Biology subjects

Jana, S.

Publications and source records attributed to Jana, S..

At least 19 recordsLinked to original sources

bioRxiv2026

The enemy of my enemy: A bacterial antagonist rescues Chlamydomonas by inactivating a toxin from a rival

Microbial interactions are shaped by multiple community members, yet the underlying mechanisms remain poorly understood. Here, we investigated a tripartite interaction between the microalga Chlamydomonas reinhardtii and two Pseudomonas bacteria that coexist in the same soil habitat. In pairwise cultures, all three interactions are amensalistic (-like): Pseudomonas protegens Pf-5 antagonizes both C. reinhardtii and Pseudomonas fluorescens HKI0770 without benefit, while P. fluorescens inhibits C. reinhardtii without profiting from the alga. However, in a tripartite culture, P. fluorescens protects C. reinhardtii from P. protegens and promotes algal recovery. This protection is not stopped when the used carbon source, acetate, is supplemented. The P. fluorescens rescue effect is associated with reduced levels of the polyyne toxin protegencin produced by P. protegens. Protegencin levels, detected by Raman microspectroscopy, are decreased by application of spent medium or protein extracts from P. fluorescens, while heat- and protease-treatment abolish this activity. A heat-sensitive, proteinaceous factor, likely an enzyme(s), appears to contribute to protegencin inactivation. Our results show that P. fluorescens rescues C. reinhardtii by inactivating protegencin, despite being inhibited by P. protegens. These findings demonstrate the importance of investigating higher-order effects, since additional community members can transform pairwise antagonistic interactions into conditional protection through toxin inactivation.

microbiology↗

bioRxiv2026

MPGEM: A harmonized and transcriptome-complete resource for large-scale reuse of legacy human microarray data

Abstract Background: Legacy microarray datasets provide an extensive record of human transcriptomic biology, but their reuse is constrained by differences in platform design, preprocessing, measurement scale, and gene coverage. Platforms measuring only subsets of genes cannot readily be integrated with higher-coverage platforms, limiting large-scale analysis and computational modeling. Results: We developed Multi-Platform Gene Expression Matrix (MPGEM), a computational framework and resource for harmonizing and completing gene-expression profiles across heterogeneous microarray platforms. MPGEM uses a Reference Quantile Distribution (RQD) and generalized Reference Subset Quantile Distribution (RSQD) framework to transform profiles with different gene coverage onto a common quantitative scale. The MPGEM Engine, a multilayer perceptron, predicts expression of unmeasured genes from genes shared across platforms. Applied to Affymetrix GPL570, GPL571, and GPL96, MPGEM uses GPL570 as a 19,320- gene reference space comprising 12,712 predictor and 6,608 target genes. The resulting resource contains 207,135 human gene-expression profiles across 19,320 genes. Evaluation using masked GPL570 profiles yielded mean sample-wise Pearson and Spearman correlations of 0.944 and 0.939, respectively, and mean gene-wise correlations of 0.830 and 0.825. The lowest-performing 5% of target genes achieved a mean Pearson correlation of 0.683. MPGEM showed comparable or higher predictive performance than baseline mean imputation and K-nearest-neighbor approaches. Conclusions: MPGEM transforms heterogeneous, partially measured legacy microarray profiles into a harmonized, transcriptome-complete representation, facilitating their reuse for large-scale transcriptomic analysis, biomarker discovery, systems biology, and machine learning. The framework, trained models, and expression resource are provided as open-source resources.

bioinformatics↗

bioRxiv2026

Organizational principles governing synapse types in a whole-brain connectome

Neurons are classically viewed as polarized elements in which dendrites receive synaptic input and axons provide output. However, many neurons contain intermingled presynaptic and postsynaptic sites, enabling non-canonical forms of connectivity beyond axo-dendritic interactions. How such mixed polarity shapes connectivity across an entire nervous system remains unknown. Here, using the complete Drosophila connectome of an adult brain (approximately 140,000 neurons and >80 million synapses), we systematically mapped synapse types across neuronal compartments, cell types, and brain regions. We found that mixed polarity is widespread yet highly structured across neuronal populations. Approximately one third of synapses between polarized neurons are non-canonical, including axo-axonic, dendrodendritic, and dendro-axonic interactions. Remarkably, synapse type can be predicted from a simple axis of neuronal complexity derived from morphology and connectivity. Low-complexity neurons, which dominate early visual circuits, preferentially interact through dendrites, whereas larger and more highly connected neurons enriched in central brain regions preferentially interact through axons. Mixed polarity also strongly promotes reciprocal connectivity, which is dominated by non-canonical motifs. Finally, we identified postsynaptic terminals on presynaptic boutons as a widespread anatomical motif associated with local reciprocal and non-canonical interactions. Together, these findings reveal organizational principles linking neuronal architecture, synapse type, and circuit connectivity across a complete brain.

neuroscience↗

bioRxiv2026

SIRT5 acts in the tumor microenvironment via endothelial cell metabolism to support breast cancer growth

Triple-negative breast cancer (TNBC) is characterized by aggressive progression and poor prognosis, partly due to abnormal angiogenesis. While the metabolic reprogramming of tumor cells is well characterized, the metabolic regulation of tumor-associated endothelial cells (ECs) remains unclear. Here, we identified the mitochondrial deacylase SIRT5, which has established tumor-promoting roles in TNBC cells, as a key regulator of endothelial metabolic homeostasis and tumor angiogenesis. SIRT5-deficient host mice showed significant defects in supporting the growth of orthotopic SIRT5-proficient mammary tumor transplants, and the resulting neoplasms showed defects in tumor vascularization. In a 3D microfluidic vessel-on-chip model, SIRT5 loss compromised vascular barrier integrity and EC sprouting. Mechanistically, SIRT5-deficient ECs exhibited diminished mitochondrial respiratory capacity but apparently normal glycolysis. SIRT5 loss also caused increased mitochondrial reactive oxygen species levels, and a mitochondrial antioxidant rescued the endothelial cell defects following SIRT5 loss, indicating that SIRT5-mediated mitochondrial redox homeostasis in the tumor microenvironment is necessary to maintain vascular function. Orthotopic co-transplantation of TNBC and EC cells with or without SIRT5 knockdown demonstrated that endothelial SIRT5 promotes increased tumor growth in vivo. These results suggest that targeting SIRT5 offers a potential therapeutic strategy to disrupt tumor angiogenesis and suppress TNBC progression by targeting the metabolic vulnerabilities of the tumor endothelium.

cancer biology↗

bioRxiv2026

Identification of Siderophores with Unexpected Antibacterial Properties from Actinoplanes teichomyceticus

Actinoplanes teichomyceticus is a well-established producer of bioactive secondary metabolites, including the glycopeptide antibiotic teicoplanin. Although its antibiotic biosynthetic capacity has been extensively investigated, its siderophore diversity and any additional biological functions of these iron-chelating metabolites remain comparatively underexplored. We identified a reproducibly bioactive, teicoplanin-independent fraction that inhibited Bacillus spizizenii. Molecular networking applied to this fraction identified hydroxamate ferrioxamine and desferrioxamine-type siderophores as the dominant metabolites, including acylated analogs detected as Al3+- and Fe3+-chelated species. Robust siderophore secretion was confirmed by the CAS assay. Notably, siderophore-enriched fractions exhibited selective antibacterial activity against Gram-positive bacteria, with minimum inhibitory concentrations of approximately 16 {micro}g/mL against B. spizizenii and partial inhibition of Staphylococcus aureus, while no activity was observed against Escherichia coli. Synthetic C7 and C9 acyl-desferrioxamine analogs showed enhanced antibacterial activity upon Al3 chelation, indicating a metal-dependent bioactivity. These findings reveal an unexpected antibacterial role for ferrioxamine-type siderophores produced by A. teichomyceticus, extending their function beyond iron acquisition, possibly through a "Trojan horse" (or "Trojan metal") mechanism.

biochemistry↗

bioRxiv2026

Reciprocal Fronto-Parietal Interactions Support Motor Anticipation during Sequential Reaching

Motor planning is a highly flexible process that adapts to changing behavioral demands. Frontal and parietal circuits are considered as critical nodes that support planning processes. However, it remains unclear whether the coordination of motor and parietal areas is preserved or adjusted in response to varying behavioral contexts. To address this open question, we trained two rhesus macaques to perform a visually-guided sequential reaching task, in which they could adapt their behavior to varying degrees of target predictability within the sequence. Analysis of eye and hand movements revealed distinct visuomotor strategies that were reflected in corresponding neural activity patterns. During motor anticipation, the direction of the upcoming reach toward predictable targets could be decoded from preparatory neural activity prior to target onset both in motor regions (PMd/M1) and parietal area 7A, whereas during reactive movements directional information emerged only after target onset. Using feature-specific information transfer analysis, we found that information about the upcoming movement direction was transmitted between 7A and PMd/M1 through bidirectional interactions. This pattern of interactions was preserved during motor anticipation and shifted earlier in time with respect to target onset. Our findings thus support a reciprocal fronto-parietal network that flexibly adjusts the timing of preparatory activity as different strategies are employed under varying behavioral constraints. Contrary to classical hierarchical models predicting serial activation across parietal and motor areas, parietal-to-motor interactions did not precede motor-to-parietal interactions, consistent with a heterarchical organization of cortical processing during flexible motor planning.

neuroscience↗

bioRxiv2026

Dimerization of MilM is essential for catalyzing the pyridoxal-5'-phosphate (PLP)-dependent Cγ-hydroxylation of L-arginine during mildiomycin biosynthesis

MilM from the mildiomycin biosynthetic pathway is a PLP-dependent enzyme, previously annotated as an aminotransferase, but has recently been demonstrated as L-arginine oxidase cum C-hydroxylase. Here, we report detailed biochemical, biophysical, structural modeling, and molecular dynamics simulation-based investigations of MilM from Streptoverticillium rimofaciens B-98891 to elucidate the mechanisms of substrate binding, catalysis, and the role of the active site residues involved in these processes. Our experimental findings confirmed that MilM functions as a stable homodimer, requiring the PLP cofactor and molecular oxygen to transform the L-arginine substrate into 5-guanidino-4-hydroxy-2-oxovaleric acid and 5-guanidino-2-oxovaleric acid through the intermediacy of a possible superoxide radical anion species, while generating H2O2 and NH3 as reaction by-products. Our labeling studies also established that the hydroxyl group in the product is obtained from the solvent, water. The structure-based three-dimensional modeling and simulation of MilM coupled with site-directed mutagenesis further confirmed that, in addition to the catalytic residues Lys232 and His31, active site residues from both the protomers are crucial for stabilization of the PLP cofactor (Ser92, Phe116, Asn164, Asp195, Lys240 from chain A and Tyr89 from chain B) and the substrate (Thr14, Glu17, Asn118, and Arg364 from chain A and Thr259, Ser260 from chain B). Moreover, the molecular dynamics simulation uncovered a dimer-mediated alternating lid mechanism in which large-scale, concerted motions of the dimer interface helices reciprocally expose and occlude the two active sites. This see-saw-like dynamics controls the substrate entry and product release through transiently formed tunnels, while preserving a catalytically protective environment, a critical phenomenon previously left unnoticed in similar PLP-dependent oxidases/hydroxylases. Overall, these findings provide new insights into the substrate/cofactor stabilization and the catalytic mechanism of MilM, a recent member of an emerging family of remarkable PLP-dependent oxidases and help us decode a key puzzle in the mildiomycin biosynthetic pathway.

biochemistry↗

bioRxiv2026

An Indole Dimer Antifungal Metabolite from a Rice Seed Endophyte Inhibits Ergosterol Biosynthesis in Fungal Pathogens

The increasing prevalence of fungal phytopathogens and the widespread emergence of fungicide resistance necessitate alternative antifungal strategies with reduced environmental impact. Here, we report the isolation and characterization of a novel antifungal metabolite, SM06, produced by the rice seed-associated endophytic bacterium Phytobacter sp. RSE02. SM06 exhibited broad-spectrum antifungal activity against plant and human pathogenic fungi, including Curvularia lunata, Fusarium oxysporum, and Candida albicans. In vitro assays and micromorphological analyses revealed that SM06, an indole dimer, disrupts fungal cell membrane integrity, while in planta experiments demonstrated significant suppression of brown leaf spot disease in tomato and rice. Molecular docking suggested that SM06 binds to lanosterol 14-demethylase (ERG11), a key enzyme in fungal sterol biosynthesis. Consistent with this prediction, LC-MS-based analyses confirmed a significant reduction in ergosterol content in SM06-treated fungal cells. Together, these findings identify SM06 as a biologically active antifungal metabolite produced by a plant-associated bacterium and highlight its potential application in sustainable fungal disease management. IMPORTANCEFungal diseases cause major losses in crop-production and contribute to the growing challenge of antifungal resistance, underscoring the need for sustainable alternatives to chemical fungicides. This study identifies SM06, a novel indole dimer produced by the rice seed endophyte Phytobacter sp. RSE02, with strong antifungal activity against economically important plant pathogens and clinically relevant fungi. Through integrated chemical, cellular, and in planta analyses, we demonstrate that SM06 disrupts fungal membrane integrity by inhibiting ergosterol biosynthesis. The compound is biocompatible, stable, and effective in plant disease suppression, highlighting its translational potential for crop protection. These findings reveal seed endophytes as an important yet underexplored sources of antifungal metabolites and provide a mechanistic foundation for developing eco-friendly biocontrol strategies with implications beyond agriculture. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/697688v1_ufig1.gif" ALT="Figure 1"> View larger version (86K): org.highwire.dtl.DTLVardef@c7da68org.highwire.dtl.DTLVardef@d3f1a1org.highwire.dtl.DTLVardef@9cab82org.highwire.dtl.DTLVardef@1c01141_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Synergy unleashed: Leishmania donovani GP63 paralogues cooperatively function to drive parasite infectivity at various pathogenic stages and regulate host survival mechanisms

Highly plastic genome of Leishmania is thought to underlie its remarkable ability to adapt to diverse host microenvironments, yet how this plasticity translates into dermal versus visceral persistence remains unclear. GP63, a zinc metalloprotease, is a well-established determinant of promastigote infectivity, particularly in cutaneous leishmaniasis. Despite this, its role in amastigote stage and in visceral disease caused by Leishmania donovani remains undefined. Here, using an integrated approach combining phylogenetics, CRISPR-mediated genome editing, and biochemical analyses, we demonstrate that GP63 paralogues in L. donovani have undergone independent evolutionary diversification to support visceral infection. While GP63-paralogues encoded on chromosome 10, previously implicated in cutaneous manifestation caused by L. major, were found severely truncated and dispensable in L. donovani, two distinct GP63-paralogues encoded on chromosome 28 and 31 were found essential to establish visceral infection. LdGP63_31 facilitates promastigote attachment to macrophages by promoting lipid raft engagement and complement inactivation, thereby enabling host entry and subsequent amastigote differentiation. LdGP63_28 is essential for intracellular amastigote survival by suppressing host inflammatory pyroptosis. Structural and enzymatic analyses revealed preferential host localization and substrate specificities, possibly resulting from distinct amino acid substitution within conserved motifs of these proteases, indicating independent evolutionary adaptations. Importantly, loss of the LdGP63_28 more severely impaired amastigote reinfection than loss of its counterpart LdGP63_31. Together, these findings reveal coordinated functional specialization of GP63-paralogues ensuring visceral adaptation.

microbiology↗

bioRxiv2025

Inference of VEGFR2 dimerization kinetics on the cell surface by integrating single-molecule imaging and mathematical modeling

Inter-receptor interactions play a key role in receptor signaling, which is the first step in cell signaling in response to external stimuli. In the case of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), dimerization is necessary for activation. VEGFR2 undergoes reversible interactions also in the absence of ligand. For a quantitative understanding of transmembrane signal transduction, it is necessary to quantify the interaction kinetics of VEGFR2 on the cell surface. Live-cell single-molecule imaging (SMI) has the powerful ability to capture receptor interaction events in their native cellular environment with high spatiotemporal resolution. However, it reveals these interactions for only the labeled subset, which is a small fraction of the full population of receptors. We have previously shown that mathematical modeling, combined with SMI data, offers a route to compensate for this lost information and infer the population-level receptor interaction kinetics from SMI data. Here, we applied this approach to VEGFR2, both wildtype full length VEGFR2 (FLR2) and a truncated mutant consisting of its extracellular and transmembrane domains (ECTM), which has been shown to exhibit reduced homotypic interactions in the absence of ligand. We developed a stochastic mathematical model mimicking VEGFR2 diffusion and interactions and determined the unknown model parameters through a combination of direct experimental measurements and stochastic model calibration. We found that a model of dimerization was sufficient to describe VEGFR2 interactions in the absence of ligand. While ECTM was primarily monomeric, FLR2 exhibited a substantial fraction of dimers. Our inference revealed that the difference between FLR2 and ECTM was primarily in the dimer association rate constant, which was about an order of magnitude lower for ECTM than for FLR2. To our knowledge, this is the first time that the interaction kinetics of VEGFR2 have been calculated in live cells. Author summaryInteractions between receptors on the cell surface play a large role in the initiation and modulation of cell signaling. For example, the receptor VEGFR2 in vascular endothelial cells must dimerize as a first step toward signaling. However, many interactions, including VEGFR2 dimerization, also take place in the absence of ligand stimulation. Therefore, elucidating when and where receptor interactions take place, and what their kinetics are, is critical for understanding receptor and cell signaling, especially on a quantitative level. Determining interaction kinetics is particularly challenging, given that membrane proteins do not express well in vitro, and are thus not amenable to traditional biophysical methods that quantify molecular interactions. In addition, the membrane and cellular environment is much more complex than an in vitro setting. To address these challenges, we combine single-molecule imaging, stochastic mathematical modeling, and rigorous model calibration to derive the dimerization kinetics of VEGFR2 (both full-length and a truncation mutant with reduced dimerization) on the surface of live cells. To our knowledge, this is the first time that the interaction kinetics of VEGFR2 have been calculated in live cells. Our approach is generalizable to many other cell surface proteins, opening an avenue for quantifying molecular interaction kinetics in situ.

biophysics↗

bioRxiv2025

Tuning Encodable Tetrazine Chemistry for Site-Specific Protein Bioorthogonal Ligations

Using genetic code expansion (GCE) to encode bioorthogonal chemistry has emerged as a promising method for protein labeling, both in vitro and within cells. Here, we demonstrate that tetrazine amino acids incorporated into proteins are highly tunable and have extraordinary potential for fast and quantitative bioorthogonal ligations. We describe the synthesis and characterize reaction rates of 29 tetrazine amino acids (20 of which are new) and compare their encoding ability into proteins using evolved Tet ncAA encoding tRNA/RS pairs. For these systems, we characterized on-protein Tet stability, reaction rates, and ligation extents, as the utility of a bioorthogonal labeling group depends on its stability and reactivity when encoded into proteins. By integrating data on encoding efficiency, selectivity, on-protein stability, and in-cell labeling for Tet tRNA/RS pairs, we developed the smallest, fastest, and most stable Tet system to date. This was achieved by introducing fluorine substituents to Tet4, resulting in reaction rates at the 10 M-{superscript 1}s-{superscript 1} level while minimizing degradation. This study expands the toolbox of bioorthogonal reagents for Tet-sTCO-based, site-specific protein labeling and demonstrates that the Tet-ncAA is a uniquely tunable, highly reactive, and encodable bioorthogonal functional group. These findings provide a foundation to further explore Tet-ncAA encoding and reactivity.

biochemistry↗

bioRxiv2025

Integrins and tetraspanins mediate CD36-actin coupling and regulate CD36 organization and signaling

The actin cortex plays a large role in regulating the dynamic organization of cell surface receptors, which in turn regulates their signaling. However, many receptors have short intracellular domains and no known link to cortical actin. In this work, we identified the {beta}1-integrin subunit and several tetraspanins - CD9, CD81 and CD151 - as part of the hitherto unknown molecular link between the surface receptor CD36 and cortical actin. We found that CD36 in vascular endothelial cells is recruited into complexes/nanodomains containing these proteins, with stronger recruitment near the cell edge. Perturbing this recruitment via the mutation G12V in the N-terminal transmembrane domain of CD36 alters the dynamic organization of CD36 on the vascular endothelial cell surface and weakens its coupling to cortical actin dynamics. Moreover, perturbing this recruitment abolishes thrombospondin-1-induced CD36 signaling through the Src family kinase Fyn. Given their many interactions with other transmembrane proteins, tetraspanins and integrins may provide a ubiquitous mechanism for plasma membrane-cortical actin coupling.

cell biology↗

bioRxiv2025

Dysregulation of neuronal activity-dependent immediate early genes in a mouse model of Angelman syndrome

Angelman syndrome (AS) is a debilitating neurodevelopmental disorder triggered by impaired function of the maternal UBE3A gene, which codes a protein that functions as a ubiquitin ligase and transcriptional coactivator. Ube3a maternal deficient mice replicate many key behavioral deficits associated with AS; however, the underlying molecular mechanisms remain poorly understood. Using an RT2 Profiler PCR array that summarizes the expression of 84 genes regulating synaptic plasticity, we identified a number of dysregulated genes in the visual cortex of AS mice brains at postnatal day 25 (P25) compared to wild-type animals. In-depth analysis revealed that various immediate early genes (IEGs), like Arc, Egr1-4, and Homer, are dramatically down-regulated in the visual cortex of AS mice with regard to wild-type controls at P25. Moreover, the dark rearing of wild-type mice considerably decreased the levels of these IEGs to nearly the same level as those found in AS mice, along with the down-regulation of Ube3a. Furthermore, a significant reduction in the expression of these IEGs can also be observed in the hippocampus of AS mice. Finally, we demonstrate that the augmented activity of HDAC2 in the AS mice brain might be connected with the down-regulation of various IEGs and other synaptic plasticity-regulating genes. These findings indicate that the deregulated expression of neural activity-dependent IEGs could be linked with abnormal synaptic plasticity and associated behavioral deficits observed in AS mice.

neuroscience↗

bioRxiv2025

Short-term alterations in dietary amino acids override host genetic susceptibility and reveal mechanisms of Salmonella Typhimurium small intestine colonization

In addition to individual genetics, environmental factors (e.g., dietary changes) may influence host susceptibility to gastrointestinal infection through unknown mechanisms. Herein, we developed a model in which CBA/J mice, a genetically resistant strain that tolerates intestinal colonization by the enteric pathogen Salmonella Typhimurium (S. Tm), rapidly succumb to infection after exposure to a diet rich in L-amino acids (AA). In mice, S. Tm-gastroenteritis is restricted to the large intestine (cecum), limiting their use to understand S. Tm small intestine (ileum) colonization, a feature of human Salmonellosis. Surprisingly, CBA mice fed AA diet developed ileitis with enhanced S. Tm ileal colonization. Using germ-free mice and ileal-fecal slurry transplant, we found diet-mediated S. Tm ileal expansion to be microbiota-dependent. Mechanistically, S. Tm relied on Fructosyl-asparagine utilization to expand in the ileum during infection. We demonstrate how AA diet overrides host genetics by altering the gut microbiotas ability to prevent S. Tm ileal colonization.

microbiology↗

bioRxiv2024

Active EB1 surges promote tubulin influx into the growing outer segments of the bipartite olfactory cilia in Drosophila

Like a photoreceptor cilium, the sensory cilia have a complex bipartite architecture containing 9+0 connecting cilium at the base and a singlet microtubule-supported, highly membranous outer segment, essential for the receptor display. How such diverse cilia morphology and underlying microtubule cytoskeleton develops remains unclear. Here we show that individual olfactory cilium, inside the large basiconic sensilla in developing Drosophila antenna, grows in episodic steps following several pulsatile influxes of tubulin. Each tubulin influx event is preceded by transient elevations of a microtubule-stabilising protein, the End-binding protein 1 (EB1). Additionally, EB1 is found to specifically interact with the tail domain of Drosophila KLP68D, an orthologue of the kinesin-2{beta} motor subunit, in vitro. Finally, the loss of EB1 in olfactory neurons preceding the growth surges reduces the tubulin influx as well as arrests the olfactory cilia assembly and stability. These findings suggest a novel mechanism of bipartite cilia assembly.

cell biology↗

bioRxiv2024

Intra-cluster receptor density (IRD) dictates TNFR1 clusters' signaling efficacy

Tumor Necrosis Factor Receptor 1 (TNFR1) signaling regulates cell fate in inflammation, immune responses, and tumorigenesis. While TNF--mediated TNFR1 pathways are well known, the role of receptor clustering remains unclear. Utilizing homo-FRET using fluorescence anisotropy, we show that intra-cluster receptor density (IRD) governs TNFR1 signaling outcomes. Soluble TNF- (sTNF-) increases IRD at cluster cores but decreases it at rims via receptor reorganization. Reducing IRD through membrane tension, zafirlukast, actin depolymerization, or cholesterol depletion suppresses sTNF- signaling, whereas increasing IRD by lowering membrane tension or exposing cells in a 3D gel-like microenvironment triggers ligand-independent activation. These findings reveal IRD as a key regulator of receptor signaling, with potential relevance across related receptor families and innovative strategies in modulating TNFR1 signaling. TeaserIntra-cluster receptor density (IRD) is critical for TNFR1 signal modulation, with higher IRD activating and lower IRD impairing the TNFR1 signaling.

biophysics↗

bioRxiv2024

Differential requirement of m6A reader proteins, IGF2BP2 and HNRNPA2B1 for the processing of N6-methyladenosine modified H19 lncRNA: Stability versus miR-675 biogenesis

H19, a lnc-pri-miRNA that encodes miR-675, is dysregulated in numerous cancers. However, the specific mechanisms underlying H19 processing, particularly miR-675 formation, remain unclear. Our study reveals that H19 is highly expressed and m6A modified in a METTL3-dependent manner in glioblastoma (GBM) and glioma stem cells (GSCs). Silencing METTL3 reduced both H19 and miR-675 levels, whereas overexpressing METTL3 promoted miR-675 processing without affecting H19 levels. Further, miR-675 derived from exogenously expressed H19 was affected considerably more in METTL3 silenced glioma cells compared to H19 levels, suggesting differential requirements in the processing of m6A modified H19 transcript. We demonstrate that H19 interacts with m6A reader proteins, IGF2BP2 and HNRNPA2B1, and silencing either reduced H19 and miR-675 levels. However, a high level of miR-675 seen in METTL3 overexpressing cells is severely affected in HNRNPA2B1-silenced compared to IGF2BP2-silenced glioma cells. Interestingly, IGF2BP2 silencing more significantly affected H19 stability from exogenous H19 construct, while HNRNPA2B1 silencing severely impacted miR-675 processing. Site-directed mutagenesis confirmed the presence of two m6A sites in the first exon of H19, with site #1 facilitating HNRNPA2B1 interaction to promote miR-675 processing. In contrast, the IGF2BP2 interaction is promoted by site #2, resulting in enhanced H19 stability. H19-METTL3-HNRNPA2B1-miR675 axis inhibited Calneuron 1 (CALN1), a known target of miR-675, to promote glioma cell migration. Notably, a low CALN1/high H19 predicted a poor prognosis in GBM patients and was further exacerbated by a high METTL3 or HNRNPA2B1 but not IGF2BP2 transcript levels. Thus, we found that the H19 transcript is highly expressed in GBM and m6A modified, and the m6A reader proteins, IGF2BP2 and HNRNPA2B1, regulate the H19 processing differently to promote glioma cell migration.

cancer biology↗

bioRxiv2024

Early 2022 breakthrough infection sera from India target the conserved cryptic class 5 epitope to counteract immune escape by SARS-CoV-2 variants

During the COVID-19 pandemic, the vast majority of epitope mapping studies have focused on sera from mRNA-vaccinated populations from high-income countries. In contrast, here we report an analysis of 164 serum samples isolated from breakthrough infection patients in India during early 2022 who received two doses of the ChAdOx viral vector vaccine. Sera were screened for neutralization breadth against wildtype, Kappa, Delta, and Omicron BA.1 viruses. Three sera with the highest neutralization breadth and potency were selected for epitope mapping using charged scanning mutagenesis coupled to yeast surface display and Next Generation Sequencing. All of these sera primarily targeted the recently identified class 5 cryptic epitope along with the class 1 and class 4 epitopes, targeted to a lesser extent. The class 5 epitope is completely conserved across all SARS-CoV-2 variants and for the majority of the Sarbecoviruses. In line with these observations, a major fraction of the serum samples, including the selected three, show broad neutralizing activity against recent Omicron subvariants including XBB.1.5. This is in contrast with the results obtained with the sera from individuals receiving multiple doses of original and updated mRNA vaccines, where impaired neutralization of XBB and later variant of concerns was observed. Our study demonstrates that two doses of the ChAdOx vaccine in a highly exposed population was sufficient to drive substantial neutralization breadth against emerging and upcoming variants of concern, thus paves the path towards the development of future vaccine candidates. ImportanceWorldwide implementation of COVID19 vaccine and parallel emergence of newer SARS-CoV-2 variants has shaped the humoral immune response in a population specific manner. While characterization of this immune response is important for monitoring the disease progression at population level, it is also imperative for the development of effective countermeasures in the form of novel vaccines and therapeutics. India has implemented the worlds second largest COVID19 vaccination drive and also encountered large number of post vaccination "breakthrough" infections. From a cohort of breakthrough infection patients, we identified individuals whose sera showed broadly cross-reactive immunity against different SARS-CoV-2 variants. Interestingly, these sera primarily target a novel cryptic epitope which was not identified in previous population level studies conducted in western countries. This rare cryptic epitope remains conserved across all SARS-CoV-2 variants including the recently emerged ones and also for the SARS-like coronaviruses that may cause future outbreaks, thus representing a potential target for future vaccines.

molecular biology↗