Biology subjects

Mishra, S.

Publications and source records attributed to Mishra, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Conserved Roles of Sp1 in Zebrafish Development and Early Organogenesis

Transcription factors (TFs) function in combinatorial networks to orchestrate gene expression during development and disease. Yet, the mechanisms underlying their context-specific regulatory roles remain poorly understood. Here, we investigate Specificity Protein 1 (Sp1), the first sequence-specific transcription factor identified and a key regulator of TATA-less gene promoters as a model to elucidate conserved and lineage-specific functions in vertebrate development. While Sp1 is extensively studied in mammalian systems, particularly in cancer progression, its evolutionary acquisition and developmental roles in early vertebrates remain unexplored. Using zebrafish as a model, we characterized the spatiotemporal expression dynamics and functional significance of sp1 during embryogenesis. Loss-of-function analyses revealed profound morphological defects across multiple organ systems, including skeletal, neural, and gastrointestinal tissues. Complementary stage-specific transcriptomic profiling uncovered widespread dysregulation of lineage-defining genes and perturbations in core cellular pathways related to the cell cycle, DNA metabolism, and apoptosis. Together, our findings establish Sp1 as a pivotal transcriptional regulator in early vertebrate development and reveal mechanistic insights into how conserved TF networks coordinate developmental programs and context-dependent disease states. Summary statementThis study characterizes the expression profile and establishes the conserved role of SP1 in specific processes during zebrafish development, which were previously unreported.

developmental biology↗

bioRxiv2025

WRN helicase regulates mitophagy by resolving an intricate nexus of G-quadruplexes-R loops-ATG7 pre-mRNA maturation in cancer

Werner syndrome (WS) is a progeroid and cancer-predisposition disorder caused by loss of the Werner RECQ helicase-exonuclease (WRN), a key genome-maintenance enzyme essential for replication-stress signalling and DNA-repair. WS patients also develop metabolic abnormalities, including fatty-liver and diabetes, suggesting a link between WRN-deficiency and mitochondrial-dysfunction. WRN is frequently epigenetically silenced in cancers, yet its precise role in mitochondrial homeostasis in cancer remains unclear. Here, we define a role for WRN in regulating mitophagy and autophagy in cancer. WRN-deficient cells show defective mitochondrial respiration, morphology, and mitophagosome/autophagosome-maturation under basal and cisplatin-induced stress. Mechanistically, WRN-loss causes strong reduction of ATG7-protein, compromising autophagosome-biogenesis. Chromatin immunoprecipitation reveals accumulation of unresolved G-quadruplex structures (G4-DNA) across the ATG7-locus in WRN-deficient cells. Paradoxically, ATG7-mRNA expression is elevated despite reduced ATG7-protein in WRN-deficient cells, indicating a post-transcriptional defect. Further, we show that WRN resolves G4-DNA which prevent R-loops formation and interacts with the mRNA-processing factor U2AF35, independent of its helicase-exonuclease functions, to promote maturation of nascent ATG7 transcripts. In cancer patients, WRN level also inversely correlated with post-transcriptional defects in ATG7 mRNA. Collectively, our findings suggest pivotal association of WRN-loss in autophagy fidelity, which may further contribute to oncogenic transformation in WRN-deficient tissues and exacerbate cancer susceptibility in WS-patients. Significance statementWRN is well recognized for its roles in DNA repair and genome maintenance, which are essential for suppressing tumorigenesis and Werner syndrome (WS)-associated premature-aging. However, its functions in mitochondrial regulation remain underexplored, despite WS patients exhibiting severe metabolic defects and increased cancer risk. Here, we uncover a mechanistic link between WRN and autophagy/mitophagy, showing that WRN resolves G-quadruplexes and R-loops to enable proper post-transcriptional processing and translation of ATG7, a key autophagy enzyme. WRN-loss associated defective-autophagy may heighten the initiation of tumorigenesis in both WS-patients and in normal individual with mutated WRN in different tissue-types. As WRN is actively pursued as a synthetic-lethal target and multiple WRN inhibitors progress through clinical-development, our findings highlight mitochondrial quality-control defects as an additional determinant of WRN-targeted therapeutic-response.

cell biology↗

bioRxiv2025

PARP1 deficiency induces aging-associated cardiac failure via activation of Akt signalling

PARP1, a poly-ADP-ribose transferase, plays a critical role in maintaining genomic stability, transcription, cellular metabolism, and cell death. PARP1 inhibition protects cardiomyocytes against oxidative and genotoxic stress. However, the role of PARP1 in aging-associated heart failure remains poorly explored. In the current study, we report that PARP1 levels are downregulated in aging mouse hearts, and PARP1 deficiency induces aging-related cardiac remodelling and contractile dysfunction in mice. PARP1 deficient mice hearts exhibit spontaneous activation of the Akt signalling pathway, leading to the development of aging-related cardiac hypertrophy and fibrosis. Our findings reveal two distinct mechanisms by which PARP1 regulates Akt signalling, direct interaction of PARP1 with Akt and the transcriptional regulation of phosphatases like PTEN, a negative regulator of Akt signalling. PARP1 binds and inhibits Akt by poly-ADP-ribosylation at E40 and E49 residues, which impairs Akt membrane recruitment and subsequent activation. Inhibition of Akt reversed hypertrophy in PARP1-depleted cardiomyocytes and improved the contractile dysfunction in PARP1-deficient hearts. These findings reveal a previously unrecognized regulatory role for PARP1 in aging-associated cardiac failure.

physiology↗

bioRxiv2025

A clinical-stage oncology compound selectively targets drug-resistant cancers

Re-evaluating existing clinical compounds can uncover previously unrecognized mechanisms that reshape a drugs therapeutic potential. The small molecule Procaspase-Activating Compound 1 (PAC-1) entered oncology testing as a proposed activator of caspase-driven apoptosis. Here, we show that PAC-1-driven cytotoxicity occurs in the absence of executioner caspase expression, demonstrating that its anti-cancer activity occurs via an alternative mechanism. We provide genetic, biochemical, and biophysical evidence demonstrating that PAC-1 functions as a highly selective iron chelator that eliminates cancer cells by disrupting iron homeostasis. Unexpectedly, we discovered that expression of the key chemotherapy-resistance pump MDR1 confers marked hypersensitivity to PAC-1 treatment. While PAC-1 is only weakly effluxed by MDR1 under basal conditions, this process is potentiated when PAC-1 is bound to iron. Consequently, PAC-1 induces progressive iron depletion and selective cytotoxicity in otherwise drug-resistant MDR1-expressing cancer cells. Together, these findings redefine PAC-1s mechanism-of-action and establish a framework for exploiting multidrug resistance as a therapeutic vulnerability through targeted iron starvation.

cancer biology↗

bioRxiv2025

What kind of birds are more susceptible to avian malaria? A global analysis based on interpretable machine learning approach

AbstractO_ST_ABSAimC_ST_ABSAvian malaria (genus Plasmodium) is a mosquito-transmitted parasitic disease of birds. It has a wide distribution across the world, infecting more than 2,400 bird species, posing a great threat to bird health. However, over half of bird species have a cumulative sample size of fewer than 20 individuals, leading to a limited understanding of the global patterns and mechanisms of their susceptibility to avian malaria. Our aim is to use interpretable machine learning to identify the global ecological and evolutionary drivers shaping species-level avian malaria susceptibility in birds. Location Global. Time period 1994-2023. Major taxa studied Global bird species and their malaria parasites (genus Plasmodium) MethodsBased on infection data and traits from 72,406 birds of 2,544 species worldwide, we developed an interpretable machine learning (IML) approach to identify the global drivers of species-level susceptibility and their trends. We further applied our model to predict the susceptibility of bird species with a sample size of less than 30 and tested multiple hypotheses related to differences in parasite susceptibility in birds. ResultsOur model distinguished susceptible birds with moderate accuracy (F1 score = 0.72) and predicted 752 bird species to be highly susceptible to avian malaria, including 16 threatened species. Susceptibility showed a moderate but significant phylogenetic signal, with most susceptible species belonging to Passeriformes. Highly susceptible species were generally characterized by larger body size, omnivory, ground-foraging behavior, wider geographic ranges, and medium diversification rates. Main ConclusionsWe show that ecological and evolutionary factors together shape species-level susceptibility of birds to avian malaria. Interpretable machine learning integrating host traits offers a global insights into infection mechanisms and supports the development of more precise surveillance and control strategies.

ecology↗

bioRxiv2025

Molecular mechanism of naturally-encoded signaling-bias at the complement anaphylatoxin receptors

The conceptual framework of biased signaling has revolutionized our understanding of GPCR signaling and regulatory paradigms, and greatly impacted the efforts focused on the discovery of GPCR-targeted therapeutics. However, the mechanistic basis of biased signaling remains primarily defined based on synthetic ligands and receptor mutants with relatively limited progress in understanding naturally-encoded signaling-bias. Here, we present fundamental molecular and structural insights into naturally-encoded signaling-bias at the complement anaphylatoxin C5a receptors namely, C5aR1 and C5aR2. We first discover that C5a-d-Arg, the naturally-occurring version of C5a lacking the terminal arginine, exhibits robust G-protein signaling-bias at C5aR1, characterised by attenuated {beta}arr recruitment. This signaling-bias manifests in both cytokine release from primary human immune cells, and in vivo, during neutrophil mobilization. We combine the cryo-EM structures of C5a/C5a-d-Arg-C5aR1 complexes with MD simulation, site-directed mutagenesis, and cellular experiments to elucidate that the G-protein-bias exhibited by C5a-d-Arg results from a distinct orientation of TM7 and helix 8 in C5aR1 leading to inefficient GRK recruitment and receptor phosphorylation. Next, we determine the first cryo-EM structures of C5aR2, a naturally-encoded {beta}-arrestin-biased receptor, in an apo state, complexed with the natural agonists C5a and C5a-d-Arg, and three peptide agonists including a first-in-class, newly discovered C5aR2-selective agonist, R8Y. These structural snapshots reveal key differences between the binding of C5a and C5a- d-Arg to C5aR1 and C5aR2, and provide a molecular basis of functional specialization at these two receptors. Moreover, the structural insights also allow us to decipher the molecular basis of naturally-encoded signaling-bias at C5aR2 originating from a shallower cytoplasmic interface with hydrophobic interior pocket that is not permissive to efficient G-protein-coupling and activation. Finally, we also engineer and characterize loss-of-function and gain-of-function variants of C5aR1 and C5aR2, which in turn corroborate and validate the structural observations presented here. Collectively, our findings offer crucial insights into previously lacking molecular mechanisms of the naturally-encoded signaling-bias at GPCRs, which have broad implications not only for the general framework of biased-signaling, but also for novel therapeutic design.

biochemistry↗

bioRxiv2025

Atypical activation and molecular glue-like dimerization mechanism of an intrinsically-biased chemokine receptor

CXCR7, also known as atypical chemokine receptor 3 (ACKR3), is a naturally-biased, {beta}-arrestin-coupled seven transmembrane receptor (7TMR) that lacks productive coupling with heterotrimeric G-proteins. Despite a critical involvement in cancer metastasis, cardiovascular pathophysiology, and inflammatory disorders, the molecular basis of non-canonical activation and functional divergence of CXCR7 remains elusive. Here, we present a complete landscape of CXCR7 activation using a series of cryo-EM structures, and discover an atypical activation mechanism that is distinct from prototypical GPCRs. CXCR7 is maintained in a basal conformation by a unique tripartite ionic-lock involving TM5-TM6, in contrast to a broadly conserved TM3-TM6 ionic-lock in GPCRs, which is disrupted upon receptor activation. Importantly, activation of CXCR7 results in a constricted pocket and distinct surface topology on the intracellular side compared to prototypical GPCRs. Serendipitously, we capture novel dimeric arrangements of CXCR7 with an inter-protomer stitching by a native phospholipid serving as a molecular glue, and identify previously unanticipated extrahelical allosteric sites on the receptor. Surprisingly, in an intermediate state structure of CXCR7, the second extracellular loop (ECL2) displays a self-blocking conformation, in stark contrast to ECL2-mediated self-activating mechanism reported recently for some orphan GPCRs. Finally, we unequivocally establish CXCR7 as an atypical opioid receptor via a large peptide library screening and structure elucidation in complex with distinct opioid peptides imparting full receptor activation. In summary, our study elucidates an atypical mechanism of CXCR7 activation, and establishes it as an alternative, non-canonical opioid receptor target with potential for novel pain therapeutics.

biochemistry↗

bioRxiv2025

Alternative splicing of synaptotagmin 7 regulates oligomerization and short-term synaptic plasticity

Synaptic plasticity is crucial for learning and memory. The presynaptic calcium sensor synaptotagmin 7 (syt7) regulates aspects of short-term plasticity (STP), but the underlying mechanisms remain unclear. Here, we show that alternative splicing of the syt7 juxtamembrane linker acts as a molecular switch at both biochemical and functional levels. The and {beta} variants undergo liquid-liquid phase separation to form condensates, while the {gamma} variant forms aggregates. Using iGluSnFR imaging, we found that when expressed at equal levels, these three isoforms also diverge regarding their abilities to regulate two key aspects of STP: paired-pulse facilitation and synaptic depression. STED microscopy showed that all three isoforms form active zone-associated clusters that colocalize with syt1, while MINFLUX super-resolution microscopy resolved syt7 clusters within the active zone, well-positioned to directly control synaptic vesicle dynamics. Thus, alternative splicing might fine-tune STP by differentially impacting syt7 oligomerization.

neuroscience↗

bioRxiv2025

Pleckstrin homology domain-containing serine/threonine kinase plays a crucial role in the survival and phagocytosis of Entamoeba histolytica.

Kinases are critical regulators of parasite survival, cytoskeletal dynamics, and endocytic processes. Entamoeba histolytica encodes 372 kinases, many of which remain functionally uncharacterized. This study characterizes EhPHDK, an AGC-family kinase essential for pseudopod formation, phagocytosis, and pinocytosis. Structural modelling suggests an autoregulatory mechanism via its PH domain, potentially modulating kinase activity. Bio-layer interferometry identified a crucial amino acid sequence facilitating interaction with EhCaBP1, implicating EhPHDK in endocytic regulation. Live-cell imaging and immunostaining confirmed its dynamic recruitment to the plasma membrane, colocalizing with EhCaBP1 during phagocytosis initiation and progression. Gene silencing severely impaired parasite viability, reducing growth by 90%, while RBC uptake assays demonstrated its indispensable role in phagocytosis. The regulatory interplay between EhPHDK and cytoskeletal components underscores its function in actin-driven processes. Given its divergence from host kinases, EhPHDK presents a promising therapeutic target for amebiasis. This study provides novel insights into kinase-mediated cytoskeletal remodelling, advancing our understanding of E. histolytica pathogenesis and offering potential avenues for intervention in parasitic survival mechanisms. HighlightsO_LIEhPHDK kinase regulates pseudopod formation, phagocytosis, and pinocytosis. C_LIO_LIStructural modelling suggests PH domain autoregulation of kinase activity. C_LIO_LIEhPHDK interacts with EhCaBP1, influencing endocytosis and cytoskeletal dynamics. C_LIO_LIGene silencing reduces parasite survival by 90%, impairing phagocytosis. C_LIO_LIEhPHDK is a potential therapeutic target, diverging from host kinases. C_LI

cell biology↗

bioRxiv2025

A Tonic Signaling Code Predicts CAR-T Cell Efficacy in Diffuse Midline Glioma

Diffuse midline glioma (DIPG/DMG) is a uniformly fatal pediatric brain tumor with no effective cure. Although CAR T-cell therapy shows promise, clinical outcomes remain inconsistent due to limited persistence and premature exhaustion. Reliable predictive biomarkers are lacking, and proposed exhaustion or stemness markers provide limited utility. Here, we systematically compare multiple CAR-T constructs targeting clinically-relevant antigen B7-H3 and identify antigen-independent CAR activation, or tonic signaling, as a key determinant of therapeutic performance. We find that B7-H3 CAR-T cells with restrained tonic signaling display superior tumor killing, persistence, and resistance to exhaustion, along with reduced CAR membrane clustering, in patient-derived DIPG models. Integrated multi-omics and single-cell profiling further reveal a CAR-T tonic signaling-associated gene signature that outperforms conventional exhaustion or stemness markers in predicting therapeutic efficacy across multiple clinical trials, including DIPG and other tumor types. Together, these findings define a mechanistic and predictive framework to guide CAR design and improve clinical outcomes.

cancer biology↗

bioRxiv2025

Genes required for Mycobacterium tuberculosis to survive the transition from aerosol to pulmonary alveolar lining fluid and early infection in a model of transmission

Mycobacterium tuberculosis (Mtb) must survive multiple changes in environment for aerosol transmission. Our genome-wide screen for rehydration of modeled aerosols in surrogate pulmonary alveolar lining fluid identified a survival-sustaining role for 22 genes not required for survival during earlier stages of transmission, 20 of which are non-essential in routine culture. Thirteen genes were also needed for Mtb to survive in alveolar macrophages after passing through the earlier stages of transmission. Nine 9 of the 13 were needed for full infectivity of aerosols in mice. Seven of the genes sustaining the intra-alveolar survival of aerosolized Mtb are likely to regulate Mtbs uptake, catabolism or synthesis of lipids as regulated by cAMP. Our results reveal a dynamic form of conditional essentiality that only emerges after bacteria experience a sequence of other pathophysiologically relevant conditions. These findings enlarge Mtbs candidate transmission survival genome with stage-specific genes encoding potential targets for blocking tuberculosis transmission.

microbiology↗

bioRxiv2025

Nuclear Fetuin-A Drives Adipocyte Senescence via HIF-1 α During Obesity

Fetuin-A (FetA), a liver derived glycoprotein, has emerged from genome-wide association studies and epidemiological surveillance as a serum biomarker linked to obesity-driven type 2 diabetes mellitus (T2D), primarily due to its contribution to adipose tissue dysfunction. Here, we uncovered an eccentric role of nuclear FetA in visceral white adipocytes of obese T2D conditions. Hypoxia-inducible factor-1 (HIF-1) facilitates the nuclear translocation of FetA via direct interaction, a process that promotes the emergence of a senescence-associated secretory phenotype (SASP). While nuclear co-localization of FetA and HIF-1 strongly promotes adipocyte senescence, silencing FetA alone is sufficient to prevent senescence, even in conditions of HIF-1 overexpression or lipid-rich hypoxic stress. Although nuclear FetA does not directly bind to DNA, it enhances HIF-1 transcriptional activity, potentiating the activation of senescence markers such as {beta}-galactosidase and p53. Selective knockdown of FetA in obese mice notably reduced adipocyte senescence in visceral white adipose tissue (vWAT) and improved fasting glycemic control. Collectively, our findings reveal a previously unrecognized nuclear function for FetA in orchestrating adipocyte senescence in obesity, establishing nuclear FetA as a potential therapeutic target for obesity related metabolic diseases.

cell biology↗

bioRxiv2025

Breast cancer cell migration is potentiated by associated fibroblasts through a laminin-511-driven cortical localization of Arp2/3

Invading cancer cells are intermingled with fibroblasts that interact with them to regulate their migration, although the precise motility-driving signaling mechanisms initiated upon such interactions remain ill-understood. We constitute time-lapse-tractable 3D pathotypic cultures with breast cancer cell lines with fibroblasts enriched from invasive ductal carcinoma biopsies. Herein, cancer-associated fibroblasts (CAFs) enhanced breast cancer migration relative to non-cancerous fibroblasts (NFs), also observed through greater infiltration of orthotopically injected cancer cells within murine inguinal lymph nodes. The enhancement was also seen in cancer cells exposed to CAF-secreted medium. CAFs phenocopied laminin-rich matrix in compacting cancer cell clusters. A subsequent immunocytochemical screen identified laminins 5, -{beta}1, and -{gamma}1 to be highly expressed in enriched CAFs populations as well as in stromal areas of breast cancer sections. Depleting laminin-5, -{beta}1, and -{gamma}1 in CAFs downregulated 3D cancer cell migration. Cancer cells cultivated on Laminin-511 substrata showed increased adhesion, faster and persistent migration, enhanced shape polarization, and deformability of cancer cells relative to Laminin-211 controls. Observation of higher invadopodial F-actin dynamics on Laminin-511 led us to assay for and demonstrate anisotropically corticalized Arp 2/3 in cancer cells. An integrin antibody screen showed Integrin 6{beta}1 inhibition specifically nullified Laminin-511-driven enhancement of morphomigrational traits of cancer cells, similar to Arp2/3 inhibition. Moreover, cells on Laminin-511 showed enhanced Integrin 6 localization to their invadopodia. We propose that activated fibroblasts use Laminin-511 to localize cognate integrin receptors, and Arp2/3 of cancer cells to their invadopodia, resulting in higher Arp2/3-driven actin remodeling and enhanced cell migration.

cancer biology↗

bioRxiv2025

The fate of horizontally acquired genes: rapid initial turnover followed by long-term persistence

A major driver of bacterial evolution is horizontal gene transfer (HGT), the acquisition of genes from other strains or species. Transfers between closely related taxa are more likely to succeed, while the pervasive deletion bias of bacterial genomes drives frequent turnover of horizontally acquired genes. However, whether the rate of gene loss after acquisition is constant across lineages or time remains unclear. Here, we analyze a comprehensive dataset of bacterial genomes to infer the frequency, distribution, and retention of inter-phylum HGT events. The retention of inter-phylum gene transfers is highly skewed, with only a small subset of bacterial genomes accounting for the majority of such events. Most transferred genes are lost rapidly. Those genes that survive the initial purging are retained over long periods, are biased toward functions such as transport and metabolism, and have larger numbers of protein-protein interactions.

evolutionary biology↗

bioRxiv2025

Ceramide transfer protein regulates G-protein coupled phospholipase signalling in Drosophila photoreceptors.

The non-vesicular transfer of lipids between organelles at membrane contact sites (MCS) has been proposed as a key principle in the regulation of cell physiology. While several proteins with lipid transfer activity have been identified and localized to MCS, their functional significance for supporting physiology is poorly understood. Ceramide transfer protein (CERT) is one such molecule that can transfer ceramide between membranes in vitro. However, evidence for the mechanism and in vivo significance of CERT function is limited. In this study, we have analyzed the function of the only gene (dcert) encoding CERT in Drosophila. We find that loss of function alleles of dcert (dcert1), show elevated levels of short chain ceramide species along with a reduction in the levels of its metabolite phosphatidyl ethanolamine ceramide. Physiological analysis of dcert1 mutant alleles showed reduced electrical responses in the eye to light stimulation although photoreceptors did not undergo retinal degeneration, and this phenotype could be rescued by reconstitution of dcert1 with the wild type gene. The altered light response in dcert1 was associated with a reduction in the rate of phosphatidylinositol 4,5 bisphosphate (PIP2) resynthesis following light induced phospholipase C (PLC) stimulation. The reduced electrical response of dcert1 could be suppressed by reducing ceramide synthesis at the ER. Taken together, our findings suggest that ceramide synthesized at the ER and transferred to the Golgi by CERT regulates G-protein coupled phospholipase C signaling in vivo.

cell biology↗

bioRxiv2025

On-target toxicity limits the efficacy of CDK11 inhibition against cancers with 1p36 deletions

The cyclin-dependent kinase CDK11 is an understudied kinase that has been the subject of conflicting reports regarding its function in cancer. Here, we combine genetic and pharmacological approaches to demonstrate that CDK11 is a critical regulator of cancer cell survival that is required for RNA splicing and the expression of homologous recombination genes. Inhibition of CDK11 disrupts genome stability, promotes the retention of intronic sequences in mature mRNAs, and induces synthetic lethality with PARP inhibitors. Through integrative analysis of functional genomics datasets, we identify heterozygous deletions of chromosome 1p36 - which encompasses CDK11 and its activating cyclin CCNL2 - as a recurrent and predictive biomarker of sensitivity to CDK11 inhibition. To assess the therapeutic potential of CDK11, we develop MEL-495R, a selective and orally bioavailable CDK11 inhibitor. Additionally, we establish a genetically-engineered mouse model that allows us to differentiate between the on-target and off-target effects of CDK11 inhibitors in vivo. Using this platform, we demonstrate that MEL-495R induces widespread on-target toxicity, revealing a narrow therapeutic index. Together, these findings define CDK11 as a core cancer dependency, uncover a chromosomal deletion that sensitizes tumors to CDK11 inhibition, and provide a generalizable strategy for deconvolving drug efficacy and toxicity in vivo for novel oncology targets.

cancer biology↗

bioRxiv2025

Promoter evolution in HIV-1C establishes latent reservoirs highly resistant to reversal

Latent viral reservoirs remain a major barrier to curing HIV-1, with the long-terminal repeat (LTR) and Tat playing crucial roles in regulating viral transcription. Subtype-specific transcription factor binding site (TFBS) variations within the LTR significantly influence latency and reservoir stability. In earlier work, we identified HIV-1C LTR variants with duplicated TFBS motifs, including NF-{kappa}B, AP1, RBEIII, and TCF-1/LEF-1. Using five cell models, including Jurkat and primary CD4 T cells, we compared canonical R-LTR and variant R2-LTR strains. Across sub-genomic reporters, single-round infections, and full-length viral vectors, we found that the balance between RBEIII and NF-{kappa}B motifs governs stability of latency. The two-viruses-one-cell system that normalized confounding environmental factors further revealed that latency is primarily controlled by intrinsic transcriptional circuits rather than external stimuli. In longitudinal studies of HIV-1 individuals from acute and chronic infection phases, we observed dominant R strains during early infection and the spontaneous emergence of R2 strains in nearly half of chronic-phase subjects, a process accelerated by ART. Upon CD4 T cell activation, R strains preferentially rebounded, while R2 strains showed strong resistance to reversal, even in subjects harbouring a co-infection. Together, these findings establish the clinical significance of LTR variation in latency regulation and identify the R2 phenotype as a critical determinant of reservoir persistence. These results underscore the importance of addressing reservoir heterogeneity in cure strategies, particularly in HIV-1C-prevalent regions.

microbiology↗

bioRxiv2025

Membrane curvature during membrane rupture and formation of pentagonal pyramidal superassemblies by a pore-forming toxin, Vibrio cholerae Cytolysin, using single particle cryo-EM

In this cryo-electron microscopy study, we provide mechanistic insights into how an archetypical {beta}-barrel pore-forming toxin ({beta}-PFT), Vibrio cholerae Cytolysin (VCC), ruptures the membrane lipid bilayer by inducing membrane curvature. We demonstrate how VCC oligomers cluster together and drastically increase local membrane curvature, thereby causing membrane blebbing. In addition, we also show how these PFTs after rupturing the host membrane tend to form symmetric super-molecular assemblies to stabilize their hydrophobic transmembrane rim domains. We further provide another example of membrane rupture with gamma hemolysin, a Staphylococcal bicomponent {beta}-PFT. These insights will usher new studies on membrane curvature due to protein crowding and broaden our mechanistic understanding of how this largest class of bacterial protein toxins induce host cellular death.

biophysics↗