \"weak\" Acr proteins predominantly benefit Acr-positive phages only and therefore provide a greater fitness advantage during competition with Acr-negative phages. This work further helps to explain what defines the strength of an Acr protein, how selection acts on different Acr types in a phage community context, and how this can shape the dynamics of phage populations in natural communities.

microbiology↗

bioRxiv2018

ROSeq: A rank based approach to modelling gene expression in single cells

1Systematic delineation of complex biological systems is an ever-challenging and resource-intensive process. Single cell transcriptomics allows us to study cell-to-cell variability in complex tissues at an unprecedented resolution. Accurate modeling of gene expression plays a critical role in the statistical determination of tissue-specific gene expression patterns. In the past few years, considerable efforts have been made to identify appropriate parametric models for single cell expression data. The zero-inflated version of Poisson/Negative Binomial and Log-Normal distributions have emerged as the most popular alternatives due to their ability to accommodate high dropout rates, as commonly observed in single cell data. While the majority of the parametric approaches directly model expression estimates, we explore the potential of modeling expression-ranks, as robust surrogates for transcript abundance. Here we examined the performance of the Discrete Generalized Beta Distribution (DGBD) on real data and devised a Wald-type test for comparing gene expression across two phenotypically divergent groups of single cells. We performed a comprehensive assessment of the proposed method, to understand its advantages as compared to some of the existing best practice approaches. Besides striking a reasonable balance between Type 1 and Type 2 errors, we concluded that ROSeq, the proposed differential expression test is exceptionally robust to expression noise and scales rapidly with increasing sample size. For wider dissemination and adoption of the method, we created an R package called ROSeq, and made it available on the Bioconductor platform.

genomics↗

bioRxiv2018

Cholesterol depletion by MβCD enhances membrane tension, its heterogeneity and affects cellular integrity.

Cholesterol depletion in cells by M{beta}CD remodels the plasma membranes mechanics and its interactions with the underlying cytoskeleton. Decoupling the two effects and studying various alterations to the membranes mechanical parameters is important for understanding cholesterols role in cellular response to stress. By mapping membrane height fluctuations in single cells, we report that M{beta}CD treatment reduces temporal fluctuations and flattens out the membrane - but does not supress activity-driven fluctuations. We find that membrane tension increase contributes most to the altered fluctuations, among the multiple mechanical parameters computed. Maps also reveal an enhanced long-range heterogeneity within single cells, both in amplitude of fluctuations and membrane tension on cholesterol depletion. To check if this alters the tenacity of membrane to mechanical stress we use hypo-osmotic shock. We find that on M{beta}CD treatment, cells are more prone to rupture than control cells, and this is not hindered by actomyosin perturbations. We report increased rupture sizes on cholesterol depletion and argue that, together, this indicates decreased lysis and line tension. Therefore, we show that cholesterol depletion directly affects cell membranes not only by enhancing membrane-cytoskeleton interactions, but also by increasing membrane tension while reducing lysis tension - hence making cells prone to rupture.

biophysics↗

bioRxiv2017

DIVERSITY In Binding, Regulation, And Evolution Revealed From High-Throughput ChIP

A high-throughput chromatin immunoprecipitation (ChIP) experiment is like a black-box: it reports all regions that are associated with the profiled protein based on the initial cross-linking step. These regions can be a highly diverse set of DNA sequences, with some making direct contact with the protein, some binding through intermediaries, and some being a result of long-range interactions involving the protein. We present O_SCPLOWDIVERSITYC_SCPLOW, a method that identifies the distinct components of such a mixture, leaving no data behind, while at the same time, using no prior motif knowledge. Using the example of the REST protein, we show that these different components give insights into the various complexes that may be forming along the chromatin and their regulatory functions.\n\nhttp://diversity.ncl.res.in/ (webserver)\n\nhttps://github.com/NarlikarLab/DIVERSITY (standalone for Mac OSX/Linux)

bioinformatics↗