Biology subjects

Chatterjee, S.

Publications and source records attributed to Chatterjee, S..

At least 127 records · Page 7Linked to original sources

bioRxiv2023

Atheroprone shear stress stimulates noxious endothelial extracellular vesicle uptake by MCAM and PECAM-1 cell adhesion molecules

Atherosclerotic lesions mainly form in arterial areas exposed to low shear stress (LSS), where endothelial cells express a senescent and inflammatory phenotype. Conversely, high shear stress (HSS) has atheroprotective effects on the endothelium. Endothelial cell-derived extracellular vesicles have been shown to regulate inflammation, senescence and angiogenesis and therefore play a crucial role in vascular homeostasis and disease. While previous studies have shown links between hemodynamic forces and extracellular vesicle release, the exact consequences of shear stress on the release and uptake of endothelial EVs remains elusive. Our aim is therefore to decipher the interplay between these processes in endothelial cells exposed to atheroprone or atheroprotective shear stress. Confluent human umbilical vein endothelial cells (HUVEC) were exposed to either LSS or HSS for 24 hours. Large and small EVs were isolated from conditioned medium by sequential centrifugation and size exclusion chromatography. They were characterized by TEM, Western blot analysis of EV markers, tunable resistive pulse sensing, flow cytometry and proteomics. Uptake experiments were performed using fluorescently-labeled EVs and differences between groups were assessed by flow cytometry and confocal microscopy. We found that levels of large and small EVs in HUVEC conditioned media were fifty and five times higher in HSS than in LSS conditions, respectively. In vivo and in vitro uptake experiments revealed greater EV incorporation by cells exposed to LSS conditions compared to HSS. Additionally, endothelial LSS-EVs appeared to have a greater affinity for HUVECs than HSS-EVs or EVs derived from platelets, red blood cells, granulocytes and peripheral blood mononuclear cells. Proteomic analysis revealed that LSS-EVs were enriched in adhesion proteins such as PECAM1, MCAM, which were involved in EV uptake by endothelial cells. LSS-EVs also carried mitochondrial material, which may be involved in elevating reactive oxygen species levels in recipient cells. These findings suggest that endothelial shear stress has a significant impact during EV biogenesis and uptake. Given the major role of EVs and shear stress in vascular health, deciphering the relation between these processes may yield innovative strategies for the early detection and treatment of endothelial dysfunction.

cell biology↗

bioRxiv2022

CLASPs stabilize the intermediate state between microtubule growth and catastrophe

CLASPs regulate microtubules in many fundamental cellular processes. CLASPs stabilize dynamic microtubules by suppressing catastrophe and promoting rescue, the switch-like transitions between microtubule growth and shrinkage. However, the molecular mechanisms underlying CLASPs activity are not understood. Here, we investigate the effects of CLASPs on distinct microtubule substrates in the absence of tubulin to gain insight into how CLASPs regulate microtubule dynamics. Surprisingly, we find that human CLASP1 depolymerizes stable microtubules in the presence of GTP, but not in the absence of nucleotide. Conversely, CLASP1 stabilizes dynamic microtubules upon tubulin dilution in the presence of GTP. Our results demonstrate that CLASP1 drives microtubule substrates with different inherent stabilities into the same slowly-depolymerizing state in the absence of tubulin in a nucleotide-dependent manner. We interpret this state as the pre-catastrophe intermediate state between microtubule growth and shrinkage. Thus, we conclude that CLASPs stabilize the intermediate state between microtubule growth and shrinkage to suppress microtubule catastrophe and promote rescue.

cell biology↗

bioRxiv2022

Study of droplet generation in an E-shaped microchannel using two-phase level-set method

Droplet-based microfluidics is the study of microfluidic device, where droplets are generated mainly using two methods i.e., active, and passive methods. The manipulation of these droplets in microfluidic channels is an immensely useful technology in various scientific fields such as biological, biomedical studies, and drug delivery. Recently droplets with a core-shell structure have achieved considerable interest due to their unique properties and varied applications. Droplet splitting as an important feature of droplet-based microfluidic systems has been widely used. In the present paper, two-dimensional numerical simulations have been done to examine the droplet splitting process in an E Junction geometry microchannel. The two-phase level set method (LSM) has been used to analyze the mechanism of droplet formation and droplet splitting in an immiscible liquid/liquid two-phase flow. Governing equations on the flow field have been studied and solved using COMSOL Multiphysics software. The model was developed to simulate the mechanism of droplet splitting at the E-junction microchannel. This study provides a passive technique to split up microdroplets at the E-junction.

bioengineering↗

bioRxiv2022

Discovery, Synthesis, and Optimization of 1,2,4-Triazolyl Pyridines Targeting Mycobacterium tuberculosis

Tuberculosis (TB) results in 1.5 million deaths every year. The rise in multi-drug resistant TB underscores the urgent need to develop new antibacterials, particularly those with new chemical entities and/or novel mechanisms of action that can be used in combination therapy with existing drugs to prevent the rapid emergence of resistance. Herein, we report the discovery and synthesis of a new series of compounds containing a 3-thio-1,2,4-triazole moiety that show inhibition of Mycobacterium tuberculosis (Mtb) growth and survival. Structure-activity relationship studies led us to identify potent analogs displaying nanomolar inhibitor activity, specifically against Mtb. These potent analogs exhibit a promising ADME/pharmacokinetic profile and no cytotoxicity in mammalian cells at over 100 times the effective dose in Mtb. Our preliminary investigations into the mechanism of action suggest this series is not engaging promiscuous targets and, thereby, could be acting on a novel target. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/516356v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1fd7a78org.highwire.dtl.DTLVardef@738c7dorg.highwire.dtl.DTLVardef@c7957org.highwire.dtl.DTLVardef@f26ae3_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

Breast cancer progression and metastasis to lymph nodes reveals cancer cell plasticity and MHC class II-mediated immune regulation

Tumor-draining lymph nodes are critical sites for generating tumor antigen-specific T cells and are associated with durable immune responses. However, lymph nodes are often the first site of metastasis and lymph node metastases portend worse outcomes. Through cross-species single cell gene expression analysis of breast cancer progression and metastasis to lymph nodes, we uncovered features that define the heterogeneity, plasticity, and immune evasion of cancer cells. Notably, a subpopulation of metastatic cancer cells in the lymph node were marked by high levels of MHC class II (MHC-II) gene expression both in mice and humans. Mechanistically, the IFN-{gamma} and JAK/STAT signaling pathways mediate MHC-II expression in cancer cells. Ablation of IFNGR1/2 or CIITA, the transactivator of MHC-II, in cancer cells prevented tumor progression. Interestingly, MHC-II+ cancer cells lacked co-stimulatory molecule expression, engendered the expansion of regulatory T cells and blunted CD4+ effector T cells in the tumor-draining lymph nodes and favor tumor progression. Overall, our data suggests that cancer cell plasticity during breast cancer progression and metastasis to lymph nodes endows metastatic cells with the ability to avoid immune surveillance. These data provide the basis for new opportunities to therapeutically stimulate anti-cancer immune responses against local and systemic metastases.

cancer biology↗

bioRxiv2022

RET enhancer haplotype-dependent remodeling of the human fetal gut development program

Hirschsprung disease (HSCR) is associated with deficiency of the receptor tyrosine kinase RET, resulting in loss of cells of the enteric nervous system (ENS) during fetal gut development. The major contribution to HSCR risk is from common sequence variants in RET enhancers with additional risk from rare coding variants in many genes. Here, we demonstrate that these RET enhancer variants specifically alter the human fetal gut development program through significant decreases in gene expression of RET, members of the RET-EDNRB gene regulatory network (GRN), other HSCR genes, and an altered transcriptome with 2,382 differentially expressed genes with diverse neuronal and mesenchymal functions. A parsimonious hypothesis for these results is that beyond RETs direct effect on its GRN, it also has a major role in enteric neural crest derived cell (ENCDC) precursor proliferation, its deficiency reducing ENCDCs with relative expansion of non-ENCDC cells. Thus, genes reducing RET proliferative activity can potentially cause HSCR. One such class is the 23 RET-dependent transcription factors enriched in early gut development. We show that their knockdown in human neuroblastoma SK-N-SH cells reduces RET and/or EDNRB gene expression, expanding the RET-EDNRB GRN. The human embryos we studied had major remodeling of the gut transcriptional but were unlikely to have had HSCR: thus, genetic changes in addition to those in RET are required for a significant enough reduction in ENCDCs to cause aganglionosis.

genetics↗

bioRxiv2022

A home-cage, video monitoring-based mouse frailty index detects age-associated morbidity in the absence of handler-induced stress.

Frailty indexes provide quantitative measurements of non-specific health decline and are particularly useful as longitudinal monitors of pre-mortal morbidity in aging studies. For mouse studies, frailty assessments can be taken non-invasively, but they require handling and direct observation that is labor-intensive to the scientist and stress-inducing to the animal. Here, we implement, evaluate, and provide a digital frailty index composed entirely of computational analyses of home-cage video and compare it to manually obtained frailty scores in genetically diverse mice. We show that the frailty scores assigned by our digital index correlate with both manually obtained frailty scores and chronological age. Thus, we provide a tool for frailty assessment that reduces stress to the animal and can be collected consistently, at scale, without substantial labor cost.

animal behavior and cognition↗

bioRxiv2022

Losartan controls immune checkpoint blocker-induced edema and improves survival in glioblastoma

Immune checkpoint blockers (ICBs) have failed in all Phase III glioblastoma trials. Here, we found that ICBs induce cerebral edema in some patients and mice with glioblastoma. Through single-cell RNA sequencing, intravital imaging, and T cell blocking studies in mice, we demonstrated that this edema results from an inflammatory response following anti-PD1 antibody treatment that disrupts the blood-tumor-barrier. Used in lieu of immunosuppressive corticosteroids, the angiotensin receptor blocker losartan prevented this ICB-induced edema and reprogrammed the tumor microenvironment, curing 20% of mice which increased to 40% in combination with standard of care treatment. Using a bihemispheric tumor model, we identified a "hot" tumor immune signature prior to losartan+anti-PD1 therapy that predicted long-term survival. Our findings provide the rationale and associated biomarkers to test losartan with ICBs in glioblastoma patients. One-Sentence SummaryLosartan prevents immunotherapy-associated edema and enhances the outcome of immunotherapy in glioblastoma.

cancer biology↗

bioRxiv2022

A non-classical mechanism of β-lactam resistance in Methicillin-Resistant Staphylococcus aureus (MRSA) and its effect on virulence

Methicillin-Resistant Staphylococcus aureus (MRSA) are pathogenic bacteria that are infamously resistant to {beta}-lactam antibiotics, a property attributed to the mecA gene. Recent studies have reported that mutations associated with the promoter region of pbp4 demonstrated high levels of {beta}-lactam resistance, suggesting the role of PBP4 as an important non-mecA mediator of {beta}-lactam resistance. The pbp4 promoter-associated mutations have been detected in strains with or without mecA. Our previous studies that were carried out in strains devoid of mecA described that pbp4 promoter-associated mutations lead to PBP4 overexpression and {beta}-lactam resistance. In this study, by introducing various pbp4 promoter-associated mutations in the genome of an MRSA strain, we demonstrate that PBP4 overexpression can supplement mecA-associated resistance in S. aureus and can lead to increased {beta}-lactam resistance. The promoter and regulatory region of pbp4 is shared with a divergently transcribed gene, abcA, which encodes for a multidrug exporter. We demonstrate that the promoter mutations caused an upregulation of pbp4 and downregulation of abcA, confirming that the resistant phenotype is associated with PBP4 overexpression only. PBP4 has also been associated with staphylococcal pathogenesis, however, its exact role remains unclear. Using a C. elegans model, we demonstrate that strains having increased PBP4 expression are less virulent compared to wild-type strains, suggesting that {beta}-lactam resistance mediated via PBP4 likely comes at the cost of virulence. ImportanceOur study demonstrates the ability of PBP4 to be an important mediator of {beta}-lactam resistance in not only Methicillin-susceptible Staphylococcus aureus (MSSA) background strains as previously demonstrated, but also in MRSA strains. When present together, PBP2a and PBP4 overexpression can produce increased levels of {beta}-lactam resistance, causing complications in treatment. Thus, this study suggests the importance of monitoring PBP4-associated resistance in clinical settings, as well as understanding the mechanistic basis of associated resistance, so that treatments targeting PBP4 may be developed. This study also demonstrates that S. aureus strains with increased PBP4 expression are less pathogenic, providing important hints about the role of PBP4 in S. aureus resistance and pathogenesis.

microbiology↗

bioRxiv2022

Circadian Control of Pulmonary Endothelial Signaling occurs via NADPH oxidase 2-NLRP3 pathway

Circadian rhythms are endogenous oscillations that occur with a 24-hr periodicity. These rhythms are ubiquitous and thus, vascular endothelial cells that line the vascular bed are also subjected to circadian regulation. While the circadian control of vascular function has been demonstrated in the context of various pathologies, the relevance and functional implication of clock control over pulmonary vasculature has never been investigated. As the pulmonary endothelium is a crucial site for the hosts inflammatory response to a lung specific pathogen, we investigated the role of the circadian clock in mediation the response of the pulmonary endothelium to inflammation. We hypothesized that the pulmonary endothelium is under circadian control and that the clock serves to curb inflammatory signaling. MethodsCircadian rhythms were monitored in pulmonary artery segments and endothelial cells isolated from mPer2luciferase transgenic mice in the presence of an inflammatory stimuli (LPS). Reactive oxygen species (ROS) production in LPS treated cells was measured by fluorescence microscopy using the cell permeant dye CellROX Green. NLRP3 inflammasome was monitored post-mortem (0-72 h post LPS instillation) by measuring the expression of the NLRP3 subunit in wild type and Bmal1-/- and Cry1/2-/- mice. Inflammation was quantified in these mice by measuring PMN adherence and intercellular adhesion molecule (ICAM-1). ResultsWe observed that the circadian rhythm of the pulmonary vasculature was altered LPS. LPS also led to ROS production in these cells; ROS increased 3 h post LPS treatment, peaked by 36 h and returned to baseline values by 72 h. ROS were inhibited by pretreating the cells with the NADPH oxidase 2 (NOX2) inhibitor dipheneylene iodonium (DPI). Addition of DPI, prior to LPS pretreatment also restored the circadian rhythmicity of the pulmonary endothelium. The increase in NLRP3 along the vessel wall (post LPS treatment) was resolved by 72 h in lungs of wild type mice but not in Bmal1-/- and Cry1-/-Cry2-/- lungs. Inflammation (ICAM-1 and PMN) was also resolved in wild type but not in mice wherein the circadian clock had been disrupted genetically. ConclusionOur data indicate that pro-inflammatory stimuli reprogram circadian rhythms in the pulmonary endothelium via ROS via the NOX2-NLRP3 pathway. Disruption of the clock mediates a sustained increase in ROS via this Nox2-NLRP3 pathway in endothelial cells, thus offering a novel mechanism for mitigating the effects of clock disruption.

physiology↗

bioRxiv2022

LDL1 and LDL2 histone demethylases interact with FVE to regulate flowering in Arabidopsis

In higher plants, epigenetic modifications provide a stage for both transient and permanent cellular reprogramming required for vegetative to reproductive phase transition. Arabidopsis LSD1-like 1 (LDL1), a histone demethylase positively regulates floral transition, but the molecular and biochemical nature of LDL1 mediated flowering is poorly understood. Here we have shown that LDL1 mediated regulation of flowering is dependent on MADS AFFECTING FLOWERING 4 (MAF4) and MAF5 floral repressors. LDL1 binds on the chromatin of MAF4 and MAF5 and removes H3K4me2 activation marks to repress their expression. Further we show that LDL2 negatively regulates the expression of MAF4 and MAF5 redundantly with LDL1. Both LDL1 and LDL2 interact with an autonomous flowering pathway protein, FLOWERING LOCUS VE (FVE), to regulate the floral transition and thus could be a part of the FVE-corepressor complex. We show that MAF5 interacts with other floral repressors FLC and SHORT VEGETATIVE PHASE (SVP) and repress the expression of FT to delay floral transition. Thus, our results deepen the mechanistic understanding of LDL1/LDL2-FVE mediated floral transition in Arabidopsis.

plant biology↗

bioRxiv2022

Thermal endurance by a hot-spring-dwelling phylogenetic relative of the mesophilic Paracoccus

High temperature growth/survival was revealed in a phylogenetic relative (strain SMMA_5) of the mesophilic Paracoccus isolated from the 78-85{degrees}C water of a Trans- Himalayan sulfur-borax spring. After 12 h at 50{degrees}C, or 45 minutes at 70{degrees}C, in mineral salts thiosulfate (MST) medium, SMMA_5 retained [~]2% colony-forming units (CFUs), whereas comparator Paracoccus had 1.5% and 0% CFU left at 50{degrees}C and 70{degrees}C respectively. After 12 h at 50{degrees}C, the thermally-conditioned sibling SMMA_5_TC exhibited [~]1.5 time increase in CFU-count; after 45 minutes at 70{degrees}C, SMMA_5_TC had 7% of the initial CFU-count intact. 1000-times diluted Reasoners 2A medium, and MST supplemented with lithium, boron or glycine-betaine (solutes typical of the SMMA_5 habitat), supported higher CFU-retention/CFU-growth than MST. With or without lithium/boron/glycine-betaine in MST, a higher percentage of cells always remained viable (cytometry data), compared with what percentage remained capable of forming single colonies (CFU data). SMMA_5, compared with other Paracoccus, contained 335 unique genes, mostly for DNA replication/recombination/repair, transcription, secondary metabolites biosynthesis/transport/catabolism, and inorganic ion transport/metabolism. Its also exclusively enriched in cell wall/membrane/envelope biogenesis, and amino acid metabolism, genes. SMMA_5 and SMMA_5_TC mutually possessed 43 nucleotide polymorphisms, of which 18 were in protein-coding genes with 13 nonsynonymous and seven radical amino acid replacements. Such biochemical and biophysical mechanisms could be involved in thermal stress mitigation which streamline the cells energy and resources towards system-maintenance and macromolecule-stabilization, thereby relinquishing cell-division for cell-viability. Thermal conditioning apparently helped memorize those potential metabolic states which are crucial for cell-system maintenance, while environmental solutes augmented the indigenous stability-conferring mechanisms. IMPORTANCEFor a holistic understanding of microbial lifes high-temperature adaptation it is imperative to explore the biology of the phylogenetic relatives of mesophilic bacteria which get stochastically introduced to geographically and geologically diverse hot spring systems by local geodynamic forces. Here, in vitro endurance of high heat up to the extent of growth under special (habitat-inspired) conditions was discovered in a hot- spring-dwelling phylogenetic relative of the mesophilic Paracoccus species. Thermal conditioning, extreme oligotrophy, metabolic deceleration, presence of certain habitat- specific inorganic/organic solutes, and typical genomic specializations were found to be the major enablers of this conditional (acquired) thermophilicity. Feasibility of such phenomena across the taxonomic spectrum can well be paradigm-changing for the established scopes of microbial adaptation to the physicochemical extremes. Applications of conditional thermophilicity in microbial process biotechnology may be far reaching and multi-faceted.

microbiology↗

bioRxiv2022

Dauer quiescence as well as continuity of the life cycle after dauer-exit in Caenorhabditis elegans are dependent on the endoribonuclease activity of XRN-2

Caenorhabditis elegans embarks on a quiescent dauer state upon exposure to unfavourable conditions and can sustain for a very long period without food, but it returns to continuous life cycle upon arrival of suitable conditions. Thus, dauer state plays a critical role in its adaptive fitness and survival. ATP-independent endoribonuclease activity of XRN-2 has been implicated in dauer microRNA metabolism, perturbation of which causes their collapse within a very short span of time. Here, we present a detailed comparative analyses of dauer transcriptomes from a conditional mutant strain for the endoribonuclease activity of XRN-2, maintained under control and experimental conditions. We observed that even a limited disruption of microRNA homeostasis in experimental dauers results in deregulation of a large number of mRNA targets. Our bioinformatic analyses, supported by morphological, physiological, and behavioral evidence further demonstrate critical changes in metabolism leading to a state unsupportive of dauer maintenance, alongside potential defects in multiple neuronal activities, which might have caused an overall disruption of dauer plasticity. We explore a possible role of this endoribonuclease activity towards the maintenance of chromatin architecture and transposon expression that in turn might affect the transcriptional program critically required for the maintenance of non-aging, long-lived dauers. Finally, we also demonstrate that perturbation of the endoribonuclease activity during the dauer state exerts drastic adverse effects on the continuity of life cycle after dauer-exit. They not only fail to recapitulate the wild type events of germline development and embryogenesis, but also present traits of very old worms and formation of tumor-like structures in the proximal gonad.

molecular biology↗

bioRxiv2022

Diversity of Secretion System Apparatus in Tomato Wilt Causing Ralstonia solanacearum Strains: a Comparative Analysis Using in-silico Approach

Ralstonia solanacearum (Rs) species is the leading cause of bacterial wilt disease in a wide range of host plants worldwide. In the present study, secretion system analysis of five tomato pathogenic Rs strains was carried out in-silico. This paper describes a new protocol to identify the secretion system components i.e. SSCs (T1SS-T6SS, Flg, T4P, and Tad-Tat). A total of 865 SSCs were identified using the new protocol. Contributions of SSCs into core-secretion system apparatus (i.e. SSA) were also studied. Synteny was discovered among the secretion system apparatus (SSA) where relative frequency of SSCs to core-SSA is high (>20%) which includes T1SS, T2SS, T5SS, T4P, and Tad-Tat, but excludes T3SS, T4SS, and Flg. To the best of our knowledge, this is the first report indicating that during the evolution of Rs, most of the secretion system apparatus (T1SS, T2SS, T5SS, T4P, and Tad-Tat) were highly conserved and came from a single ancestor, while T3SS and T6SS may have arrived later, probably from horizontal gene transfer.

microbiology↗

bioRxiv2022

DNA damage response signaling is crucial for effective Chikungunya virus replication

Viruses utilize a plethora of strategies to manipulate the host pathways and hijack its machineries for efficient replication. Several DNA as well as handful of RNA viruses are reported to interact with proteins involved in DNA damage responses (DDR). As the DDR pathways have never been explored in Alphaviruses, this investigation intended to determine the importance of the DDR pathways in CHIKV infection through in vitro, in vivo and ex vivo models. The study reveals that CHIKV infection activates the Chk2 and Chk1 proteins associated with DDR signaling pathways and increases DNA damage by 95%. Inhibition of both ATM-ATR kinases by ATM/ATR kinase inhibitor (AAKi) shows drastic reduction in viral particle formation in vitro. Next, the treatment of mice with this drug has been shown to reduce the disease score substantially in CHIKV-infected C57BL/6 mice with 71% decrement in the viral copy and the same has been established in hPBMC-derived monocyte-macrophage populations. Additionally, gene silencing of Chk2 and Chk1 reduces viral progeny formation around 73.7% and 78% respectively. Moreover, it has been demonstrated that CHIKV-nsP2 interacts with Chk2 and Chk1 during CHIKV infection and docking analysis depicts the specific amino acids responsible for these interactions. Further, the data suggests that CHIKV infection induces cell cycle arrest in G1 and G2 phases. In conclusion, this work demonstrated for the first time the mechanistic insight of the induction of DDR pathways by CHIKV that might contribute to the designing of effective therapeutics for the control of this virus infection in future. IMPORTANCEViruses being intra-cellular parasite, need several host cell machineries so as to achieve effective replication of their own genome, along with virus-encoded enzymes. One of the strategies is to hijack the DDR pathways. Several DNA as well as handful of RNA viruses interact with the cellular proteins involved in DDR pathways, however, reports with respect to the association of Chk2 and Chk1 in alphavirus infection are scanty. Hence, this study is amongst the first to report that modulation of DDR pathways is crucial for effective CHIKV infection. This work also shows that there is interaction of CHIKV-nsP2 with two crucial host factors, Chk2 and Chk1 for efficient viral infection. Interestingly, CHIKV infection was found to cause DNA damage and arrest cell cycle in G1 and G2 phases to facilitate viral infection. This information might facilitate to develop effective therapeutics for the control of the CHIKV infection in future.

microbiology↗

bioRxiv2022

NEUROeSTIMator: Using Deep Learning to Quantify Neuronal Activation from Single-Cell and Spatial Transcriptomic Data

Neuronal activity-dependent transcription directs molecular processes that regulate synaptic plasticity, brain circuit development, behavioral adaptation, and long-term memory. Single cell RNA-sequencing technologies (scRNAseq) are rapidly developing and allow for the interrogation of activity-dependent transcription at cellular resolution. Here, we present NEUROeSTIMator, a deep learning model that integrates transcriptomic signals to estimate neuronal activation in a way that we demonstrate is associated with Patch-seq electrophysiological features and that is robust against differences in species, cell type, and brain region. We demonstrate this methods ability to accurately detect neuronal activity in previously published studies of single cell activity-induced gene expression. Further, we applied our model in a spatial transcriptomic study to identify unique patterns of learning-induced activity across different brain regions. Altogether, our findings establish NEUROeSTIMator as a powerful and broadly applicable tool for measuring neuronal activation, whether as a critical covariate or a primary readout of interest.

bioinformatics↗

bioRxiv2022

Bidirectional regulation between AP-1 and SUMO genes modulates inflammatory signaling during Salmonella Typhimurium infection

Gram-negative bacterium Salmonella Typhimurium (STm) is the causative agent of gastroenteritis. Among the various gut pathogens, STm is still one of the most frequent culprits posing a significant health challenge. STm utilizes its effector proteins to highjack host cell processes. Alteration of SUMOylation, a post-translational modification mechanism, is one such change caused by STm. STm mediated simultaneous downregulation of SUMO-pathway genes, Ubc9 and PIAS1, is required for an efficient infection. In the present study, the regulation of SUMO pathway genes during STm infection was investigated. Promoters of both UBC9 and PIAS1, were seen to harbor binding motifs of AP-1, Activator protein-1 (c-Jun:c-Fos heterodimers or c-Jun:c-Jun homodimers). Using electrophoretic mobility shift assays, a direct binding of c-Fos to the identified motifs was observed. Perturbation of c-Fos led to changes in expression of Ubc9 and PIAS1, while its SUMO-modifications resulted in differential regulation of its target genes. In line with this, STm infection of fibroblasts with SUMOylation deficient c-Fos (c-FOS-KOSUMO-def-FOS) resulted in uncontrolled activation of target genes, as revealed by 3mRNA-Seq analysis and mathematical modelling, resulting in massive activation of inflammatory pathways. Infection of c-FOS-KOSUMO-def-FOS cells favored STm replication, indicating misdirected immune mechanisms in these cells. Finally, chromatin Immuno-precipitation assays confirmed a context dependent differential binding and release of AP-1 to/from target genes due to its Phosphorylation and SUMOylation respectively. Overall, our data point towards existence of a bidirectional cross-talk between c-Fos and the SUMO pathway and highlighting its importance in AP-1 function relevant to STm infections and beyond. Author summaryFood borne infections caused Salmonella Typhimurium pose a major health challenge in developing and developed world. Unfortunately, many aspects of Salmonella-host crosstalk still remain unknown. In the current work, using sophisticated computational tools along with cell culture experiments and mathematical modeling, we demonstrate how Salmonella controls SUMOylation, a post-translational modification (PTM) pathway of host. SUMOylation governs fundamental processes of the host cell, and its alteration is required for a successful Salmonella infection. We show that SUMO-pathway genes, Ubc9 and Pias1, are direct target genes of AP-1 transcription factor. C-Fos, a component of AP-1 transcriptionally regulates SUMO-genes by binding to their promoters. During Salmonella infection, a selective activation of target genes of c-Fos was observed. The selective regulation of target genes relied on c-fos PTMs. Experimental perturbation of c-Fos PTMs led to global transcriptional dysregulation including immune hyperactivation. Thus, we show existence of a complex interplay between the SUMO-pathway genes and AP-1 transcription factors which mediate selective gene regulation during Salmonella infection.

microbiology↗

bioRxiv2021

Ret loss-of-function decreases enteric neural crest progenitor proliferation and restricts developmental fate potential during enteric nervous system development

The receptor tyrosine kinase gene RET plays a critical role in the fate specification of enteric neural crest-derived cells (ENCDCs) during enteric nervous system (ENS) development. Pathogenic RET loss of function (LoF) alleles are associated with Hirschsprung disease (HSCR), which is marked by aganglionosis of the gastrointestinal (GI) tract. ENCDCs invade the developing GI tract, proliferate, migrate caudally, and differentiate into all of the major ENS cell types. Although the major phenotypic consequences and the underlying transcriptional changes from Ret LoF in the developing ENS have been described, its cell type and state-specific effects are unknown. Consequently, we performed single- cell RNA sequencing (scRNA-seq) on an enriched population of ENCDCs isolated from the developing GI tract of Ret null heterozygous and homozygous mouse embryos at embryonic day (E)12.5 and E14.5. We demonstrate four significant findings: (1) Ret-expressing ENCDCs are a heterogeneous population composed of ENS progenitors as well as glial and neuronal committed cells; (2) neurons committed to a predominantly inhibitory motor neuron developmental trajectory are not produced under Ret LoF, leaving behind a mostly excitatory motor neuron developmental program; (3) HSCR-associated and Ret gene regulatory network genes exhibit distinct expression patterns across Ret-expressing ENCDC with their expression impacted by Ret LoF; and (4) Ret deficiency leads to precocious differentiation and reduction in the number of proliferating ENS precursors. Our results support a model in which Ret contributes to multiple distinct cellular phenotypes associated with the proper development of the ENS, including the specification of inhibitory neuron subtypes, cell cycle dynamics of ENS progenitors, and the developmental timing of neuronal and glial commitment. Summary StatementRet LoF affects proper development of the mouse ENS through multiple distinct cellular phenotypes including restriction of neuronal fate potential, disruption of ENCDC migration, and modulation of progenitor proliferation rate.

developmental biology↗