Biology subjects

Bansal, A.

Publications and source records attributed to Bansal, A..

At least 19 recordsLinked to original sources

bioRxiv2026

A CTCF-LDHB axis converts nuclear lactate availability into an H3K18 acylation switch that directs alternative splicing in hypoxic breast cancer

Lactate accumulating in hypoxic tumours lactylates histones, coupling metabolic state to chromatin. Why it differs between tumours of comparable glycolytic rate has remained unresolved. Here we show that nuclear, rather than total intracellular, lactate availability determines histone H3K18 lactylation in breast cancer, and that CCCTC-binding factor (CTCF) and lactate dehydrogenase B (LDHB) jointly set it. CTCF sustains glycolytic gene expression under hypoxia and drives LDHB in triple-negative breast cancer (TNBC), whereas a CTCF-ER complex represses LDHB in non-TNBC. LDHB consumes lactate favouring H3K18 acetylation; where LDHB is low, CTCF-dependent MCT1 delivers lactate into the nucleus and sustains lactylation. At a CTCF site downstream of TACC3 exon 3, an invariant polymerase II pause couples kinetic and adaptor control of splicing, with the mark deciding the outcome: H3K18la recruits SMARCA4 and hnRNPA1 to skip the exon, generating an isoform that drives EMT, whereas H3K18ac recruits TRIM33 and SRSF10 to include it, generating one that confers stemness. Inhibiting LDHB restores exon skipping and suppresses tumour growth in vivo. Nuclear lactate availability therefore acts as a metabolic switch that converts hypoxic stress into subtype-specific splicing decisions and identifies LDHB as a tractable target in TNBC.

cancer biology↗

bioRxiv2026

A propensity to hyperinsulinaemia underpins diet-induced diabetes in NODk mice

Aims/hypothesisHeterogeneity in the pathophysiology of type 2 diabetes is increasingly being realised. The currently available rodent models of type 2 diabetes all have limitations and do not accurately reflect all human type 2 diabetes subtypes. NOD.BR-H2k /Wicker mice (NODk), derived from the non-obese diabetic (NOD) mouse, are type 1 diabetes resistant. However, transgene induced beta-cell stress in male NODk mice induces hyperinsulinaemia followed by diabetes. Here we have investigated the propensity of NODk mice to develop a Western-diet (WD) induced hyperinsulinaemic subtype of type 2 diabetes. Comparator mouse strains used were BALB/c and B10.BR-H2k /SgSnJ mice (B10k). MethodsIn the longer-term studies (14-24 weeks), NODk, B10k and BALB/c mice were randomised to receive Chow or WD from 4 weeks of age, followed by serial measurement of body weight and fed-state blood glucose. IPGTT and IPITT tests were conducted at 13 weeks of age. Blood and pancreas were harvested for further analyses at 14 and 24 weeks of age, or sooner if diabetes developed (blood glucose concentrations [≥]20 mmol/l on two consecutive days). In the acute studies, metabolic characteristics of the three strains at 8 weeks of age, continued on Chow or after a 5-day WD challenge (WDC) were assessed, along with harvesting pancreas on day 5 for ex vivo islet insulin secretion, electron microscopy, and bulk islet transcriptomics analyses. ResultsMale WD-fed NODk mice became markedly hyperinsulinaemic, gained excess weight and developed a severe type 2 diabetes phenotype. Emergence of diabetes was associated with islet endocrine cell apoptosis and loss of beta-cell mass, without evidence of insulitis. Insulin resistance on IPITT testing, however, was not evident in Chow-fed NODk mice. In contrast, male B10k mice already had poor glucose tolerance on Chow diet and, despite having a hypoinsulinaemic phenotype, were resistant to WD-induced diabetes. BALB/c mice developed very mild glucose intolerance and hyperinsulinaemia in response to the WD. Female NODk mice were diabetes resistant. At 8 weeks of age, male Chow-fed NODk mice were mildly hyperinsulinaemic despite relative hypoglycaemia compared to the other strains. The acute 5-day WDC markedly increased hyperinsulinaemia in NODk mice. Transcriptomics analyses identified robust strain-specific differences, including altered islet cell differentiation, energy metabolism, endoplasmic reticulum to golgi vesicle transport and insulin processing. Conclusions/interpretationNODk mice, which exhibit mild hyperinsulinaemic hypoglycaemia on Chow diet and rapidly develop marked hyperinsulinaemia on WD, are type 2 diabetes prone. In contrast, B10k mice have poor glucose tolerance on Chow diet and no or limited capacity to increase insulinaemia in response to WD, are diabetes resistant. These findings support the hypothesis that hyperinsulinaemia is upstream to insulin resistance in the pathogenesis of severe insulin resistant subset of type 2 diabetes for which the WD-fed NODk mouse is a suitable new mouse model. Research in ContextO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIWhich of insulin hypersecretion and insulin resistance are upstream in the pathogenesis of the severe insulin resistant subtype of type 2 diabetes continues to be debated C_LIO_LIRodent models of type 2 diabetes do not accurately reflect all human subtypes of type 2 diabetes C_LIO_LINODk mice, derived from the non-obese diabetic (NOD) mouse, are type 1 diabetes resistant, but with transgene induction of islet beta-cell stress develop hyperinsulinaemia, followed by type 2 diabetes C_LI What is the key question?O_LICould Western-diet fed NODk mice be developed as a model of severe insulin resistant type 2 diabetes and shed light on its upstream pathogenesis? C_LI What are the new findings?O_LIMale NODk mice tend to hyperinsulinaemic hypoglycaemia on Chow diet, rapidly develop marked hyperinsulinaemia on Western-diet feeding, and then develop type 2 diabetes C_LIO_LIMale B10k mice (one of two comparator strains (B10k and BALB/c)) have poor glucose tolerance on Chow diet, limited capacity to increase insulinaemia in response to Western-diet feeding, but are resistant to develop Western-diet induced type 2 diabetes C_LIO_LIIsolated islet findings show strain differences that favour intrinsic hyper-responsiveness and hypo-responsiveness of islet beta-cells of NODk and B10k mice, underpinning their respective metabolic phenotypes C_LI How might this impact on clinical practice in the foreseeable future? O_LIThe findings are in support of the insulin hypersecretion hypothesis for severe insulin resistant type 2 diabetes, such that therapies to limit islet beta-cell hyperresponsiveness to prevent and treat this subtype of diabetes warrant investigation C_LI

physiology↗

bioRxiv2026

The Plasmodium falciparum Raf Kinase Inhibitor Protein is Essential for Red Blood Cell Invasion and Can Be Functionally Substituted by Host RKIP

Raf kinase inhibitor protein (RKIP) plays a key role in regulating critical signaling pathways in higher eukaryotes. We previously demonstrated that Plasmodium falciparum RKIP (PfRKIP) modulates the activity of PfCDPK1, a key regulator of red blood cell invasion. Here, we have used a pharmacological approach to investigate the function of PfRKIP. Locostatin, a mammalian RKIP inhibitor, shows a dose-dependent decrease in RBC invasion. Mechanistically, locostatin increases the interaction between PfRKIP and PfCDPK1, thereby sequestering PfCDPK1 in a complex preventing substrate phosphorylation. Surprisingly, PfRKIP could be knocked out from the parasite without any perceptible growth defect. Interestingly, the PfRKIP-deficient parasites show an increase in the import of host RKIP. Like the parasite RKIP, the host RKIP interacts with PfCDPK1 and is associated with high-molecular-weight complexes comprising PfCDPK1 suggesting functional complementation by the host RKIP in the PfRKIP null parasites. As expected, locostatin shows similar inhibitory effect on the knock-out parasites as the WT. Our study reveals a novel host-parasite interaction wherein the parasite co-opts host proteins to maintain critical signaling pathways. Targeting the PfRKIP signaling axis, along with the host proteins, represents a potential strategy for anti-malarial drug development that is conceptually less prone to parasite-driven resistance development.

microbiology↗

bioRxiv2026

Enriched-GF: A Reproducible High-Yield Autologous Blood-Derived Growth Factor Method for Regenerative Medicine

BackgroundPlatelet-derived Growth factors play key roles in tissue repair and regeneration, yet conventional platelet-rich plasma (PRP) formulations release these mediators inconsistently in vivo due to variability in platelet yield and activation dynamics. To overcome this limitation, direct administration of concentrated platelet-derived growth factor preparations has gained interest, though current manufacturing approaches for human platelet lysate (hPL), growth factor concentrates (GFC), and conditioned serum remain constrained by batch variability, incomplete platelet degranulation, and reliance on anticoagulants. Here, we examine alternative platelet activation workflows to establish a standardized, efficient, and reproducible method for high-yield growth factor recovery suitable for translational and clinical applications. MethodsNine GFC production protocols were compared, employing different combinations of freeze-thaw (FT) cycling, glass bead (GB) agitation, calcium (Ca2) activation, and a novel Enriched Growth Factor (Enriched-GF) method. The objective was to identify a protocol capable of maximizing growth factor yield within a three-hour workflow. Optimal Ca2 concentrations and GB conditions were determined from prior optimization studies and integrated into the Enriched-GF processing scheme. Platelet concentrates (n = 10 per protocol) were processed under each condition, and growth factor levels were quantified using ELISA. ResultsGrowth factor yields differed significantly across protocols. The greatest and most consistent increases in growth factor release were observed with the Enriched-GF method combining GB activation, FT cycling, and Ca2 stimulation. This approach resulted in markedly elevated concentrations of key regenerative mediators, including enhanced EGF release, a 4.5-fold increase in PDGF, maximal TGF-{beta} liberation, and a four-fold increase in FGF2 relative to conventional platelet lysate or conditioned serum preparations. These results were reproducible across independent donor pools, demonstrating robustness and batch-to-batch consistency. ConclusionWe describe a rapid and reproducible method for producing highly concentrated platelet-derived growth factors using a combined GB-FT-Ca2 activation strategy. The Enriched-GF protocol consistently outperformed existing platelet lysate, conditioned serum, and conventional GFC preparation methods, yielding a standardized product with enhanced growth factor content. This Enriched-GF approach offers a clinically practicable solution for applications in regenerative medicine requiring reliable and high-yield growth factor delivery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/712883v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@ac60c3org.highwire.dtl.DTLVardef@104d8eborg.highwire.dtl.DTLVardef@13d5afcorg.highwire.dtl.DTLVardef@120645c_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic overview of platelet concentrate preparation from whole blood and the generation of different platelet lysates and growth factor-enriched serum using freeze-thaw, calcium gluconate, and glass bead activation methods.

biochemistry↗

bioRxiv2026

Oncogenic chromodomain mutations allosterically impair TIP60 acetyltransfearse function preventing activation of DNA repair genes under genotoxic stress

TIP60 is a tumor suppressor with histone acetyltransferase activity that regulates chromatin accessibility in diverse processes including DNA repair, apoptosis, mitosis, transcription, and autophagy. Structurally TIP60 contains a C-terminal MYST domain that mediates its HAT activity, while the N-terminal chromodomain are conserved modules that facilitate its association with chromatin by recognizing histone modifications. Mutations within the chromodomain have been implicated in various cancers, yet their functional consequences remain poorly understood, particularly with respect to TIP60s role in maintaining genomic integrity. Here, we uncover a novel allosteric mechanism whereby cancer-associated chromodomain mutations impair TIP60s catalytic activity without disrupting its chromatin binding, underscoring critical interdomain communication between the chromodomain and the MYST domain. Through structural modelling and molecular dynamics simulations, we identified two missense mutations (R53H and R62W) in TIP60s chromodomain that not only altered TIP60s conformation but also destabilized its trimeric assembly, thereby impairing acetyl-CoA docking. Importantly, we found that TIP60 engages acetyl-CoA exclusively in its trimeric state, and the R62W mutation perturbs the trimeric interface, thereby altering the docking sites for acetyl-CoA. Consistent with these structural changes, biochemical assays revealed that chromodomain mutant TIP60 variants, while retaining chromatin loading, exhibited markedly reduced autoacetylation and histone acetyltransferase activity. Moreover, these mutants failed to activate the p21 gene in response to DNA damage, thereby predisposing the genome to the accumulation of mutations and leaving cells unable to arrest the cell cycle for repair of genomic lesions. Together, our findings establish that distal chromodomain mutations allosterically destabilize TIP60 oligomerization, impair acetyl-CoA utilization, and compromise DNA damage responses. The mechanism establishes a link between chromodomain mutations and genomic instability, shedding light on how reader domain alterations may underlie cancer progression.

molecular biology↗

bioRxiv2025

Calcium Dependent Protein Kinase 3 is not essential for the asexual replication of Plasmodium falciparum

Calcium dependent protein kinases are essential at various stages of malaria parasite development within the vertebrate host and the mosquitoes. In the rodent malaria parasite, Plasmodium berghei, CDPK3 is essential for the sexual stage development of the parasite within mosquitoes. However, the function of CDPK3 in the most lethal human malaria parasite, P. falciparum remains unexplored. Moreover, the calcium dependent kinase activity of CDPK3 has not been experimentally tested. With the aim to biochemically characterize CDPK3 and perform its functional evaluation in the asexual replication of P. falciparum, here we have expressed the full-length CDPK3 in E. coli and demonstrated that it is a bona fide calcium dependent kinase using a luminescence-based assay and a semi-synthetic epitope approach. Complete knock-out of cdpk3 using CRISPR/Cas9 did not show any defect in the asexual replication of the parasite and the knockout parasites look morphologically similar to the wild type. Our study suggests that cdpk3 is not essential for the asexual replication of the parasite under in vitro culture conditions. Perhaps CDPK3 is required for the sexual development of P. falciparum like its ortholog in the rodent malaria parasite. CDPK3 may be a good target for the development of transmission blocking drugs.

microbiology↗

bioRxiv2025

Diverse ancestral myosin motors generate and segregate distinct types of nanocluster-rich domains at the plasma membrane

Molecular organization of the plasma membrane (PM) at nano and micron scales is critical for its function in all living cells. This emerges not only from the self-assembly of lipids and proteins but also from active forces originating in the underlying cytoskeletal cortex. These forces drive membrane molecules into non-equilibrium steady state patterns such as nanoclusters. However, the molecular agents connecting membrane organization with cytoskeletal dynamics and stresses have remained unknown. Here we show that two classes of ubiquitous ancestral non-muscle myosins are deployed for the organization of different types of membrane components. Inner-leaflet localized Class I myosins link outer-leaflet GPI-anchored molecules to juxta-membrane actin-filaments, whereas the more cortically-localized Class II myosins operate on transmembrane proteins endowed with actin-binding capacity. Consistent with an active Flory Huggins theory for phase separation, these observations show that the distinct motor-driven membrane molecules generate spatially segregated mesoscale domains, enriched in nanoclusters derived from different myosin classes. Moreover, chemically reversible post-translational modifications such as palmitoylation enable concatenation of these domains by enhancing affinity of the membrane domain constituents for each other. We anticipate that the segregation potential of the ATP-fueled cell membrane is made available for the crucial purpose of modulating information transduction because it can be regulated in space and time during the construction of signaling cascades, underpinning functional plasma membrane organization.

cell biology↗

bioRxiv2025

SMTrackR: a R/Bioconductor package for mapping protein binding at individual DNA molecules

Single-molecule assays like NOMe-seq, dSMF and Nanopore are superior to DNase-seq and ATAC-seq as they do not nibble down DNA. Thus, they enable quantification of all three i.e., protein-free, Transcription Factor-bound and histone-complex-bound states. But a user-friendly tool to visualize and quantify such states is lacking. Here, we present, SMTrackR, a Bioconductor package to visualize protein-DNA binding states on individual sequenced DNA molecules. SMTrackR queries the single-molecule footprint database we built and hosted at Galaxy Server. It comprises of BigBed files generated from NOMe-seq, dSMF, and Nanopore (SMAC-seq) datasets. SMTrackR exploits UCSC REST API to query a BigBed file and plot footprint heatmap categorized in different binding states as well as report their occupancies. Additionally, this package generates a Gviz-enabled script to visualize these single-molecules on gene tracks. Availability and implementationThe SMTrackR tool is implemented the statistical programming language R and is available at the GitHub developer site, https://github.com/satyanarayan-rao/SMTrackR. The tool is also available in a web version https://smtrackr.iitr.ac.in. A function is provided to use local BigBed file for users who wish to use unpublished data. A fully automated pipeline to generate such BigBed files is available at https://github.com/satyanarayan-rao/SMF_for_SMThub and https://github.com/satyanarayan-rao/dSMF_for_SMThub.

bioinformatics↗

bioRxiv2025

How the characteristics of a virtual environment affects the perception of travel distance through it

Although simulated self-motion through virtual environments has been widely used to investigate perceptual odometry, the characteristics of the virtual environments used, and the reported results have varied greatly. Here, we systematically vary the characteristics of the environment through which observers are moved in order to explore the effect of (1) the structure of an environment including the presence and texture of a ground surface, (2) the naturalism and scale of an environment, (3) colour, and (4) the density of a starfield and how it might affect perceived travel distance. In all four experiments, participants were visually moved forwards through a virtual environment and perceived travel distance was estimated by either (1) stopping at the location of a previously seen target (the Move-To-Target Task) or (2) adjusting the position of a target to indicate a previously travelled distance (the Adjust-Target Task). Data were analyzed in terms of gain (perceived travel distance/actual travel distance). Results show no significant differences that depended on the structure of an environment or on the presence or absence of a ground surface (Experiment 1), or on the naturalism of the environment (Experiment 2), or on whether the environment was in colour or in black and white (Experiment 3). However, there was a small effect of the texture of the ground surface and of the scale of the environment. In Experiment 4, we show that there may be a very low ceiling effect in the density of a starfield needed to accurately estimate travel distance. Together these experiments have implications for the design of real and virtual environments where perceived motion is important and will enable us to further predict our perception of moving through an environment.

neuroscience↗

bioRxiv2025

Inflammatory cytokines promote interferon regulatory factor (IRF) transcriptional activity in human pulmonary epithelial cells through the induction of IRF1 by nuclear factor-κB

Transcription factors of the interferon regulatory factor (IRF) family play key roles during viral and bacterial infections. However, IRF regulation by inflammatory cytokines, including interleukin (IL)-1{beta} and tumor necrosis factor (TNF) , remains underexplored. Since airway epithelial cells (AECs) modulate lung inflammation, IRF expression was characterized in pulmonary A549 and bronchial BEAS-2B epithelial cells, plus primary human AECs grown in submersion or highly differentiated air-liquid interface culture. Whereas most IRF mRNAs were expressed across models, IRF4 and IRF8 showed consistently low expression, and IRF6 was only expressed highly in primary cells. IRF3 and IRF9 were highly expressed in each model, and while protein expression was confirmed in A549 cells, this remained primarily cytoplasmic post-IL-1{beta} treatment. IRF2 was constitutively nuclear in A549 cells and showed moderate/high mRNA expression in each model. IRF1 mRNA was induced by IL-1{beta}-in primary and A549 cells, where IL-1{beta} and TNF dramatically increased IRF1 protein expression. TNF also increased IRF1 protein in BEAS-2B cells. In A549 cells, an IRF reporter system combined with RNA silencing did not support transcriptional roles for IRF2 or IRF3 but suggested a modest role for IRF9 following IL-1{beta} stimulation. IL-1{beta}-induced IRF1 localized to the nucleus of A549 cells revealing a major transcriptional role. IRF1 expression induced by IL-1{beta}/TNF required NF-{kappa}B and four novel 5' enhancer regions to IRF1 bound the NF-{kappa}B subunit, p65. These regions drove IL-1{beta}/TNF-inducible reporter activity via consensus NF-{kappa}B motifs. Three such regions recruited RNA polymerase-2 and were flanked by the active chromatin mark, histone 3 lysine 27 acetylation which, given the high levels of genetic conservation, support involvement in IRF1 transcription. Finally, IRF1 expression, transcription rate, and enhancer activity induced by IL-1{beta}/TNF were relatively unaffected by glucocorticoid co-treatment. IRF1-dependent gene expression could therefore be insensitive to glucocorticoid and may contribute to glucocorticoid-resistant diseases such as severe asthma.

molecular biology↗

bioRxiv2023

Plasmodium falciparum Raf kinase inhibitor is a lipid binding protein that interacts with CDPK1 and regulates its activity in asexual blood stage.

Raf Kinase Inhibitor Protein (RKIP) is an important regulator of MAPK signaling pathway in multicellular eukaryotes. Plasmodium falciparum RKIP (PfRKIP) is a putative phosphatidylethanolamine binding protein (PEBP) that shares limited similarity with Homo sapiens RKIP (HsRKIP). Interestingly, critical components of MAPK pathway are not expressed in malaria parasite and the physiological function of PfRKIP remains unknown. PfRKIP is expressed throughout the asexual schizogony with maximum expression in late schizonts. Interestingly, PfRKIP and HsRKIP show pH dependent differential interaction profiles with various lipids. At physiological pH, PfRKIP show interaction with PE and lipids containing phosphorylated phosphatidylinositol group; however, HsRKIP show no interaction under the same conditions. Mutation of conserved residues in the PEBP domain of PfRKIP decreases its interaction with PI(3)P. Furthermore, our results suggest that PfRKIP leads to increase in the autophosphorylation of PfCDPK1 that leads to transphosphorylation of substrates by PfCDPK1. Using various in vitro and in vivo experiments we have demonstrated the interaction of PfRKIP with PfCDPK1 and have also identified key residues in PfRKIP that play important role in this interaction. Interestingly, locostatin, a specific inhibitor of mammalian RKIP increased the interaction of PfRKIP with PfCDPK1 that perhaps leads to the sequestration of PfCDPK1 in a heterodimeric complex. Importantly, treatment of malaria parasite with locostatin shows dose dependent inhibition of parasite growth. This study suggests that specific inhibitors that modify PfRKIP leading to increase in its interaction with PfCDPK1 may be designed and explored as novel anti-malarial compounds to inhibit malaria parasite growth.

biochemistry↗

bioRxiv2022

Electroencephalogram Data Collection for Student Engagement Analysis with Audio-Visual Content

Recognizing and monitoring students attention during learning is crucial to successful knowledge acquisition since it influences cognitive function. As a result, gaining a precise picture of a learners mental state may enable interactive learning systems to alter tutoring content, devise effective help tactics, and improve learning outcomes. In computer-based learning environments, keeping track of students mental states is vital. Investigating the feasibility of utilizing active learning in enhancing student engagement index when exposed to various visual stimuli is the genesis of this work. The research includes collecting EEG data from 20 participants (ten males, ten females) while resting and being subjected to various virtual infotainment/educational content. The EEG data were collected using the Allengers Neuro PLOT, a 40-channel wet electrode system. The work includes raw and pre-processed EEG data under quiescent and audio-visual continuous cues. The recorded data is accommodated by a sophisticated EEG data pre-processing pipeline and will be available to the research community for usage. Specifications Table O_TBL View this table: org.highwire.dtl.DTLVardef@340852org.highwire.dtl.DTLVardef@e5c51org.highwire.dtl.DTLVardef@cefb1borg.highwire.dtl.DTLVardef@c7d208org.highwire.dtl.DTLVardef@ae4824_HPS_FORMAT_FIGEXP M_TBL C_TBL

neuroscience↗

bioRxiv2021

Active Segregation Dynamics in the Living Cell

In this paper, we bring together our efforts in identifying and understanding nonequilibrium phase segregation driven by active processes in the living cell, with special focus on the segregation of cell membrane components driven by active contractile stresses arising from cortical actomyosin. This also has implications for active segregation dynamics in membraneless regions within the cytoplasm and nucleus (3d). We formulate an active version of the Flory-Huggins theory that incorporates a contribution from fluctuating active stresses. Apart from knitting together some of our past theoretical work in a comprehensive narrative, we highlight some new results, and establish a correspondence with recent studies on Active Model B/B+. We point to the many unusual aspects of the dynamics of active phase segregation, such as (i) anomalous growth dynamics, (ii) coarsening accompanied by propulsion and coalescence of domains that exhibit nonreciprocal effects, (iii) segregation into mesoscale domains, (iv) emergence of a nonequilibrium phase segregated steady state characterised by strong macroscopic fluctuations (fluctuation dominated phase ordering (FDPO)), and (v) mesoscale segregation even above the equilibrium Tc. Apart from its implications for actively driven segregation of binary fluids, these ideas are at the heart of an Active Emulsion description of the lateral organisation of molecules on the plasma membrane of living cells, whose full molecular elaboration appears elsewhere.

biophysics↗

bioRxiv2021

Dissection of function and recognition mechanism of M. tuberculosis ESX-1 secreted virulence factor EspC

Mycobacterium tuberculosis uses the ESAT-6 system-1/type VII (ESX-1) system for secretion of virulence proteins into the host cell, however the mechanism of virulence proteins secretion, molecular components and regulation of ESX-1 system are only partly understood. In the current study, we have analyzed the biological function and recognition mechanism between ESX-1 virulence EspC and EccA1 ATPase proteins. The EspC enters into A549 human lung carcinoma cells and exhibited cytotoxicity, as observed in MTT Assay. To understand the recognition mechanism between EspC and EccA1 ATPase, the EspC and EccA1 mutants were generated based on EspC~EccA1 interactions, as observed in molecular modeling. Binding analysis shows that EspC export arm interacts specifically to the {beta}-hairpin insertion motif of the TPR domain of EccA1 ATPase. Mutations in these epitopes lead to significant decrease/or abolish the binding between EspC and EccA1 ATPase. Our study provides insight into biological function and recognition mechanism between EspC and EccA1 ATPase, which can be used as target to prevent EspC secretion/ or in general virulence factor secretion by mycobacterial ESX-1 system.

biophysics↗

bioRxiv2020

Cryo-EM reveals structural breaks in a patient-derived amyloid fibril from systemic AL amyloidosis

Systemic AL amyloidosis is a debilitating and potentially fatal disease that arises from the misfolding and fibrillation of immunoglobulin light chains (LCs). The disease is patient-specific with essentially each patient possessing a unique LC sequence. In this study, we present the first ex vivo fibril structures of a {lambda}3 LC. The fibrils were extracted from the explanted heart of a patient (FOR005) and consist of 115 residues, mainly from the LC variable domain. The fibril structures imply that a 180{degrees} rotation around the disulfide bond and a major unfolding step are necessary for fibrils to form. The two fibril structures show highly similar fibril protein folds, differing in only a 12-residue segment. Remarkably, the two structures do not represent separate fibril morphologies, as they can co-exist at different z-axial positions within the same fibril. Our data imply the presence of structural breaks at the interface of the two structural forms.

molecular biology↗

bioRxiv2020

Remarkable recent changes in genetic diversity of the avirulence gene AvrStb6 in global populations of the wheat pathogen Zymoseptoria tritici

Septoria tritici blotch (STB), caused by the fungus Zymoseptoria tritici, is one of the most economically important diseases of wheat. Recently, both factors of a gene-for-gene interaction between Z. tritici and wheat, the wheat receptor-like kinase Stb6 and the Z. tritici secreted effector protein AvrStb6, have been identified. Previous analyses revealed a high diversity of AvrStb6 alleles present in historic Z. tritici isolate collections, with up to ~ 18% of analysed isolates possessing the avirulence isoform of AvrStb6 identical to that originally identified in the reference isolate IPO323. With Stb6 present in many commercial wheat cultivars globally, we aimed to assess potential changes in AvrStb6 genetic diversity and the incidence of alleles allowing evasion of Stb6-mediated resistance in more recent Z. tritici populations. Here we show, using targeted re-sequencing of AvrStb6, that this gene is universally present in field isolates sampled from major wheat-growing regions of the world between 2013-2017. However, in contrast to the data from studies of historic isolates, our study revealed a complete absence of the originally described avirulence isoform of AvrStb6 amongst modern Z. tritici isolates. Moreover, a remarkably small number of alleles, each encoding AvrStb6 protein isoforms conditioning virulence on Stb6-containing wheat, were found to predominate among modern Z. tritici isolates. A single virulence isoform of AvrStb6 was found to be particularly abundant throughout the global population. These findings indicate that, despite the ability of Z. tritici to sexually reproduce on resistant hosts, AvrStb6 avirulence alleles tend to be eliminated in subsequent populations.

microbiology↗

bioRxiv2020

ATM-mediated DNA damage response in macrophages primes phagocytosis and immune checkpoint regulation

Genotoxin-based cancer therapies trigger a DNA damage response (DDR) to eliminate cancer cells but similarly exert profound alterations to the immune cell compartment. Macrophages in the tumor microenvironment are important and multifaceted components in actively sustaining tumor progression but also in clearance of tumor cells and play an important role in the outcome of chemotherapy. Here, we report that post neo-adjuvant chemotherapy macrophages in tumor samples display elevated expression of the immune checkpoint PD-L1. We determined that chemotherapy or direct application of DNA damage to macrophages in vitro triggers a specific MDDR polarization phenotype characterized by increased phagocytic activity, elevated PD-L1 expression, and induction of endotoxin tolerance. Phospho-proteomics analysis revealed epigenetic alterations and ATM-dependent induction of senescence in macrophages upon DNA damage. We propose that ATM-induced senescence and PD-L1 immune checkpoint induction mediate a specific MDDR macrophage polarization that might contribute to chemotherapy responses. Furthermore, our results clarify the functional role of tumor associated macrophages in the context of DNA damage and combination therapies including checkpoint inhibition.

immunology↗

bioRxiv2019

Ectopic hepatocyte transplantation cures the pig model of tyrosinemia

The effectiveness of cell-based therapies to treat liver failure is limited by the diseased liver environment. Herein we provide preclinical proof-of-concept for the treatment of liver failure through hepatocyte transplantation into lymph nodes in a large-animal model of hereditary tyrosinemia type 1 (HT1), a metabolic liver disease caused by deficiency of fumarylacetoacetate hydrolase (FAH) enzyme. FAH-deficient pigs received autologous hepatocyte transplantation into mesenteric lymph nodes after ex vivo transduction with a lentiviral vector carrying the pig Fah gene. Hepatocytes showed early (6 hour) and durable (8 month) engraftment in lymph nodes, with reproduction of vascular and hepatic microarchitecture. Subsequently, hepatocytes migrated to and repopulated the native diseased liver. The corrected cells generated enough liver mass to clinically ameliorate disease as early as 97 days post-transplantation, with complete normalization of tyrosine levels and liver function tests. Integration site analysis defined the corrected hepatocytes in the liver as a subpopulation of hepatocytes in the lymph nodes, indicating that the lymph nodes served as a source for healthy hepatocytes to repopulate a diseased liver. Ectopic transplantation of hepatocytes cures the pig model of HT1 and presents a promising approach to the treatment of liver disease in patients with pre-existing liver damage and fibrosis.\n\nOne Sentence SummaryTransplantation of corrected hepatocytes in mesenteric lymph nodes can cure fatal metabolic liver disease by providing organized liver tissue and by repopulating the diseased liver in the pig tyrosinemia model.

molecular biology↗