Biology subjects

Yadav, S.

Publications and source records attributed to Yadav, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Cellular Traction and Morphological Adaptation to Cell-Scale Stiffness Gradients on Length-Encoded Micropillar Arrays

In tumour microenvironments, cancer cells routinely encounter abrupt and spatially heterogeneous stiffness variations at the scale of individual cells. How cells sense and respond to such steep, local stiffness gradients is not well understood. Here, we developed length-encoded micropillar arrays in which stiffness is set by the effective pillar length, while the pillar tops remain at the same height and spacing. Pillars with diameters of 2, 2.5 and 3 {micro}m gave stiffness values of 4.0-82.6 nN/{micro}m (effective modulus 2.9-39.4 kPa), varying periodically over distances comparable to the size of individual cancer cells. This platform allows simultaneous measurement of cell morphology, alignment, spreading, pillar displacement, and traction forces. In MDA-MB-231 breast cancer cells, pillar displacement showed a biphasic response. It decreased with increasing stiffness up to about 10 nN/{micro}m and then remained close to 0.5 {micro}m. In contrast, traction forces increased across the full stiffness range. Cells contacted soft pillars more than stiff pillars, with about 23% greater coverage of soft regions. Cells also avoided large stiffness differences along their length: among cells whose two ends sat on pillars of equal stiffness, 48-50% lay along a single stiffness row, compared with about 14% expected by chance. Cell alignment was enriched along the two principal array directions (27-31% of cells within 10{degrees}) and increased slightly with pillar diameter. Length-encoded micropillar arrays therefore provide a platform for studying how cells respond to cell-scale mechanical heterogeneity, with relevance to cancer invasion.

bioengineering↗

bioRxiv2026

Functional dissection of ultraconserved non-coding regions present near the Hoxd complex in vertebrates

Ultraconserved non-coding elements (UCEs) exhibit near-perfect sequence conservation across vertebrate genomes, implying severe evolutionary constraint and essential biological functions. Near the vertebrate Hoxd complex, a master regulator of axial patterning and limb morphogenesis, three Conserved Regions (CR1, CR2, and CR3), each longer than 200 bp, show exceptional sequence conservation and drive robust regulatory activity in transient reporter assays. To investigate their endogenous in vivo functions, we used CRISPR-Cas9 genome editing to generate a series of deletions of varying sizes spanning CR2 and CR3, individually and in combination, within hoxd complex in zebrafish. Screening thousands of founder embryos revealed a clear size and conservation-dependent relationship between deletion extent and viability: complete deletion of core CR2, CR3, or the combined CR2/3 loci could not be transmitted through the germline, whereas the smaller and least-conserved deletion, a 626-bp subregion of CR2, was the only allele to yield a stable, homozygous-viable line. Our results indicate that the core ultraconserved sequences are developmentally indispensable, whereas flanking, less conserved sequences can be removed without consequence. Detailed phenotypic and molecular characterization of the mutant embryos revealed normal primary and secondary body axis formation, intact somite segmentation, and unperturbed craniofacial and pectoral fin development. Expression of hoxd complex genes showed no significant change in mutant embryos by RNA-ISH or RT-qPCR, although RT-qPCR revealed a consistent, modest upward trend at 24 and 48 hpf, raising the possibility that the deleted subregion normally contributes a weak repression of overall hoxd complex genes transcription. While this study reveals some functional aspects of ultraconserved elements, much more remains to be uncovered.

developmental biology↗

bioRxiv2026

High-yield expression and purification of the non-phosphorylated form of Human CaMKIIα Kinase

Human Ca2+/calmodulin (CaM)-dependent protein kinase II alpha (CaMKII) is a highly abundant and conserved protein in the brain. It is a multifunctional serine/threonine kinase that phosphorylates cellular substrates across subcellular compartments, activating signaling pathways essential for long-term memory storage. However, a detailed understanding of protein structure and enzyme-substrate interaction mechanisms require a method to produce active, stable, and pure proteins at scale. Since phosphorylation can alter catalytic activity and substrate affinity, examining the non-phosphorylated state enables an unbiased characterization of basal enzymatic function. Therefore, producing the non-phosphorylated form of the kinase is important for biochemical and structural studies aimed at understanding kinase activation mechanisms. We describe a detailed protocol for the expression and high-yield purification of full-length, non-phosphorylated human CaMKII in bacterial cells. CaMKII was co-expressed with {lambda}-phosphatase to maintain it in a non-phosphorylated state. Protein expression was induced by autoinduction at low temperature to enhance the stability and solubility of CaMKII. Recombinant CaMKII was purified by Ni-NTA affinity chromatography, followed by gel filtration chromatography. Although CaMKII has been considered difficult to purify in its non-phosphorylated form, we obtained it in high yield, and the protein was predominantly non-phosphorylated. Our results show that combining co-expression with autoinduction improves both the solubility and yield of the homogeneous non-phosphorylated form of CaMKII, which could be suitable for subsequent biochemical and structural studies.

biochemistry↗

bioRxiv2026

BLTP3A dysfunction unleashes lysosomal stress-induced antitumor immunity through STING

High-grade serous ovarian cancer progresses within a lipid-rich ascites microenvironment that engages stress-adaptation programs supporting malignant growth, yet how failed stress resolution influences tumor progression remains incompletely defined. Here, we show that unresolved lysosomal stress restrains tumor progression by sustaining tumor cell-intrinsic STING activity and reorganizing antitumor immunity. We identify BLTP3A as a lysosomal stress-resolution factor in ovarian cancer and show that the common germline missense variant BLTP3A M1098T increases stress-induced lysosomal damage. Patients carrying M1098T had longer overall survival and tumors enriched for cytotoxic T cell-myeloid neighborhoods. In syngeneic tumors, Bltp3a loss or orthologous variant expression delayed progression and generated Cxcl9+ antigen-presenting macrophage niches; M1098T-expressing human tumors similarly promoted interferon-responsive myeloid and T-cell enrichment in humanized mice. Mechanistically, ascites-induced lysosomal stress activated a STING-ATF4-BLTP3A feedback program. BLTP3A dysfunction disrupted CASM-associated lysosomal stress resolution, resulting in prolonged STING activity and sustained tumor-derived IFN-{lambda} without increased cGAMP. Tumor-cell STING and IFN-{lambda} were required for immune remodeling and tumor control. Thus, BLTP3A-dependent lysosomal stress resolution constrains the persistence of stress-induced STING signaling, whereas failure of this response sustains a tumor-intrinsic inflammatory program that organizes protective antitumor immunity.

cancer biology↗

bioRxiv2026

Bio-mimicked Leaf-Imprinted Topographies: Pattern Characterization and Cell Response

Proper alignment of cells is crucial for functioning of various tissues such as skeletal muscle tissues, neural cells, adipose-derived stem cells, etc. Current in-vitro fabrication methods to replicate the cellular environment, e.g., photolithography and 3D printing, are not cost-effective and cannot capture the complexity of the surfaces to which these cells are exposed to. In this work, we used bio-mimicked leaf templates to closely resemble the in-vivo environment the muscle cells and cultured C2C12 cells, myoblast cell lines, on modified PDMS substrates fabricated using these leaf templates. Using image analysis software, we analyzed the degree of alignment of cells, aspect ratio and the area projected by individual cells cultured on these surfaces. The C2C12 cells cultured on the PDMS substrates formed utilizing the front and back sides of the leaves of Musaceae Banana were found to have an Aspect Ratio of 6.3 and 8.3, the highest among the surfaces studied in this paper. C2C12 cells cultured had the highest degree of alignment on the negative replica of the back side of Dracaena Sanderiana. Due to the availability of a wide range of leaf templates and bio-mimicked surface structures to measure cell response, it is difficult to find the optimal design. Hence, we have also tried to create a catalog using 15 different leaf surfaces and characterized these surfaces into various categories based on the grooves on the surfaces to provide a more comprehensive set of surface designs for studying cell behavior. To quantitatively analyze the groove pattern, 2D FFT analysis was also performed to find the dominant wavelength of the grooves. In surface characterization, hydrophobicity is also a parameter that needs to be considered; hence, the water contact angle of these surfaces was also measured. Our findings highlight the importance of surface topography and hydrophobicity in influencing cell alignment and can contribute to developing biomimetic surfaces for tissue engineering applications.

bioengineering↗

bioRxiv2026

Dysregulated neutrophil extracellular trap formation is a novel driver of clonogenicity and therapeutic vulnerability in CMML

Chronic myelomonocytic leukemia (CMML) is an aggressive hematologic malignancy characterized by excess inflammatory signaling and clonal myeloproliferation. The relative contribution of neutrophils (PMNs) to the inflammatory milieu in CMML is poorly understood. In this study we sought to understand whether neutrophil extracellular trap (NET) formation, a key mediator of neutrophilic inflammation, is dysregulated in CMML and can be therapeutically targeted with a novel peptide inhibitor of NETosis called neonatal NET-inhibitory factor (nNIF). Here, we demonstrate that baseline NET formation is aberrantly increased in primary CMML PMNs transcriptionally primed for NETosis, and that soluble factors produced during CMML NET formation promote clonogenicity in CMML CD34+ hematopoietic cells matched to the same patient. Further, we show that nNIF and clinical agents under investigation in CMML effectively inhibit NETosis, warranting further study of NET inhibitory agents in this rare disease with limited treatment options.

cancer biology↗

bioRxiv2026

Damaging RBM20 E-rich domain variants are not rescued by gene replacement

The promise of precision therapeutics in genetic cardiomyopathies relies on linking specific therapies to variant mechanisms. Missense variants in the cardiac splice regulator RBM20 cause a highly penetrant and arrhythmogenic dilated cardiomyopathy. Disease-causing variants in RBM20s arginine-serine rich (RS) domain act via formation of toxic gain of function cytoplasmic granules, but this is not true for a small number of clinically adjudicated pathogenic variants in its glutamate(E)-rich domain. To better define the effects of E-rich domain variants, we developed a scalable screen based on induced pluripotent stem cell (iPSC) cardiomyocyte differentiation that identified several additional damaging variants. Several of these reduced RBM20 protein abundance and stability. We therefore hypothesized that, unlike RS domain variants, these E-rich variants might be rescued by RBM20 overexpression. To test this hypothesis, we generated induced pluripotent stem cells (iPSCs) from a patient with a pathogenic E-rich domain variant (p.E913K), and confirmed reduced RBM20 protein expression in these RBM20+/p.E913K cells after differentiation to iPSC-derived cardiomyocytes (iPSC-CM, vs. engineered isogenic RBM20+/+). These iPSC-CMs also displayed aberrant transcriptional splicing, reduced contractility, increased calcium-induced calcium release, and nuclear localization of RBM20 protein, often with more than the two expected RBM20-centric splice factories. AAV-based overexpression of RBM20 reversed some, but not all of the mis-splicing events identified in RBM20+/p.E913K iPSC-CMs, and did not improve their abnormal contractility, calcium handling or supernumerary RBM20 nuclear granules. In summary, our data indicate that pathogenic E-rich domain variants reduce RBM20 protein abundance, but that their mechanism is unlikely to be explained by haploinsufficiency alone.

genetics↗

bioRxiv2026

Vitamin D mitigates Inflammatory Bone Loss in Postmenopausal Osteoporosis via modulating the Gut-Immune-Bone axis

Osteoporosis is a prevalent skeletal disorder characterized by deterioration of bone microarchitecture and loss of bone mineral density, leading to increased fracture risk and substantial health and economic burdens, particularly among older adults. Bone remodeling is orchestrated by a complex interplay of systemic and local regulators, among which vitamin D plays a central role in maintaining skeletal homeostasis. Although numerous studies have examined the effects of vitamin D on bone metabolism, outcomes have been inconsistent across populations, dosing regimens, and experimental models. To clarify the net skeletal impact of vitamin D, we investigated its effects in postmenopausal osteoporosis (PMO). Vitamin D (1,25-dihydroxyvitamin D3- active form of vitamin D) supplementation effectively prevented bone loss in ovariectomized mice, at both lower and higher concentrations. Mechanistically, vitamin D promoted osteoclast differentiation in vitro, consistent with its RANKL-dependent pro-osteoclastogenic activity, yet paradoxically conferred bone protection in vivo. This discrepancy was explained by vitamin Ds profound immunomodulatory effects, which reshaped both innate and adaptive immune responses to suppress osteoclast formation and function. Concurrently, vitamin D improved intestinal barrier integrity and restored gut microbial composition, thereby stabilizing the gut-immune-bone axis and reducing pro-resorptive inflammatory signaling. Together, these findings demonstrate that vitamin D prevents bone loss through the coordinated regulation of immune and gut homeostasis, reconciling its apparent pro-resorptive effects in vitro with its overall anti-resorptive outcomes in vivo. This integrated mechanism highlights immune-gut microbial modulation as a key mediator of vitamin D-induced bone preservation and supports the development of vitamin D as an immunotherapeutic adjunct for the prevention and management of PMO. Altogether, our findings for the first time dissect the paradox surrounding the osteoprotective property of vitamin D supplementation. O_FIG O_LINKSMALLFIG WIDTH=186 HEIGHT=200 SRC="FIGDIR/small/735433v1_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@1550497org.highwire.dtl.DTLVardef@a06ab4org.highwire.dtl.DTLVardef@1bd55e5org.highwire.dtl.DTLVardef@1526acd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Vitamin D strengthens intestinal barrier integrity and restores gut microbial composition, which stabilizes the gut-bone axis and suppresses pro-resorptive inflammatory signaling. Vitamin D enhances osteoclastogenesis both directly and through ILC3- and Th17-mediated pathways, while inhibiting osteoclastogenesis via Treg- and Breg-dependent mechanisms. In circulation, vitamin D maintains the homeostasis between IL-10 and IL-17 cytokines. Collectively, these results indicate that vitamin D helps prevent bone loss by orchestrating the immune system and maintaining gut homeostasis. C_FIG

immunology↗

bioRxiv2026

Head-to-head organized segmental paralogs AtOFP2 and AtOFP17 exhibit differential, spatio-temporal partitioning of function, and negative regulation of multiple developmental traits including seed-yield and root architecture

Ovate Family Proteins (OFPs) are a class of plant-specific, negative nuclear transcriptional regulators characterized by conserved C-terminal OVATE domain. This study on comparative functional characterization of two head-to-head arranged OFPs - AtOFP2 (Ovate-OFP with full ovate domain) and AtOFP17 (Ovate-Like OFP with partial ovate domain) provides critical insight into how structural variations in ovate domain leads to functional divergence. Detailed phenotypic analysis of 28 physical and physiological traits of loss- and gain-of-function mutants revealed that both genes act as broad, pleotropic repressors of plant growth and development. Removal of repression in knock-down mutants of both genes exhibited reduced duration of seed dormancy, faster rate of germination and growth, bigger plants and significantly higher seed yield. In contrast, constitutive over-expression showed a generalized repressive nature of both genes, with nuanced differences for fine tuning of specific traits. For example, both genes showed antagonistic behaviours on root hair architecture. AtOFP2 act as a strong repressor of root hair development whereas AtOFP17 is a stronger repressor of hypocotyl and root cell architecture. AtOFP17 owing to partial ovate domain exerts a mild level of repression throughout life span as indicated by smaller plants and lesser yield in knock-down AtOFP17 mutants. On the contrary, AtOFP2 exerted a much stronger repressor effect in which > 90% over-expression mutants died at the juvenile stage ; the survival of remaining 10% is probably owing to activation of dosage-dependent feedback loop mechanism as indicated by normal growth of mature plants, and is also evident by transcriptome data. Transcriptome analysis of roots of 7-day old seedling of knock-down and over-expression mutants of AtOFP2 showed downregulation of OFP2 in over-expressed mutants. However, severely stunted phenotype indicated presence of stable OFP2 protein to exert effects. Analysis of DEGs in OFP2 mutants revealed that it acts as an important regulator working at intersection of hormonal signalling affecting critical genes required for auxin, cytokinin, GA, BR and ABA functioning. Perturbations across hormonal signalling pathways affects cell wall remodelling factors such as EXPANSINS, Xyloglucan hydrolases (XTHs) and cellulose synthases (CSLs) causing overall stunted growth; and epidermal patterning genes such as WER, GL1, EGL3, TTG1 leading to severely reduced root length and root hairs. Significantly, functional analysis of this master regulator highlighted a significant economic potential. Knockdown of both these genes relieves their natural repression on reproductive traits, leading to longer siliques, bigger and heavier seeds, and substantially increased overall seed yield, positioning AtOFP2 and AtOFP17 as highly valuable targets for agricultural crop improvement.

plant biology↗

bioRxiv2026

Metagenomic prediction of methane emissions in sheep using single- and multi-matrix BLUP models with taxonomic and functional microbial features

BackgroundEnteric methane emissions from ruminant livestock represent a major greenhouse gas contributor, yet identification of high- and low-emitting ruminants remains expensive and logistically challenging for agricultural methane mitigation strategies. Ruminal microbial profiles derived from long-read sequencing technology provide a potential proxy to predict methane production. The optimal bioinformatic pipelines for processing long-read metagenomic data to perform methane predictions have yet to be determined. Here we evaluated how different metagenomic analysis pipelines affect methane predictive model accuracy in grazing sheep. ResultsWe applied three bioinformatic pipelines to characterize the taxonomic and functional features of rumen microbiomes from 396 sheep. Functional abundance features were annotated from Clusters of Orthologous Genes (COG) or Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The single-matrix model using COG features achieved the highest microbiability (m2 = 0.942: proportion of variance component explained by microbial features) and predictive accuracy (5-fold cross validation r = 0.609: Pearsons correlation between predicted and observed values). Both functional features outperformed all taxonomic features across all three pipelines in predictive accuracy. The multi-matrix models combined functional and taxonomic features slightly improved methane predictive accuracy across both 5-fold cross-validation and leave-one-day-out validation compared to the models using functional features alone. ConclusionsThese findings demonstrate the potential advantages of using long-read metagenomic data to predict enteric methane emissions in ruminants. COG-based functional features achieved the highest predictive accuracy among all feature types, suggesting that functional annotation of existing long-read sequences is sufficient for accurate methane prediction without requiring complementary taxonomic data.

genomics↗

bioRxiv2026

Spin-Dependent Extracellular Respiration

Biological energy conversion relies on highly efficient electron transfer. The chirality induced spin selectivity (CISS) effect, which couples electron spin to momentum in chiral molecules, is hypothesized to promote this efficiency. While observed in isolated biomolecules, the physiological relevance of CISS during active cellular metabolism remains unknown. Here, we demonstrate that CISS influences extracellular electron transfer in living Geobacter sulfurreducens biofilms. Cultivation on ferromagnetic electrodes yields a significant asymmetry in respiratory current between opposite substrate spin states. Furthermore, in situ magnetization reversal induces reversible changes in respiratory flux. These results provide the first in vivo demonstration that spin selectivity directly impacts respiration. By revealing a quantum feature of extracellular respiration, our findings offer a strategy to exploit the spin degree of freedom in bioelectronics.

biophysics↗

bioRxiv2026

Residue-specific dominant-negative mutant of ubiquitin reveals functional selectivity as Ubp14 deubiquitinase inhibitor

Multifunctional proteins encode specificity through nuanced molecular interactions that operate in the presence of abundant wild-type molecules. Previous studies on multifunctional proteins have shown how certain regulated interactions parse complex biological activities into discrete functional modules. Discrete interaction edges can function as regulatory nodes whose perturbations selectively remodel proteostasis output, especially under disease conditions. However, whether such interaction specificity can be harnessed to selectively manipulate functional modules and reveal residue-level control principles, remains largely unexplored. Here, we screened for dominant negative mutations that impact partner-selective functions linked to Leu8, a critical binding residue on ubiquitin. Our results reveal that the Leu8Ala mutation specifically leads to accumulation of polyubiquitinated proteins and decreased levels of free ubiquitin suggesting loss of deubiquitinase function in yeast cells. Cellular and biochemical analyses establish that the Leu8Ala variant of ubiquitin specifically inhibits the yeast deubiquitinase, Ubp14, and its human homolog, USP5. We further demonstrate remodelling of the binding interface with increased interface contacts for the variant and Ubp14 complex. The variant shows a higher inhibitory potency compared to the wild-type ubiquitin and can inhibit Ubp14 both as unconjugated and as conjugated ubiquitin chain indicating the strength of its inhibitory function. Our results provide mechanistic insight into how edge perturbations in ubiquitin can reveal critical nodes that impact selective functions and thus fine-tune the cellular proteostasis network for therapeutic benefit.

biochemistry↗

bioRxiv2026

Resting State Somatomotor Functional Brain Networks using Empirical Mode Decomposition and Hilbert Transformation

The goal of the resting-state functional connectivity studies is to determine the inherent dynamics of the brain networks while the body is at rest. These networks get differentially activated when the brain is involved in various tasks such as processing of sensory inputs, initiating motor activities, or various cognitive tasks. Resting state functional connectivity networks are commonly revealed by determining Pearson Correlation Coefficients of the Blood Oxygenation Level Dependent (BOLD) signals collected from different brain regions using functional Magnetic Resonance Imaging (fMRI) while the subject is not actively performing any task. However, the functional connectivity thus determined does not correlate well with the known structural connectivity between different brain regions. Here, we used Empirical Mode decomposition (EMD), followed by Hilbert Transformation (HT), to determine the resting state functional connectivity in the human brains. We show the advantage of using this EMD-HT method using somatomotor network as an example. We show that the time series data decomposed by this method improves correlation of the derived functional connectivity with the known structural connectivity (especially for low -TR fMRI data) as compared to the methods commonly used.

neuroscience↗

bioRxiv2026

Chemical Genetic Screen Identifies PSD3 as a Direct Substrate of NUAK1 that Regulates Dendritic Spine Maturation

Dysregulation of signaling by Novel (Nua) Kinase 1 (NUAK1) is associated with autism spectrum disorder, tissue fibrosis, and cancer progression. Direct phosphorylation targets of NUAK1 in the brain are unknown, hindering mechanistic understanding of its role in neurodevelopment. Here, we demonstrate that autism-associated NUAK1 variants differentially impact catalytic activity and subcellular distribution. We engineered ATP-analog sensitive NUAK1 and utilized its specificity towards bulky analogs to identify over 30 hitherto unknown direct phosphorylation targets of NUAK1 in the brain. We demonstrate that Pleckstrin Homology and Sec7-domain containing protein 3 (PSD3) is a bona fide phosphorylation target of NUAK1. PSD3, a guanine nucleotide exchange factor (GEF) for ARF6 GTPase, is phosphorylated by NUAK1 at Ser476. In neurons, expression of phosphodeficient PSD3 (S476A) leads to enhanced dendritic spine maturation in an ARF6-dependent fashion. Mechanistically, NUAK1 suppresses ARF6 activation through PSD3 phosphorylation. Abolishing Ser476 phosphorylation leads to aberrant ARF6 activation and increased generation of PI(4,5)P2. Our study reveals direct neuronal substrates of an autism risk gene NUAK1 and further delineates a mechanism through which NUAK1 phosphorylation of PSD3 controls ARF6 activation and dendritic spine maturation.

neuroscience↗

bioRxiv2026

Proviral dynamics and HIV-1C viral diversity in the context of HIV-TB co-infection

BackgroundART effectively suppresses HIV replication and restores CD4+ T cells; however, long-lived HIV latent reservoirs enable viral persistence. Tuberculosis (TB) co-infection further impacts HIV latency and enhances viral replication. Given the high prevalence of latent TB infection (LTBI) in TB-endemic settings, understanding its impact on HIV biology is critical. Our study aims to investigate the influence of TB co-infection on HIV reservoir dynamics, viral diversity, and drug resistance mutations in ART-naive individuals. MethodologySamples from 90 ART-naive HIV-1C individuals, stratified based on IGRA and TB diagnosis, were used in this study. Plasma and PBMCs were isolated for viral RNA and DNA extraction respectively. Total proviral DNA was quantified using gag PCR. Full-length env and pol genes were amplified, purified and sequenced using ONT and Illumina platforms. Pol sequences were subjected to Drug Resistance Mutation (DRM) analysis via Stanford HIVdb with a minimum threshold mutation frequency of [≥]10%. Full length env sequences were used for phylogenetic analysis by aligning with Indian Subtype C reference sequence and phylogenetic tree was generated using ggplot2. ResultProviral load analysis showed no significant differences across HIV+LTBI-, HIV+LTBI+, and HIV+TB+ groups, although a trend toward higher levels was observed in HIV+TB+ individuals. Correlation analysis revealed distinct immune associations, with HIV+LTBI+ individuals showing positive correlations with activation and PD-1 expression. Longitudinal analysis of proviral loads demonstrated a modest decline in proviral load post-ART but remained persistent for up to 18-20 months following initiation of ART accompanied by low level ongoing viral replication. DRM analysis revealed a 33% prevalence in ART-naive individuals, with higher occurrence in HIV+LTBI+ group. Of the identified DRMs, 38% (5/13) and 71% (5/7) in sequences obtained from PBMC and plasma respectively were attributed to polymorphic mutations associated with Integrase strand transfer inhibitors (INSTIs). DRMs within plasma and PBMC derived viruses showed high concordance. Phylogenetic analysis of env sequences indicated overlapping viral populations between the 3 groups, with greater diversity in PBMCs compared to plasma. ConclusionThe study highlights that HIV reservoir dynamics, drug resistance, and viral diversity are significantly influenced by TB co-infection. While proviral loads were comparable, LTBI-associated immune activation and granuloma niches may have driven viral diversification and DRM emergence. High concordance between compartments and presence of transmitted resistance underscore the need for baseline screening, multi-compartment analysis, and sustained surveillance.

microbiology↗

bioRxiv2026

The Human Male Mammary Gland has Similar Epithelial Populations to Female but Distinct Composition and Transcriptional Properties

The normal adult male breast has not been characterized at single-cell resolution, leaving the cellular basis of male breast cancer (MBC) biology undefined. Here we present an integrated single-cell RNA sequencing atlas of the adult human breast comprising 174,471 cells from 17 donors (3 male, 14 female), including 18,117 male-derived cells. This revealed that the male breast retains all three epithelial populations, basal (BC), luminal progenitor (LP), and luminal committed cells (LC), but with an increase in LC at the expense of BC and LP across all three male donors. Male LC were distinguished from female by elevated ESR1 and PGR mRNA, enrichment of RNA processing and ribosome biogenesis programs, reduced inflammatory cytokine and growth factor signaling, elevated estradiol gene set enrichment scores, and higher inferred activity of developmental patterning transcription factors. This pattern was observed across differential expression, gene ontology, ligand profiling, and regulon-based analyses, and was not restricted to sex chromosome-linked gene expression. This is consistent with the near-universal estrogen receptor (ER) positivity that characterizes MBC clinically. This atlas provides the first cellular and transcriptional reference for the normal male breast and a resource for investigating sex differences in mammary biology, germline susceptibility variant interpretation, and modeling breast malignancies.

cancer biology↗

bioRxiv2026

Highly Stable Mn(V)-Nitrido and Nitrogen-Atom Transfer Reactivity within a De Novo Protein

High-valent metal-nitrido species are powerful nitrogen-atom transfer intermediates but remain difficult to access and control due to intrinsic instability and bimolecular N-N coupling pathways. Herein, we report the first formation of a high-valent Mn(V)-nitrido complex within a de novo designed protein scaffold and demonstrate that a reactive precursor to this species can be catalytically intercepted for enantioselective aziridination. A Mn(V){equiv}N unit derived from an abiological diphenyl porphyrin is confined within a designed helical bundle protein, where the protein environment suppresses bimolecular decay and enables detailed spectroscopic characterization. Electron paramagnetic resonance, resonance Raman, and circular dichroism spectroscopies confirm formation of a low-spin Mn(V)-nitrido species that is stable for weeks at room temperature and exhibits minimal perturbation of the Mn{equiv}N unit upon modulation of the axial histidine ligand, while catalytic activity and stereochemical outcome are sensitive to its presence. Mechanistic studies identify monochloramine (NH2Cl) as the operative nitrogen-atom donor and support the involvement of a transient Mn-bound N-transfer intermediate en route to nitrido formation. Under catalytic conditions, this intermediate is inter-cepted to perform aziridination with TON {approx} 180 and an enantiomeric ratio of 65:35. Together, these results establish de novo protein design as a platform for stabilizing high-valent metal-nitrido species and harnessing their reactivity for nitrogen-atom transfer chemistry beyond the limits of natural metalloenzymes and small-molecule catalysts.

biochemistry↗

bioRxiv2026

Ribosome Processing Factor-2 Interacts with RPL10A to Regulate Selective Translation during Plant Immunity and Drought Stress

Processing of ribosomal RNA (rRNA) is essential for ribosome biogenesis, translation, plant development, and stress adaptation. Ribosome processing factor-2 (RPF2), which plays a role in the later stages of rRNA maturation, interacts with ribosomal protein L10A (RPL10A). RPF2 overexpression in Arabidopsis and Nicotiana benthamiana showed enhanced plant growth and trichome development due to increased gibberellic acid (GA) levels. Conversely, RPF2-silenced and mutant plants had a dwarf phenotype, reduced stomatal apertures, and decreased glucosinolate accumulation. RPF2 silenced and mutant plants also showed compromised nonhost disease resistance, whereas RPF2 overexpression lines exhibited enhanced disease resistance to both host and nonhost pathogens. RPL10A and RPF2 overexpression lines were sensitive to abscisic acid (ABA) and tolerant to drought, which is attributed to their unique roles in translation regulation. Despite having larger stomatal apertures, RPF2 overexpression plants displayed low pathogen multiplication rates and reduced water loss, indicating independent resistance mechanisms associated with ribosomal functions in translation regulation. Although both RPL10A and RPF2 proteins interact with each other and are involved in translation regulation, proteomic analysis suggests that they regulate the translation of distinct sets of genes during pathogen or drought stress. These findings indicate that RPF2 and RPL10A play independent roles in the regulation of unique protein translation.

plant biology↗