Biology subjects

Agarwal, P.

Publications and source records attributed to Agarwal, P..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Innate immune epigenomic landscape following controlled human influenza virus infection

Viral infections can induce changes in innate immunity that persist after virus clearance. Here, we used blood samples from a human influenza H3N2 challenge study to perform comprehensive multi-omic analyses. We detected remodeling of immune programs in innate immune cells after resolution of the infection that was proportional in magnitude to the level of prior viral load. We found changes associated with suppressed inflammation including decreased cytokine and AP-1 gene expression as well as decreased accessibility at AP-1 targets and interleukin-related gene promoter regions. We also found decreased histone deacetylase gene expression, increased MAP kinase gene expression, and increased accessibility at interferon-related gene promoter regions. Genes involved in inflammation and epigenetic-remodeling showed modulation of gene-chromatin site regulatory circuit activity. These results reveal a coordinated rewiring of the epigenetic landscape in innate immune cells induced by mild influenza virus infection.

immunology↗

bioRxiv2024

Comprehensive Guide of Epigenetics and Transcriptomics Data Quality Control

Host response to environmental exposures such as pathogens and chemicals can cause modifications to the epigenome and transcriptome. Analysis of these modifications can reveal signatures with regards to the agent and timing of exposure. Exhaustive interrogation of the cascade of the epigenome and transcriptome requires analysis of disparate datasets from multiple assay types, often at single cell resolution, from the same biospecimen. Improved signature discovery has been enabled by advancements in assaying techniques to detect RNA expression, DNA base modifications, histone modifications, and chromatin accessibility. However, there remains a paucity of rigorous quality control standards of those datasets that reflect quality assurance of the underlying assay. This guide outlines a comprehensive suite of metrics that can be used to ensure quality from 11 different epigenetics and transcriptomics assays. Recommendations on mitigation approaches to address failed metrics and poor quality data are provided. The workflow consists of assessing dataset quality and reiterating benchwork protocols for improved results to generate accurate exposure signatures.

bioinformatics↗

bioRxiv2024

Evidence of centromeric histone 3 chaperone involved in DNA damage repair pathway

The centromeric protein-A (CENP-A) is an evolutionary conserved histone H3 variant that marks the identity of the centromeres. Several mechanisms regulate the centromeric deposition of CENP-A as its mislocalization causes erroneous chromosome segregation, leading to aneuploidy-based diseases, including cancers. The most crucial deposition factor is a CENP-A specific chaperone, HJURP (Scm3 in budding yeast), which specifically binds to CENP-A. However, the discovery of HJURP as a DDR (DNA damage repair) protein and evidence of its binding to Holliday junctions in vitro indicate a CENP-A-deposition-independent role of these chaperones. In this study, using budding yeast, we demonstrate that Scm3 is crucial for the DDR pathway as scm3 cells are sensitive to DNA damage. We further observe that the scm3 mutant interacts with the rad52 DDR mutant and is compromised in activating DDR-mediated arrest. We demonstrate that Scm3 associates with the DNA damage sites and undergoes posttranslational modifications upon DNA damage. Overall, from this report and earlier studies on HJURP, we conclude that DDR functions of CENP-A chaperones are conserved across eukaryotes. Thus, the revelation that these chaperones confer genome stability in more than one pathway has clinical significance.

cell biology↗

bioRxiv2024

PLAbDab-nano: a database of camelid and shark nanobodies from patents and literature

Nanobodies are essential proteins of the adaptive immune systems of camelid and shark species, complementing conventional antibodies. Properties such as their relatively small size, solubility and high thermostability make VHH and VNAR modalities a promising therapeutic format and a valuable resource for a wide range of biological applications. The volume of academic literature and patents related to nanobodies has risen significantly over the past decade. Here, we present PLAbDab-nano, a nanobody complement to the Patent and Literature Antibody Database (PLAbDab). PLAbDab-nano is a selfupdating, searchable repository containing approximately 5000 annotated VHH and VNAR sequences. We describe the methods used to curate the entries in PLAbDab-nano, and highlight how PLAbDab-nano could be used to design diverse libraries, as well as find sequences similar to known patented or therapeutic entries. PLAbDab-nano is freely available as a searchable web server (opig.stats.ox.ac.uk/webapps/plabdab-nano/). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/604232v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@5d81d4org.highwire.dtl.DTLVardef@f69f21org.highwire.dtl.DTLVardef@1493c54org.highwire.dtl.DTLVardef@117e3c5_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

A novel repressor-activator-competitor module comprising C2H2 zinc finger and NAC transcription factors regulates rice grain development

Grain size and quality are crucial agronomic traits. We have characterized a seed-preferential C2H2 zinc finger transcriptional repressor, ZOS1-15. Its overexpression, knock-down and knock-out plants indicated a negative control over grain size due to altered cell expansion. ZOS1-15 homodimerized and directly interacted with co-repressor TOPLESS and histone deacetylases to form a repression complex. ZOS1-15 also interacted with Mediator subunit MED14_1 and a seed-preferential transcriptional activator, ONAC024, with three alternatively spliced isoforms. The ectopic expression of ONAC024 negatively affected plant growth and development. Seed-preferential overexpression and knock-down plants showed ONAC024 as a positive regulator of grain length due to increased cell proliferation and expansion. CRES-T generated transgenic rice plants indicated a functional divergence amongst ONAC024 isoforms. Tandem interactions were observed between ONAC024-ONAC023-ONAC026-ONAC020. ZOS1-15 and ONAC024 functioned antagonistically to regulate grain amylose and SSP accumulation while ONAC023 affected only amylose. ZOS1-15 and ONAC024 directly regulated the expression of two SSP encoding genes. Binding of ONAC024 was competed by ONAC025-MADS29 complex. The seed-preferential overexpression of SS1/ ONAC025 resulted in decreased grain size and amylose content, but higher yield. This study proposes a repressor-activator-competitor module, wherein ZOS1-15, ONAC024, ONAC023, ONAC025 along with their interactors synergistically and antagonistically regulate multiple aspects of rice grain development.

plant biology↗

bioRxiv2024

A zinc finger transcriptional repressor ZOS5-09 regulates rice grain size, starch and protein biosynthesis

HighlightA rice seed-preferential transcriptional repressor, ZOS5-09 controls grain size by affecting cell proliferation and expansion; and regulates starch and protein biosynthesis. It acts as a repressor by deacetylation. Grain size is the one of the key determinants of grain yield. Transcription factors form an important class of regulatory proteins that play crucial roles in regulating developmental responses. Our study focuses on a novel seed-preferential C2H2 zinc finger transcription factor, ZOS5-09 (LOC_Os05g38600) that plays an important role in regulating grain related traits in rice. Rice plants with ZOS5-09 promoter::GUS construct showed its high expression in rice endosperm. In planta reporter effector assays and localization studies showed that ZOS5-09 is a nuclear localized transcriptional repressor. It has a C-terminal NoRS (nucleolar retention signal). Ectopic and seed-preferential overexpression of ZOS5-09 resulted in lethality. Seed-preferential overexpression without NoRS was detrimental to grain filling. Rice plants with knock-down or CRISPR based knock-out of ZOS5-09 displayed reduced grain length and weight but increased grain width. Plant height, second leaf width, and panicle number were reduced and second leaf length were increased. The change in grain size was due to lower cell proliferation and increased cell size in the transverse direction due to down regulation of cell cycle related genes and increased expression of expansins. Decreased expression of ZOS5-09 also resulted in reduced total starch and protein content and higher endosperm chalkiness, thus, negatively affecting grain quality. ZOS5-09 directly regulated a seed storage protein encoding gene, GLU6 by binding to the zinc finger binding site in its promoter. It acted as a repressor by promoting deacetylation upon interaction with a histone deacetylase. In summary, our results indicate that optimum expression of ZOS5-09 is essential for proper rice grain development. Our study highlights the role of a transcriptional repressor in regulating rice grain traits and improves our understanding of the transcriptional regulatory networks affecting grain size.

plant biology↗

bioRxiv2024

The Observed T cell receptor Space database enables paired-chain repertoire mining, coherence analysis and language modelling

T cell activation is governed through T cell receptors (TCRs), heterodimers of two sequence-variable chains (often an alpha [] and beta [{beta}] chain) that recognise linear antigen fragments presented on the cell surfaces. Early sequencing technologies limited the study of immune repertoire TCRs to unpaired transcripts, leading to extensive analysis of {beta}-chain data alone as its greater sequence diversity suggested it should dominate antigen recognition. Over time, structural data has revealed that both and {beta} chains contribute to binding most antigens and highthroughput single-cell handling technologies have been increasingly applied to obtain samples of complete TCR variable region sequences from repertoires. Despite this, there is currently no repository dedicated to the curation of publicly available paired TCR sequence data. We have addressed this gap by creating the Observed T cell receptor Space (OTS) database, a source of consistently processed and annotated, full-length, paired-chain TCR sequencing data from 50 studies and at least 75 individuals. Currently, OTS contains 5.35M redundant (1.63M nonredundant) predominantly human TCR sequences and, based on recent data availability trends, will grow rapidly. We perform an initial analysis of OTS, leading to the identification of pairing biases, public TCRs, and distinct chain coherence patterns relative to antibodies. We also harness the data to build a publicly available paired-chain TCR language model, providing paired embedding representations and a method for residue in-filling that is conditional on the partner chain. OTS will be updated and maintained as a central community resource and is freely downloadable and available as a web application at https://opig.stats.ox.ac.uk/webapps/ots.

immunology↗

bioRxiv2024

A Tunable and Druggable Mechanism to Delay Forgetting of Olfactory Memories in C. elegans

The poet W.B Yeats wrote that "All that is personal soon rots, it must be packed in ice or salt". Here we show that in Caenorhabditis elegans nematodes, simple animals with just 302 neurons, memories are preserved on ice and in lithium salt. C. elegans nematodes can form associative memories, which are typically forgotten quickly. We discovered that when placed on ice, worms delay forgetting of specific olfactory memories by at least 8-fold. Delayed forgetting was canceled completely when the worms were gradually adapted to low temperatures, owing to a genetically-encoded program that turns acclimated worms cold-tolerant. RNA-seq, mutant analyses, and pharmacological assays revealed that regulation of membrane properties switches cold-induced delayed forgetting ON and OFF, and, remarkably, that lithium delays forgetting only in cold-sensitive but not cold-tolerant worms. We found that downregulation of the diacylglycerol pathway in the AWC sensory neurons is essential for lithium-mediated delayed forgetting, and using neuronal activity recordings located the memory trace to the downstream AIY interneurons. We suggest that the awesome genetic tractability of C. elegans might be harnessed to study the effects of lithium and cold temperatures on the brain, why it influences psychiatric disorders, and even more fundamentally how memory is stored and lost.

neuroscience↗

bioRxiv2023

Evidence of 14-3-3 proteins contributing to kinetochore integrity and chromosome congression during mitosis

The 14-3-3 family of proteins are conserved across eukaryotes and serve myriad important regulatory functions of the cell. Homo/heterodimers of these protein homologs, majorly recognize their ligands via conserved motifs from a plethora of cellular proteins to modulate the localization and functions of those effector ligands. In most of the genetic backgrounds of Saccharomyces cerevisiae, disruption of both 14-3-3 homologs (Bmh1 and Bmh2) are either lethal or survive with severe growth defects showing gross chromosomal missegregation and prolonged cell cycle arrest. To elucidate their contributions to chromosome segregation, in this work we investigated their centromere/kinetochore-related functions. Analysis of appropriate deletion mutants shows that Bmh isoforms have cumulative and unshared isoform-specific contributions in maintaining the proper integrity of the kinetochore ensemble. Consequently, bmh mutant cells exhibited perturbations in kinetochore-microtubule (KT-MT) dynamics, characterized by kinetochore declustering, mis-localization of kinetochore proteins, and Mad2-mediated transient G2/M arrest. These defects also caused an asynchronous chromosome congression in bmh mutants during metaphase. In summary, this report advances the knowledge on contributions of budding yeast 14-3-3 proteins in chromosome segregation by demonstrating their roles in kinetochore integrity and chromosome congression.

cell biology↗

bioRxiv2023

A non-canonical Arp2/3 complex is essential for Plasmodium DNA segregation and transmission of malaria.

The malaria-causing parasite Plasmodium has a complex life cycle involving both vertebrate and mosquito hosts. Sexual stages or gametocytes are the only stage competent for transmission to mosquitoes. Formation of flagellated male gametes from gametocytes requires rapid rounds of genome replication. Here we discovered a non-canonical Plasmodium actin-related protein 2/3 (Arp2/3) complex essential for DNA segregation during male gametogenesis. Plasmodium Arp2/3 dynamically localizes within the nucleus to the endomitotic spindles and interacts with a kinetochore protein. Deletion of key Arp2/3 subunits or interfering with actin polymerisation leads to the formation of sub-haploid male gametes and a complete block in transmission through delayed developmental arrest at the oocyst stage. Our work identified an evolutionary divergent protein complex in malaria parasites that offers potential targets for transmission-blocking interventions.

microbiology↗

bioRxiv2023

Higher meiotic chromosome condensation: a potential function of kinetochore through polo-like kinase

Chromosome condensation plays a pivotal role during faithful chromosome segregation, hence understanding the factors that drive condensation is crucial to get mechanistic insight into chromosome segregation. Previously we showed that in budding yeast, the absence of the non-essential kinetochore proteins affects chromatin-condensin association in meiosis but not in mitosis. A differential organization of the kinetochores, that we and others observed earlier during mitosis and meiosis may contribute to the meiotic-specific role. Here, with our in-depth investigation using in vivo chromosome condensation assays in cells sans a non-essential kinetochore protein, Ctf19, we establish that these proteins have roles in achieving a higher meiotic condensation without influencing much of the mitotic condensation. We further observed an accumulation of the polo-like kinase Cdc5 owing to its higher protein stability in ctf19{Delta} meiotic cells. High Cdc5 activity causes hyper-phosphorylation of the condensin resulting in its reduced stability and concomitant decreased association with the chromatin. Overall, our findings highlight the role of Ctf19 in promoting meiotic chromosome condensation by influencing the activity of Cdc5 and thereby affecting the stability and association of condensin with the chromatin.

cell biology↗

bioRxiv2023

Elevated temperature fatally disrupts nuclear divisions in the early Drosophila embryo.

Temperature variations can challenge animal survival, with elevated temperatures presenting distinct vulnerabilities at different stages of the animal life cycle. Embryonic development is known to be especially vulnerable, though the molecular mechanisms that underlie this vulnerability remain unclear. Here we investigate how elevated temperature affects embryogenesis in Drosophila melanogaster, an insect model known for extensively characterized genetic makeup and developmental processes. We identify the first three hours as a critical window in which Drosophila embryos are particularly vulnerable to elevated temperature. This period includes the formation of a syncytial blastoderm, which involves 4 rounds of meta-synchronous nuclear divisions at the embryonic cortex, and cellularization that leads to the formation of the cellular blastoderm. Embryos exposed to elevated temperature during this period subsequently exhibited developmental defects at the gastrulation stage, leading to increased lethality. At elevated temperature, we observed an increase in mitotic failures causing a loss of cortical nuclei during cellularization. There is also a local crowding of nuclei and an increase in asynchrony between the nuclear cycles in the center vs. the poles. Interestingly, these features cooperatively amplify the frequency of mitotic failures, leading to holes in the blastoderm epithelium. Further mechanistic analysis of mitotic failures revealed that they trigger DNA damage response. Focusing on cell biological causes of mitotic failures, we found that the interaction between F-actin and microtubules is weakened at elevated temperature. We performed functional tests to determine whether known regulators of mitosis could rescue mitotic failures, blastoderm hole formation, and improve embryo survival. We further analyzed genomic datasets from wild populations to determine whether these regulators also carry signatures of adaptation in wild populations. Our genetic rescue experiments show that in Drosophila embryos, the interaction between cortical F-actin and astral microtubules of mitotic spindle is vulnerable to disruption at elevated temperatures, leading to mitotic failures that can be potentially rescued by modulating the expression of just a few factors. We propose that levels of expression of corresponding genes could be used as indicators to predict the effects of increasing temperature variations on insect populations.

developmental biology↗

bioRxiv2023

Schizophrenia Risk Mapping and Functional Engineering of the 3D Genome in Three Neuronal Subtypes

Common variants associated with schizophrenia are concentrated in non-coding regulatory sequences, but their precise target genes are context-dependent and impacted by cell-type-specific three-dimensional spatial chromatin organization. Here, we map long-range chromosomal conformations in isogenic human dopaminergic, GABAergic, and glutamatergic neurons to track developmentally programmed shifts in the regulatory activity of schizophrenia risk loci. Massive repressive compartmentalization, concomitant with the emergence of hundreds of neuron-specific multi-valent chromatin architectural stripes, occurs during neuronal differentiation, with genes interconnected to genetic risk loci through these long-range chromatin structures differing in their biological roles from genes more proximal to sequences conferring heritable risk. Chemically induced CRISPR-guided chromosomal loop-engineering for the proximal risk gene SNAP91 and distal risk gene BHLHE22 profoundly alters synaptic development and functional activity. Our findings highlight the large-scale cell-type-specific reorganization of chromosomal conformations at schizophrenia risk loci during neurodevelopment and establish a causal link between risk-associated gene-regulatory loop structures and neuronal function.

genomics↗

bioRxiv2023

Confined environment facilitates stacked conformations in Holliday Junction

Holliday Junction (HJ) is an important intermediate for homologous recombination and is involved in DNA break repair. It is a highly dynamic structure and transitions between two stacked isomers through an intermediate open conformer. In a constrained cellular environment, HJ is expected to behave differently than in vitro. This can affect target recognition by junction-binding proteins and their subsequent functions. However, the studies on the effect of the confined environment on HJ is extremely scanty. In this work, by employing fluorescence-based techniques we investigated the effect of confinement on HJ conformation using reverse micelle encapsulation as a model for confined space. We observed that inside reverse micelle, HJ prefers to adopt stacked conformation. Most strikingly, even at lower divalent cation concentrations, the preference for stacked conformation is prevalent over an open conformation. Our finding suggests that such confinement-induced changes in the conformer population might affect the interaction and activity of the HJ-recognizing proteins in the cellular environment.

biophysics↗

bioRxiv2022

Emerging variants of SARS-CoV-2 NSP10 highlight strong functional conservation of its binding to two non-structural proteins, NSP14 and NSP16

The coronavirus SARS-CoV-2 protects its RNA from being recognized by host immune responses by methylation of its 5 end, also known as capping. This process is carried out by two enzymes, non-structural protein 16 (NSP16) containing 2-O-methyltransferase and NSP14 through its N7 methyltransferase activity, which are essential for the replication of the viral genome as well as evading the hosts innate immunity. NSP10 acts as a crucial cofactor and stimulator of NSP14 and NSP16. To further understand the role of NSP10, we carried out a comprehensive analysis of >13 million globally collected whole-genome sequences (WGS) of SARS-CoV-2 obtained from the Global Initiative Sharing All Influenza Data (GISAID) and compared it with the reference genome Wuhan/WIV04/2019 to identify all currently known variants in NSP10. T12I, T102I, and A104V in NSP10 have been identified as the three most frequent variants and characterized using X-ray crystallography, biophysical assays and enhanced sampling simulations. In contrast to other proteins such as spike and NSP6, NSP10 is significantly less prone to mutation due to its crucial role in replication. The functional effects of the variants were examined for their impact on the binding affinity and stability of both NSP14-NSP10 and NSP16-NSP10 complexes. These results highlight the limited changes induced by variant evolution in NSP10 and reflect on the critical roles NSP10 plays during the SARS-CoV-2 life cycle. These results also indicate that there is limited capacity for the virus to overcome inhibitors targeting NSP10 via the generation of variants in inhibitor binding pockets. Significance StatementThe SARS-CoV-2 proteins have constantly been evolving. These variants assist the virus to survive, adapt and evade the host immune responses. While the main focus has been on structural proteins like Spike, there is very limited structural and functional information on the effects of emerging mutations on other essential non-structural viral proteins. One such protein is NSP10, an essential cofactor for NSP14 and NSP16. This study demonstrates that NSP10 is more resistant to genetic variations than other SARS-CoV-2 non-structural proteins and that the presence of mutations conserve structural and dynamic changes in NSP10. The effects of naturally occurring mutations reflect the evolutionary relationship between structurally conserved essential cofactors, their function and the role they play in the survival of the virus.

biophysics↗

bioRxiv2022

Future COVID-19 surges prediction based on SARS-CoV-2 mutations surveillance

COVID19 has aptly revealed that airborne viruses such as SARS-CoV-2 with the ability to rapidly mutate, combined with high rates of transmission and fatality can cause a deadly world-wide pandemic in a matter of weeks.1 Apart from vaccines and post-infection treatment options, strategies for preparedness will be vital in responding to the current and future pandemics. Therefore, there is wide interest in approaches that allow predictions of increase in infections ("surges") before they occur. We describe here real time genomic surveillance particularly based on mutation analysis, of viral proteins as a methodology for a priori determination of surge in number of infection cases. The full results are available for SARS-CoV-2 at http://pandemics.okstate.edu/covid19/, and are updated daily as new virus sequences become available. This approach is generic and will also be applicable to other pathogens.

genomics↗

bioRxiv2022

Intrinsic disorder in CENP-ACse4 tail and its chaperone facilitates synergistic association for kinetochore stabilization

The kinetochore is a complex multiprotein network that assembles at a specialized DNA locus called the centromere to ensure faithful chromosome segregation. The centromere is epigenetically marked by a histone H3 variant - the CenH3. The budding yeast CenH3, called Cse4, consists of an unusually long and disordered N-terminal tail that has a role in kinetochore assembly. Its disordered chaperone, Scm3 is involved in its centromeric deposition as well as in the maintenance of a segregation-competent kinetochore. The dynamics of the Cse4 N-tail and chaperone interaction have not been studied, leaving a gap in our understanding of their roles at the centromere. Previously, we had shown that Scm3 is an intrinsically disordered protein. Here, using NMR and a variety of biophysical and bioinformatics tools, we show that Cse4 N-tail is also disordered, the two proteins interact with each other at multiple sites, and this interaction reduces the disorder in Scm3; the chain opens up relative to the native state ensemble and the conformational exchange is reduced. Interestingly, this interaction between the two intrinsically disordered protein is fairly specific as seen by positive and negative controls, and is majorly driven by electrostatics as both the proteins have multiple acidic and basic regions. The complex retains a fair amount of disorder, which facilitates a synergistic association with the essential inner kinetochore Ctf19-Mcm21-Okp1-Ame1 complex; a model has been suggested to this effect. Given the abundance of intrinsic disorder in the kinetochore proteins, this type of interaction and adaptation may be prevalent in other proteins as well for mediating kinetochore assembly. Thus, the present study, on one hand, provides significant structural and mechanistic insights into the complex and dynamic process of kinetochore assembly, and on the other hand, illustrates a mechanism that intrinsically disordered proteins would adapt to mediate the formation of complex multiprotein networks, in general. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/504061v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@8fe5cforg.highwire.dtl.DTLVardef@138afb1org.highwire.dtl.DTLVardef@96943forg.highwire.dtl.DTLVardef@1deb673_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Model-based intensification of CHO cell cultures: one-step strategy from fed-batch to perfusion

There is a growing interest in continuous processing of the biopharmaceutical industry. However, the technology transfer from traditional batch-based processes is considered a challenge as protocol and tools still remain to be established for their usage at the manufacturing scale. Here, we present a model-based approach to design optimized perfusion cultures of CHO cells using only the knowledge captured during small-scale fed-batch experiments. The novelty of the proposed model lies in the simplicity of its structure. Thanks to the introduction of a new catch-all variable representing a bulk of by-products secreted by the cells during their cultivation, the model was able to successfully predict cellular behavior under different operating modes without changes in its formalism. To our knowledge, this is the first experimentally validated model capable, with a single set of parameters, to capture culture dynamic under different operating modes and at different scales.

bioengineering↗