Biology subjects

Sharma, S.

Publications and source records attributed to Sharma, S..

At least 127 records · Page 7Linked to original sources

bioRxiv2025

Antifungal therapeutic potential of Candida albicans Fun30: screening and validation of novel inhibitors against Candida albicans

WHO has listed Candida albicans as a fungal pathogen of priority. With the increasing incidence of C. albicans infection and limited treatment strategies, the identification of new drug targets and inhibitor molecules is of global concern. ATP-dependent chromatin remodelling proteins play a crucial role in regulating gene expression. Fun30 from C. albicans (CaFun30) mediates DNA end-resection during DNA double-strand break repair. It also transcriptionally co-regulates the expression of DNA damage response genes and genes involved in white-opaque switching. The gene is not essential for viability; however, the null mutant shows increased sensitivity to genotoxic agents. We hypothesised that the protein could be a potential therapeutic target against C. albicans. The structural model of CaFun30 was generated using homology modelling. Screening of a small molecule library identified 10 potential molecules, of which F1853-0039 was found as the lead inhibitor candidate. In vitro studies showed that the molecule had an MIC in the micromolar range against C. albicans with minimal effect on human THP-1 cells. Finally, coculture experiments showed that the inhibitor molecule prevented the formation of hyphae of C. albicans upon infecting THP-1 cells leading us to conclude that CaFun30 could potentially be developed as a therapeutic target against C. albicans.

pharmacology and toxicology↗

bioRxiv2025

Microbial Scl1 Activates TGFBR1 receptor kinase signalling to Drive Fibrosis-Inflammation Axis in Alcohol-Associated Liver Disease

Background and AimsAlcohol consumption alters gut microbiota, which can affect metabolism, immune regulation, and signalling pathways and lead to alcohol-associated liver disease (ALD). We investigated how alcohol-associated gut microbiota (AGMs) modulate liver kinome signalling to drive inflammation and fibrosis in ALD. MethodLiver kinome and metaproteome changes were studied in rats (n=6/group) colonized with stool of severe alcohol-related hepatitis (SAH) patients (SAH[->]healthy-rats) or healthy human donors (HD[->]ALD-rats). AGM-associated liver kinome changes were cross-correlated with bacterial genera. A bacterial protein; Streptococcal collagen-like protein 1 (Scl1) was identified with affinity for TGF{beta}R1. This was validated by molecular docking and immunoprecipitation-LCMS assay. Expression of Streptococcus pneumonia and pyogenes was done in stool of SAH patients (n=10). Also, Scl1 was quantified in patient stool (n=24) and liver tissue (n=19) samples. Validation was performed by assessing Scl1 level in plasma and Streptococcus level in stool samples before and after fecal microbiota transplantation (FMT) in SAH patients. ResultsStool metaproteomics showed significant increase in 10 bacterial genera (Streptococcus, Staphylococcus, and Clostridium) in SAH[->]Healthy-rats, mirroring changes seen in ALD-rats (FC>1.5, p<0.05). Clusters of Orthologous Groups analysis indicated increased post-translational modifications (PTMs) and decreased lipid metabolism in ALD-rats and SAH[->]Healthy-rats. Liver kinome profiling showed 85 upregulated kinases in SAH[->]Healthy-rats, with 34 overlapping with ALD-rats, associated with inflammation (Mapk14, Map3k10 and others), fibrosis (Tgfbr1, Igf1r, and others), lipid metabolism (Cdk14, Cdk18) and regeneration (Met, Bmpr1a and others). FMT from healthy human-donors to ALD-rats reversed the expression of Streptococcus (10-fold), Staphylococcus (3-fold), and Clostridium (5-fold), along with 18 kinases associated with inflammation (Btk, Camk4, Nuak1) and fibrosis (Tgfbr1, Col4a1, Fgfr2 and others). Strong association (r{superscript 2}>0.9, p<0.05) was seen between Streptococcus abundance and fibrosis and inflammation-related kinases. Level of Scl1 was significantly high in SAH and ALD-rat stool samples (FC>2, p<0.05). It showed strong affinity for TGF{beta}R1, as validated by molecular docking (>86%confidence) and immunoprecipitation assays (>100FC,p<0.05), indicating it as a potential ligand for TGF{beta}R1. Concordantly, expression of Scl1 was highest amongst all the known ligands for TGF{beta}R1 (p<0.05) suggesting Scl1 is a major contributor for TGF{beta}R1 activation and its downstream signalling in these patients. Following fecal microbiota transplantation, expression of Scl1 in plasma and Streptococcus levels in stool were significantly reduced by 2.1-folds and 1.9 folds respectively in SAH patients. Further, Scl1 levels SAH plasma is capable of predicting poor therapeutic response and 30-day mortality with high accuracy (AUC=0.92, cutoff >70 normalized abundance). ConclusionAlcohol induced gut dysbiosis alters the liver kinome, promoting inflammation, fibrosis, and lipid dysregulation. Streptococcal Scl1 is identified as a bacterial protein mimicking human collagen and capable of activating fibrotic signalling pathway through TGF{beta}R1in liver and is capable of predicting poor response in SAH. FMT from healthy donor restores microbial imbalance and harmful microbial-host interactions.

microbiology↗

bioRxiv2025

Taurine Transporter SLC6A6 Expression Promotes Mesenchymal Stromal Cell Function

Mesenchymal stromal cell (MSC) differentiation is critical for the development, maintenance, and repair of bone tissue. This occurs within the bone marrow microenvironment, which consists of various stromal populations and structural components that support skeletal growth, repair and homeostasis. MSCs also promote self-renewal of hematopoietic stem cells and regulate their differentiation. Analysis of our own and publicly available single-cell RNA sequencing datasets of the murine non-immune bone and bone marrow indicates that Slc6a6 expression is enriched in MSCs. Slc6a6 encodes for an ion-dependent transporter of taurine (TauT). Although taurine supplements have been shown to mitigate the onset of bone defects in aged populations, the absence of TauT expression in osteolineage cells suggests that taurines effect on bone may be secondary to its function in other populations, such as MSCs. Using young TauT genetic loss-of-function murine models we find that TauT loss impacts MSC populations in vivo and impairs MSC osteogenic differentiation in vitro. This correlates with decreased bone mineral density and bone strength in young TauT knockout mice. Importantly, shRNA-based knockdown of SLC6A6 expression in primary human MSCs reduces osteogenic differentiation, indicating a key role of taurine uptake in human MSC function. Consistent with a decline in MSC function with TauT loss, we find that TauT null MSCs are unable to support self-renewal and expansion of co-cultured hematopoietic stem/progenitor populations. Mechanistically, our RNA-sequencing analysis identifies downregulation of Wnt/beta-catenin signaling in MSCs in the absence of TauT. These cells also show reduced oxidative phosphorylation, and increased ROS levels, indicating that impaired Wnt signaling and elevated oxidative stress may contribute to the observed defects in osteogenic differentiation capacity. Collectively, our data identify taurine uptake as a key regulator of mesenchymal stromal cell maintenance and osteogenic fate determination.

cell biology↗

bioRxiv2025

Functional genomics and tumor microenvironment analysis reveal prognostic biological subtypes in Mantle cell lymphoma

Mantle cell lymphoma (MCL) is a genetically and clinically heterogeneous B-cell malignancy. We studied two MCL cohorts with differing treatment patterns: one enriched for immunochemotherapy, the other for chemotherapy alone. TP53 alterations were consistently associated with poor prognosis, whereas ATM mutations correlated with improved outcomes following rituximab-based chemotherapy. Based on recurrent genetic events, six clusters were identified and refined into three prognostic groups: high-risk (TP53 mutations and deletions at 17p13.3, 13q14.2, and 19p13.3), intermediate-risk (ATM and epigenetic regulator mutations, or gains at 8q/17q/15q), and low-risk (lacking TP53 alterations, rare ATM mutations without 11q deletions, gains at 3q, deletions at 6q). Transcriptomic analysis revealed enrichment of proliferation, metabolism-promoting gene signatures in high-risk; angiogenesis and NOTCH signaling in intermediate-risk; and proinflammatory-related (i.e., IFN, TNF) in low-risk MCLs. Multi-proteomic spatial profiling using imaging mass cytometry (IMC) demonstrated enrichment of CD8 T cells with high expression of exhaustion markers and dominant population of myeloid cells skewed toward an M2-like phenotype. Compared to ATM-perturbed tumors, TP53-perturbed tumors exhibited enriched SOX11 tumor cells and enhanced tumor-immune cell interactions. Functional analysis revealed that p53 represses BCR signaling through PTPN6 activation. Collectively, these findings highlight distinct molecular and immune landscapes and reveal therapeutic vulnerabilities in high-risk TP53-altered MCL.

cancer biology↗

bioRxiv2025

Serum exosome miR-150-5p in rheumatic heart disease-associated angiogenesis

The focus of this study is on Rheumatic heart disease (RHD), an autoimmune consequence of rheumatic fever leading to mitral valve damage. Aim is to explore the role of exosomes derived from RHD patients in promoting angiogenesis during RHD pathogenesis. Exosomes were isolated via column chromatography from the pericardial fluid (PF) and serum of RHD patients, pericardial fluid from non-RHD patients undergoing coronary artery bypass grafting (CABG) surgery, and serum of healthy individuals. These exosomes were characterized by transmission electron microscopy (TEM) and western blotting. Human umbilical vein endothelial cells (HUVECs) cultured from umbilical cord(s), were treated with purified exosomes from RHD, CABG patients or healthy individuals. Exosome treatment induced morphological changes in HUVECs, including elongation and tube-like structure formation. The qRT-PCR gene expression analysis revealed significant upregulation of angiogenic markers (VEGF-A 2.7-fold, MMP2 1.5-fold) and inflammatory cytokine (IL-4 15.17-fold), in HUVECs stimulated with RHD serum exosomes. Conversely, there was downregulation of anti- angiogenic genes (RASA-1 0.65-fold and endostatin 0.4-fold). Notably, elevated levels of miR- 150-5p (34.29 + 9.524-fold mean difference; p= 0.0007) were detected in serum-derived exosomes from RHD patients compared to healthy controls. Stimulation of HUVECs with RHD serum exosomes resulted in reduction in the expression of TP53 (0.227-fold; p= 0.006), a known target of miR-150-5p. Furthermore, transfection of HUVECs with miR-150-5p mimics induced the formation of angiogenic endothelial tubes, underscoring the role of miR-150-5p in promoting angiogenesis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/659484v1_ufig1.gif" ALT="Figure 1000"> View larger version (27K): org.highwire.dtl.DTLVardef@1520e92org.highwire.dtl.DTLVardef@3aa03aorg.highwire.dtl.DTLVardef@ed6e1dorg.highwire.dtl.DTLVardef@1d85231_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Protein folding stress transcriptionally reprograms muscle metabolism

Cellular stress responses crosstalk with many physiological and metabolic pathways. Muscle cells constantly respond to various endogenous stressors while actively maintaining critical metabolic functions for the tissue and whole animal. The molecular mechanisms of how muscle stress responses transcriptionally reprogram metabolic networks are complex and inadequately understood. Using a multi-omics approach of metabolomics, lipidomics, and single-nuclei RNA-sequencing in Drosophila, we reconstructed the physiological landscape of muscle during chronic activation of endoplasmic reticulum unfolded protein response (UPR), a stress response that ensures the secretion of vital proteins from muscle, known as myokines. By ectopically expressing a constitutively active form of X-box binding protein 1 (Xbp1), a highly conserved transcription factor (TF) and UPR effector, we found that UPR downregulates key metabolic pathways in muscle, including carbohydrate and purine metabolism, while upregulating a robust lipogenic program enriched for phospholipids and several antioxidant metabolic pathways. Using gene regulatory network (GRN) analysis, we linked these metabolic changes to distinct TF regulon activities. The activation of a single TF, Xbp1, increased the activity of other stress response TFs in muscle, including cap-n-collar (cnc/Nrf2), cryptocephal (crc/Atf4), and sterol regulatory element binding protein (SREBP). Simultaneously, we observed decreased activity of TFs, namely Forkhead box O (FoxO), that resulted in downregulated metabolic pathways critical to muscle function, including oxidative phosphorylation and glycolysis. We propose that these GRNs antagonize each other downstream of UPR to reprogram muscle metabolism away from carbohydrates and towards lipogenesis, offering novel insight into how metabolic rewiring can be transcriptionally controlled in response to chronic tissue damage, even to the detriment of organ function.

cell biology↗

bioRxiv2025

The Mycobacterium smegmatis bd-II terminal oxidase employs a 1 carboxylate shift mechanism

Cytochrome bd is a terminal oxidase expressed under low oxygen conditions and central for the survival of many pathogens. Here we characterise the first qOR-2 type bd oxidase, the cyt bd-II from Mycobacterium smegmatis, by combining biochemical studies with cryo-electron microscopy (cryo-EM), and multiscale simulations. By over-expressing the appCB operon in its native host, we produce a highly active bd-II (kcat=30 e-s-1) that together with a high-resolution (2.8 [A]) cryo-EM structure and multiscale simulations reveal unique proton pathways and oxygen channels responsible for its function. We propose that O2-scavenging activates a pH-dependent molecular switch, involving coordination changes of heme d and surrounding bulky residues that regulate substrate access into the active site. Taken together, our findings provide detailed mechanistic insight of qOR-2 type bd oxidases, and a basis for understanding the evolution of the superfamily.

biochemistry↗

bioRxiv2025

Metabolomic Profiling of Ashwagandha (Withania somnifera): Linking Bioactive Compounds to Functional Therapeutic Roles

IntroductionWithania somnifera (Ashwagandha) is a widely used medicinal plant with established roles in traditional systems of medicine. Known for diverse pharmacological effects, including neuroprotection, hepatoprotection, enhancement of male fertility, and anti-inflammatory activity. Despite its longstanding use, a deeper understanding of its phytochemical composition and bioactive mechanisms is required to bridge traditional knowledge with modern therapeutic applications. MethodsA comprehensive metabolomics profiling of Withania somnifera was performed to identify and quantify its bioactive secondary metabolites. Focus was placed on major classes such as alkaloids, phenolic compounds, and terpenoids. Known compounds were annotated and categorized based on their abundance, chemical class, and reported biological functions. Particular attention was given to compounds implicated in neuroprotection, liver health, reproductive support, and inflammation-modulation. ResultsThe analysis revealed a rich but low-abundance of known metabolites. Alkaloids such as Gentianaine (8.44%) and Sauroxine (2.94%), and phenolics like Mulberrofuran A (14.58%) and Sulphuretin (9.61%), were among the most prominent. These compounds are associated with antioxidative, anti-inflammatory, and neuroregenerative activities. Terpenoids including Pinocarvone and Ergosterol contributed to hepatoprotective and anti-inflammatory properties. Compounds such as Gomphrenin-I, Sitosterol, Pfaffic acid, beta-Carotene and others (>3%) supported spermatogenesis, while Embelin and Kievitone enhanced oxidative stress response. Although most identified compounds were individually present in low percentages (<0.30%), their collective activity suggests synergistic effects. ConclusionMetabolomic analysis of Withania somnifera confirms high abundance of bioactive secondary metabolites that have hepatoprotective, neuroprotective, anti-oxidant and spermatogenesis-enhancing effects. These findings justify protective and beneficial effect of Withania in promoting stress relief, energy, and overall wellness. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/659832v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1ae8e5forg.highwire.dtl.DTLVardef@105300corg.highwire.dtl.DTLVardef@1958320org.highwire.dtl.DTLVardef@d3ba13_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

AI-Guided Design and AKT3 Degradation Synergize to Enhance Bispecific and Trispecific CAR-T Cell Persistence and Overcome Antigen Escape

The structural design of chimeric antigen receptors (CARs) is critical for achieving robust and durable anti-tumor responses, particularly when targeting multiple antigens to prevent tumor antigen escape. However, increasing CAR complexity can introduce structural vulnerabilities, leading to antigen-independent T cell activation, activation-induced cell death, and reduced CAR-T cell persistence. To overcome these challenges, we designed 10,824 CAR molecules across diverse formats and screened 1,452 constructs in-vitro to develop an artificial intelligence model, termed CAR-Mediated Self-Destruction (CARMSeD), which predicts CAR designs susceptible to self-activation and dysfunction. Guided by CARMSeD and structural CAR-CAR interaction modeling, we identified optimized CAR architectures incorporating ICOS and 4-1BB co-stimulatory domains. Humanized bispecific CARs targeting CD20/CD19 and CD22/CD19 demonstrated superior anti-tumor efficacy and persistence both in-vitro and in various xenograft mice models. To further extend CAR-T cell persistence, we engineered bispecific CARs integrated with an AKT3-targeted PROTAC strategy. Targeted degradation of AKT3 enhanced anti-tumor potency, promoted memory T cell formation, and enabled sustained responses even under tumor rechallenge and CD19 antigen-loss conditions. Mechanistically, these effects were mediated by metabolic reprogramming involving FOXO4; notably, FOXO4-deficient CAR-T cells exhibited impaired long-term persistence. Leveraging these mechanistic insights, we developed a trispecific CAR-T cell platform incorporating a bispecific T cell engager (BiTE) targeting CD22/CD3, combined with AKT3 PROTACs. These trispecific CAR-T cells achieved potent tumor eradication, even against malignancies lacking both CD19 and CD20 expression. Collectively, this study presents a comprehensive strategy combining structure-based design, AI-guided screening, and targeted protein degradation to engineer next-generation bi and trispecific CAR-T cells with enhanced persistence, broad antigen coverage, and superior therapeutic durability.

cancer biology↗

bioRxiv2025

Chenodeoxycholic Acid Modulation via Bacteroides intestinalis AM1 underscores a Novel Approach in Acute Liver Failure

BackgroundAcute liver failure (ALF) is associated with rapid and progressive hepatocellular injury, and severe metabolic-microbial derangements. We investigated early metabolic markers of non-survival, and a potential microbial intervention using Bacteroides intestinalis-AM1, to improve outcomes in ALF. MethodPlasma metabolomics and meta-proteomics were performed in 40 ALF patients and 5 healthy controls (training cohort). A non-survival marker panel was identified and validated in 270 ALF patients (test cohort) using high resolution mass spectrometry and machine learning. It was functionally validated in acetaminophen-induced ALF mouse model. B. intestinalis-AM1 was used to study alteration of gut bacteria and amelioration of liver injury. ResultsALF non-survivors showed a distinct metabolomic signature with elevated primary bile acids {chenodeoxycholic acid (CDCA), cholic acid (CA)}, tryptophan, tyrosine, and enrichment of pathways linked to inflammation, cell death, and stress response (p<0.01, FDR<0.01, FC>1.5). Non-survivors had higher alpha/beta diversity (p<0.05) with increase in Proteobacteria, Firmicutes, Actinobacteria (p<0.05); functionally associated with energy, amino acid and xenobiotic metabolism (p<0.05). A gut microbiota derangement in converting primary to secondary bile acids was evident as CDCA and cytotoxic metabolites (4-(2-Amino phenyl)-2,4-dioxobutanoate, L-Tyrosine) were higher. Elevated CDCA (logFC>10) levels correlated with mortality in ALF patients as well as in mouse model. In the later, administration of B. intestinalis-AM1 bacteria, (10^9) reduced CDCA and CA levels by enhancing FXR, FGF15, SLC10A1 gene expression, attenuating inflammation (IL-1beta, TLR4-signalling), necroptosis, and modulating glutathione(oxidative-repair), tryptophan(inflammation), and histidine (tissue repair) metabolism. ConclusionHigh levels of chenodeoxycholic acid (CDCA) represent a poor prognostic indicator in ALF patients. B. intestinalis-AM1, a primary-to-secondary bile acid converter, effectively reduce CDCA levels, activated FXR, reduced inflammation and protected hepatocytes, highlighting its therapeutic potential in ALF.

microbiology↗

bioRxiv2025

Metabolic tuning enables immediate adaptation to energy stress in yeast

In Saccharomyces cerevisiae, glucose depletion induces metabolic reprogramming through widespread transcriptional and translational reorganization. We report that initial, very rapid translational silencing is driven by a specialized metabolic mechanism. Following glucose withdrawal, intracellular NTP levels drop drastically over 30 sec, before stabilizing at a regulated, post-stress set-point. Programmed translational control results from the differential NTP affinities of key enzymes; ATP falls below the (high) binding constants for DEAD-box helicase initiation factors, including eIF4A, driving mRNA release and blocking 80S assembly. Contrastingly, GTP levels always greatly exceed the (low) binding constants for elongation factors, allowing ribosome run-off and orderly translation shutdown. Translation initiation is immediately lost on all pre-existing mRNAs, before being preferentially re-established on newly synthesized, upregulated stress-response transcripts. We conclude that enzymatic constants are tuned for metabolic remodeling. This response counters energy depletion, rather than being glucose-specific, allowing hierarchical inhibition of energy-consuming processes on very rapid timescales.

molecular biology↗

bioRxiv2025

Global Landscape of Human Kinase Motifs in Viral Proteomes

Viruses are classically viewed as targets of host sensing, yet whether they also sense and respond to host cues remains largely unexplored. We propose that host-driven post-translational modification of viral proteins allows viruses to dynamically sense host cellular states. We annotated human kinase motifs in 1,505 viral proteomes and discovered an enrichment for stress, inflammation, and cell-cycle kinases. Mapping kinase motifs onto 21,606 viral protein structures and integrating with phosphoproteomics of infected cells revealed surface-accessible residues were preferentially phosphorylated, showed greater kinase specificity, and were under positive selection for stress and immune kinase motifs. Temporal phosphoproteomics of alphavirus-infected cells confirmed stress kinase activation and viral protein phosphorylation, and MAP kinase inhibition reduced alphavirus replication and phosphorylation of ERK and JNK motifs on viral proteins. Our findings suggest that viruses evolved as biosensors of the host signaling state, unveiling new antiviral opportunities aimed at disrupting virus decision-making.

microbiology↗

bioRxiv2025

Mapping allosteric rewiring in related protein structures from collections of crystallographic multiconformer models

How do related proteins with a common fold perform diverse biological functions? Although the average structure may be similar, structural excursions from this average may differ, giving rise to allosteric rewiring that enables differential activity and regulation. However, this idea has been difficult to test in detail. Here we used the qFit algorithm to model "hidden" alternate conformations from electron density maps for an entire protein family, the Protein Tyrosine Phosphatases (PTPs), spanning 26 enzymes and 221 structures. To interrogate these multiconformer models, we developed a new algorithm, Residue Interaction Networks From Alternate conformations In RElated structures (RINFAIRE), that calculates networks of interactions between flexible residues and quantitatively compares them. We show that PTPs share a common allosteric network which rewires dynamically in response to catalytic loop motions or active-site vs. allosteric ligand binding, but also that individual PTPs have unique allosteric signatures. As experimental validation, we show that targeted mutations at residues with varying sequence conservation but high network connectivity modulate enzyme catalysis, including a surprising enhancement of activity. Overall, our work provides new tools for understanding how evolution has recycled modular macromolecular building blocks to diversify biological function. RINFAIRE is available at https://github.com/keedylab/rinfaire.

biophysics↗

bioRxiv2025

Predicting targeted- and immunotherapeutic response outcomes in melanoma with single-cell Raman Spectroscopy and AI

PURPOSEIdentifying reliable predictors of immunotherapeutic response in melanoma remains an outstanding challenge. Existing transcriptomic and proteomic profiling methods for the tumor-immune microenvironment (TIME) are costly and may not faithfully capture modifications actively impacting tumor behavior. Here, we present a non-destructive, single-cell approach combining Raman spectroscopy and machine learning (ML) that enables rapid cell profiling and therapeutic response prediction. METHODSWe analyzed single-cell Raman spectra of mouse and human melanoma cell lines alongside nine melanoma patient-derived samples with known resistance profiles to targeted and immunotherapeutic inhibitors bemcentinib, cabozantinib, dabrafenib, nivolumab, and a combination of nivolumab and relatlimab. We assessed cell phenotyping classification and treatment resistance using random forests and feature importance analysis. For patient samples, we constructed a two-stage evaluation workflow to determine clinical drug resistance through aggregated single-cell predictions and identified corresponding highly variant spectral signatures using computational methods adapted from single-cell RNA sequencing methods. RESULTSIn cell lines, our approach achieved >96% differentiation accuracy across tumor microenvironment cell types and induced functional phenotypes. Persistent (drug-resistant) cells formed subclusters based on genetic mutations rather than sample origin, with Raman signatures reflecting biochemical changes relevant to therapeutic pathways. For patient samples, our workflow correctly inferred resistance likelihoods for 30 of 33 clinically-relevant patient-drug combinations (91% accuracy). CONCLUSIONSingle-cell Raman spectroscopy combined with machine learning offers a scalable, prognostic platform to predict therapeutic resistance likelihood, with further potential to advance clinical, multi-omic biomarker efforts for melanoma. Our approach may improve first-and second-line therapy selection assessments for precision medicine by providing rapid, non-destructive prediction of therapeutic response based on cellular spectral profiles. Context SummaryO_ST_ABSKey objectiveC_ST_ABSCan label-free, single-cell Raman spectroscopy and machine learning approach accurately profile melanoma cell states and therapeutic resistance likelihood to targeted and immunotherapeutic agents? Knowledge generatedRaman spectroscopy with machine learning differentiated tumor microenvironment cell types and functional phenotypes with >96% accuracy in cell lines. When applied to patient-derived metastatic melanoma samples, the approach correctly inferred patient response to a panel of targeted and immunotherapeutic inhibitors with 91% accuracy (30 of 33 cases). High-likelihood persistent and sensitive cells across diverse patients exhibited recurrent spectral features. RelevanceSingle-cell Raman-based profiling supports functional-diagnostic assessment or resistance likelihood and may contribute to improved therapeutic selection and precision oncology strategies for melanoma patients.

cancer biology↗

bioRxiv2025

Mapping Macaque to Human Cortex with Natural Scene Responses

Neuroscience has long relied on macaque studies to infer human brain function, yet identifying functionally corresponding brain regions across species and measurement modalities remains a fundamental challenge. This is especially true for higher-order cortex, where functional interpretations are constrained by narrow hypotheses and anatomical landmarks are often non-homologous. We present a data-driven approach for mapping functional correspondence across species using rich, naturalistic stimuli. By directly comparing macaque electrophysiology with human fMRI responses to 700 natural scenes, we identify fine-grained alignment based on response pattern similarity, without relying on predefined tuning concepts or hand-picked stimuli. As a test case, we examine the ventral face patch system, a well-studied but contested domain in cross-species alignment. Our approach resolves a longstanding ambiguity, yielding a correspondence consistent with full-brain anatomical warping but inconsistent with prior studies limited by narrow functional hypotheses. These findings show that natural image-evoked response patterns provide a robust foundation for cross-species functional alignment, supporting scalable comparisons as large-scale primate recordings become more widespread.

neuroscience↗

bioRxiv2025

Uridine cytidine kinases dictate the therapeutic response of molnupiravir via its bioactivation

Molnupiravir is a nucleoside analogue antiviral drug with activities against a broad spectrum of RNA viruses, including its clinical indication SARS-CoV-2. Whilst its antiviral mechanism-of-action is well defined, host pharmacogenetic factors that regulate its therapeutic responses - efficacy as well as selectivity - have not been mechanistically deciphered and characterized. Here we identified that uridine cytidine kinase (UCK), the first and rate-limiting kinase of the pyrimidine salvage pathway, could effectively phosphorylate N4-hydroxycytidine (NHC), the active compound of molnupiravir, and thereby dictate its anti-SARS-CoV-2 efficacy, as well as its selectivity index. Using target engagement and enzyme kinetic assays, we demonstrate that recombinant UCK isoform 1 (UCK1) and 2 (UCK2) effectively bind and phosphorylate NHC, with UCK2 displaying 9-fold higher catalytic efficiency. Accordingly, in SARS-CoV-2-infected cells, downregulation of UCK2 via siRNA hampered the intracellular accumulation of the triphosphate antiviral metabolite of NHC, resulting in 10-fold reduction of antiviral efficacy. Furthermore, we could recapitulate this using a pan-UCK small molecule inhibitor. Critically, both UCK downregulation and inhibition significantly reduced the selectivity index of molnupiravir/NHC. Altogether, this work underscores the pivotal roles of UCK enzymes in upholding molnupiravir efficacy and therapeutic window, and furthermore, highlights their potential as pharmacologically tractable targets for tailoring the therapeutic response to this antiviral agent.

microbiology↗

bioRxiv2025

The Consortium for Genomic Diversity, Ancestry, and Health in Colombia (CODIGO): building local capacity in genomics, bioinformatics, and precision medicine

The Consortium for Genomic Diversity, Ancestry, and Health in Colombia (CODIGO) aims to build a community of Colombian researchers in support of local capacity in genomics, bioinformatics, and precision health. Here, we present the first CODIGO data release and the consortium web platform, including annotations for more than 95 million genetic variants from 1,441 samples representing 14 populations from across the country. CODIGO samples show a wide range of African (16.7%), European (50.6%), and Indigenous American (32.8%) genetic ancestry components, with five distinct ancestry clusters. Thousands of ancestry-enriched variants, with divergent allele frequencies across clusters, show pharmacogenomic and clinical genetic associations. Examples include African ancestry-enriched variants associated with fast metabolism of the immunosuppressive drug tacrolimus and malaria resistance and European ancestry-enriched variants associated with nicotine dependence and hereditary hemochromatosis. CODIGO reveals the nexus between ancestry and health in Colombia and underscores the utility of collaborative genome sequence analysis efforts.

genomics↗

bioRxiv2025

Immunogenicity and Structural Features of a Stabilized Clade-C HIV-1 Env Derived from a Pediatric Elite-neutralizer

The envelope (Env) glycoprotein derived from circulating viruses in elite-neutralizers (who naturally develop broadly neutralizing antibodies (bNAbs)), serves as potential template for HIV-1 vaccine design. Herein, we report the structure and immunogenicity of soluble clade-C HIV-1 Env trimer (330) derived from a pediatric elite-neutralizer (AIIMS_330). Using SOSIP, NFL and ferritin-nanoparticle (NP) platforms, we engineered immunogens that preserved native-like Env conformation, exhibited high thermostability and nanomolar affinity for diverse bNAbs, with minimal reactivity to non-neutralizing antibodies. Cryo-EM structure solved at 5 [A] resolution of 330-SOSIP trimer-autologous bNAb 44m complex revealed interactions at GDIR motif, N332 supersite along with additional contacts at E293, K337, K446 and glycans N326, N442, N448, as compared to our previously reported BG505-44m structure. Rabbit immunizations with soluble and NP-displayed formats elicited autologous, and heterologous neutralization of tier-1 clade-C viruses. Herein we define a structurally resolved HIV-1 clade C Env that supports multivalent vaccine strategies and provides mechanistic insights toward rational HIV-1 immunogen design.

immunology↗