Biology subjects

Mandal, P.

Publications and source records attributed to Mandal, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Gut bacterium of Leishmania donovani-non-transmitting sand fly induces IL-23~Th17 axis to protect hosts from drug-resistant visceral leishmaniasis

Background: Visceral leishmaniasis (VL) is caused by Leishmania donovani and differentiates under the influence of biotope-specific gut bacteria in the Leishmania donovani-transmitting sand fly vector, Phlebotomus argentipes, which coexists with the Leishmania donovani-non-transmitting Sergentomyia species in the Indian subcontinent. Hypothesis: Sergentomyia are primarily herpetophilic. Interestingly, we identified a specific population of anthropophilic Sergentomyia babu babu, a proven naturally non-competent vector for transmitting Leishmania donovani, with high abundance and human-bite incidences in VL-non-endemic regions, compared with VL-endemic regions of India. Thus, the possibility of egesting gut bacteria to humans from VL-non-transmitting Sergentomyia and inducing downstream immune cascades remains undeniable. Innovation: We characterized gut bacteria from anthropophilic Sergentomyia babu babu in both VL-endemic and VL-non-endemic regions of India and found that a heat-inactivated formulation of Heyndrickxia sporothermodurans [NCBI Sequence Read Archive (SRA): PRJNA1403236], isolated from anthropophilic Sergentomyia babu babu from a VL-non-endemic region, prevented and cured L. donovani infection by inducing IL-23~Th17. More importantly, it also prevented the initial parasite establishment by inducing phagocytosis, ROS, and TNF- in circulatory Ly6G-CD11b+Ly6Chi monocytes. To the best of our knowledge, this is the first report confirming that the gut bacteria of VL-non-transmitting sand flies are critical and need to be investigated in depth for developing therapeutics and/or prophylactics against visceral leishmaniasis (VL), and suggests that possible bite-transfer of gut bacteria from disease-non-transmitting vectors to hosts may restrict disease pathogenicity. Further clinical trials are needed to warrant (Indian patent: 202511041013, April 28, 2025).

immunology↗

bioRxiv2026

Gene therapy with doxycycline-controlled expression of human Kv1.1 reduces neuronal excitability and increases sociability of Scn2a-deficient mice

Genetic loss-of-function (LoF) variants in SCN2A, a gene encoding the voltage-gated sodium channel Nav1.2, have been identified as one of the foremost monogenic causes of autism spectrum disorder (ASD). ASD encompasses a broad spectrum of behavioral phenotypes, with impaired sociability as a core characteristic. We have established Scn2a-deficient mice (Scn2agt/gt) to model Scn2a-related ASD and found that this model recapitulates social impairment, exhibiting severe social deficits. Scn2agt/gt mice exhibit neuronal hyperexcitability and a marked global reduction in potassium channel expression, which plays a crucial role in maintaining the resting membrane potential and repolarizing neurons after an action potential. Among these downregulated potassium channels, potassium voltage-gated channel subfamily A member 1 (Kv1.1) was one of the most affected. To explore whether Kv1.1 could be a potential therapeutic target in SCN2A-related ASD, we evaluated the in vivo efficacy of a genetic construct driven by the CaMKII promoter that allows for exogenous expression of human Kv1.1 (hKv1.1) in principal neurons. In Scn2agt/gt mice, we found that hKv1.1 expression normalizes neuronal hyperexcitability. Importantly, doxycycline-induced hKv1.1 expression enhances sociability in Scn2a-deficient mice without influencing social behavior in wild-type mice, and this effect was reversed upon doxycycline withdrawal. Overall, we demonstrate the successful use of an inducible AAV-mediated gene delivery system to supplement hKv1.1 expression to mitigate neuronal hyperexcitability and ameliorate social impairments in a mouse model of SCN2A-related ASD. These findings highlight the contribution of Kv1.1 to SCN2A-associated pathophysiology and its potential as a therapeutic target for severe social deficits.

neuroscience↗

bioRxiv2025

Mitochondrial bioenergetic signatures differentiate asymptomatic from symptomatic Alzheimer's disease

Asymptomatic Alzheimers disease (AsymAD) refers to individuals who, despite exhibiting amyloid-{beta} plaques and tau pathology comparable to Alzheimers disease (AD), maintain cognitive performance similar to cognitively normal individuals. The resilience mechanism in these AsymAD individual remains understudied. We performed a systematic analysis comparing AsymAD and AD across multiple cohorts (ROSMAP, Banner and Mount Sinai), brain regions (BA6, BA9, BA36 and BA37) and neuronal and glial cell types using proteomics and transcriptomics data. AsymAD brains exhibited preserved mitochondrial bioenergetics, characterized by enhanced oxidative phosphorylation (OXPHOS), electron transport chain (ETC) activity, fatty acid and lipid metabolism, and branched-chain amino acid (BCAA) utilization. Pathways regulating mitochondrial complex biogenesis and calcium homeostasis were also upregulated. Key mitochondrial proteins such as MRPL47, CPT2, BCAT2, and IDH2, were consistently upregulated in AsymAD, whereas MACROD1 was downregulated. At the cellular level, excitatory neurons, including superficial, mid-layer, and deep-layer subtypes, exhibited the most preserved mitochondrial function, whereas vulnerable inhibitory subtypes, including PVALB and SST neurons, showed increased cellular abundance and bioenergetic activity. In contrast, microglia and oligodendrocytes proportions were reduced in AsymAD relative to AD. Our findings identify preserved mitochondrial bioenergetics as a defining feature of resilience in AD and suggest that enhancing NADH metabolism via NAD+ precursor-based interventions may potentially help in maintaining cognitive function despite amyloid and tau pathology.

neuroscience↗

bioRxiv2025

Single-nucleus transcriptomics reveal disrupted pathways in the prefrontal cortex of Scn2a-deficient mice

Truncating variants in SCN2A, which encodes the NaV1.2 sodium channel critical for action potential initiation and propagation, are associated with autism spectrum disorder (ASD) and epilepsy. To investigate SCN2A deficiency-related phenotypes, we developed a preclinical mouse model with under 50% NaV1.2 expression, exhibiting neuronal hyperexcitability and social deficits. However, the neuronal populations and molecular alterations underlying these phenotypes at single-cell resolution have not been investigated. In this study, we conducted single-nucleus RNA sequencing (snRNA-seq) of wild-type (WT), homozygous Scn2a-deficient (HOM) mice, and HOM mice with Scn2a restoration (HOM-FlpO) to examine the effects of Scn2a level on gene expression in the medial prefrontal cortex (mPFC), a critical brain region related to ASD development. Differential expression analysis in GABAergic and glutamatergic neurons between genotypes revealed gene expression enriched in neurotransmitter activity regulation and synapse organization. Lastly, snRNA-seq results in HOM-FlpO identified genes that were rescued after Scn2a restoration. These results reveal that reduced Scn2a expression disrupts RNA transcriptomes in multiple neuronal subtypes, providing insight into cell type-specific mechanisms underlying SCN2A-related disorders.

neuroscience↗

bioRxiv2025

Gene therapies alleviate absence epilepsy associated with Scn2a deficiency in DBA/2J mice

Mutations in the voltage-gated sodium channel gene SCN2A, which encodes the NaV1.2 channel, cause severe epileptic seizures. Patients with SCN2A loss-of-function (LoF) mutations, such as protein-truncating mutations, often experience later-onset and drug-resistant epilepsy, highlighting an urgent unmet clinical need for new therapies. We previously developed a gene-trap Scn2a (Scn2agt/gt) mouse model with a global NaV1.2 reduction in the widely used C57BL/6N (B6) strain. Although these mice display multiple behavioral abnormalities, EEG recordings indicated only mild epileptiform discharges, possibly attributable to the seizure-resistant characteristics associated with the B6 strain. To enhance the epileptic phenotype, we derived congenic Scn2agt/gt mice in the seizure-susceptible DBA/2J (D2J) strain. Notably, we found that these mice exhibit prominent spontaneous absence seizures, marked by both short and long spike-wave discharges (SWDs). Restoring NaV1.2 expression in adult mice substantially reduced their SWDs, suggesting the possibility of SCN2A gene replacement therapy during adulthood. RNA sequencing revealed significant alterations in gene expression in the Scn2agt/gt mice, in particular a broad downregulation of voltage-gated potassium channel (KV) genes, including KV1.1. The reduction of KV1.1 expression was further validated in human cerebral organoids with SCN2A deficiency, highlighting KV1.1 as a promising therapeutic target for refractory seizures associated with SCN2A dysfunction. Importantly, delivery of exogenous human KV1.1 expression via adeno-associated virus (AAV) in D2J Scn2agt/gt mice substantially reduced absence seizures. Together, these findings underscore the influence of mouse strain on seizure severity and highlight the potential of targeted gene therapies for treating SCN2A deficiency-related epilepsies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/657652v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@35374eorg.highwire.dtl.DTLVardef@1c22197org.highwire.dtl.DTLVardef@2b4582org.highwire.dtl.DTLVardef@e06e68_HPS_FORMAT_FIGEXP M_FIG C_FIG In briefScn2a deficiency leads to absence seizures in D2J mice and neuronal hyperexcitability with compensatory KV reduction; restoring NaV1.2 or introducing human KV1.1 reduces seizure burden. HighlightsO_LIScn2a deficiency induces robust absence seizures in the DBA/2J but not the C57BL/6N strain. C_LIO_LICortical neurons in adult DBA/2J mice with Scn2a deficiency exhibit intrinsic hyperexcitability. C_LIO_LISevere Scn2a deficiency leads to downregulation of multiple potassium channel genes. C_LIO_LIGenetic restoration of NaV1.2 expression alleviates spike-wave discharges (SWDs). C_LIO_LIAAV-mediated human KV1.1 delivery substantially reduced absence seizures, demonstrating the therapeutic potential of targeted gene therapy. C_LI

neuroscience↗

bioRxiv2025

A protein-DNA surface hydrogel mechanically protects the cell nucleus

The nuclear envelope protects the genome from mechanical stress during processes such as migration, division, and compression1-6, but how it buffers forces at the scale of DNA remains unclear. Here, we utilize optical tweezers to show that a multivalent protein-DNA co-condensate containing the nuclear envelope protein LEM27,8 and the DNA-binding protein BAF 9,10 shield DNA beyond its melting point at 65 pN11. Under load, their collective assembly induces an unconventional DNA stiffening effect that provides mechanical reinforcement, dependent on the intrinsically disordered region (IDR) of LEM2. At the nuclear surface, these components form an elastic surface hydrogel in which LEM2 IDR-IDR interactions contract the surface hydrogel relative to its relaxed state, introducing a pre-stress in the lamin network. Inside cells, this surface hydrogel model can recapitulate elastic properties of the nuclear envelope measured via AFM indentation experiments as well as nuclear morphology, using parameters obtained at the molecular scale by use of optical tweezers. Disruption of the surface hydrogel increases DNA damage and micronuclei formation during nuclear deformation. These findings reveal a load-bearing, mesoscale surface hydrogel that reinforces the nucleus and expands the functional repertoire of biomolecular condensates to include DNA protection under mechanical stress.

biophysics↗

bioRxiv2025

Cancer Drug Bortezomib, a Proteasomal Inhibitor, Triggers Cytotoxicity in Microvascular Endothelial Cells via Multi-Organelle Stress

Proteasomes maintain cellular homeostasis by degrading abnormal proteins, while cancer cells exploit them for survival, becoming a key chemotherapeutic target. Bortezomib (BTZ), a reversible proteasomal inhibitor, is a front-line treatment for multiple myeloma, mantle cell lymphoma, and non-small cell lung cancer. However, its efficacy is limited by severe side effects, including neurotoxicity and cardiovascular distress, with its toxicity mechanisms largely unexplored. Here, we discover that Bortezomib (BTZ), is cytotoxic to non-cancerous cells distinctly from Carfilzomib (CFZ), the second-line irreversible PI. BTZ or CFZ is administered intravenously, impacting blood vessel (vascular) endothelial cells. We used human pulmonary microvascular endothelial cells (HPMECs) to demonstrate that BTZ but not CFZ elicits endoplasmic reticulum (ER) stress, mitochondrial membrane compromise, mitochondrial reactive oxygen species (ROS) accumulation, and Caspase (CASP)9 activation (mediator of Intrinsic apoptosis) within fifteen hours of treatment. By twenty-four hours, BTZ-treated cells display cleavage of CASP8 (mediator of extrinsic apoptosis), activation of CASP3 (terminal executioner of apoptosis), cell-death and vascular barrier loss. Pan-caspase inhibitor zVAD significantly rescues BTZ-treated cells from cytotoxicity. Both BTZ and CFZ effectively kill MM cells. These findings reveal novel insights into fundamental signaling of regular cells where reversible inhibition of the proteasome dictates a unique cascade of stress distinct from irreversible inhibition. These harmful effects of BTZ emphasize the need to re- evaluate its use as a frontline chemotherapy for MM. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/645304v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1b423dborg.highwire.dtl.DTLVardef@19e07f1org.highwire.dtl.DTLVardef@1b84f9org.highwire.dtl.DTLVardef@b70afa_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIReversible proteasomal inhibitor Bortezomib is cytotoxic to non-cancerous, microvascular endothelial cells C_LIO_LIIn endothelial cells, Bortezomib, but not irreversible inhibitor Carfilzomib, activates temporal cascade of caspases (Caspase-9, Caspase-8, Caspase-3) triggering apoptosis C_LIO_LICaspase activation results from ER stress (via the IRE1-CHOP) pathway and mitochondrial stress (ROS accumulation) independently from contribution from extrinsic signal via TNF C_LIO_LIBortezomib-dependent cytotoxicity compromises endothelial barrier potential C_LI

cell biology↗

bioRxiv2024

Phosphatidylinositol-3,5-bisphosphate mediated vacuolar morphology modulation is integral to ethanol stress response

Vacuoles enlarge in response to ethanol. We used quantitative imaging to show that even brief exposure to ethanol causes a rapid transition from multilobed vacuoles to a single compartment. Vacuole unlobing is essential as mutants restricted for vacuole fusion exhibit greater ethanol sensitivity. This response involves the inhibition of vacuole fission via downregulation of the activity of Fab1, a lipid kinase generating the signaling lipid phosphatidylinositol-3,5-bisphosphate. Ethanol exposure results in the redistribution of a phosphatidylinositol-3,5-bisphosphate sensor from the perivauolar dots to the cytoplasm and the dissociation of the effector of this lipid, Atg18, from the vacuolar membrane to the cytoplasm. Impaired Atg18 membrane recruitment also entails a loss of interaction between a scaffold of the Fab1 complex, Vac14, and Atg18. A hyperactive Fab1 mutant, with elevated pools of phosphatidylinositol-3,5-bisphosphate, shows delayed vacuole enlargement; the partial relocation of the phosphatidylinositol-3,5-bisphosphate probe to the perivacuolar dots and of Atg18 to the vacuole membrane. Since Atg18 is responsible for vacuole fragmentation, our results show that, under ethanol stress, cells actively regulate vacuole morphology by modulating phosphatidylinositol-3,5-bisphosphate levels.

cell biology↗

bioRxiv2024

Divergent Functions of Late ESCRT Components in Giardia lamblia: Insights from Subcellular Distributions and Protein Interactions

Giardia lamblia, a human gut pathogen, possesses a minimal ESCRT (Endosomal Sorting Complex Required for Transport) machinery. Paradoxically, there are multiple paralogs of some late-ESCRT components. There are three paralogs for Vps4, GlVps4a, GlVps4b, and GlVps4c, and two for Vps46, GlVps46a, and GlVps46b. This study addressed whether these paralogs discharge overlapping and/or distinct cellular functions by determining the sub-cellular distribution of the paralogs in trophozoites and during encysting. Consistent with the distribution of orthologs in model organisms, most of these components were found to be associated with various cellular membranes, particularly in regions of acute membrane bending. Some of these paralogs are also associated with microtubule structures, such as cytoplasmic axonemes and the median body. Considering their diverse sub-cellular distributions, it is likely that they perform non-overlapping functions within the cell. Furthermore, their redistribution during encystation indicates that they may play a role in the morphological and functional changes accompanying this transition. The study also characterized GlIst1, an ESCRT-III accessory protein that undergoes unique post-translational myristoylation at lysine 43, potentially aiding its membrane recruitment. GlIst1 selectively interacts with GlVps4b through non-canonical MIT-MIM interactions. GlIst1 also exhibits selective interaction with GlVps46b. Such selective interaction of GlIst1 with only specific paralogs of GlVps4 and GlVps46 further underscores the distinct cellular roles of these paralogs. Author SummaryGiardia lamblia, a unicellular protozoan parasite, is the causative agent of giardiasis, a water-transmitted disease affecting millions globally. This disease poses a substantial threat to public health, especially in less developed countries, where clean water and proper sanitation are scarce. The parasite manifests in two morphologically distinct forms, trophozoites and cysts. Transformation between these forms is essential for the organisms survival, spread, and infection processes. Trophozoites, the active and motile form of Giardia, inhabit the small intestine of the host and trigger infections. These trophozoites can transform into cysts through encystation, enabling the parasite to endure harsh external environments and spread between hosts through contaminated water or food sources. The transition between these states necessitates extensive membrane restructuring. Such changes are likely to involve the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, as it has been shown to participate in both prokaryotic and eukaryotic membrane remodeling events. Our research sheds light on the ESCRT machinery in G. lamblia, a crucial membrane-shaping system that may facilitate the transition between trophozoites and cysts. The ESCRT machinery in G. lamblia is distinct from that in yeast and humans, representing one of the most basic ESCRT systems. Our investigation provides valuable information about the intracellular distribution of various late-ESCRT components under different conditions, their potential functions, and their interactions with other late-ESCRT components. These findings may contribute significantly to our understanding of the basic operation of the ESCRT machinery in this parasite.

cell biology↗

bioRxiv2024

Two paralogues of N-ethylmaleimide sensitive factor: An exception to the minimal vesicular trafficking machinery of Giardia

Vesicular trafficking plays a critical role in the survival of the human gut pathogen Giardia lamblia as it drives nutrient uptake and morphological stage transition. Unlike most eukaryotes, Giardia has a minimal vesicular trafficking machinery. Herein, we report a rare exception to this minimalism wherein two paralogues of NSF, a crucial factor driving vesicular trafficking by uncoupling the cis-SNARE bundle, are present in this unicellular parasite. While GlNSF114776 and GlNSF112681 share very high sequence homology, they are likely to have distinct cellular roles as they exhibit differences in their affinities towards the Gl-SNAPs and display non-overlapping distribution in encysting trophozoites. Under multiple stress conditions (nutritional, oxidative and nitrosative), while GlNSF112681 remains at peripheral vesicles, GlNSF114776 relocalizes to the anterior flagella-associated striated fibres, indicating a possible role in regulating flagellar motility. At this location, GlNSF114776 is likely to perform a 20S complex independent function as neither Gl-SNAPs nor GlSNAREs are present there. The two paralogues are likely needed for stress adaptation as both copies have also been retained in Giardia genomes isolated from clinical samples. This non-canonical function of the GlNSF114776 may have evolved to support the unique architecture and motility of the anterior flagella.

cell biology↗

bioRxiv2023

WELPCR: Low-cost polymerase chain reaction (PCR) thermal cycler for nucleic acid amplification and sensing

The COVID-19 pandemic highlighted the need for sensitive and cost-effective diagnostics. The gold standard test to diagnose infectious diseases such as COVID-19 is quantitative polymerase chain reaction (qPCR). Although there have been multiple advances in nucleic acid testing, PCR thermocyclers are expensive and typically not affordable for offering hands-on training to PCR in instructional laboratory courses in undergraduate colleges. We introduce a low-cost, portable, and automated real-time PCR device designed at the Wadhwani Electronics Lab (WEL) at IIT Bombay, christened WELPCR. WELPCR is an open-source design, and capable of reverse transcription PCR (RT-PCR) for complementary DNA synthesis, as well as end-point PCR of small amount of DNA in a test sample. The WELPCR design enables ramp rate up to 2 {degrees}C/s, weighs less than 2 kg, and is easy to assemble. The built-in LED display and keypad provides an intuitive and seamless user interface to configure and set up WELPCR. With bill of materials (BOM) cost less than INR Rs. 10 000, WELPCR is an attractive solution for bringing PCR technology to instructional and research laboratories in a cost-effective manner.

molecular biology↗

bioRxiv2023

Unraveling the Interplay of Different Traits and Parameters Related to Nitrogen Use Efficiency in Wheat: Insights for Grain Yield Influence

Enhancing Nitrogen Use Efficiency (NUE) in wheat to optimize grain yield is a significant challenge. To address this challenge, a comprehensive study was conducted to investigate various morphological, biochemical, molecular parameters, and agronomic traits related to NUE. By examining various traits under both optimum-N (ON) and stressed-N (SN) conditions, the study explores the interrelationships among these traits, providing novel insights not previously reported. A set of 278 diverse wheat genotypes were assessed, encompassing eight NUE-related traits: Grain Yield, Biomass, Grain nitrogen, N at head, N at harvest, N-uptake, Nitrogen Uptake Efficiency, Nitrogen Utilization Efficiency, and NUE under both ON and SN conditions in the field. The findings demonstrated a significant positive correlation between grain yield and all NUE-related traits, highlighting their significance in comprehending the biological NUE of wheat plants. Notably, the study identified N-uptake and N-uptake related traits as key factors influencing the impact of soil nitrogen status on yield and associated parameters. These traits hold particular importance for selecting wheat genotypes with optimal yield and NUE in wheat cultivation. To complement the field data, representative genotypes were further subjected to a hydroponics experiment under absolute nitrogen control. This experiment provided insights into the effects of nitrogen stress on morphological parameters and the performance of eight essential nitrogen and carbon metabolizing enzymes. Correlation analysis highlighted the substantial influence of four key N-metabolizing enzymes, namely Nitrate Reductase, Glutamine Synthetase, Glutamate Oxo-Glutarate Amino Transferase, and Glutamate Dehydrogenase, on grain yield. Additionally, this study underscored the direct and indirect associations between seedling parameters and field traits, emphasizing the significance of shoot and root length parameters in nitrogen acquisition under nitrogen stress. In conclusion, these findings offer valuable insights into the intricate network of traits and parameters that influence wheat grain yield under varying nitrogen regimes. This knowledge can aid in the selection of wheat genotypes with enhanced NUE and grain yield, particularly in scenarios of reduced nitrogen application. Key PointsO_LIA comprehensive study in field and hydroponics conditions revealed differential responses of various morphological, biochemical, molecular parameters, and agronomic traits to different nitrogen levels. C_LIO_LIN-uptake related traits in field condition and chlorophyll content and morphological parameters in hydroponics condition were found as essential factors contributing to variations under both optimum and stressed N conditions. C_LIO_LIAmong the parameters observed in the seedling stage, SL and RL, along with the enzymes NR, GS, GOGAT, and GDH, demonstrated their influence on GY. C_LI

plant biology↗

bioRxiv2022

Zinc ions prevent a-synuclein aggregation by enhancingchaperone function of human serum albumin

Metal ions present in cellular microenvironment have been implicated as drivers of aggregation of amyloid forming proteins. Zinc (Zn2+) ions have been reported to directly interact with -synuclein (AS), a causative agent of Parkinsons disease and other neurodegenerative diseases, and promote its aggregation. AS is a small intrinsically disordered protein (IDP) i.e., understanding molecular factors that drive its misfolding and aggregation has been challenging since methods used routinely to study protein structure are not effective for IDPs. Here, we report the atomic details of Zn2+ binding to AS at physiological conditions using proton-less NMR techniques that can be applied to highly dynamic systems like IDPs. We also examined how human serum albumin (HSA), the most abundant protein in human blood, binds to AS and whether Zn2+ and/or ionic strength affect this. We conclude that Zn2+ enhances the anti-aggregation chaperoning role of HSA that relies on protecting the hydrophobic N-terminal and NAC regions of AS, rather than polar negatively charged C-terminus. This suggested a previously undocumented role of Zn2+ in HSA function and AS aggregation.

biophysics↗

bioRxiv2021

Molecular basis of UHRF1 allosteric activation for synergistic histone modification binding by PI5P

Chromatin marks are recognized by distinct binding modules many of which are embedded in multidomain proteins or complexes. How the different protein functionalities of complex chromatin modulators are regulated is often unclear. Using a combination of biochemical, biophysical and structural approaches we delineated the regulation of the H3unmodified and H3K9me binding activities of the multidomain UHRF1 protein. The phosphoinositide PI5P interacts with two distant flexible linker regions of UHRF1 in a mode that is dependent on the polar head group and the acyl part of the phospholipid. The associated conformational rearrangements stably position the H3unmodified and H3K9me3 histone recognition modules of UHRF1 for multivalent and synergistic binding of the H3 tail. Our work highlights a novel molecular function for PI5P outside of the context of lipid mono- or bilayers and establishes a molecular paradigm for the allosteric regulation of complex, multidomain chromatin modulators by small cellular molecules.

biochemistry↗

bioRxiv2020

Vinculin mediated axon growth requires interaction with actin but not talin

Axon growth requires coordination of the actin cytoskeleton by actin-binding proteins in the extending neurites. Vinculin is a major constituent of focal adhesion but its role in neuronal migration and axon growth is poorly understood. We found that vinculin deletion in mouse neocortical neurons attenuated axon growth both in vitro and in vivo. Using different functional mutants of vinculin, we found that expression of a constitutively active vinculin significantly enhanced axon growth while the head-neck domain had a moderate inhibitory effect. Interesting, we found that vinculin-talin interaction was dispensable for axon growth and neuronal migration. Strikingly, expression of the tail domain delayed migration, increased branching and stunted axon. Inhibition of the Arp2/3 complex or abolishing the tail domain interaction with actin completely reversed the branching phenotype caused by tail domain expression without affecting axon length. Super-resolution microscopy showed increased mobile fraction of actin in tail domain expressing neurons. Our results provide novel insights into the role of vinculin and its functional domains in regulating neuronal migration and axon growth.

neuroscience↗

bioRxiv2020

Novel role of AGT gene in aplastic anaemia among paediatric patients based on gene expression profiling

ObjectivesSevere aplastic anemia is characterized by a hypocellular bone marrow and peripheral cytopenia. Mesenchymal stem cells (MSCs) play a crucial role in haematopoietic stem cells (HSCs) development and the microenvironment suitable for haematopoiesis. Investigation of the therapeutic targets by paediatric patient-specific gene expression analysis of the MSCs can be important for diagnosis. MethodsThe study was based on freely available miRNA and host gene expression in NCBI GEO dataset. Microarray based gene expression profiles (GSE33812) of MSCs for five paediatric aplastic anaemia patients and healthy controls were generated using Agilent-014850 platform and the data was downloaded from the database. ResultsMSCs gene expression profiling distinguished between healthy controls, children with aplastic anemia. Angioteninogen (AGT) gene involved in ERK1/ERK2 cascade, cyotokine secretion, metabolic processes was strongly down-regulated among all the patients with aplastic anemia. Emerging role of various transcription factors binding to this gene was identified as a new avenue of therapeutic application. ConclusionsAs a potential diagnostic tool, patient-specific gene expression profiling of MSCs made it possible to make the difficult diagnosis of most patients with aplastic anemia.

bioinformatics↗

bioRxiv2019

Conformational switching of CD81 controls its function as a receptor for hepatitis C virus

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational opening of the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol sensing mechanism; however, the relevance of this for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81-cholesterol association, but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol unbound) or closed (cholesterol bound) conformation. The open mutant of CD81 exhibited reduced receptor activity whereas the closed mutant enhanced activity. These data are consistent with CD81 cholesterol sensing resulting in a switch between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81-partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry and CD81s function as a molecular scaffold; these insights are relevant to CD81s varied roles in both health and disease.

molecular biology↗

bioRxiv2019

\"Risk Factors for Visceral Leishmaniasis in Selected High Endemic Areas of Morang District, Nepal\": A case control Study

Visceral leishmaniasis is a major public health concern in Nepal. During the last few years, several KA outbreaks have been reported from Tarai region including Morang district. A case control study was conducted to assess the risk factors associated with VL in 5 endemic VDC of Morang district with 62 cases already treated from BPKIHS and Koshi zonal hospital and 248 controls selected randomly from the same village. Data collected using semi structured questionnaire from September to November 2013. This study revealed that people living in thatched house, sleeping in ground floor, ownership of animal, history of migration to India (Bihar and Jharkhand) and proximity to other KA cases within 50 m distance of household were strong risk factors for VL. Education remains protective (OR 0.39,95 % CI 0.19-0.79). The association with socioeconomic status showed clear dose - response effect. The odds for VL consistently decreased as the level of socioeconomic status increased (OR 4.26, 3.81). Strengthening surveillance system for early diagnosis and treatment, awareness programme and further extensive study is needed on risk factor, vector and control measures. Author SummaryThis study aims to explore the risk factors for visceral leishmaniasis. Based on findings there is a need to educate people in high-transmission areas how to realize, reduce or avoid environmental factors that favor the survival of the vector in the community. Similarly introduction of more exact surveillance tools in order to improve morbidity and mortality surveillance by health sector. People living in mud thatched houses need to be aware about cracks and crevices in the mud walls, their function as breeding places and how they can be controlled, for example by plastering with lime and mud.. However, a primary condition is that people need to understand the purpose of all these efforts in order to be motivated to put them into practice.

epidemiology↗