Biology subjects

Verma, N.

Publications and source records attributed to Verma, N..

At least 19 recordsLinked to original sources

bioRxiv2026

Structural mechanism governing radiationless energy transfer in Renilla bioluminescence

The nonradiative transport of electronic excitation from one chromophore to another, known as resonance energy transfer, lies at the root of photochemical processes in biology. Unlike photosynthesis, bioluminescence converts chemical energy into light through an enzymatic oxygenation of an energy-rich luciferin. In glowing cnidarians, the energy is relocated from an excited oxyluciferin to a fluorescent protein, shifting the colour and enhancing the quantum yield of a photogenic reaction. How protein-chromophore complexes assemble during this interplay in real space, and what this association entails for function, are unknown. Here, we report co-crystal structures of a 120-kilodalton energy-transfer complex from the luminescent soft coral Renilla reniformis. We find a heterotetrameric 2:2 assembly composed of two coelenteramide-loaded luciferases (RrLuc) docked at opposite sides of a head-to-tail dimer of green fluorescent protein (RrGFP). The edge-to-edge distance between donor and acceptor chromophores is below 3 nm, favouring the Forster-type radiationless energy transfer. Furthermore, RrGFP serves not only as a colour-switchable antenna and luminescence amplifier but also tunes the efficiency of luciferase catalysis by controlling its inherent dynamics. Our results provide detailed spatial information about intermolecular dipole-dipole coupling in Renilla bioluminescence, including the arrangement of donor-acceptor pairs that secure excited-state energy transfer with exquisite precision.

biochemistry↗

bioRxiv2026

Open or closed? Exploring the conformational heterogeneity of human plasminogen across multiple resolution scales

Plasminogen (Plg), the zymogen of the fibrinolytic protease plasmin, is a multidomain, conformationally-rich protein that plays a crucial role in targeted thrombolysis for stroke and heart attack treatments. Yet, the conformational state that affects binding to Plg activators and fibrinolytic activity is rarely validated, contributing to poor reproducibility and failures in drug development. Here, we establish a structural biology workflow to characterize human Plg suspended in an aqueous solution, preserving it in a near-native state and eliminating the need for crystal growth. Two variants, cleavage-resistant Plg-RV (R561A) and catalytically inactive Plg-CAT (S741A), were expressed in mammalian cells. Both variants were correctly folded and stable (Tm {approx} 60.6{degrees}C) and, unlike commercial plasma-derived Plg, were resistant to staphylokinase-mediated activation. Small-angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry revealed substantial conformational heterogeneity. The recombinantly-produced Plg variants adopted a closed conformation, exhibiting a good fit to the closed structure determined by X-ray diffraction. Conversely, the plasma-derived Plg populated an extended, open-like state. Cryo-EM analysis of the Plg-RV variant yielded a 4.4 [A] resolution map, and a rigid-body-fitted model revealed the closed-state architecture. Our findings demonstrate that rigorous structural validation of Plg is essential for future functional studies and rational development of next-generation thrombolytic agents.

biochemistry↗

bioRxiv2026

Molecular Alterations of Bovine Serum Albumin Induced by the Food Dye Acid Yellow 23: A Mechanistic Study

The widespread use of synthetic food dyes, such as Acid Yellow 23 (AY 23), in the food, cosmetics, and pharmaceutical industries raises questions about their potential effects on biological systems and public health. The concentration-dependent interaction between AY 23 and bovine serum albumin (BSA), a crucial model protein for understanding pharmacokinetics and protein-ligand behaviour, was examined in this study. We demonstrate that, under physiological conditions, increasing dye concentrations from 50 {micro}M to 200 {micro}M results in notable conformational changes, increased surface hydrophobicity, and protein aggregation using a multimodal biophysical approach that includes fluorescence spectroscopy. Direct visualisation verified these structural changes and aggregate formation, whereas hemolytic assay confirmed the high hemolytic nature of AY 23-induced fibrils. Additionally, this study provides a mechanistic basis for the toxicological effects of AY 23, underscoring the implications of food dyes for public health. HighlightsO_LIAcid Yellow 23 (AY 23) modulates Bovine Serum Albumin (BSA) structure and leads to aggregation under physiological conditions. C_LIO_LIStructural alteration is followed by binding of AY 23 at the hydrophobic regions of BSA, perturbing the globular protein into fibrillar aggregates. C_LIO_LI{middle dot} Confirmational changes induced by AY 23 in the BSA via interaction with Asp108, Pro110 and Ala193. C_LIO_LIFibrils formed after AY 23 interaction are observed to be hemolytic in nature. C_LIO_LIMolecular mechanism of AY 23-mediated fibrillation of BSA was assessed. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/737154v2_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@241c95org.highwire.dtl.DTLVardef@d0a03aorg.highwire.dtl.DTLVardef@c7445borg.highwire.dtl.DTLVardef@58cf7c_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

Cervical spinal cord stimulation disrupts proprioception yet improves voluntary arm reaching

Whether proprioception is necessary for upper-limb motor control has been debated for decades. Classic studies in deafferented animals and humans suggested that proprioception may be dispensable for rapid, goal-directed movements. However, chronic sensory loss conflates the absence of proprioceptive input with years of compensatory adaptation. As a result, the field has lacked a strong causal test of proprioceptions contribution to motor control. Here, we leveraged a clinical trial of cervical spinal cord stimulation (SCS) in individuals with chronic post-stroke hemiparesis to study if electrical stimulation of sensory afferents causally perturbs proprioception and affects arm reaching. We found that turning SCS ON causally impaired proprioceptive perception and postural stabilization in response to force perturbations, and enhanced adaptation to visual errors during implicit learning. Yet visually and non-visually rapid, goal-directed reaching improved in smoothness, straightness and spatial accuracy. These findings provide strong causal evidence that proprioception is not required for rapid, goal-directed action, helping resolve a decades-long debate regarding its necessity for effective movement.

bioengineering↗

bioRxiv2026

LizardLens: A Two-Stage Deep Learning Pipeline for Detecting and Classifying Similar Species in Visually Complex Environments

Community science platforms like iNaturalist generate unprecedented volumes of biodiversity data, but their scientific utility depends critically on accurate species identification--a persistent challenge when contributors often lack taxonomic expertise. We developed "LizardLens", a two-stage machine learning pipeline that decouples object detection from species classification to enable fine-grained identification of morphologically similar organisms in visually complex field photographs. Using 10,000 verified iNaturalist images of five Anolis lizard species in Florida, we trained specialized YOLO-based detection and Swin Transformer classification models and compared performance against state-of-the-art single-stage architectures. Our two-stage pipeline achieved 83.0% Top-1 accuracy and a macro-averaged F1-score of 89.0%, indicating strong precision-recall performance across species and outperforming single-stage YOLOv8 and YOLOv12 models across all evaluation metrics for all species, with relative improvements ranging from 10.5% to 13.2%. Gradient-weighted Class Activation Mapping (Grad-CAM) indicated that the models predictions were consistently associated with regions corresponding to diagnostic morphological (e.g., head shape, feet, and limb lengths) and pattern features (e.g., ocular rings and body patterning), providing evidence that LizardLens leverages biologically relevant visual cues consistent with those used by expert taxonomists. Error analysis identified partial occlusion and multiple proximate individuals as primary sources of missed detections, while spurious detections of lizard-like environmental features (e.g., sticks, bark) represented the dominant false positive error mode. We deployed LizardLens as an accessible web application featuring interactive bounding box correction, ranked species predictions with confidence scores, directly supporting the "Lizards on the Loose" middle school community science initiative. By combining technical advances in fine-grained visual classification with user-centered design, LizardLens demonstrates how machine learning can simultaneously enhance data quality for biodiversity monitoring and provide authentic scientific experiences for student participants. Our approach is generalizable to other small-bodied organisms in complex habitats and provides a framework for translating computer vision advances into practical tools for community science and conservation.

ecology↗

bioRxiv2026

Uncovering Functional Distant Mutations by Ultra-High-Throughput Screening of Dehalogenases

Conformational dynamics play a central role in enzyme function by controlling substrate access and productive binding. Yet mutations that beneficially modulate these properties are difficult to identify. Here, we used ultrahigh-throughput fluorescence-activated droplet sorting (FADS) with a bulky fluorogenic substrate derived from coumarin (COU-3) to impose steric selection pressure on the haloalkane dehalogenase LinB. Screening a focused library yielded five single substitutions located 11.5-15.5 [A] from the catalytic centre. Variant I138N showed a fourfold increase in catalytic efficiency toward COU-3 through reduced KM and increased kcat, associated with increased cap-domain flexibility and facilitated substrate entry. In contrast, variant P208S markedly reduced substrate inhibition and shifted specificity toward bulkier iodinated haloalkanes by reshaping its tunnel environment. Integrated kinetic and structural analyses revealed that screening with bulky substrates directs selection toward distal regions controlling substrate access and unproductive binding. These findings demonstrate that ultrahigh-throughput FADS can reveal dynamic mechanisms of enzyme adaptation that remain difficult to predict by rational design. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/713925v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@782038org.highwire.dtl.DTLVardef@8b43f3org.highwire.dtl.DTLVardef@11a403eorg.highwire.dtl.DTLVardef@6fcaea_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Investigating the Conformational Flexibility of Staphylokinase Across Multiple Time Scales

Cardiovascular diseases, including ischemic stroke, necessitate improved thrombolytic agents. A microbe-encoded plasminogen activator staphylokinase (SAK) is a promising alternative to the widely used tissue plasminogen activator (tPA) due to its high fibrin specificity and low production cost. To overcome potential immunogenicity hampering its use in clinical settings, the low-immunogenic variants SAK SY155 and SAK THR174 were previously engineered. However, the molecular basis underlying their reduced immunogenicity is not understood and requires detailed elucidation. Here, we determine molecular structures and compare flexibility between low-immunogenic and immunogenic SAK variants, using a combination of experimental and computational structural techniques. Our analyses show that all variants share the canonical SAK fold and retain similar plasminogen activation kinetics, despite the number of introduced substitutions. Crucially, the low-immunogenic variants exhibit distinct flexibility profiles, with SAK THR174 showing substantially increased flexibility in the H1 helix and B3 region. SAK SY155 exhibits an increased flexibility in the H1-B3 loop and propensity to homodimerize. These flexibility changes are found in the known immunogenic epitopes. Our multi-scale flexibility analysis provides the molecular explanation for the reduced immunogenicity, altered thermostability, and retained fibrinolytic function of the engineered variants. This information is critical for the design of next-generation thrombolytics.

biochemistry↗

bioRxiv2026

Enabling Skilled Human-Computer Interaction After Paralysis via a Wearable sEMG Interface

Most individuals with tetraplegia retain some myoelectric function in their forearms, which offers the possibility of using surface electromyographic (sEMG) control for human-computer interaction (HCI). We demonstrate the potential of this approach by showing that people with motor-complete (n=5) and motor-incomplete (n=2) tetraplegia can accurately control myoelectric activity in their forearm to perform discrete button-click and continuous positioning tasks. These control inputs were mapped to the firing rate of motor units detected by a wireless wristband sensor designed for everyday use. Participants completed four testing sessions to assess their speed and accuracy. Motor units that displayed a wide dynamic range in their firing rate performed best during tasks requiring continuous, single-axis control. Interestingly, the level of impairment did not affect performance on the clicking and 1D cursor control tasks. However, those with motor-incomplete injuries showed greater independent control over two motor units than participants with motor-complete injuries, who exhibited stronger coupling between units. Participants also confirmed the practical utility of the device, successfully placing and removing the sEMG wristband on their own and consistently rating it as comfortable and easy to manage. These findings are significant because they offer the first demonstration of motor unit-based control in individuals with cervical spinal cord injury (SCI) using a fully wearable wristband interface, highlighting the feasibility of moving these systems out of the lab and into daily life. One-Sentence SummaryPeople with tetraplegia used a wristband sensor to detect forearm motor unit firing and perform human-computer interaction tasks.

bioengineering↗

bioRxiv2026

CAR-T-drug conjugate against solid tumor

Chimeric antigen receptor (CAR)-T cell therapy has been clinically successful in hematologic cancers but faces challenges in solid tumors, primarily due to limited tumor infiltration, immunosuppressive tumor microenvironments (TME), and antigen heterogeneity. While combining CAR-T cell therapy with chemotherapy can enhance antitumor activity, this often leads to substantial systemic toxicity. In this study, we introduce CAR-T-drug conjugate (CAR-T-D-C), a new class of dual-functional therapeutics that effectively addresses these obstacles by integrating click chemistry for precise conjugation of cytotoxic agents onto antigen-specific CAR-T cells. By transforming the hostile TME into an ally, this approach facilitates localized delivery of cytotoxic payload directly to the tumor site, enhancing the overall effectiveness of CAR-T therapy in solid tumors. CAR-T-D-Cs with different CAR-T cell binders exhibit robust antitumor activity across diverse solid tumor models, including both human tumor xenografts and syngeneic models. Spatial transcriptomic studies reveals that CAR-T-D-C achieves improved CAR-T tumor infiltration and functional activation within the TME. Compared to conventional CAR-T therapy, CAR-T-D-C markedly enhances immune cell infiltration, augments effector functions, promotes antigen spreading, amplifies systemic immune responses, and improves overall anti-tumor immunity. CAR-T-D-C represents a versatile therapeutic concept that combines the potency of small molecule drugs and the specificity of CAR-T cells as a 2-in-1 immunochemotherapy for treatment of solid tumors.

immunology↗

bioRxiv2025

Crosstalk Between Phosphorylation and Ubiquitination Controls the Fate and Function of Strigolactone Signal Transducers

Strigolactones (SL) are pivotal plant hormones that sculpt plant architecture by modulating shoot branching, root development, and meristem activity. While transcriptional responses downstream of SL perception have been well explored, the role of post-translational regulation fine-tuning these responses remains less understood. In this study, we identify a dual-layered regulatory module involving MPK4-mediated phosphorylation and MAX2-dependent ubiquitination that synergistically control the stability and function of BRC1, a key SL-responsive transcription factor. Phosphorylation by MPK4 stabilizes BRC1, enhancing its activity and SL sensitivity, whereas loss of phosphorylation leads to BRC1 degradation and functional inactivation. BRC1, in turn, directly activates MPK4 transcription, establishing a positive feedback loop that amplifies SL signaling. Genetic analyses of the brc1-2 x mpk4 double mutant reveals phenotypic defects and SL insensitivity additive to those observed in the brc1-2 and mpk4 single mutants, indicating that MPK4 and BRC1 act in parallel yet converging pathways downstream of SL. Additionally, MAX2 functions as a regulatory checkpoint that degrades non-phosphorylated MPK4 and BRC1, thereby resetting the signaling circuit to ensure accurate and timely response. Together, these findings illuminate a finely-tuned regulatory module integrating phosphorylation and ubiquitination to control the intensity and duration of SL responses, suggesting a model for hormone-driven developmental plasticity in plants.

molecular biology↗

bioRxiv2025

Taurine Inhibits Apolipoprotein E4 Aggregation

Apolipoprotein E4 (ApoE4) is a major genetic risk factor in many neurodegenerative diseases, yet effective therapeutic strategies targeting its associated pathologies remain unresolved. The aggregation of ApoE4, a key pathological feature, can be attenuated by tramiprosate and its metabolite 3-sulfopropanoic acid. In this study, we investigated the potential of taurine, a close chemical analogue of tramiprosate, to modulate ApoE4-mediated pathological processes. Using an integrated approach--including molecular dynamics simulations, static light scattering, mass spectrometry, and cerebral organoid models--we investigated taurines effects on ApoE4 aggregation. We found that taurine effectively inhibits ApoE4 aggregation. Notably, taurine significantly ameliorates the pathophysiological characteristics of ApoE4, bringing its phenotype closer to the more benign ApoE3 variant. By leveraging its neuroprotective properties, taurine may offer effects comparable to tramiprosate and 3-sulfopropanoic acid, positioning it as an accessible and promising candidate for mitigating neurodegeneration, particularly in individuals with the high-risk ApoE4/E4 genotype.

biochemistry↗

bioRxiv2025

Spatial Analysis of Placentae During Congenital Cytomegalovirus Infection Reveals Distinct Cellular Profiles in Immune Cells and Trophoblasts

Withdrawal StatementThe authors have withdrawn the manuscript because the data it contained has been updated. Consequently, they request that this work should not be cited as a reference for any projects to ensure the use of the most current information. For any questions or further clarifications, readers are encouraged to contact the corresponding author directly

immunology↗

bioRxiv2024

TCPGdb: A comprehensive T cell perturbation genomics database for identification of critical T cell regulators

CAR-T therapies utilizing T cells engineered with chimeric antigen receptors (CARs) have revolutionized the treatment of hematologic and immune-related malignancies. However, the anti-tumor capability of engineered CAR-T cells are often not persistent, which greatly dampen its clinical efficacy. To address this limitation, massive parallel genetic screens were widely used to identified novel targets and regulators that enhance T cell anti-tumor capability and persistence in tumor microenvironment. We hypothesized that by combining the pooled screen data from multiple independent genetic screens we could provide a systematic, comprehensive, and robust analysis of the effect of gene perturbation on T-cell based immunotherapies. After collecting data from previously published T cell screens, including CRISPR-based and ORF-based screens, through Gene Expression Omnibus (GEO), we reprocessed the gene hits summary and conducting a pathway enrichment analysis. A T cell screen perturbation score (TPS) was employed to quantifies the impact of the gene on T cell function. Additionally, gene expression data (both bulk RNA level and single-cell RNA level) from autoimmune disease and T cell-derived cancers were analyzed to pick up gene perturbations that potentially augment T cell proliferation. We integrated all data and analysis on 27 T-cells screens into our state-of-the-art T cell perturbation genomics database (TCPGdb), which is made easily accessible through our webserver and allows users to interactively explore the impact of query genes on T cell function based on prior screen data and our TPS scoring module. TCPGdb is publicly accessible at http://tcpgdb.sidichenlab.org/.

bioinformatics↗

bioRxiv2024

Widespread Prevalence of CD19 Exon 5-6 Skipping In Indian Pediatric B-cell Acute Lymphoblastic Leukemia Patients

B-cell acute lymphoblastic leukemia (B-ALL) is characterized by the malignant burgeoning of abnormal B-cell lymphoblasts. In recent years, the use of CART therapy which targets CD19 antigen present on the surface of B-cells, has gained significant attention as a treatment option against aggressive and refractory forms of B-ALL. However, the loss of CD19 antigen on B-cell surface due to aberrant splicing under therapy pressure has been suggested as one of the main factors for the emerging CART therapy resistance. Herein, using RT-PCR based splice assays we examined CD19 splicing patterns in 43 primary pediatric B-ALL patient samples spread across various subtypes. We observed that CD19 isoform lacking exon 5-6 exists in [~] 55% of pediatric patients at the initial diagnosis stage itself. Using in-silico analysis, we identified RNA binding proteins, RC3H1 and MBNL1, as potential regulators of exon 5-6 skipping. Furthermore, qRT-PCR analysis in patient samples revealed that RC3H1 and MBNL1 are significantly upregulated in samples exhibiting exon 5-6 skipping. Taken together, we for the first time report the existence of aberrantly spliced CD19 isoform lacking exon 5-6 in primary pediatric patients, and this occurrence could potentially result from RC3H1 and MBNL1 dysregulation.

cancer biology↗

bioRxiv2024

Sensitive detection of synthetic response to cancer immunotherapy driven by gene paralog pairs

Emerging immunotherapies such as immune checkpoint blockade (ICB) and chimeric antigen receptor T-cell (CAR-T) therapy have revolutionized cancer treatment and have improved the survival of patients with multiple cancer types. Despite this success many patients are unresponsive to these treatments or relapse following treatment. CRISPR activation and knockout (KO) screens have been used to identify novel single gene targets that can enhance effector T cell function and promote immune cell targeting and eradication of tumors. However, cancer cells often employ multiple genes to promote an immunosuppressive pathway and thus modulating individual genes often has a limited effect. Paralogs are genes that originate from common ancestors and retain similar functions. They often have complex effects on a particular phenotype depending on factors like gene family similarity, each individual genes expression and the physiological or pathological context. Some paralogs exhibit synthetic lethal interactions in cancer cell survival; however, a thorough investigation of paralog pairs that could enhance the efficacy of cancer immunotherapy is lacking. Here we introduce a sensitive computational approach that uses sgRNA sets enrichment analysis to identify cancer-intrinsic paralog pairs which have the potential to synergistically enhance T cell-mediated tumor destruction. We have further developed an ensemble learning model that uses an XGBoost classifier and incorporates features such as gene characteristics, sequence and structural similarities, protein-protein interaction (PPI) networks, and gene coevolution data to predict paralog pairs that are likely to enhance immunotherapy efficacy. We experimentally validated the functional significance of these predicted paralog pairs using double knockout (DKO) of identified paralog gene pairs as compared to single gene knockouts (SKOs). These data and analyses collectively provide a sensitive approach to identify previously undetected paralog pairs that can enhance cancer immunotherapy even when individual genes within the pair has a limited effect.

bioinformatics↗

bioRxiv2024

Epidural Spinal Cord Recordings (ESRs): Sources of Artifact in Stimulation Evoked Compound Action Potentials

IntroductionEvoked compound action potentials (ECAPs) measured using epidural spinal recordings (ESRs) during epidural spinal cord stimulation (SCS) can help elucidate fundamental mechanisms for the treatment of pain, as well as inform closed-loop control of SCS. Previous studies have used ECAPs to characterize the neural response to various neuromodulation therapies and have demonstrated that ECAPs are highly prone to multiple sources of artifact, including post-stimulus pulse capacitive artifact, electromyography (EMG) bleed-through, and motion artifact resulting from disturbance of the electrode/tissue interface during normal behavior. However, a thorough characterization has yet to be performed for how these sources of artifact may contaminate recordings within the temporal window commonly used to determine activation of A-beta fibers in a large animal model. MethodsWe characterized the sources of artifacts that can contaminate the recording of ECAPs in an epidural SCS swine model using the Abbott Octrode lead. Muscle paralytics were administered to block muscle activation preventing EMG from contaminating the recorded ECAPs. Concurrent EMG recordings of the longissimus, a long muscle of the back, were used to confirm a 2-4 millisecond (ms) latency source of EMG bleed-through that frequently contaminated the A-beta temporal window. Additionally, we obtained recordings approximately 5-10 minutes post-mortem after clear evoked A-beta and associated EMG responses ceased to characterize the representation of stimulation artifact across the array. ResultsSpinal ECAP recordings can be contaminated by capacitive artifact, short latency EMG from nearby long muscles of the back, and motion artifact from multiple sources. In many cases, the capacitive artifact can appear nearly identical in duration and waveshape to evoked A-beta responses. These sources of EMG can have phase shifts across the electrode array, very similar to the phase shift anticipated by propagation of an evoked A-beta fiber response across the array. This short latency EMG is often evident at currents similar to those needed to activate A-beta fibers associated with the treatment of pain. Changes in cerebrospinal fluid between the cord and dura, and motion induced during breathing created a cyclic oscillation in all evoked components of the recorded ECAP signal. ConclusionCareful controls must be implemented to accurately separate neural signal from the sources of artifact in spinal cord ECAPs. To address this, we suggest experimental procedures and associated reporting requirements necessary to disambiguate the underlying neural response from these confounds. These data are important to better understand the conceptual framework for recorded ESRs, with components such as ECAPs, EMG responses and artifacts, and have important implications for closed-loop control algorithms to account for transient motion such as postural changes and cough.

neuroscience↗

bioRxiv2024

Genome scale CRISPR screens identify actin capping proteins as key modulators of therapeutic responses to radiation and immunotherapy

Radiotherapy (RT), is a fundamental treatment for malignant tumors and is used in over half of cancer patients. As radiation can promote anti-tumor immune effects, a promising therapeutic strategy is to combine radiation with immune checkpoint inhibitors (ICIs). However, the genetic determinants that impact therapeutic response in the context of combination therapy with radiation and ICI have not been systematically investigated. To unbiasedly identify the tumor intrinsic genetic factors governing such responses, we performed a set of genome-scale CRISPR screens in melanoma cells for cancer survival in response to low-dose genotoxic radiation treatment, in the context of CD8 T cell co-culture and with anti-PD1 checkpoint blockade antibody. Two actin capping proteins, Capza3 and Capg, emerged as top hits that upon inactivation promote the survival of melanoma cells in such settings. Capza3 and Capg knockouts (KOs) in mouse and human cancer cells display persistent DNA damage due to impaired homology directed repair (HDR); along with increased radiation, chemotherapy, and DNA repair inhibitor sensitivity. However, when cancer cells with these genes inactivated were exposed to sublethal radiation, the persistence of DNA damage promoted activation of the STING pathway, induction of inhibitory CEACAM1 ligand expression and resistance to CD8 T cell killing. Patient cancer genomics analysis reveals an increased mutational burden in patients with inactivating mutations in CAPG and/or CAPZA3, at levels comparable to other HDR associated genes. There is also a positive correlation between CAPG expression, CD8 T cell tumor infiltration and response to immunotherapy among patient datasets. Our results unveil the critical roles of actin binding proteins for efficient HDR within cancer cells and demonstrate a previously unrecognized regulatory mechanism of therapeutic response to radiation and immunotherapy.

genetics↗

bioRxiv2023

The five homologous CiaR-controlled Ccn sRNAs of Streptococcus pneumoniae modulate Zn-resistance.

Zinc is a vital transition metal for Streptococcus pneumoniae, but is deadly at high concentrations. In S. pneumoniae, elevated intracellular free Zn levels result in mis-metallation of key Mn-dependent metabolic and superoxide detoxifying enzymes resulting in Zn intoxication. Here, we report our identification and characterization of the function of the five homologous, CiaRH-regulated Ccn sRNAs in controlling S. pneumoniae virulence and metal homeostasis. We show that deletion of all five ccn genes (ccnA, ccnB, ccnC, ccnD, and ccnE) from S. pneumoniae strains D39 (serotype 2) and TIGR4 (serotype 4) causes Zn hypersensitivity and an attenuation of virulence in a murine invasive pneumonia model. We provide evidence that bioavailable Zn disproportionately increases in S. pneumoniae strains lacking the five ccn genes. Consistent with a response to Zn intoxication or relatively high intracellular free Zn levels, expression of genes encoding the CzcD Zn exporter and the Mn-independent ribonucleotide reductase, NrdD-NrdG, were increased in the {Delta}ccnABCDE mutant relative to its isogenic ccn+ parent strain. The growth inhibition by Zn that occurs as the result of loss of the ccn genes is rescued by supplementation with Mn or OxyraseTM, a reagent that removes dissolved oxygen. Lastly, we found that the Zn-dependent growth inhibition of the {Delta}ccnABCDE strain was not altered by deletion of sodA, whereas the ccn+{Delta}sodA strain phenocopied the {Delta}ccnABCDE strain. Overall, our results indicate that the Ccn sRNAs have a crucial role in preventing Zn intoxication in S. pneumoniae. AUTHOR SUMMARYZn and Mn are essential micronutrients for many bacteria, including Streptococcus pneumoniae. While Zn performs vital structural or catalytic roles in certain proteins, in excess, Zn can inhibit Mn uptake by S. pneumoniae and displace, but not functionally replace Mn from key enzymes including superoxide dismutase A (SodA). Here, we show that the Ccn small regulatory RNAs promote S. pneumoniae resistance to Zn intoxication. Furthermore, we demonstrate that these small regulatory RNAs modulate the ability of S. pneumoniae to cause invasive pneumonia. Altogether, these findings reveal a new layer of regulation of S. pneumoniae Zn homeostasis and suggest that there are factors in addition to known transporters that modulate intracellular, bioavailable Zn levels.

microbiology↗