Biology subjects

Malhotra, N.

Publications and source records attributed to Malhotra, N..

18 recordsLinked to original sources

bioRxiv2026

Intranasal Delivery of Hypoxia Primed Whartons Jelly MSC Derived sEVs Reprograms Neuroimmune Signalling and Ameliorates Behavioural Deficits in a Valproic Acid Induced Mouse Model of Autism Spectrum Disorder

BackgroundBeyond disrupted neurodevelopment, autism spectrum disorder (ASD) encompasses progressive neurodegenerative alterations that remain refractory to standard of care behavioral therapies, highlighting the need for biologically targeted adjunctive interventions. Although, Mesenchymal stem cell (MSC) derived small extracellular vesicles (sEVs) have emerged as potent neuroprotective agents; however, the optimal MSC tissue source and strategies enhancing their efficacy for clinical translation remain undefined. Hypoxic preconditioning potentiates their therapeutic effects, however its specific impact on ASD-related impairments is yet to be established. This study conducts a head to head evaluation of sEVs from bone marrow (BM) and Whartons jelly (WJ) MSCs under normoxic and hypoxic conditions to delineate their comparative neuroregenerative efficacy in ASD. MethodsWe investigated the neuroprotective efficacy of sEVs derived from human tissue-specific mesenchymal stem cells (BM/WJ) cultured under normoxic (21% O2) and hypoxic (1% O2) conditions. In vitro assays evaluated sEV uptake, cell proliferation, neurodifferentiation, mitochondrial health, and immunomodulatory effects using neural stem cell (C17.2) and microglial (N9) cell lines. For in vivo studies, ASD-like phenotypes were induced in C57BL/6 mice via prenatal exposure to valproic acid (600 mg/kg), followed by the biodistribution of fluorescently labeled hypoxia-primed WJ MSCs sEVs (WJ-H-sEVs) following intranasal administration was assessed using IVIS imaging. Behavioural outcomes after intranasal treatment were evaluated. Additionally, post-mortem brain tissues were analyzed for neuroinflammation, oxidative stress, synaptic integrity, and downstream signalling pathways using immunofluorescence, Western blotting, and qRT-PCR, while systemic cytokine pro-inflammatory and anti-inflammatory levels were quantified by ELISA. Mechanistic involvement of Nrf2 and NF-{kappa}B signaling pathways was examined using pharmacological inhibitors. ResultsComparative analyses revealed that hypoxic priming significantly enhanced sEV yield and enriched neuroprotective miRNAs, with WJ-H-sEVs exhibiting elevated levels of miR-125b-5p, miR-21a-5p, and miR-145a-5p. Consistent with this enrichment, WJ-H-sEVs demonstrated superior cellular uptake as observed in neural stem cells and microglia, enhanced neurodifferentiation, robust antioxidant activity, effective immunomodulation, and improved mitochondrial health, thereby outperforming their normoxic and BM-derived counterparts. Intranasally administered WJ-H-sEVs efficiently localized to the brain within 12 h and significantly improved anxiety-like behavior, recognition memory, and spatial learning in ASD mice. These effects were accompanied by reduced neuroinflammation and oxidative damage, enhanced neuronal survival, decreased serum IL-6 levels, and increased IL-10 and TGF-{beta}. Mechanistically, therapeutic benefits were mediated through activation of the Nrf2 antioxidant pathway and suppression of NF-{kappa}B and JAK-STAT signaling. ConclusionIn this study, hypoxia emerges as a clinically exploitable priming strategy to enhance the neuroregenerative performance of tissue-specific MSC-derived sEVs, with potential applicability across MSC sources and other neurological disorders. Moreover, intranasal delivery of WJ-H-sEVs demonstrates translational feasibility, reinforcing their potential as an adjunctive, cell-free, pediatric-friendly neurotherapeutic modality for ASD management. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/721255v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@dd0f7forg.highwire.dtl.DTLVardef@1247d24org.highwire.dtl.DTLVardef@93e6deorg.highwire.dtl.DTLVardef@1a349d_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2026

A Network-Based Approach for Prioritizing Candidate Genes in Alzheimer's Disease

Alzheimers disease (AD) is a multifactorial neurodegenerative disorder characterized by coordinated dysregulation of multiple genes, requiring system-level approaches to elucidate underlying molecular mechanisms. While existing computational studies largely focus on differential expression analysis or machine-learning-based feature selection, they often overlook inter-gene relationships and network topology, limiting biological interpretability. In this study, we present a network-based framework for prioritizing candidate genes in Alzheimers disease by integrating gene co-expression network analysis with multiple centrality measures. Transcriptomic data comprising approximately 39,000 genes across 324 Alzheimers and control samples were preprocessed using log-transformation, variance filtering and Z-score normalization, followed by LASSO-based feature selection to retain phenotype-associated genes. A weighted gene co-expression network was constructed using Pearson correlation to capture coordinated transcriptional activity. Network topology was analyzed using degree, betweenness and eigenvector centrality to identify genes that are highly connected, act as information brokers or occupy influential positions within the network. A consensus ranking was derived by merging these centrality measures, enabling robust prioritization of candidate genes. The results highlight a subset of highly central genes, including several small nucleolar RNAs and regulatory transcripts implicated in RNA processing, synaptic function and neurodegenerative pathways. By jointly leveraging co-expression structure and complementary centrality metrics, the proposed framework provides an interpretable and reproducible strategy for identifying biologically meaningful gene candidates, offering potential utility for biomarker discovery and therapeutic target prioritization in Alzheimers disease.

systems biology↗

bioRxiv2025

The N-terminal Segment of the Human ANKZF1 negatively regulates its internal mitochondrial targeting signal to prevent its mitochondrial localization

ANKZF1, the human orthologue of yeast Vms1, is a multi-domain cytosolic protein. Occasionally, the protein is also found in mitochondria, although the reason for its mitochondrial localization and the mechanism of its mitochondrial targeting remains unclear. Despite the absence of any predicted Mitochondrial Targeting Sequence or Domain (MTS/MTD) in the protein, ANKZF1 possesses multiple internal Matrix-targeting sequence-like sequences (iMTS-Ls). In this study, we show that ANKZF1s N-terminal 73 residues negatively regulate its mitochondrial targeting. Interestingly, 1-72-ANKZF1 peptide hinders the mitochondrial targeting of {Delta}73-ANKZF1 upon co-expression. Using a series of truncation mutants of ANKZF1, we further show that iMTS-Ls constituted by residues 231-240 of ANKZF1 is indispensable for its mitochondrial localization. The sequence of 231-240 residues is extremely conserved across different organisms, indicating the importance of this segment for conditional mitochondrial targeting of the protein. Importantly, 231-324 residues containing two consecutive predicted iMTS-Ls constitute an independent mitochondrial signal sequence and can target Green Fluorescent Protein (GFP) to mitochondria when fused to N-terminus. Further, by Molecular Dynamic simulation, we show that the deletion of N-terminal 74 amino acids of ANKZF1 leads to a massive structural rearrangement within the protein leading to the opening of its C-terminal part and solvent exposure of the 231-240 residues. We posit that these structural rearrangements and exposure of the internal mitochondrial targeting sequence in the absence of N-terminal segment of ANKZF1 leads to its mitochondrial translocation.

cell biology↗

bioRxiv2025

Chaperone-mediated autophagy substrates bind to lid domain of Hsc70: Exploration using integrated approach

Chaperone-mediated autophagy (CMA) is a selective lysosomal degradation pathway crucial for cellular homeostasis. Its dysfunction is linked to cancer and neurodegenerative disorders. Hsc70 mediates CMA selectivity by recognizing proteins with KFERQ-type sequences, but the structural basis for substrate recognition and the substrate-binding mechanism of Hsc70 remain unclear. This study integrates literature, bioinformatics, AlphaFold 3 predictions, and 44 s of molecular dynamics (MD) simulations to elucidate the lid domain interactions of Hsc70 with fifteen experimentally verified CMA substrate segments. Structural and thermodynamic residue-level analyses suggest stable binding of substrates in two distinct sites. The first site resides in a hydrophobic cavity formed by alpha helix 1 and 3 in the lid domain of Hsc70, with complex stabilization primarily driven by hydrophobic residues within KFERQ-type motif and its flanking regions. The second stable site was observed to be canonical hydrophobic cleft in Hsc70, with complex stabilization primarily driven by hydrophobic residues within KFERQ-type motif as well as the through water-mediated interactions by the lid domain. The robustness of our findings is confirmed by consistent convergence across force fields, substrates, and supporting literature, all identifying the same site. We propose these sites may facilitate upstream or downstream recognition and activation events. Author SummaryCells must constantly remove damaged or unnecessary proteins in order to stay healthy. One important pathway that performs this task is chaperone-mediated autophagy. When this pathway fails, it has been linked to conditions such as cancer and neurodegenerative diseases. A key player in this process is the protein Hsc70, which recognizes short sequence signatures in target proteins and directs them for degradation. However, how Hsc70 physically recognizes these signatures has remained poorly understood. In this study, we investigated how Hsc70 interacts with a range of confirmed target protein segments. We combined information from prior experimental studies, with computational structure prediction and extensive molecular simulations to explore these interactions in detail. Our results indicate that Hsc70 can bind target proteins at two distinct regions, each stabilized mainly by hydrophobic amino acids within and/or around the recognition signal, with additional contribution from water-mediated interactions in the latter site. Notably, these binding modes were consistent across different targets and computational conditions. Together, our findings provide a clearer structural picture of how selectivity is achieved in this degradation pathway. This improved understanding may help guide future studies on how chaperone-mediated autophagy is regulated and how it becomes disrupted in disease.

biophysics↗

bioRxiv2025

Bioenergetic reprogramming of macrophages reduces drug tolerance in Mycobacterium tuberculosis

Eradication of Mycobacterium tuberculosis (Mtb) requires strategies targeting bacteria inside the host. Mtb exhibits heterogeneity in redox metabolism inside macrophages to evade killing by anti-TB drugs. If and how macrophage physiology correlates with bacterial redox heterogeneity and drug tolerance remains unclear. Using a fluorescent reporter of mycobacterial redox potential, flow sorting, and RNA sequencing of infected macrophages, we characterized transcriptional and metabolic responses of macrophages harboring redox-diverse Mtb populations. We found that macrophages with suppressed glycolysis and elevated oxidative phosphorylation (OXPHOS) correlated with Mtb populations exhibiting reductive stress and drug tolerance. Conversely, macrophages with elevated glycolysis and suppressed OXPHOS displayed higher mitochondrial reactive oxygen species through reverse electron transport, resulting in oxidative stress in Mtb and enhancing drug efficacy. Computational and genetic approaches identified Nrf2 as a key regulator of macrophage bioenergetics driving redox heterogeneity and drug tolerance in Mtb. Redirecting macrophage metabolism from OXPHOS to glycolysis using an FDA- approved antiemetic drug, meclizine, subverted redox heterogeneity and diminished drug tolerance in macrophages and mice. The pharmacological profile of meclizine (Cmax and AUClast) indicated no adverse interactions with first-line anti-TB drugs in mice. Our data demonstrate the feasibility of reprogramming macrophage metabolism to reduce drug tolerance in Mtb infection.

microbiology↗

bioRxiv2024

Hydrogen sulfide (H2S) coordinates redox balance, carbon metabolism, and mitochondrial bioenergetics to suppress SARS-CoV-2 infection.

Viruses exploit host metabolism for replication and immune-regulation. Understanding how SARS-CoV-2 alters the host metabolism may lead to treatments for COVID-19. We report that a ubiquitous gaseous molecule, hydrogen sulfide (H2S), regulates redox, metabolism, and mitochondrial bioenergetics to control SARS-CoV-2. Virus replication is associated with down-regulation of the H2S-producing enzymes cystathionine-{beta}-synthase (CBS), cystathionine-{gamma}-lyase (CTH), and 3-mercaptopyruvate sulfurtransferase (3-MST), resulting in diminished endogenous H2S levels. Inhibition of CTH resulted in SARS-CoV-2 proliferation. A slow-releasing H2S donor, GYY4137, diminished virus replication by inducing Nrf2/Keap1 pathway, restoring redox balance and mitochondrial bioenergetics. Treatment of SARS-CoV-2-infected animals with GYY4137 suppressed viral replication, ameliorated respiratory pathology, and restored antioxidant gene expression. Notably, whole-body plethysmography showed improved pulmonary function variables, including pulmonary obstruction and end-expiratory pause upon GYY4137 treatment in vivo. Data extend our understanding of H2S-mediated regulation of viral-infections, and open new avenues for investigating the pathogenic mechanisms and therapeutic opportunities for coronavirus-associated disorders.

microbiology↗

bioRxiv2024

Environmental sphagnum-associated fungi target thiol homeostasis to compete with Mycobacterium tuberculosis.

Mycobacterial species in nature are found in abundance in sphagnum peat bogs where they compete for nutrients with a variety of microorganisms including fungi. We screened a collection of fungi isolated from sphagnum bogs by co-culture with Mycobacterium tuberculosis (Mtb) to look for inducible expression of antitubercular agents and identified five fungi that produced cidal antitubercular agents upon exposure to live Mtb. Whole genome sequencing of these fungi followed by fungal RNAseq after Mtb exposure allowed us to identify biosynthetic gene clusters induced by co-culture. Three of these fungi induced expression of patulin, one induced citrinin expression and one induced the production of nidulalin A. The biosynthetic gene clusters for patulin and citrinin have been previously described but the genes involved in nidulalin A production have not been described before. All three of these potent electrophiles react with thiols and treatment of Mtb cells with these agents followed by Mtb RNAseq showed that these natural products all induce profound thiol stress suggesting a rapid depletion of mycothiol. The induction of thiol-reactive mycotoxins through three different systems in response to exposure to Mtb suggests that fungi have identified this as a highly vulnerable target in a similar microenvironment to that of the caseous human lesion.

microbiology↗

bioRxiv2024

Uncovering the bequeathing potential of Apoptotic Mesenchymal Stem Cells via small Extracellular Vesicles for its enhanced immunomodulatory ability

Small Extracellular Vesicles (sEVs) derived from Mesenchymal Stem Cells (MSCs) have emerged as a promising avenue for cell-free therapeutics in regenerative medicine. These vesicles, endowed with regenerative cargo inherited from their parent cells, have attracted attention for their role in immunomodulation and ROS alleviation. Notably, the deliberate induction of apoptosis in MSCs prior to Extracellular Vesicles (EVs) isolation has been identified as a strategy to augment the regenerative capabilities of MSCs-EVs, as certain reports have suggested that MSCs undergo apoptosis to exert their therapeutic effect post-transplantation. Moreover, selecting an optimal tissue source for deriving MSC-sEVs is equally crucial to ensure consistent and improved clinical outcomes. Multiple attributes of MSCs like their antioxidant, Immunomodulatory & regenerative properties make them particularly appealing for clinical studies, wherein mechanisms such as paracrine secretions and efferocytosis play pivotal roles. This investigation meticulously explores the comparative immunomodulatory & antioxidant capabilities of Apoptotic sEVs (Apo-sEVs) with Viable sEVs (V-sEVs) obtained from both Bone Marrow (BM) and Whartons Jelly (WJ)-derived MSCs, using an in vitro liver injury model. The findings from the present study contribute valuable insights into the comparative efficacy of Apo-sEVs and V-sEVs. This will aid in addressing a critical gap in understanding the role of apoptosis in enhancing the reparative capability of MSCs-sEVs. It also aims to shed light on the optimal source of MSCs for generating Apo-sEVs in translational applications. GRAPHICAL ABSTARCT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/590581v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1392183org.highwire.dtl.DTLVardef@1c72d6borg.highwire.dtl.DTLVardef@176ee90org.highwire.dtl.DTLVardef@128559e_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Determining the defining lengths between mature microRNAs/small interfering RNAs and tinyRNAs

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are loaded into Argonaute (AGO) proteins, forming RNA-induced silencing complexes (RISCs). The assembly process establishes the seed, central, 3' supplementary, and tail regions across the loaded guide, enabling the RISC to recognize and cleave target RNAs. This guide segmentation is caused by anchoring the 3' end at the AGO PAZ domain, but the minimum guide length required for the conformation remains to be studied because there was no method by which to do so. Using a 3'[->]5' exonuclease ISG20, we determined the lengths of AGO-associated miR-20a and let-7a with 3' ends that no longer reach the PAZ domain. Unexpectedly, miR-20a and let-7a needed different lengths, 19 and 20 nt, respectively, to maintain their RISC conformation. This difference can be explained by the low affinity of the PAZ domain for the adenosine at g19 of let-7a, suggesting that the tail-region sequence slightly alters the minimum guide length. We also present that 17-nt guides are sufficiently short enough to function as tinyRNAs (tyRNAs) whose 3 ends are not anchored at the PAZ domain. Since tyRNAs do not have the prerequisite anchoring for the standardized guide segmentation, they would recognize targets differently from miRNAs and siRNAs.

biochemistry↗

bioRxiv2022

Manganese-dependent microRNA trimming by 3'->5' exonucleases generates 14-nucleotide or shorter tiny RNAs

MicroRNAs (miRNAs) are about 22-nucleotide (nt) non-coding RNAs forming the effector complexes with Argonaute (AGO) proteins to repress gene expression. Although tiny RNAs (tyRNAs) shorter than 19 nt have been found to bind to plant and vertebrate AGOs, their biogenesis remains a long-standing question. Here, our in vivo and in vitro studies show several 3[->]5 exonucleases, such as interferon-stimulated gene 20 kDa (ISG20), three prime repair exonuclease 1 (TREX1), and ERI1 (enhanced RNAi, also known as 3hExo), capable of trimming AGO-associated full-length miRNAs to 14 nt or shorter tyRNAs. Their guide trimming occurs in a manganese-dependent manner but independently of the guide sequence and the loaded four human AGO paralogs. We also show that ISG20-mediated guide trimming makes Argonaute3 (AGO3) a slicer. Given the high Mn2+ concentrations in stressed cells, virus-infected cells, and neurodegeneration, our study sheds light on the roles of the Mn2+-dependent exonucleases in remodeling gene silencing.

molecular biology↗

bioRxiv2022

Replication-dependent organisation constrains positioning of long DNA repeats in bacterial genomes

Bacterial genome organisation is primarily driven by chromosome replication from a single origin of replication. However chromosomal rearrangements, which can disrupt such organisation, are inevitable in nature. Long DNA repeats are major players mediating rearrangements, large and small, via homologous recombination. Since changes to genome organisation affect bacterial fitness - and more so in fast-growing than slow-growing bacteria - and are under selection, it is reasonable to expect that genomic positioning of long DNA repeats is also under selection. To test this, we identified identical DNA repeats of at least 100 base pairs across ~6,000 bacterial genomes and compared their distribution in fast and slow growing bacteria. We find that long identical DNA repeats are distributed in a non-random manner across bacterial genomes. Their distribution differs in their overall number, orientation and proximity to the origin of replication, in fast and slow growing bacteria. We show that their positioning - which might arise from a combination of the processes that produce repeats and selection on rearrangements that recombination between repeat elements might cause - permits minimum disruption to the replication-dependent genome organisation of bacteria, thus acting as a major constraint.

genomics↗

bioRxiv2021

Emergence of networks of shared restriction-modification systems in phage-bacteria ecosystems

1Restriction-modification (RM) systems are the most ubiquitous bacterial defense system against bacteriophages and an important part of controlling phage predation. Using genomic sequence data, we show that RM systems are often shared among bacterial strains in a structured way. Examining the network of interconnections between bacterial strains within each genus, we find that in many genera strains share more RM systems than expected from a random network. We also find that many genera have a larger than expected number of bacterial strains with unique RM systems. We use population dynamics models of closed and open phage-bacteria ecosystems to qualitatively understand the selection pressures that could lead to these non-random network structures with enhanced overlap or uniqueness. In our models we find that the phages impose a pressure that favours bacteria with more RM systems, and more overlap of RM systems with other strains, but in bacteria dominated states this is opposed by the increased cost to the growth rate of these bacteria. Similar to what we observe in the genome data, we find that two distinct bacterial strategies emerge - strains either have a larger overlap than expected, or they have more unique RM systems than one expects from a null model. The former strategy appears to dominate when the repertoire of available RM systems is smaller but the average number of RM systems per strain is larger.

microbiology↗

bioRxiv2021

Identification of SGLT2 inhibitor Ertugliflozin as a treatment for COVID-19 using computational and experimental paradigm

Drug repurposing can expedite the process of drug development by identifying known drugs which are effective against SARS-CoV-2. The RBD domain of SARS-CoV-2 Spike protein is a promising drug target due to its pivotal role in viral-host attachment. These specific structural domains can be targeted with small molecules or drug to disrupt the viral attachment to the host proteins. In this study, FDA approved Drugbank database were screened using a virtual screening approach and computational chemistry methods. Five drugs were short listed for further profiling based on docking score and binding energies. Further these selected drugs were tested for their in vitro biological activity. There was significant correlation between the prediction from computational studies and the actual RBD-ACE2 binding inhibition by the drugs. Then, we performed a series of studies that mimic some of the biological events seen in COVID-19 patients such as secretion of IL1{beta}, presentation of a more thrombogenic endothelium by production of thrombomodulin and accumulation of inflammatory cells such as monocytes in the lungs. Of all the drugs, most promising drug was Ertugliflozin which is used for type-2 diabetes. This drug possesses several desired properties and may be a good candidate for immediate repurposing for treatment of COVID-19.

pharmacology and toxicology↗

bioRxiv2021

Tyrosine Kinase Inhibitor Family of Drugs as Prospective Targeted Therapy for COVID-19 Based on In Silico And 3D-Human Vascular Lung Model Studies

COVID-19 pandemic has ravaged the world and vaccines have been rapidly developed as preventive measures. But there is no target-based therapy which can be used if infection sets in. Remdesiver and dexamethasone were not designed to combat COVID-19 but are used clinically till better targeted therapies are available. Given this situation target based therapies that intervene in the disease pathway are urgently needed. Since COVID-19 genesis is driven by uncontrolled inflammation/thrombosis and protein kinases are critical in mounting this response, we explored if available tyrosine kinase inhibitors (TKIs) can be used as intervention. We profiled four TKIs namely; Lapatinib, Dasatinib, Pazopanib and Sitravatinib which inhibit tyrosine kinases but are completely distinct in their chemical structures. We demonstrate using in silico and an in vitro 3D-human vascular lung model which profiles anti-inflammatory and anti-thrombogenic properties that all four TKIs are active in varying degrees. Our findings that chemically different TKIs which share kinase inhibition as the common mechanism of action are active, strongly indicates that its a tyrosine kinase target-based activity and not off-target arbitrary effect. We propose that TKIs, approved for human use and widely available, can be rapidly deployed as specific target-based therapy for COVID-19.

pharmacology and toxicology↗

bioRxiv2021

In silico and in vitro Demonstration of Homoharrintonine Antagonism of RBD-ACE2 Binding and its Anti-inflammatory and anti-thrombogenic Properties in a 3D human vascular lung model

Since 2019 the world has seen severe onslaught of SARS-CoV-2 viral pandemic. There is an urgent need for drugs that can be used to either prevent or treat the potentially fatal disease COVD-19. To this end, we screened FDA approved antiviral drugs which could be repurposed for COVID-19 through molecular docking approach in the various active sites of receptor binding domain (RBD). The RBD domain of SARS-CoV-2 spike protein is a promising drug target due to its pivotal role in viral-host attachment. Specifically, we focussed on identifying antiviral drugs which could a) block the entry of virus into host cells, b) demonstrate anti-inflammatory and/or anti-thrombogenic properties. Drugs which poses both properties could be useful for prevention and treatment of the disease. While we prioritized a few antiviral drugs based on molecular docking, corroboration with in vitro studies including a new 3D human vascular lung model strongly supported the potential of Homoharringtonine, a drug approved for chronic myeloid leukaemia to be repurposed for COVID-19. This natural product drug not only antagonized the biding of SARS-CoV-2 spike protein RBD binding to human angiotensin receptor 2 (ACE-2) protein but also demonstrated for the first time anti-thrombogenic and anti-leukocyte adhesive properties in a human cell model system. Overall, this work provides an important lead for development of rapid treatment of COVID-19 and also establishes a screening paradigm using molecular modelling and 3D human vascular lung model of disease to identify drugs with multiple desirable properties for prevention and treatment of COVID-19.

pharmacology and toxicology↗

bioRxiv2020

Nitric oxide-inducible Fe-S cluster biogenesis enables Mycobacterium tuberculosis to persist in mice

The persistence of Mycobacterium tuberculosis (Mtb) is a major problem in managing tuberculosis (TB). Host-generated nitric oxide (NO) is perceived as one of the signals by Mtb to reprogram metabolism and respiration for persistence. However, the mechanisms involved in NO sensing and reorganizing Mtbs physiology are not fully understood. Since NO damages Fe-S clusters of essential enzymes, the mechanism(s) involved in regulating iron-sulfur (Fe-S) cluster biogenesis could help Mtb persist in host tissues. Here, we show that a transcription factor SufR (Rv1460) senses NO via its 4Fe-4S cluster and promotes persistence of Mtb by mobilizing the Fe-S cluster biogenesis system; suf operon (Rv1460-Rv1466). Analysis of anaerobically purified SufR by UV-visible spectroscopy, circular dichroism, and iron-sulfide estimation confirms the presence of a 4Fe-4S cluster. Atmospheric O2 and H2O2 gradually degrade the 4Fe-4S cluster of SufR. Furthermore, electron paramagnetic resonance (EPR) analysis demonstrates that NO directly targets SufR 4Fe-4S cluster by forming a protein-bound dinitrosyl-iron-dithiol complex. DNase I footprinting, gel-shift, and in vitro transcription assays confirm that SufR directly regulates the expression of the suf operon in response to NO. Consistent with this, RNA- sequencing of Mtb{Delta}sufR demonstrates deregulation of the suf operon under NO stress. Strikingly, NO inflicted irreversible damage upon Fe-S clusters to exhaust respiratory and redox buffering capacity of Mtb {Delta}sufR. Lastly, Mtb{Delta} sufR failed to recover from a NO-induced non-growing state and displayed persistence defect inside immune-activated macrophages and murine lungs in a NO-dependent manner. Data suggest that SufR is a sensor of NO that supports persistence by reprogramming Fe-S cluster metabolism and bioenergetics. HighlightsO_LIMycobacterium tuberculosis (Mtb) induces the expression of suf operon for Fe-S cluster biogenesis in response to nitric oxide (NO). C_LIO_LIWe found that a transcription factor SufR senses NO via its 4Fe-4S cluster and regulates the expression of the suf operon for Fe-S cluster biogenesis. C_LIO_LISufR-regulated Fe-S cluster biogenesis confers respiratory and redox features that promote recovery of Mtb from NO stress. C_LIO_LISufR activity is required to support the NO-dependent persistence of Mtb in macrophages and mice. C_LI

microbiology↗

bioRxiv2019

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

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

genomics↗

bioRxiv2019

Neonatal-derived IL-17 producing dermal gd T cells are required to prevent spontaneous atopic dermatitis

Atopic Dermatitis (AD) is a T cell-mediated chronic skin disease and is associated with altered skin barrier integrity. Infants with mutations in genes involved in tissue barrier fitness are predisposed towards inflammatory diseases, but most do not develop or sustain the diseases, suggesting that there exist regulatory immune mechanisms to repair tissues and/or prevent aberrant inflammation. The absence of one single murine dermal cell type, the innate neonatal-derived IL-17 producing {gamma}{delta} T (T{gamma}{delta}17) cells, from birth resulted in spontaneous, highly penetrant AD with all the major hallmarks of human AD. In T{gamma}{delta}17 cell-deficient mice, basal keratinocyte transcriptome was altered months in advance of AD induction. Fulminant disease is driven by skin commensal bacteria dysbiosis and highly expanded dermal {beta} T clonotypes that produce the type three cytokines, IL-17 and IL-22. These results demonstrate that neonatal T{gamma}{delta}17 cells are innate skin regulatory T cells. The bifurcation of type 3 cytokine producing skin T cells into the homeostatic, early innate and pathogen-sensing, late adaptive T cell compartments underpin healthy skin and accounts for the dual function of type 3 cytokines in skin maintenance and inflammation.

immunology↗