Biology subjects

Roy, S.

Publications and source records attributed to Roy, S..

At least 19 recordsLinked to original sources

bioRxiv2018

Haplotype assignment of virus NGS data using co-variation of variant frequencies

Longitudinal deep sequencing of viruses can provide detailed information about intra-host evolutionary dynamics including how viruses interact with and transmit between hosts. Many analyses require haplotype reconstruction, identifying which variants are co-located on the same genomic element. Most current methods to perform this reconstruction are based on a high density of variants and cannot perform this reconstruction for slowly evolving viruses. We present a new approach, HaROLD (HAplotype Reconstruction Of Longitudinal Deep sequencing data), which performs this reconstruction based on identifying co-varying variant frequencies using a probabilistic framework. We test this method with synthetic data sets of mixed cytomegalovirus and norovirus genomes, demonstrating high accuracy when longitudinal samples are available.

genomics↗

bioRxiv2018

Mcidas mutant mice reveal a two-step process for the specification and differentiation of multiciliated cells in mammals

Motile cilia on multiciliated cells (MCCs) function in fluid clearance over epithelia. Studies with Xenopus embryos and patients with the congenital respiratory disorder reduced generation of multiple motile cilia, have implicated the nuclear protein MCIDAS (MCI), in the transcriptional regulation of MCC specification and differentiation. Recently, a paralogous protein, GMNC, was also shown to be required for MCC formation. Surprisingly, and in contrast to the presently held view, we find that Mci mutant mice can specify MCC precursors. However, these precursors cannot produce multiple basal bodies, and mature into single ciliated cells. We show that MCI is required specifically to induce deuterosome pathway components for the production of multiple basal bodies. Moreover, GMNC and MCI associate differentially with the cell-cycle regulators E2F4 and E2F5, which enables them to activate distinct sets of target genes (ciliary transcription factor genes versus genes for basal body generation). Our data establish a previously unrecognized two-step model for MCC development: GMNC functions in the initial step for MCC precursor specification. GMNC induces Mci expression, which then drives the second step of basal body production for multiciliation.\n\nSUMMARY STATEMENTWe show how two GEMININ family proteins function in mammalian multiciliated cell development: GMNC regulates precursor specification and MCIDAS induces multiple basal body formation for multiciliation.

developmental biology↗

bioRxiv2018

TCR-pMHC bond length controls TCR ligand discrimination

T-cell receptors (TCRs) detect specifically and sensitively a small number of agonist peptide-major histocompatibility complexes (pMHCs) from an ocean of structurally similar self-pMHCs to trigger antigen-specific adaptive immune responses1-4. Despite intense efforts, the mechanism underlying TCR ligand discrimination remains a major unanswered question in immunology. Here we show that a TCR discriminates between closely related peptides by forming TCR-pMHC bonds with different lengths, which precisely control the accessibility of CD3{zeta} immunoreceptor tyrosine-based activation motifs (ITAMs) for phosphorylation. Using in situ fluorescence resonance energy transfer (FRET)3,5, we measured the intermolecular length of single TCR-pMHC bonds and the intramolecular distance of individual TCR-CD3{zeta} complexes at the membrane of live primary T cells. We found that an agonist forms a short TCR-pMHC bond to pull the otherwise sequestered CD3{zeta} off the inner leaflet of the plasma membrane, leading to full exposure of its ITAMs for strong phosphorylation. By contrast, a structurally similar weaker peptide forms a longer bond with the TCR, resulting in partial dissociation of CD3{zeta} from the membrane and weak phosphorylation. Furthermore, we found that TCR-pMHC bond length determines 2D TCR binding kinetics and affinity, T-cell calcium signaling and T-cell proliferation, governing the entire process of signal reception, transduction and regulation. Thus, our data reveal the fundamental mechanism by which a TCR deciphers the structural differences between foreign antigens and self-peptides via TCR-pMHC bond length to initiate different TCR signaling for ligand discrimination.

immunology↗

bioRxiv2018

In silico prediction of high-resolution Hi-C interaction matrices

The three-dimensional organization of the genome plays an important role in gene regulation by enabling distal sequence elements to control the expression level of genes hundreds of kilobases away. Hi-C is a powerful genome-wide technique to measure the contact count of pairs of genomic loci needed to study three-dimensional organization. Due to experimental costs high resolution Hi-C datasets are available only for a handful of cell lines. Computational prediction of Hi-C contact counts can offer a scalable and inexpensive approach to examine three-dimensional genome organization across many cellular contexts. Here we present HiC-Reg, a novel approach to predict contact counts from one-dimensional regulatory signals such as epigenetic marks and regulatory protein binding. HiC-Reg exploits the signal from the region spanning two interacting regions and from across multiple cell lines to generalize to new contexts. Using existing feature importance measures and a new matrix factorization based approach, we found CTCF and chromatin marks, especially repressive and elongation marks, as important for predictive performance. Predicted counts from HiC-Reg identify topologically associated domains as well as significant interactions that are enriched for CTCF bi-directional motifs and agree well with interactions identified from complementary long-range interaction assays. Taken together, HiC-Reg provides a powerful framework to generate high-resolution profiles of contact counts that can be used to study individual locus level interactions as well as higher-order organizational units of the genome.

bioinformatics↗

bioRxiv2018

Chemical Characterization of Interacting Genes in Few Subnetworks of Alzheimer’s Disease

A number of genes have been identified as a key player in Alzheimers disease (AD). Topological analysis of co-expression network reveals that key genes are mostly central or hub genes. The association between a hub gene and its neighbour genes can be derived easily using relative abundance of their expression levels. However, it is still unexplored fact that whether any hub and its neighbour genes within a sub-network exhibits any kind of proximity with respect to their chemical properties of the DNA sequences or not, that code for a sequence of amino acids.\n\nIn this work, we try to make a quantitative investigation of the underlying biological facts in DNA sequential and primary protein level in mathematical paradigm. It may gives a holistic view of the interrelationships existing between hub genes and neighbour genes in few selective AD subnetworks. We define a mapping model from physicochemical properties of DNA sequence to chemical characterization of amino acid sequences. We use distribution of chemical groups present in a sequence after decoding into corresponding amino acids to investigate the fact that whether any hub genes are associated closely with its neighbour genes chemically in the subnetworks. Interestingly, our preliminary results confirm the fact the dependent genes that are coexpressed with its hub gene are also having proximity with respect to their amino acid chemical group distributions.\n\nCCS Concepts*Applied computing [->] Computational genomics;

synthetic biology↗

bioRxiv2018

A CRISPR/Cas9 based strategy to manipulate the Alzheimer’s amyloid pathway

The gradual accumulation of amyloid-{beta} (A{beta}) is a neuropathologic hallmark of Alzheimers disease (AD); playing a key role in disease progression. A{beta} is generated by the sequential cleavage of amyloid precursor protein (APP) by {beta}- and {gamma}-secretases, with BACE-1 ({beta}-site APP cleaving enzyme-1) cleavage as the rate limiting step 1-3. CRISPR/Cas9 guided gene-editing is emerging as a promising tool to edit pathogenic mutations and hinder disease progression 4,5,6 However, few studies have applied this technology to neurologic diseases 7-9. Besides technical caveats such as low editing efficiency in brains and limited in vivo validation 7, the canonical approach of mutation-correction would only be applicable to the small fraction of neurodegenerative cases that are inherited (i.e. < 10% of AD, Parkinsons, ALS); with a new strategy needed for every gene. Moreover, feasibility of CRISPR/Cas9 as a therapeutic possibility in sporadic AD has not been explored. Here we introduce a strategy to edit endogenous APP at the extreme C-terminus and reciprocally manipulate the amyloid pathway - attenuating {beta}-cleavage and A{beta}, while up-regulating neuroprotective a-cleavage. APP N-terminus, as well as compensatory APP homologues remain intact, and key physiologic parameters remain unaffected. Robust APP-editing is seen in cell lines, cultured neurons, human embryonic stem cells/iPSC-neurons, and mouse brains. Our strategy works by limiting the physical association of APP and BACE-1, and we also delineate the mechanism that abrogates APP/BACE-1 interaction in this setting. Our work offers an innovative cut and silence gene-editing strategy that could be a new therapeutic paradigm for AD.

neuroscience↗

bioRxiv2018

The contribution of parent-to-offspring transmission of telomeres to the heritability of telomere length in humans

Leukocyte telomere length (LTL) is a heritable trait with two potential sources of heritability (h2): inherited variation in non-telomeric regions (e.g., SNPs that influence telomere maintenance) and variability in the lengths of telomeres in gametes that produce offspring zygotes (i.e., \"direct\" inheritance). Prior studies of LTL h2 have not attempted to disentangle these two sources. Here, we use a novel approach for detecting the direct inheritance of telomeres by studying the association between identity-by-descent (IBD) sharing at chromosome ends and phenotypic similarity in LTL. We measured genome-wide SNPs and LTL for a sample of 5,069 Bangladeshi adults with substantial relatedness. For each of the 7,254 relative pairs identified, we used SNPs near the telomeres to estimate the number of chromosome ends shared IBD, a proxy for the number of telomeres shared IBD (Tshared). We then estimated the association between Tshared and the squared pairwise difference in LTL (({Delta}LTL)2) within various classes of relatives (siblings, avuncular, cousins, and distant), adjusting for overall genetic relatedness ({phi}). The association between Tshared and ({Delta}LTL)2 was inverse among all relative pair types. In a meta-analysis including all relative pairs ({phi} >0.05), the association between Tshared and ({Delta}LTL)2 (P=0.002) was stronger than the association between {phi} and ({Delta}LTL)2 (P=0.45). Our results provide strong evidence that telomere length (TL) in parental germ cells impacts TL in offspring cells and contributes to LTL h2 despite telomere \"reprogramming\" during embryonic development. Applying our method to larger studies will enable robust estimation of LTL h2 attributable to direction transmission.

genetics↗

bioRxiv2018

PAGE4 and Conformational Switching: Insights from Molecular Dynamics Simulations and Implications for Prostate Cancer

Prostate-Associated Gene 4 (PAGE4) is a disordered protein implicated in the progression of prostate cancer. PAGE4 can be phosphorylated at two residue sites by Homeodomain-Interacting Protein Kinase 1 (HIPK1) to facilitate its binding to the Activator Protein-1 (AP-1) transcription factor. In contrast, a further hyperphosphorylation of PAGE4 by CDC-Like Kinase 2 (CLK2) reduces its binding affinity to AP-1, thus affecting the androgen receptor (AR) activity. Both SAXS and smFRET experiments have shown a structural expansion of PAGE4 upon hyperphosphorylation and a significant increase in size at its N-terminal half than that at its C-terminus. To understand the molecular mechanism underlying this structural transition, we performed a series of constant temperature molecular dynamics simulations using Atomistic AWSEM -- a multi-scale molecular model combining detailed atomistic and coarse-grained simulation approaches. Our simulations show that electrostatic interaction drives a transient formation of an N-terminal loop, which causes the change in size for different phosphorylated forms of PAGE4. Phosphorylation also changes the preference of secondary structure formation of PAGE4, which signifies a transition between states that display different degree of disorder. Finally, we construct a mechanism-based mathematical model that allows us to capture the interactions of different forms of PAGE4 with AP-1 and AR, a key therapeutic target in prostate cancer. Our model predicts intracellular oscillatory dynamics of HIPK1-PAGE4, CLK2-PAGE4 and AR activity, indicating phenotypic heterogeneity in an isogenic cell population. Thus, conformational switching among different forms of PAGE4 may potentially affect the efficiency of therapeutic targeting of AR.

biophysics↗

bioRxiv2017

Improved Aedes aegypti mosquito reference genome assembly enables biological discovery and vector control

Female Aedes aegypti mosquitoes infect hundreds of millions of people each year with dangerous viral pathogens including dengue, yellow fever, Zika, and chikungunya. Progress in understanding the biology of this insect, and developing tools to fight it, has been slowed by the lack of a high-quality genome assembly. Here we combine diverse genome technologies to produce AaegL5, a dramatically improved and annotated assembly, and demonstrate how it accelerates mosquito science and control. We anchored the physical and cytogenetic maps, resolved the size and composition of the elusive sex-determining \"M locus\", significantly increased the known members of the glutathione-S-transferase genes important for insecticide resistance, and doubled the number of chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites. Using high-resolution QTL and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. We predict that AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly arboviral vector.

genomics↗

bioRxiv2017

Formation of retinal direction-selective circuitry initiated by starburst amacrine cell homotypic contact

Impact statementSelective synapse formation in a retinal motion-sensitive circuit is orchestrated by starburst amacrine cells, which use homotypic interactions to initiate formation of a dendritic scaffold that recruits projections from circuit partners.\n\nSUMMARYA common strategy by which developing neurons locate their synaptic partners is through projections to circuit-specific neuropil sublayers. Once established, sublayers serve as a substrate for selective synapse formation, but how sublayers arise during neurodevelopment remains unknown. Here we identify the earliest events that initiate formation of the direction-selective circuit in the inner plexiform layer of mouse retina. We demonstrate that radially-migrating newborn starburst amacrine cells establish homotypic contacts on arrival at the inner retina. These contacts, mediated by the cell-surface protein MEGF10, trigger neuropil innervation resulting in generation of two sublayers comprising starburst-cell dendrites. This dendritic scaffold then recruits projections from circuit partners. Abolishing MEGF10-mediated contacts profoundly delays and ultimately disrupts sublayer formation, leading to broader direction tuning and weaker direction-selectivity in retinal ganglion cells. Our findings reveal a mechanism by which differentiating neurons transition from migratory to mature morphology, and highlight this mechanisms importance in forming circuit-specific sublayers.

neuroscience↗

bioRxiv2017

Processive movement of actin by biased polymerization: a new paradigm of axonal transport

Classic pulse-chase studies have shown that actin is conveyed in slow axonal transport, but the mechanistic basis for this movement is unknown. Recently, we reported that axonal actin was surprisingly dynamic, with focal assembly/dis-assembly events (\"hotspots\") and elongating polymers along the axon-shaft (\"trails\"). Using a combination of live imaging, super-resolution microscopy, and modeling, here we explore how these axonal actin dynamics can lead to processive transport. We found abundant actin nucleation, along with a slow, anterogradely-biased flow of actin in axon-shafts. Starting with first principles of monomer/filament assembly - and incorporating imaging data - we generated a quantitative model simulating axonal hotspots and trails. Our simulations predict that the axonal actin dynamics indeed lead to an anterogradely-biased flow of the population, at rates consistent with slow transport. Collectively, the data point to a surprising scenario where local assembly and biased polymerization generate the slow axonal transport of actin. This mechanism is distinct from polymer-sliding, and seems well suited to convey highly dynamic cytoskeletal cargoes.\n\nAcknowledgementsThis work was supported by an NIH grant to SR (R01NS075233). The authors thank Stephanie Gupton (UNC) for the Mena/Vasp constructs.

neuroscience↗

bioRxiv2017

Id proteins suppress E2A-driven innate-like T cell development prior to TCR selection

Id proteins have been shown to promote the differentiation of conventional {beta} and {gamma}{delta}T cells, and to suppress the expansion of invariant Natural Killer T (iNKT) cells and innate-like {gamma}{delta}NKT within their respective cell lineages. However, it remains to be determined whether Id proteins regulate lineage specification in developing T cells that give rise to these distinct cell fates. Here we report that in the absence of Id2 and Id3 proteins, E2A prematurely activates genes critical for the iNKT cell lineage prior to TCR expression. Enhanced iNKT development in Id3-deficient mice lacking {gamma}{delta} NKT cells suggests that Id3 regulates the lineage competition between these populations. RNA-Seq analysis establishes E2A as the transcriptional regulator of both iNKT and {gamma}{delta}NKT development. In the absence of pre-TCR signaling, Id2/Id3 deletion gives rise to a large population of iNKT cells and a unique innate-like DP population, despite the block in conventional {beta} T cell development. The transcriptional profile of these unique DP cells reflects enrichment of innate-like signature genes, including PLZF (Zbtb16) and Granzyme A (Gzma). Results from these genetic models and genome-wide analyses suggest that Id proteins suppress E2A-driven innate-like T cell programs prior to TCR selection to enforce predominance of conventional T cells.

immunology↗

bioRxiv2017

p53 suppresses mutagenic RAD52 and POLθ pathways by orchestrating DNA replication restart homeostasis

Classically, p53 tumor-suppressor acts in transcription, apoptosis, and cell-cycle arrest. Yet, replication-mediated genomic instability is integral to oncogenesis, and p53 mutations promote tumor progression and drug-resistance. By delineating human and murine separation-of-function p53 alleles, we find that p53 null and gain-of-function (GOF) mutations exhibit defects in restart of stalled or damaged DNA replication forks driving genomic instability independent of transcription activation. By assaying protein-DNA fork interactions in single cells, we unveil a p53-MLL3-enabled recruitment of MRE11 DNA replication restart nuclease. Importantly, p53 defects or depletion unexpectedly allow mutagenic RAD52 and POL{theta} pathways to hijack stalled forks, which we find reflected in p53 defective breast-cancer patient COSMIC mutational signatures. These data uncover p53 as a keystone regulator of replication homeostasis within a DNA restart network. Mechanistically, this has important implications for development of resistance in cancer therapy. Combined, these results define an unexpected role for p53 suppression of replication genome instability.

genetics↗

bioRxiv2017

Allosteric Activation of p300 Autoacetylation by the Tumor Suppressor p53

The transcriptional coactivator p300 is essential for p53 transactivation, although its precise mechanism remains unclear. We report that, p53 allosterically activates the acetyltransferase activity of p300 through the enhancement of p300 autoacetylation. Cryo-electron microscopy revealed that the domain organization of p300 is substantially altered upon binding of p53, suggesting that a structural switch may underpin the activation. Acetylated p300 accumulates near the transcription start sites accompanied by a similar enrichment of activating histone marks near those sites. Disruption of p53-p300 interaction by a site-directed peptide inhibitor abolished autoacetylated p300-mediated enhanced histone acetylation, suggesting a crucial role played by the allosteric activation in p53-mediated gene regulation. Gain-of-function mutant p53, known to impart aggressive proliferative properties in tumor cells, also activate p300 autoacetylation. The same peptide abolished many of the gain-of-functions of mutant p53 as well. We conclude that allosteric activation of p300 by p53 underpins gene regulation by p53. Reversal of gain-of-function properties of mutant p53 suggests that molecules targeting the p53-p300 interface may be good candidates for anti-tumor drugs.

molecular biology↗

bioRxiv2017

Heterologous expression of the immune receptor EFR in Medicago truncatula reduces pathogenic infection, but not rhizobial symbiosis

Interfamily transfer of plant pattern recognition receptors (PRRs) represents a promising biotechnological approach to engineer broad-spectrum, and potentially durable, disease resistance in crops. It is however unclear whether new recognition specificities to given pathogen-associated molecular patterns (PAMPs) affect the interaction of the recipient plant with beneficial microbes. To test this in a direct reductionist approach, we transferred the Brassicaceae-specific PRR ELONGATION FACTOR-THERMO UNSTABLE RECEPTOR (EFR) from Arabidopsis thaliana to the legume Medicago truncatula, conferring recognition of the bacterial EF-Tu protein. Constitutive EFR expression led to EFR accumulation and activation of immune responses upon treatment with the EF-Tu-derived elf18 peptide in leaves and roots. The interaction of M. truncatula with the bacterial symbiont Sinorhizobium meliloti is characterized by the formation of root nodules that fix atmospheric nitrogen. Although nodule numbers were slightly reduced at an early stage of the infection in EFR-Medicago when compared to control lines, nodulation was similar in all lines at later stages. Furthermore, nodule colonization by rhizobia, and nitrogen fixation were not compromised by EFR expression. Importantly, the M. truncatula lines expressing EFR were substantially more resistant to the root bacterial pathogen Ralstonia solanacearum. Our data suggest that the transfer of EFR to M. truncatula does not impede root nodule symbiosis, but has a positive impact on disease resistance against a bacterial pathogen. In addition, our results indicate that Rhizobium can either avoid PAMP recognition during the infection process, or is able to actively suppress immune signaling.\n\nSignificance StatementCrop engineering helps reducing the economic and environmental costs of plant disease. The genetic transfer of immune receptors across plant species is a promising biotechnological approach to increase disease resistance. Surface-localized pattern-recognition receptors (PRRs), which detect conserved characteristic microbial features, are functional in heterologous taxonomically-diverse plant species, and confer broad-spectrum disease resistance. It was unclear whether PRR transfer negatively impacts the association of the recipient plants with symbiotic microbes. Here, we show that a legume engineered with a novel PRR recognizing a conserved bacterial protein becomes more resistant to an important bacterial pathogen without significant impact on nitrogen-fixing symbiosis with rhizobia. This finding is of particular relevance as attempts to transfer this important symbiosis into non-legume plants are ongoing.

plant biology↗

bioRxiv2017

Bacterial Communication And Relevance Of Quantum Theory

The recent findings confirm that bacteria communicate each other through chemical and electrical signals. Bacteria use chemical signaling molecules which are called as quorum sensing molecules(QSMs) or autoinducers. Moreover, the ion channels in bacteria conduct a long-range electrical signaling within biofilm communities through propagated waves of potassium ions and biofilms attracts other bacterial species too. Both communication process are used by bacteria to make their own survival strategies. In this article, we model this bacterial communication mechanism by complex Ginzburg- Landau equation and discuss the formation of patterns depending on kinematic viscosity associated with internal noise. Again, the potassium wave propagation is described by the non-linear Schrodinger equation in a dissipative environment. By adding perturbation to non-linear Schrodinger equation one arrives at Complex Ginzburg-Landau equation. In this paper we emphasize that at the cellular level(bacteria) we use Complex Ginzburg - Landau equation as a perturbed Nonlinear Schrodinger equation to understand the bacterial communication as well as pattern formation in Biofilms for certain range of kinematic viscosity which can be tested in laboratory experiment. Here, the perturbation is due to the existence of non thermal fluctuations associated to the finite size of the bacteria. It sheds new light on the relevance of quantum formalism in understanding the cell to cell communication.

biophysics↗

bioRxiv2017

The role of coherence in bacterial communication

Bacteria within biofilms can coordinate their behavior through distinct from of communication mechanism1. The well-established cell - to - cell signaling process in bacteria is known as quorum sensing through chemical signaling molecules2-5. Recently, another cell- to - cell communication process based on ion channel mediated electrical signaling6 has also been observed. In this article, we propose a novel approach to explain the role of coherence and phase synchronization in the cell - to - cell bacterial communication. The observable long - range coherent electrical signaling is species independent and it is caused by membrane - potential - dependent modulation of tumbling frequency7-9. Moreover, noise can play a constructive role in enhancing the synchronization of chaotic bacterial communication systems and noise associated with the opening and closing the gate of ion channel induce small kinetic viscosity that make a wave-like pattern in concentration profile of quorum sensing.

biophysics↗

bioRxiv2017

Bacterial Conversations and Pattern Formation

It has long been recognized that certain bacterial groups exhibit cooperative behavioral patterns. Bacteria accomplish such communication via exchange of extracellular signaling molecules called pheromones(autoinducer or quorum sensing molecules). As the bacterial culture grows, signal molecules are released into extracellular milieu accumulate, changing water fluidity. Under such threshold conditions swimming bacterial suspensions impose a coordinated water movement on a length scale of the order 10 to 100 micrometers compared with a bacterial size of the order of 3 micrometers.Here, we investigate the non-local hydrodynamics of the quorum state and pattern formation using forced Burgers equation with Kwak transformation. Such approach resulted in the conversion of the Burgers equation paradigm into a reaction-diffusion system. The examination of the dynamics of the quorum sensing system, both analytically as well as numerically result in similar long-time dynamical behaviour.

biophysics↗