Biology subjects

Dubey, A.

Publications and source records attributed to Dubey, A..

30 records · Page 2Linked to original sources

bioRxiv2023

Direct interaction with the BRD4 carboxyl-terminal motif (CTM) and TopBP1 is required for human papillomavirus 16 E2 association with mitotic chromatin and plasmid segregation function

During the human papillomavirus 16 life cycle, the E2 protein binds simultaneously to the viral genome and host chromatin throughout mitosis, ensuring viral genomes reside in daughter cell nuclei following cell division. Previously, we demonstrated that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1, and that this interaction is required for optimum E2 mitotic chromatin association and plasmid segregation function. Others have implicated BRD4 in mediating the plasmid segregation function of E2 and we have demonstrated that there is a TopBP1-BRD4 complex in the cell. We therefore further investigated the role of the E2-BRD4 interaction in mediating E2 association with mitotic chromatin and plasmid segregation function. Using a combination of immunofluorescence and our novel plasmid segregation assay in U2OS and N/Tert-1 cells stably expressing a variety of E2 mutants, we report that direct interaction with the BRD4 carboxyl-terminal motif (CTM) and TopBP1 is required for E2 association with mitotic chromatin and plasmid segregation. We also identify a novel TopBP1 mediated interaction between E2 and the BRD4 extra-terminal (ET) domain in vivo. Overall, the results demonstrate that direct interaction with TopBP1 and the BRD4 CTM are required for E2 mitotic chromatin association and plasmid segregation function. Disruption of this complex offers therapeutic options for targeting segregation of viral genomes into daughter cells, potentially combatting HPV16 infections, and cancers that retain episomal genomes. ImportanceHPV16 is a causative agent in around 3-4% of all human cancers and currently there are no anti-viral therapeutics available for combating this disease burden. In order to identify new therapeutic targets, we must increase our understanding of the HPV16 life cycle. Previously, we demonstrated that an interaction between E2 and the cellular protein TopBP1 mediates the plasmid segregation function of E2, allowing distribution of viral genomes into daughter nuclei following cell division. Here, we demonstrate that E2 interaction with an additional host protein, BRD4, is also essential for E2 segregation function, and that BRD4 exists in a complex with TopBP1. Overall, these results enhance our understanding of a critical part of the HPV16 life cycle and presents several therapeutic targets for disruption of the viral life cycle.

cancer biology↗

bioRxiv2023

Multi-omics analysis of a Bacteroides fragilis isolate from an ulcerative colitis patient defines genetic determinants of fitness in bile

Bacteroides fragilis comprises 1-5% of the gut microbiota in healthy humans but can expand to >50% of the population in ulcerative colitis (UC) patients experiencing inflammation. The mechanisms underlying such microbial blooms are poorly understood, but the gut of UC patients has physicochemical features that differ from healthy patients and likely impact microbial physiology. For example, levels of the secondary bile acid deoxycholate (DC) are highly reduced in the ileoanal J-pouch of UC colectomy patients. We isolated a B. fragilis strain from a UC patient with pouch inflammation (i.e. pouchitis) and developed it as a genetic model system to identify genes and pathways that are regulated by DC and that impact B. fragilis fitness in DC and crude bile. Treatment of B. fragilis with a physiologically relevant concentration of DC reduced cell growth and remodeled transcription of one-quarter of the genome. DC strongly induced expression of chaperones and select transcriptional regulators and efflux systems and downregulated protein synthesis genes. Using a barcoded collection of {approx}50,000 unique insertional mutants, we further defined B. fragilis genes that contribute to fitness in media containing DC or crude bile. Genes impacting cell envelope functions including cardiolipin synthesis, cell surface glycosylation, and systems implicated in sodium-dependent bioenergetics were major bile acid fitness factors. As expected, there was limited overlap between transcriptionally regulated genes and genes that impacted fitness in bile when disrupted. Our study provides a genome-scale view of a B. fragilis bile response and genetic determinants of its fitness in DC and crude bile. ImportanceThe Gram-negative bacterium, Bacteroides fragilis, is a common member of the human gut microbiota that colonizes multiple host niches and can influence human physiology through a variety of mechanisms. Identification of genes that enable B. fragilis to grow across a range of host environments has been impeded in part by the relatively limited genetic tractability of this species. We have developed a high-throughput genetic resource for a B. fragilis strain isolated from a UC pouchitis patient. Bile acids limit microbial growth and are altered in abundance in UC pouches, where B. fragilis often blooms. Using this resource, we uncovered pathways and processes that impact B. fragilis fitness in bile and that may contribute to population expansions during bouts of gut inflammation.

microbiology↗

bioRxiv2023

Subdural CMOS optical probe (SCOPe) for bidirectional neural interfacing

Optical neurotechnologies use light to interface with neurons and can monitor and manipulate neural activity with high spatial-temporal precision over large cortical extents. While there has been significant progress in miniaturizing microscope for head-mounted configurations, these existing devices are still very bulky and could never be fully implanted. Any viable translation of these technologies to human use will require a much more noninvasive, fully implantable form factor. Here, we leverage advances in microelectronics and heterogeneous optoelectronic packaging to develop a transformative, ultrathin, miniaturized device for bidirectional optical stimulation and recording: the subdural CMOS Optical Probe (SCOPe). By being thin enough to lie entirely within the subdural space of the primate brain, SCOPe defines a path for the eventual human translation of a new generation of brain-machine interfaces based on light.

neuroscience↗

bioRxiv2023

Top-down control of exogenous attentional selection is mediated by beta coherence in prefrontal cortex

Salience-driven exogenous and goal-driven endogenous attentional selection are two distinct forms of attention that guide selection of task-irrelevant and task-relevant targets in primates. During conflict i.e, when salience and goal each favor the selection of different targets, endogenous selection of the task-relevant target relies on top-down control. Top-down attentional control mechanisms enable selection of the task-relevant target by limiting the influence of sensory information. Although the lateral prefrontal cortex (LPFC) is known to mediate top-down control, the neuronal mechanisms of top-down control of attentional selection are poorly understood. Here, using a two-target free-choice luminance-reward selection task, we demonstrate that visual-movement neurons and not visual neurons or movement neurons encode exogenous and endogenous selection. We then show that coherent-beta activity selectively modulates mechanisms of exogenous selection specifically during conflict and consequently may support top-down control. These results reveal the VM-neuron-specific network mechanisms of attentional selection and suggest a functional role for beta-frequency coherent neural dynamics in the modulation of sensory communication channels for the top-down control of attentional selection.

neuroscience↗

bioRxiv2023

Human papillomavirus 16 E2 interaction with TopBP1 is required for E2 and viral genome stability during the viral life cycle.

CK2 phosphorylation of HPV16 E2 at serine 23 promotes interaction with TopBP1, and this interaction is important for E2 plasmid segregation function. Here we demonstrate that the E2-TopBP1 interaction is critical for E2 and viral genome stability during the viral life cycle. Introduction of the S23A mutation into the HPV16 genome results in a loss of E2 expression and viral genome integration during organotypic rafting. Co-culture of N/Tert-1+E2-S23A cells with J2 fibroblasts results in E2-S23A degradation via the proteasome, wild-type E2 is not degraded. TopBP1 siRNA treatment of N/Tert-1+E2-WT cells results in E2 degradation only in the presence of J2 cells demonstrating the critical role for TopBP1 in maintaining E2 stability. The CK2 inhibitor CX4945 promotes E2-WT degradation in the presence of fibroblasts as it disrupts E2-TopBP1 interaction. siRNA targeting SIRT1 rescues E2-S23A stability in N/Tert-1 cells treated with J2 fibroblasts, with an increased E2-S23A acetylation. The results demonstrate that the E2-TopBP1 interaction is critical during the viral life cycle as it prevents fibroblast stimulated SIRT1 mediated deacetylation of E2 that promotes protein degradation. This means that the E2-TopBP1 complex maintains E2 and viral genome stability and that disruption of this complex can promote viral genome integration. Finally, we demonstrate that HPV11 E2 also interacts with TopBP1 and that this interaction is critical for HPV11 E2 stability in the presence of J2 cells. Treatment of N/Tert-1+11E2-WT cells with CX4945 results in 11E2 degradation. Therefore, CK2 inhibition is a therapeutic strategy for alleviating HPV11 diseases, including juvenile respiratory papillomatosis. ImportanceHuman papillomaviruses are pathogens that cause a host of diseases ranging from benign warts to cancers. There are no therapeutics available for combating these diseases that directly target viral proteins or processes, therefore we must enhance our understanding of HPV life cycles to assist with identifying novel treatments. In this report, we demonstrate that HPV16 and HPV11 E2 protein expression is dependent upon TopBP1 interaction in keratinocytes interacting with fibroblasts, which recapitulate stromal interactions in culture. The degradation of 16E2 promotes HPV16 genome integration, therefore the E2-TopBP1 interaction is critical during the viral life cycle. We demonstrate that the CK2 inhibitor CX4945 disrupts HPV11 interaction with TopBP1 and destabilizes HPV11 E2 protein in the presence of J2 fibroblasts; we propose that CX4945 could alleviate HPV11 disease burden.

cancer biology↗

bioRxiv2022

Glucocorticoid signaling mediates CD8+ T cell memory differentiation

We provide evidence on the TRM potentiation by glucocorticoids in stimulated CD8+ T cells. Signaling via glucocorticoid receptor reciprocally regulated short-lived effector cells and memory precursor effector cells. Influenza A virus infected mice treated transiently with dexamethasone preferentially generated multipotent TRM cells that efficiently responded to the subsequent infection. Compromised memory following abrogation of glucocorticoid signaling further confirmed their role in memory potentiation. Transcriptomic and biochemical analysis of dexamethasone treated cells revealed a metabolic switch to oxidative phosphorylation via an engagement of AMPK signaling due to reduced glucose uptake. These cells exhibited an accumulation of phosphorylated STAT3 and STAT5 to drive memory differentiation. Therefore, glucocorticoids mediate tissue homing memory T cell differentiation. One-Sentence SummaryGlucocorticoid signaling in responding CD8+ T cells promotes memory differentiation

immunology↗

bioRxiv2021

Selective estrogen receptor modulators limit alphavirus infection by targeting the viral capping enzyme nsP1

Alphaviruses cause animal or human diseases that are characterized by febrile illness, debilitating arthralgia, or encephalitis. Selective estrogen receptor modulators (SERMs), a class of FDA-approved drugs, have been shown to possess antiviral activities against multiple viruses, including Hepatitis C virus, Ebola virus, dengue virus, and vesicular stomatitis virus. Here, we evaluated three SERM compounds, namely 4-hydroxytamoxifen, tamoxifen, and clomifene, for plausible antiviral properties against two medically important alphaviruses, chikungunya virus (CHIKV) and Sindbis virus (SINV). In cell culture settings, these SERMs displayed potent activity against CHIKV and SINV at non-toxic concentrations with EC50 values ranging between 400 nM and 3.9 M. Further studies indicated that these compounds inhibit a post-entry step of the alphavirus life cycle, while enzymatic assays involving purified recombinant proteins confirmed that these SERMs target the enzymatic activity of non-structural protein 1 (nsP1), the capping enzyme of alphaviruses. Finally, tamoxifen treatment restrained CHIKV growth in the infected mice and diminished musculoskeletal pathologies. Combining biochemical, cell culture-based studies, and in vivo analyses, we strongly argue that SERM compounds, or their derivatives, may provide for attractive therapeutic options against alphaviruses.

immunology↗

bioRxiv2021

Targeting conserved viral virulence determinants by single domain antibodies to block SARS-CoV2 infectivity

We selected SARS-CoV2 specific single domain antibodies (sdAbs) from a previously constructed phage display library using synthetic immunogenic peptides of the virus spike (S) protein as bait. The sdAbs targeting the cleavage site (CS) and the receptor binding domain (RBD) in S protein efficiently neutralised the infectivity of a pseudovirus expressing SARS-CoV2 S protein. Anti-CS sdAb blocked the virus infectivity by inhibiting proteolytic processing of SARS-CoV2 S protein. Both the sdAbs retained characteristic structure within the pH range of 2 to 12 and remained stable upto 65{degrees}C. Furthermore, structural disruptions induced by a high temperature in both the sdAbs were largely reversed upon their gradual cooling and the resulting products neutralised the reporter virus. Our results therefore suggest that targeting CS in addition to the RBD of S protein by sdAbs could serve as a viable option to reduce SARS-CoV2 infectivity and that proteolytic processing of the viral S protein is critical for infection.

immunology↗

bioRxiv2020

A slow-exchange conformational switch regulates off-target cleavage by high-fidelity Cas9

The Cas9 endonuclease is broadly used for genome engineering applications by programming its single-guide RNA, where high specificity is required. Although mechanistic understanding of DNA cleavage in the CRISPR-Cas9 system has been guided by crystallographic structures and single-molecule FRET experiments, the role of conformational plasticity within its DNA recognition lobe (REC) for target accuracy remains elusive. Through NMR analysis of milliseconds-timescale dynamics, we show that the REC3 domain exchanges between a major, closed and a minor, open conformation. We find that a single mutation in the HiFi Cas9 variant (R691A) increases conformational dynamics in REC3, eliciting a global transition to the open conformation which is regulated by an intramolecular salt-bridge network. These observations suggest a mechanism for reduced off-target recognition by switching on a global conformational exchange process to allow REC3 scanning for proper base pairing. Our data establish a framework for rational engineering Cas9 variants with improved target discrimination.

biochemistry↗

bioRxiv2019

Comparison of tuning properties of gamma and high-gamma power in local field potential (LFP) versus electrocorticogram (ECoG) in visual cortex

Electrocorticogram (ECoG), obtained from macroelectrodes placed on the cortex, is typically used in drug-resistant epilepsy patients, and is increasingly being used to study cognition in humans. These studies often use power in gamma (30-70 Hz) or high-gamma (>80 Hz) ranges to make inferences about neural processing. However, while the stimulus tuning properties of gamma/high-gamma power have been well characterized in local field potential (LFP; obtained from microelectrodes), analogous characterization has not been done for ECoG. Using a hybrid array containing both micro and ECoG electrodes implanted in the primary visual cortex of two female macaques, we compared the stimulus tuning preferences of gamma/high-gamma power in LFP versus ECoG and found them to be surprisingly similar. High-gamma power, thought to index the average firing rate around the electrode, was highest for the smallest stimulus (0.3{degrees} radius), and decreased with increasing size in both LFP and ECoG, suggesting local origins of both signals. Further, gamma oscillations were similarly tuned in LFP and ECoG to stimulus orientation, contrast and spatial frequency. This tuning was significantly weaker in electroencephalogram (EEG), suggesting that ECoG is more like LFP than EEG. Overall, our results validate the use of ECoG in clinical and basic cognitive research.

neuroscience↗

bioRxiv2019

Laboratory Evolution Experiments Help Identify a Predominant Site of ΔrnhA-dnaA Constitutive Stable DNA Replication Initiation

The bacterium E. coli can initiate replication in the absence of the replication initiator protein DnaA and / or the canonical origin of replication oriC in a{Delta} rnhA background. This phenomenon, which can be primed by R-loops, is called constitutive stable DNA replication (cSDR). Whether DNA replication during cSDR initiates in a stochastic manner through the length of the chromosome or at specific sites, and how E. coli can find adaptations to loss of fitness caused by cSDR remain inadequately answered. We use laboratory evolution experiments of{Delta} rnhA-{Delta}dnaA followed by deep sequencing to show that DNA replication preferentially initiates within a broad region located [~]0.4-0.7 Mb clockwise of oriC. This region includes many bisulfite-sensitive sites, which have been previously defined as R-loop forming regions; and includes a site containing sequence motifs that favour R-loop formation. Initiation from this region would result in head-on replication-transcription conflicts at rRNA loci. Inversions of these rRNA loci, which can partly resolve these conflicts, help the bacterium suppress the fitness defects of cSDR. These inversions partially restore the gene expression changes brought about by cSDR. The inversion however increases the possibility of conflicts at essential mRNA genes, which would utilise only a miniscule fraction of RNA polymerase molecules most of which transcribe rRNA genes. Whether subsequent adaptive strategies would attempt to resolve these conflicts remains an open question. ImportanceThe bacterium E. coli can replicate its DNA even in the absence of the molecules that are required for canonical replication initiation. This often requires the formation of RNA-DNA hybrid structures, and is referred to as constitutive stable DNA replication (cSDR). Where on the chromosome does cSDR initiate? We answer this question using laboratory evolution experiments and genomics, and show that selection favours cSDR initiation predominantly at a region [~]0.6 Mb clockwise of oriC. Initiation from this site will result in more head on collisions of DNA polymerase with RNA polymerase operating on rRNA loci. The bacterium adapts to this problem by inverting a region of the genome including several rRNA loci such that head-on collisions between the two polymerases are minimised. Understanding such evolutionary strategies in the context of cSDR can provide insights into the potential causes of resistance against antibiotics that target initiation of DNA replication.

genomics↗

bioRxiv2019

Evaluating the ecological impacts of pesticide seed treatments on arthropod communities in a grain crop rotation

O_LIWhile many studies have investigated non-target impacts of neonicotinoid seed treatments (NSTs), they usually take place within a single crop and focus on specific pest or beneficial arthropod taxa.\nC_LIO_LIWe compared the impacts of three seed treatments to an untreated control: imidacloprid + fungicide products, thiamethoxam + fungicide products, and fungicide products alone in a three-year crop rotation of full-season soybean, winter wheat, double-cropped soybean and maize. Specifically, we quantified neonicotinoid residues in the soil and in weedy winter annual flower buds and examined treatment impacts on soil and foliar arthropod communities, and on plant growth and yield.\nC_LIO_LITrace amounts of insecticide were found in winter annual flowers of one species in one site year, which did not correspond with our treatments. Although low levels of insecticide residues were present in the soil, residues were not persistent. Residues were highest in the final year of the study, suggesting some accumulation.\nC_LIO_LIWe observed variable impacts of NSTs on the arthropod community; principle response curve analysis, diversity and evenness values exhibited occasional community disturbances, and treatments impacted the abundance of various taxa. Overall, imidacloprid had a greater effect than thiamethoxam, with the fungicide only treatment also occasionally impacting communities and individual taxa.\nC_LIO_LIPest pressure was low throughout the study, and although pest numbers were reduced by the insecticides no corresponding increases in yield were observed. However, the fungicide products contributed to higher yields in wheat.\nC_LIO_LISynthesis and applications. Pesticide seed treatments can disturb arthropod communities, even when environmental persistence and active ingredient concentrations are low. The foliar community in wheat and maize exhibited a trend of increasing disturbance throughout the sampling period, suggesting that recovery from the impacts of NSTs is not always rapid. Our study is among the first to demonstrate that seed applied fungicides alone can disrupt arthropod communities in agroecosystems and highlights the need for further investigation into the impacts of seed applied fungicides.\nC_LI

ecology↗