Biology subjects

Jain, A.

Publications and source records attributed to Jain, A..

13 recordsLinked to original sources

bioRxiv2026

The filopodial scaffold polyphosphate dictates cell adhesion-versus-invasion decisions

Inorganic polyphosphate (polyP) is an ancient polymer conserved across all life, serving cell type and location specific functions in every major compartment. Yet its role at the plasma membrane, where it accumulates to peak levels in many primary cells, is largely unknown. Here we identify polyP as a stabilizing component of filopodia, actin based membrane protrusions that govern cell adhesion, contact inhibition, and chemotaxis. Elevating cellular polyP increases filopodial stability and enhances cell adhesion, whereas reducing polyP accelerates filopodial disassembly and promotes cell migration. Mechanistically, we find that polyP acts as a structural filopodial scaffold, recruiting and organizing IRSp53, a membrane curvature inducing protein. We show that metastatic fibroblasts and breast cancer organoids carry markedly reduced and intracellularly reorganized polyP levels relative to their non transformed counterparts. Restoring endogenous polyP via lipid nanoparticle delivery suppresses their invasive phenotypes and reverses prometastatic gene expression signatures, implicating polyP as a primordial tumor suppressor.

cell biology↗

bioRxiv2026

Microbial valerate is associated with CAR T dysbiosis and its supplementation enhances CAR T function in B-cell lymphoma

Anaerobe-depleting antibiotic exposure is associated with inferior progression-free survival after CD19 CAR T-cell therapy in large B-cell lymphoma, yet the cellular mechanisms linking gut dysbiosis to the CAR T-cell product and whether this imprint is reversible have remained undefined. In two independent CAR-T candidate cohorts, low stool valerate at the time of CAR-T eligibility identified a multi-metabolite-deficient dysbiotic gut microbiome state marked by depletion of fiber-fermenting commensals and loss of carbohydrate-fermentation, SCFA-biosynthesis, and amino-acid metabolism pathways. Reanalysis of single-cell RNA sequencing from 42 lymphoma patients stratified by piperacillin-tazobactam/imipenem/meropenem (PIM) exposure revealed that PIM-exposed CAR T-cell products were CD4-skewed, with significantly elevated AP-1/immediate-early gene (IEG) and cellular activation signatures that together predicted inferior progression-free survival. Ex vivo conditioning of CAR T-cells with valerate produced a chromatin and transcription factor program distinct from butyrate or propionate, characterized by KLF/SP/EGR family engagement, KLF4 promoter opening, and broad induction of AP-1/IEG and MHC class II transcripts, whereas butyrate drove broader chromatin remodeling with TBX21/EOMES/NF-{kappa}B gains and KLF2 promoter closure, and propionate induced an NFY-centered program with preferential commitment to low-mitochondrial-content states. Untargeted metabolomics confirmed valerate uptake and mitochondrial {beta}-oxidation in CAR T-cells, while dietary sodium valerate supplementation in meropenem-treated mice bearing A20 lymphoma significantly reduced tumor burden and extended survival compared with CAR T-cells alone. These findings identify stool valerate as a bedside-deployable biomarker of dysbiosis-imprinted CAR T-cell dysfunction and support ex vivo or dietary valerate supplementation as a clinically tractable strategy to improve CAR-T anti-tumor function in patients with disrupted gut microbiomes.

cancer biology↗

bioRxiv2018

Transcriptome profiling of pathogen-specific CD4 T cells identifies T-cell-intrinsic caspase-1 as an important regulator of Th17 differentiation

One sentence summaryOur study revealed that DCs shape distinct pathogen-specific CD4 T cell transcriptome and from which, we discovered an unexpected role for T-cell-intrinsic caspase-1 in promoting Th17 differentiation.\n\nABSTRACTDendritic cells (DCs) are critical for priming and differentiation of pathogen-specific CD4 T cells. However, to what extent innate cues from DCs dictate transcriptional changes in T cells leading to effector heterogeneity remains elusive. Here we have used an in vitro approach to prime naive CD4 T cells by DCs stimulated with distinct pathogens. We have found that such pathogen-primed CD4 T cells express unique transcriptional profiles dictated by the nature of the priming pathogen. In contrast to cytokine-polarized Th17 cells that display signatures of terminal differentiation, pathogen-primed Th17 cells maintain a high degree of heterogeneity and plasticity. Further analysis identified caspase-1 as one of the genes upregulated only in pathogen-primed Th17 cells but not in cytokine-polarized Th17 cells. T-cell-intrinsic caspase-1, independent of its function in inflammasome, is critical for inducing optimal pathogen-driven Th17 responses. More importantly, T cells lacking caspase-1 fail to induce colitis following transfer into RAG-deficient mice, further demonstrating the importance of caspase-1 for the development of pathogenic Th17 cells in vivo. This study underlines the importance of DC-mediated priming in identifying novel regulators of T cell differentiation.

immunology↗

bioRxiv2018

Down syndrome fibroblasts exhibit diminished autophagic clearance and endosomal dysfunction after serum starvation

Down syndrome (DS) is a genetic disorder caused by trisomy of chromosome 21 (Tri21). This unbalanced karyotype has the ability to produce proteotoxic stress and dysfunction of the proteostasis network (PN), which are mechanistically associated with several comorbidities found in the DS phenotype. Autophagy is the cellular process responsible for bulk protein degradation and its impairment could negatively impact protein quality control. Based on our previous observations of PN disruption in DS, we investigated possible dysfunction of the autophagic machinery in human DS fibroblasts. Both euploid (CTL) and DS fibroblasts induced autophagy successfully through serum starvation (SS), as evidenced by increased LC3-II abundance in CTL and DS. However, DS cells displayed evidence of autophagolysosome (AL) accumulation and impaired clearance of autophagosome cargo, e.g. accumulation of p62 and NBR1. Similar observations were also present in DS cells from multiple differentiation stages, implicating impeded autophagic degradation as a possible early pathologic mechanism in DS. Lysosomal pH and cathepsin B proteolytic activity were found to not differ in CTL and DS fibroblasts after SS, indicating that lysosomal dysfunction did not appear to contribute to unsuccessful autophagic clearance. Based on these results, we hypothesized that possible interference of the endosomal system with autophagy results in autophagosome fusion with endosomal vesicles and negatively impacts degradation. Consistent with this hypothesis, we observed increased abundance of the recycling endosome marker, Rab11, after SS in DS fibroblasts. Further, colocalization of autophagosome markers with resident proteins of early endosomes, late endosomes and recycling endosomes (Rab11) further support our hypothesis. In summary, our work is consistent with impairment of autophagic flux and general PN dysfunction as candidate mechanisms for pathology in DS.

cell biology↗

bioRxiv2018

Integrin-mediated attachment of the blastoderm to the vitelline envelope impacts gastrulation of insects

During gastrulation, physical forces reshape the simple embryonic tissue to form a complex body plan of multicellular organisms1. These forces often cause large-scale asymmetric movements of the embryonic tissue2,3. In many embryos, the tissue undergoing gastrulation movements is surrounded by a rigid protective shell4,5. While it is well recognized that gastrulation movements depend on forces generated by tissue-intrinsic contractility6,7, it is not known if interactions between the tissue and the protective shell provide additional forces that impact gastrulation. Here we show that a particular part of the blastoderm tissue of the red flour beetle Tribolium castaneum tightly adheres in a temporally coordinated manner to the vitelline envelope surrounding the embryo. This attachment generates an additional force that counteracts the tissue-intrinsic contractile forces to create asymmetric tissue movements. Furthermore, this localized attachment is mediated by a specific integrin, and its knock-down leads to a gastrulation phenotype consistent with complete loss of attachment. Moreover, analysis of another integrin in the fruit fly Drosophila melanogaster suggests that gastrulation in this organism also relies on adhesion between the blastoderm and the vitelline. Together, our findings reveal a conserved mechanism whereby the spatiotemporal pattern of tissue adhesion to the vitelline envelope provides controllable counter-forces that shape gastrulation movements in insects.

developmental biology↗

bioRxiv2018

The evolutionary traceability of proteins

Orthologs document the evolution of genes and metabolic capacities encoded in extant and ancient genomes. Orthologous genes that are detected across the full diversity of contemporary life allow reconstructing the gene set of LUCA, the last universal common ancestor. These genes presumably represent the functional repertoire common to - and necessary for - all living organisms. Design of artificial life has the potential to test this. Recently, a minimal gene (MG) set for a self-replicating cell was determined experimentally, and a surprisingly high number of genes have unknown functions and are not represented in LUCA. However, as similarity between orthologs decays with time, it becomes insufficient to infer common ancestry, leaving ancient gene set reconstructions incomplete and distorted to an unknown extent. Here we introduce the evolutionary traceability, together with the software protTrace, that quantifies, for each protein, the evolutionary distance beyond which the sensitivity of the ortholog search becomes limiting. We show that the LUCA set comprises only high-traceable proteins most of which have catalytic functions. We further show that proteins in the MG set lacking orthologs outside bacteria mostly have low traceability, leaving open whether their eukaryotic orthologs have just been overlooked. On the example of REC8, a protein essential for chromosome cohesion, we demonstrate how a traceability-informed adjustment of the search sensitivity identifies hitherto missed orthologs in the fast-evolving microsporidia. Taken together, the evolutionary traceability helps to differentiate between true absence and non-detection of orthologs, and thus improves our understanding about the evolutionary conservation of functional protein networks.

evolutionary biology↗

bioRxiv2017

Content-Aware Image Restoration: Pushing the Limits of Fluorescence Microscopy

Fluorescence microscopy is a key driver of discoveries in the life-sciences, with observable phenomena being limited by the optics of the microscope, the chemistry of the fluorophores, and the maximum photon exposure tolerated by the sample. These limits necessitate trade-offs between imaging speed, spatial resolution, light exposure, and imaging depth. In this work we show how image restoration based on deep learning extends the range of biological phenomena observable by microscopy. On seven concrete examples we demonstrate how microscopy images can be restored even if 60-fold fewer photons are used during acquisition, how near isotropic resolution can be achieved with up to 10-fold under-sampling along the axial direction, and how tubular and granular structures smaller than the diffraction limit can be resolved at 20-times higher frame-rates compared to state-of-the-art methods. All developed image restoration methods are freely available as open source software in Python, FO_SCPLOWIJIC_SCPLOW, and KO_SCPLOWNIMEC_SCPLOW.

bioinformatics↗

bioRxiv2017

Ancestral Reconstruction of Gene Blocks using an Event-Based Method

Complexity is a fundamental attribute of life. Complex systems are made of parts that together perform functions that a single component, or subsets containing individual components, cannot. Examples of complex molecular systems include protein structures such as the F1Fo-ATPase, the ribosome, or the flagellar motor: each one of these structures requires most or all of its components to function properly. Given the ubiquity of complex systems in the biosphere, understanding the evolution of complexity is central to biology. At the molecular level, operons are a classic example of a complex system. An operons genes are co-transcribed under the control of a single promoter to a polycistronic mRNA molecule, and the operons gene products often form molecular complexes or metabolic pathways. With the large number of complete bacterial genomes available, we now have the opportunity to explore the evolution of these complex entities, by identifying possible intermediate states of operons. In this work, we developed a maximum parsimony algorithm to reconstruct ancestral operon states, and show a simple vertical evolution model of how operons may evolve from the individual component genes. We describe several ancestral states that are plausible functional intermediate forms leading to the full operon. We also offer Reconstruction of Ancestral Gene blocks Using Events or ROAGUE as a software tool for those interested in exploring gene block and operon evolution.\n\nThe software accompanying this paper is available under GPLv3 license in: https://github.com/nguyenngochuy91/Ancestral-Blocks-Reconstruction.\n\nAll figures in this paper are available in enlarged downloadable form from:https://github.com/nguyenngochuy91/Ancestral-Blocks-Reconstruction/tree/master/images

evolutionary biology↗

bioRxiv2017

Fast and Accurate Genomic Analyses using Genome Graphs

The human reference genome serves as the foundation for genomics by providing a scaffold for alignment of sequencing reads, but currently only reflects a single consensus haplotype, which impairs read alignment and downstream analysis accuracy. Reference genome structures incorporating known genetic variation have been shown to improve the accuracy of genomic analyses, but have so far remained computationally prohibitive for routine large-scale use. Here we present a graph genome implementation that enables read alignment across 2,800 diploid genomes encompassing 12.6 million SNPs and 4.0 million indels. Our Graph Genome Pipeline requires 6.5 hours to process a 30x coverage WGS sample on a system with 36 CPU cores compared with 11 hours required by the GATK Best Practices pipeline. Using complementary benchmarking experiments based on real and simulated data, we show that using a graph genome reference improves read mapping sensitivity and produces a 0.5% increase in variant calling recall, or about 20,000 additional variants being detected per sample, while variant calling specificity is unaffected. Structural variations (SVs) incorporated into a graph genome can be genotyped accurately under a unified framework. Finally, we show that iterative augmentation of graph genomes yields incremental gains in variant calling accuracy. Our implementation is a significant advance towards fulfilling the promise of graph genomes to radically enhance the scalability and accuracy of genomic analyses.

bioinformatics↗

bioRxiv2017

Less is More: Oligomer extraction and hydrothermal annealing increase PDMS bonding forces for new microfluidics assembly and for biological studies

Key determinants in the emergence of complex cellular morphologies and functions are cues in the micro-environment. Primary among these is the presence of neighboring cells as networks form. Therefore, for high-resolution analysis, it is crucial to develop micro-environments that permit exquisite control of network formation. This is especially true in cell science, tissue engineering, and clinical biology. We introduce a new approach for assembling polydimethylsiloxane (PDMS)-based microfluidic environments that enhances cell network formation and analyses. We report that the combined processes of PDMS solvent-extraction (E-PDMS) and hydrothermal annealing create unique conditions that produce high-strength bonds between E-PDMS and glass - properties not associated with conventional PDMS. Extraction followed by hydrothermal annealing removes unbound oligomers, promotes polymer cross-linking, facilitates covalent bond formation with glass, and retains the highest biocompatibility. Our extraction protocol accelerates oligomer removal from 5 to 2 days. Resulting microfluidic platforms are uniquely suited for cell-network studies owing to high bond strengths, effectively corralling cellular extensions and eliminating harmful oligomers. We demonstrate simple, simultaneous actuation of multiple microfluidic domains for invoking ATP- and glutamate-induced Ca2+ signaling in glial-cell networks. These low-cost, simple E-PMDS modifications and flow manipulations further enable microfluidic technologies for cell-signaling and network studies as well as novel applications.

bioengineering↗

bioRxiv2017

Stability of Commonly Used Haematological Parameters in Samples Stored at 33°C, 22°C and 4°C

AimThis study aimed to investigate the analytical bias and imprecision in haematological parameters induced by storage at 4{degrees}C, 22{degrees}C and 33 {degrees}C.\n\nMethodsThree K2EDTA anticoagulated vials of blood were collected from each of twenty blood donors and stored at 4{degrees}C, 22{degrees}C and 33{degrees}C respectively. Readings from each vial were taken at 0, 4, 6, 12, 24, 48 and 72 hours after collection on the Sysmex XP-100 analyser. The mean and median shift of the parameters relative to the baseline and the coefficient of variation for each time-temperature combination were calculated. The shift was compared to the maximum acceptable bias.\n\nResultsHaemoglobin, Red Blood Cell Count, White Blood Cell Count, Mean Corpuscular Haemoglobin were stable for at least twenty four hours at 33{degrees}C. Haematocrit, Mean Corpuscular Volume and Platelet Counts were stable for less than four hours at 33{degrees}C. All the above parameters were stable for longer at 22{degrees}C and 4{degrees}C. The three-part differential count showed instability within four hours at 33 {degrees}C.\n\nConclusionsStrict pre-analytical control is needed at 33{degrees}C or above due to the marked instability of most parameters. However, Haemoglobin, Red Blood Cell Count, White Blood Cell Count and Mean Corpuscular Haemoglobin remain relatively stable even at 33{degrees}C.\n\nKey MessageHaematology samples exposed to temperatures of 33{degrees}C or above show rapid change in MCV, HCT,MCHC, RDW, Platelet Counts and three-part differential counts. Settings where prolonged exposure to these temperatures cannot be avoided should rely on the more stable parameters of Haemoglobin, RBC Counts, MCH and WBC Counts.

pathology↗

bioRxiv2017

Tracking electron uptake from a cathode into Shewanella cells: implications for generating maintenance energy from solid substrates.

While typically investigated as a microorganism capable of extracellular electron transfer to minerals or anodes, Shewanella oneidensis MR-1 can also facilitate electron flow from a cathode to terminal electron acceptors such as fumarate or oxygen, thereby providing a model systems for a process that has significant environmental and technological implications. This work demonstrates that cathodic electrons enter the electron transport chain of S. oneidensis when oxygen is used as the terminal electron acceptor. The effect of electron transport chain inhibitors suggested that a proton gradient is generated during cathode-oxidation, consistent with the higher cellular ATP levels measured in cathode-respiring cells relative to controls. Cathode oxidation also correlated with an increase in the cellular redox (NADH/FMNH2) pool using a bioluminescent assay. Using a proton uncoupler, generation of NADH/FMNH2 under cathodic conditions was linked to reverse electron flow mediated by the proton pumping NADH oxidase Complex I. A decrease in cathodic electron uptake was observed in various mutant strains including those lacking the extracellular electron transfer components necessary for anodic current generation. While no cell growth was observed under these conditions, here we show that cathode oxidation is linked to cellular energy conservation, resulting in a quantifiable reduction in cellular decay rate. This work highlights a potential mechanism for cell survival and/or persistence in environments where growth and division are severely limited.

microbiology↗

bioRxiv2017

RNA Gelation in Repeat Expansion Disorders

Expansions of short nucleotide repeats in the protein coding and non-coding regions of >30 genes produce a variety of neurological and neuromuscular disorders including Huntingtons disease (CAG repeats), muscular dystrophy (CTG repeats) and amyotrophic lateral sclerosis (GGGGCC repeats) [1-3]. Expression of expanded repeats alone is sufficient to recapitulate disease pathology in animal models [4-6]. Repeat-containing transcripts accumulate in the nucleus as aberrant \"RNA foci\" [7-10] and sequester numerous RNA binding proteins [11,12], leading to a disruption of cellular homeostasis [13,14]. Interestingly, RNA foci, as well as the disease symptoms, only manifest at a critical threshold of nucleotide repeats: >30 for CAG/CTG expansions [1] and >7 for the GGGGCC expansion [15]. However, the reason for this characteristic threshold, as well as the molecular mechanism of foci formation, remain unresolved [16]. Here, we show that nucleotide repeat expansions in RNA create templates for multivalent Watson-Crick (CAG/CUG expansions) or Hoogsteen (GGGGCC expansion) base-pairing. These multivalent interactions cause purified RNAs containing repeat expansions to undergo a sol-gel transition and form micron-sized clusters. Reflecting an increase in the valency for intermolecular hybridization, the gelation of purified RNA only occurs above a critical number of trinucleotide or hexanucleotide repeats. These thresholds for in vitro RNA gelation are similar to those associated with manifestation of disease. By visualizing RNA in live cells, we show that nuclear foci form as a result of phase separation of the repeat-containing RNA and that these foci can be dissolved by agents that disrupt RNA gelation in vitro. Analogous to protein aggregation disorders, our results suggest that the sequence-specific gelation of RNAs could be a contributing factor to neurological disease.

biophysics↗