Biology subjects

Bhattacharya, A.

Publications and source records attributed to Bhattacharya, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Dose dependent impact of global Cc2d1a reduction on cognitive, social, and metabolic phenotypes

Background Autism spectrum disorder (ASD) and Intellectual disability (ID) share an in-creased male prevalence, but the biological mechanisms underlying this male bias remain poorly understood. Mice deficient for CC2D1A, a protein scaffold mutated in ASD/ID, have shown multiple male-specific deficits both in ASD-like behaviors and in the regulation of neuronal intracellular signaling pathways. However, previous re-search relied on conditional knockout (cKO) models due to early postnatal lethality of global Cc2d1a KO strains. Thus, the ability to define the developmental impacts of CC2D1A LOF in mice has been limited. While generating a Cc2d1a knock-in strain with V5-HA epitope tags, we found that homozygous animals (Cc2d1aVH/VH) resulted in a hypomorph line that can be used to study developmental loss of CC2D1A. Methods We assessed gross morphology, behavior, and intracellular signaling in male and female Cc2d1aVH/VH animals, where only ~20% of CC2D1A is present. We then crossed this line with a null allele to obtain a more severe hypomorph (Cc2d1a-/VH) and explore phenotypic penetrance thresholds. In addition to the previous phenotypic characterization, we also assessed body composition, feeding, and molecular hall-marks of metabolic function in both sexes. Results We found that ~80% CC2D1A reduction in Cc2d1aVH/VH mice did not recapitu-late any of the behavioral or molecular disruptions seen in cKO models. In contrast, ~90% CC2D1A reduction in Cc2d1a-/VH mice resulted in ASD/ID-like behavioral phe-notypes in males, similar to those reported in cKO lines, with distinct intracellular sig-naling disruptions. Male and female Cc2d1a-/VH mice also showed subtle but signifi-cant adult weight deficits associated with distinct metabolic changes. Limitations These models still do not fully recapitulate the complete CC2D1A LOF found in patients. Due to the analysis of two independent strains over multiple metrics, these studies are not powered to compare statistical differences between sexes and data were analyzed in males and females independently. Conclusions We identified a strikingly narrow CC2D1A expression threshold for the emer-gence of ASD/ID-like behavioral deficits in males. In addition, we revealed a novel role for CC2D1A in adult weight gain. These mouse strains will be useful to better under-stand CC2D1A LOF phenotypes.

neuroscience↗

bioRxiv2026

Molecularly Distinct Innexin Gap Junction Channels and Undocked Hemichannels Regulate Glia Morphology and Function in Caenorhabditis elegans

Glial cells across species orchestrate nervous system development, maintenance, and function through ionic and metabolic crosstalk. These cells express multiple gap junction and hemichannel components. However, the mechanisms by which glia communicate utilizing these channel components to support nervous system architecture and function remain poorly understood. By studying GLR glial cells in Caenorhabditis elegans, we show that individual glial cells employ distinct innexin channel configurations to support specific functional roles, utilizing discrete downstream cellular mechanisms. We find that while innexins UNC-7and UNC-9 function as gap junction channels, the innexin INX-18 operates independently to form undocked hemichannels in discrete domains within the same cells. In this combinatorial configuration, UNC-7/UNC-9 gap junctions specifically regulate Synaptobrevin/SNB-1 localization in RME neurons, as previously reported. However, both channel types function non-redundantly to maintain glial morphology by regulating the Calpain/CLP-4 and CDK-5 pathway through cell-autonomous modulation of intracellular calcium levels. In contrast, INX-18 hemichannels in GLR glia regulate high-salt-induced paralysis behavior through distinct downstream cellular mechanisms, potentially in conjunction with tyramine signaling. Altogether, our findings demonstrate a broader repertoire of innexin channel utilization at the individual glial cell level, supporting specific aspects of nervous system maintenance and functioning.

neuroscience↗

bioRxiv2026

Assessment of Microbial Consortia for Biogenic Mineralization Potential of Lead (Pb) using Microbiologically Induced Calcite Precipitation and Nitrate Reduction

Groundwater contamination by lead and nitrate exceeding the permissible limits of the World Health Organization (WHO) and the Bureau of Indian Standards (BIS) of 10 ppb lead and 45 to 50 ppm nitrate poses a severe health risk of systemic toxicity in humans and animals. Microbial nitrate reduction coupled with microbiologically induced calcite precipitation (MICP) proved to be a novel bioremediation strategy to transform soluble lead nitrate into insoluble lead biominerals. This study presents statistical optimization of operational parameters for nitrate reduction and co-precipitation of biogenic lead carbonate-calcium carbonate by nitrate-reducing bacterial consortia. The SEM, TEM with SEAD, and XRD analysis of the biomineral confirmed lead carbonate co-precipitation with CaCO3. A two-level factorial design was applied to screen significant variables and interactive factors that influence nitrate reduction and biomineral precipitation. The highest nitrate reduction, maximum ammonia production, and biogenic Pb-mineralization efficiency of > 90% were obtained at the optimum conditions (initial nitrate requirement of 600 ppm, temperature 37 oC, pH 8, and carbon content of 5.5 g/L) compared to those obtained under the common process parameters. ANOVA with a high correlation coefficient (R2 > 0.99) corroborated the linear model for the biogenic mineralization process. This study on the biogenic removal of Pb by nitrate-reducing bacterial consortia revealed possible opportunities for large-scale treatment of Pb-contaminated surface and subsurface water bodies.

microbiology↗

bioRxiv2026

Leucine Regulates Neuronal Health During Nutrient Deprivation in Caenorhabditis elegans

Alterations in nutrient availability induce changes in neuronal structure and connectivity across species, ultimately leading to nervous system plasticity and behavioural changes. However, it remains unclear how prolonged acute nutrient deprivation affects the integrity and function of the nervous system. Using the nematode Caenorhabditis elegans, we uncovered that prolonged macronutrient deprivation profoundly impacts neuronal health. Across developmental stages, multiple sensory neuron classes in the C. elegans nervous system, including polymodal IL2, volatile odor-sensing AWB and AWC, and CO2-sensing BAG neurons, display extensive dendritic and axonal blebbing, severely disorganized dendritic, axonal, and cell body morphologies, and severely disrupted chemical and electrical synaptic organization during prolonged nutrient deprivation. The severity of these defects increases progressively with the duration of nutrient deprivation, ultimately affecting animal behaviours. Our results show that the availability of a particular macronutrient, specifically the branched-chain amino acid leucine alone, in the absence of any other macronutrients, although insufficient to support animal growth, is sufficient to completely prevent nutrient-deprivation-mediated neuronal damage and the decline in animal behavioural responses. Moreover, leucine supplementation alone is sufficient to reverse nutrient-deprivation-mediated neuronal abnormalities. These findings suggest that the availability of leucine, rather than absolute caloric intake, is crucial for maintaining neuronal health and function under adverse conditions.

neuroscience↗

bioRxiv2026

An Improved Protocol for High-Efficiency and Cost-Effective CRISPR/Cas9-Mediated Knock-ins in C. elegans

CRISPR/Cas9-mediated homology-directed precise genome editing using long single-stranded DNA (ssDNA) donors has expanded the possibilities for generating defined genetic modifications. However, the required ssDNA donor preparation can be technically demanding and often requires extensive locus-specific sequence design. Here, we examined parameters influencing ssDNA donor-mediated genome editing and developed approaches to simplify donor preparation using {lambda}-exonuclease-mediated ssDNA generation. By evaluating donor designs across multiple genomic loci, we found that efficient genome editing can be achieved with relatively short homology regions for a range of insertion sizes. These findings provide a basis for simplifying ssDNA donor preparation and the overall gene-editing pipeline, potentially facilitating its application across species.

molecular biology↗

bioRxiv2026

Mapping Alzheimer's neuropathology signatures to the whole brain transcriptome using machine learning data fusion

In Alzheimer's disease (AD), misfolded proteins emerge across the entire brain in structured, yet not rigid, spatiotemporal patterns. Yet, a systematic bias of single-cell genomics toward sampling mostly cortical tissue limits our understanding of the whole-brain transcriptomic vulnerability to AD. Here, we develop a machine learning method to extrapolate local AD neuropathology signatures to the whole brain. By analyzing gene expression profiles of over two million cortical cells from 427 humans spanning the AD-pathology spectrum, we derive transcriptomic estimators of AD neuropathology. After extensive validations on datasets with known ground truth, we apply this framework to three million cells from 108 brain regions in the Siletti whole human brain atlas and derive an anticipated brain map of transcriptomic signatures indexing AD neuropathology. This interrogation of regions spanning the cortical, subcortical, and brainstem structures uncovers transcriptomic signatures associated with hyperphosphorylated tau in the medulla oblongata, dorsal raphe nucleus, and the tuberal and mammillary regions of the hypothalamus. At the cellular level, assessments of these signatures across 31 cell populations identify VGLUT1/2 expressing neurons, astrocytes, and microglia as key neuropathology-resembling populations. Within the hippocampus, pathology signatures surface in the rostral cornu ammonis (CA) subfields, particularly in the CA1 pyramidal neurons and dentate granule cells. {beta}-amyloid-like signatures localize to the neocortex with laminar selectivity--most prominently in upper layer somatostatin+ intratelencephalic neurons (L2-L3), but also in deep layer intratelencephalic and corticothalamic neurons (L5-L6). Neocortical astrocytes and microglia exhibiting disease associated signatures similarly demonstrate a unique laminar preference. Together, this study provides the first whole human brain map of AD pathology-associated transcriptomic signals, and exposes cell type, region, and cortex layer specific vulnerabilities.

neuroscience↗

bioRxiv2026

Membrane Mimetic-Thermal Proteome Profiling Reveals Broad, Sequence-Independent Membrane Protein Stabilization by Cholesteryl Hemisuccinate

Membrane protein stability is strongly influenced by the surrounding lipid environment, yet how individual lipid species shape membrane proteome stability remains poorly understood. Here, we systematically examined the impact of sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesteryl hemisuccinate (CHS) on membrane proteomes using membrane mimetic platforms combined with membrane mimetic thermal proteome profiling (MM-TPP). CHS shifted the proteome composition away from soluble proteins and toward integral membrane proteins, and induced concentration-dependent thermal stabilization of the mouse liver membrane proteome. Organellar membrane proteins, which displayed greater intrinsic lability than plasma membrane proteins, showed preferential stabilization by CHS. CHS supplementation of E. coli membranes similarly produced broad stabilization, indicating that this effect occurs even in cholesterol-naive systems. CHS responses were reproducible across Peptidisc and DDM and independent of CRAC/CARC motif density, supporting a broad, sequence-independent mechanism rather than selective lipid binding, although stabilization was greater among proteins with more transmembrane helices. Accordingly, individual purified proteins reconstituted with CHS exhibited only modest stabilization, consistent with a broad effect that is more apparent at the proteome scale than for any single protein examined in isolation. Together, these findings redefine CHS as a general sterol scaffold that broadly stabilizes membrane proteins and establish MM-TPP as a versatile platform for investigating lipid-dependent effects on membrane proteome stability. Subject areaIntegral Membrane Proteins, Thermal Proteome Profiling, Membrane Mimetics, Cholesterol, Lipid-Protein Interactions, Mass Spectrometry HighlightsO_LICHS broadly stabilizes membrane proteins across diverse membrane mimetics. C_LIO_LIOrganellar membrane proteins exhibit the strongest CHS-mediated stabilization. C_LIO_LICHS stabilization is conserved in cholesterol-naive E. coli. C_LIO_LIMM-TPP enables proteome-wide analysis of lipid-dependent protein stability. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/745344v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1a3e017org.highwire.dtl.DTLVardef@1cd4464org.highwire.dtl.DTLVardef@1453da7org.highwire.dtl.DTLVardef@d2dc1d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

A detergent-free workflow for native membrane proteomics using Peptergents

Quantitative membrane proteomics remains fundamentally limited by sample preparation because detergent extraction can perturb membrane protein interactions, ligand-responsive conformations, and higher-order assemblies before mass spectrometric analysis. Here, we demonstrate that peptide-based surfactants (Peptergents) enable a complete detergent-free workflow for native membrane proteomics. Membrane proteins are extracted directly from biological membranes while preserving their structural and functional integrity and remaining fully compatible with downstream LC-MS/MS workflows. Functional preservation is evidenced by maintenance of ligand-responsive conformations in the ABC transporter MsbA and the endogenous GPCR P2RY12, together with stabilization of the detergent-sensitive nine-subunit holo-translocon HTL, indicating that fragile membrane protein assemblies remain intact. At the proteome level, despite recovering fewer membrane proteins than conventional detergent extraction, Peptergent consistently generates higher peptide signal intensities, retains tissue-specific membrane proteome signatures, and preferentially enriches endoplasmic reticulum-associated metabolic networks, including cytochrome P450 enzymes and their interaction network. Together, these findings establish Peptergents as a broadly applicable membrane extraction technology for LC-MS/MS-based membrane proteomics, preserving native membrane organization and expanding the proteomics toolbox for biochemical, structural, and systems-level analyses of membrane proteins. In Brief StatementThis study establishes Peptergents as a detergent-free membrane extraction technology for LC-MS/MS-based membrane proteomics. Peptergent extraction preserves ligand-responsive membrane proteins, fragile membrane protein assemblies, and tissue-specific membrane proteome signatures while remaining fully compatible with quantitative proteomic workflows. These findings provide a broadly applicable strategy for preserving native membrane organization for biochemical, structural, and systems-level analyses of membrane proteins. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/744532v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1cef0c1org.highwire.dtl.DTLVardef@1203aeaorg.highwire.dtl.DTLVardef@fbdb47org.highwire.dtl.DTLVardef@f915e6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPeptergents preserve ligand-responsive membrane proteins. C_LIO_LISupport chemoproteomics in thermal proteome profiling assays. C_LIO_LISimplify membrane proteomics workflow. C_LIO_LIMaintain native tissue-specific membrane biology. C_LIO_LIPreserve fragile membrane protein assemblies. C_LI

biochemistry↗

bioRxiv2026

A Charge-Encoded Rheostat Permits Helix Nucleation but Limits Propagation in Skp1

Molecular recognition by intrinsically disordered regions (IDRs) is widely thought to involve coupled folding and binding, yet the sequence features that regulate this transition remain underexplored. Here we show that helix 8 (H8), a disordered C-terminal segment of the SCF ubiquitin ligase adaptor Skp1, is intrinsically prevented from forming a stable helix by its own sequence grammar. Using an integrative approach to dissect its conformational dynamics, we find that H8 frequently nucleates helical structure but rarely propagates into a fully formed stable helix, populating instead a shallow metastable basin of helical intermediates. Contrary to conventional models of helix-coil exchange, where nucleation is rate limiting, helix initiation in H8 is readily accessible, while propagation is selectively suppressed by a glutamate-rich acidic patch. This acidic segment acts as a charge-sensitive conformational rheostat that limits helix extension and maintains H8 in a predominantly disordered state. As a result, H8 transiently samples partially helical conformations on the microsecond timescale without committing to a stable fold. We propose that this propagation-limited mechanism preserves conformational flexibility while maintaining recognition competence across a structurally diverse family of F-box binding partners. More broadly, our findings suggest that charged- hydrophobic-charged sequence patterning can encode conditional, context-dependent structure as a general organisational principle in intrinsically disordered proteomes.

biophysics↗

bioRxiv2026

M1C IS NECESSARY FOR DARAXONRASIB RESISTANCE OF NSCLC KRAS(G12C) MUTANT CELLS

IntroductionThe RAS(ON) multi-selective daraxonrasib (RMC-6236) inhibitor is effective in patients with NSCLC KRAS mutant cancers. Tolerance to daraxonrasib invariably develops by mechanisms that remain unclear. There is no known involvement of the M1C oncogenic protein in daraxonrasib resistance. MethodsNSCLC H358 KRAS(G12C), H2122 KRAS(G12C) and patient derived MGH1112 KRAS(G12C) cells with acquired daraxonrasib resistance were investigated for M1C dependence in studies of SHP2, STAT1/3 and NF-KB activation, clonogenicity, and self-renewal capacity. ResultsWe demonstrate that M1C is induced as a protective response in NSCLC KRAS(G12C) mutant cells treated with daraxonrasib. We report that M1C forms novel cell membrane-associated biomolecular condensates with the SHP2 protein tyrosine phosphatase in driving daraxonrasib resistance. M1C integrates SHP2 activation with induction of (i) oncostatin-m/gp130/STAT3 signaling, and (ii) the NF-{kappa}B-mediated epithelial-mesenchymal transition (EMT) pathway. The functional significance of this M1C-driven pathway is supported by the demonstration that targeting STAT3 and NF-{kappa}B reverses daraxonrasib resistance. Consistent with M1C dependence, we also show that targeting M1C is effective against daraxonrasib-resistant NSCLC KRAS mutant cell line and tumor models. In contrast, M1C drives sotorasib resistance by STAT1-mediated inflammatory signaling, demonstrating that M1C confers resistance to KRAS(G12C)-selective and RAS(ON) tri-complex inhibitors by noncongruent mechanisms. ConclusionsThese findings demonstrate that M1C is required for daraxonrasib tolerance and is a potential target for the treatment of patients with NSCLC KRAS(G12C) mutant tumors refractory to this agent.

Cancer Biology↗

bioRxiv2026

A Conserved Obg-type GTPase sustains protein synthesis and its physiological roles in eukaryotic cells

Translation is regulated both temporally and spatially to ensure that proteins are synthesized at the correct time and delivered to the proper subcellular location. Temporary slowing or halting of ribosomal movement along an mRNA, a physiological pausing, allows cells to coordinate protein production with internal cues and external signals. However, how cells distinguish such constructive pauses from harmful pathological stalls remains a fundamental unresolved question in molecular biology. Here, we report that a ribosome-binding GTPase (Rbg in yeast, and so-called Developmentally Regulated GTP-binding (Drg) in human) promotes protein biosynthesis in halted ribosomes in an evolutionarily conserved manner. We show that the essential bacterial Obg GTPase rescues cell growth and translation in Saccharomyces cerevisiae and human cells lacking their endogenous Rbg/Drg proteins in a GTPase-dependent manner. Furthermore, Obg enhances puromycin incorporation into stalled ribosomes in a manner requiring GTP hydrolysis. We reason that Rbg/Drg proteins act to prevent premature engagement of ribosome-associated quality-control or stress-response pathways. Under physiological conditions, proteomic analyses of Rbg1-bound ribosomes in yeast reveal that Rbg GTPases associate with ribosomes translating proteins essential for diverse functions, including those of the nucleus, ER, Golgi, mitochondria, and protein transport pathways. Finally, loss of even one of the two eukaryotic Rbg/Drg paralogs impedes cell-cycle progression, disrupts DNA replication, and compromises mitochondrial function. Together, these findings suggest that Rbg/Drg proteins enable cells across different domains of life to protect physiological ribosomal pauses, revealing a previously unrecognized layer of regulation in gene expression. Significance StatementHow cells distinguish productive ribosomal pauses from harmful stalls remains unclear. We identify a ribosome-binding GTPase--Rbg in yeast and Drg in humans--as a conserved regulator that protects physiological pauses and sustains protein synthesis. Remarkably, bacterial Obg functionally substitutes for eukaryotic Rbg/Drg in both yeast and human cells, revealing deep evolutionary conservation of this mechanism. Our data show that Obg promotes continued peptide-bond formation in a GTPase-dependent manner. Positioned near the ribosomal GTPase-associated center, these GTPases are also poised to compete with stringent-response and stress-activation factors that trigger translation shutdown. Together, the findings suggest that translation is controlled not only by GTPases that drive protein synthesis forward but also by Rbg/Drg GTPases that protect the catalytic cycle against premature arrest.

biochemistry↗

bioRxiv2026

Organoid modeling of tumor-associated macrophages reveals phagocytosis checkpoint blockade-induced conversion to an immunosuppressive SPP1+ phenotype

Tumor-associated macrophages (TAM) exert essential functions during the immune response to cancer. However, investigations of TAM within a native human tumor microenvironment (TME) have been impeded by a lack of appropriate model systems. Here, patient-derived organoids (PDO) from air-liquid interface (ALI)-grown tumor fragments, containing a human TME that encompassed stroma and immune subsets, robustly preserved TAM that were maintained by endogenous CSF-1 and appropriately responded to polarization signals. Antibody blockade of the CD47 regulatory checkpoint in organoids stimulated phagocytosis and remodeled TAM cytokine secretion profiles that were confirmed in anti-CD47 phase I trial patients. Amongst PDO histologies screened, anti-CD47 tumor killing was notable in clear cell renal cell carcinoma (ccRCC) which was associated with increased TAM infiltration. PDO contained diverse previously described TAM subsets; however, anti-CD47 reprogrammed organoid TAM toward an immunosuppressive SPP1+ phenotype, highlighting a negative feedback mechanism. Our findings uncover a resistance circuit engaged by macrophage checkpoint blockade and position ALI PDO as a robust translational platform for dissecting human macrophage biology and informing precision immunotherapy.

cancer biology↗

bioRxiv2026

Axonal spike-count regimes link spinal cord stimulation periodicity to artificial sensory detection and discrimination in rodents

Electrical stimulation is widely used to evoke artificial sensation, yet how temporal stimulation patterns are transformed into neural activity and perception remains poorly understood. Recent behavioral studies have shown that increasing the aperiodicity of spinal cord stimulation pulse trains alters both detection thresholds and discrimination performance despite constant pulse counts, suggesting a role for temporal structure beyond rate alone. However, the neural mechanisms underlying these effects remain unresolved. We developed a biophysically grounded computational framework linking a finite-element model of the rodent spinal cord, conductance-based axon simulations, and observer decision models to investigate how stimulation periodicity shapes neural responses and resulting behavior. By systematically varying stimulation amplitude, frequency, and inter-pulse interval variability, we identified distinct spike-count regimes arising from interactions between stimulation timing and axonal membrane dynamics. These regimes ranged from single spikes and small volleys to sustained spike trains, and they exhibited diverse frequency- and variability-dependent trends. No single regime was able to sufficiently explain experimentally observed detection threshold trends; instead, mixtures of regimes accurately reproduced both frequency-dependent threshold behavior and trial-level variability. Extending this framework to periodicity discrimination, we show that features derived from regime mixtures contain sufficient information to recover behavioral psychometric curves. Furthermore, observer model results provide a mechanistic account of behavioral asymmetries depending on the periodicity of the reference stimulus: discrimination relative to periodic inputs relied on combined rate and timing evidence, whereas discrimination relative to aperiodic inputs was dominated by timing irregularity. These results establish a mechanistic link between stimulation temporal structure, axonal spike generation, and perceptual behavior. This framework suggests that spinal cord stimulation does not operate within a single fixed neural regime but instead engages a spectrum of spike-count regimes whose mixtures shape perception. These findings have important implications for the design of biomimetic stimulation strategies, highlighting temporal patterning as a key dimension for controlling sensory outcomes.

neuroscience↗

bioRxiv2026

Natural variation in transplacental transfer efficiency exposes distinct transcriptional network architectures of PFAS effects on birth weight and gestational age

Per- and polyfluoroalkyl substances (PFAS) are environmental contaminants that produce heterogeneous effects on perinatal outcomes despite chemical similarity. Natural variation in transplacental transfer efficiency (TPTE, the degree to which compounds cross the placental barrier) presents a mechanistic lens for understanding this heterogeneity, but whether TPTE systematically shapes the transcriptional pathways linking exposure to outcomes has not been tested. Using isoform-resolved placental transcriptomics from n = 124 term deliveries with eight PFAS measured in maternal and cord blood, validated against patient-derived placental explants (n = 18) using a tissue-specific long-read transcriptome assembly, we show that PFAS influence perinatal outcomes primarily through co-expression network hubs rather than differentially expressed features, indicating that network position, not fold-change magnitude, determines mediator importance. Leveraging TPTE as a natural experiment, we find that direct fetal exposure recruits increasingly numerous and tightly coordinated transcriptional mediators for both birth weight and gestational age, but the two outcomes diverge in network architecture: network centrality and maternal-fetal compartmentalization scale with TPTE exclusively for birth weight, while gestational age shows no such topological reorganization. These outcome-specific patterns are detectable only at the transcript level, as gene-level aggregation masks systematic TPTE-network relationships. This framework distinguishes which perinatal outcomes are mechanistically vulnerable to fetal exposure dose, with implications for risk assessment and therapeutic target identification for environmental contaminants.

developmental biology↗

bioRxiv2026

Improving isoform-level eQTL and integrative genetic analyses of breast cancer risk with long-read RNA transcript assemblies

Most eQTL and TWAS analyses quantify expression using aggregate, tissue-agnostic transcript annotations and ignore isoform-level regulation, potentially obscuring or misattributing regulatory mechanisms. Here, we developed a framework leveraging publicly available long-read RNA-seq data to perform tissue-informed inference of genetic regulation and prioritize candidate causal isoforms for breast cancer risk. We quantified gene- and isoform-level expression in breast tumor (TCGA), non-cancerous mammary tissue, and cultured fibroblasts (GTEx) using three transcriptome annotations: standard GENCODE, tissue-specific long-read-derived assemblies, and combined annotations incorporating transcript-isoforms from both. While GENCODE cataloged over 250,000 pan-tissue isoforms, the tissue-specific long-read assemblies captured reduced sets of 74,717 isoforms in tumor, 48,057 in fibroblasts, and 22,941 in healthy breast. We performed eQTL mapping and fine-mapping, followed by colocalization with overall and subtype-specific breast cancer GWAS and isoform-level TWAS. While most eGenes were concordant across annotations, approximately 1/3 of lead cis-eQTLs for shared eGenes differed between long-read assemblies and GENCODE. Further, eIsoform discovery was highly annotation-specific. In healthy breast tissue, the gold standard tissue for building gene expression prediction models for TWAS of breast cancer, 46% of eIsoforms identified by the long-read annotation were unique to that annotation even though 93.7% of them are present in GENCODE. Despite combined annotations expanding the GENCODE catalog by only 0.6-7.6% depending on tissue source, 69% of unique significant isoform-trait associations were specific to a single annotation. Long-read-informed annotations uncovered regulatory associations entirely missed by GENCODE, including a candidate regulatory isoform at the MARK1 locus captured only in fibroblasts and a previously unannotated splice variant prioritized as the likely effector transcript at NUP107. These findings demonstrate that transcript annotation is not merely a technical consideration but critically defines the biological hypothesis space for regulatory mechanisms and shapes discovery. Incorporating tissue-resolved isoform annotations from long-read RNA-seq improves the specificity of regulatory inference and enhances identification of candidate causal isoforms at GWAS loci.

genomics↗

bioRxiv2026

PEPTERGENT: A Peptide-Based Method for Detergent-Free Extraction and Purification of Membrane Proteins and Membrane Proteomes

Peptergent is a novel class of amphipathic peptides that enable detergent-free extraction and purification of membrane proteins (MPs). These designed peptides self-assemble around hydrophobic transmembrane regions of proteins, forming stable, water-soluble assemblies that can be isolated directly from biological membranes. By doing so, Peptergent bypass the limitations imposed by traditional detergents, which often destabilize proteins and restrict downstream analyses. Since detergents are completely avoided, Peptergent-isolated MPs are directly amenable to structural and mass spectrometry (MS) analysis, thereby addressing their persistent underrepresentation in proteomic datasets and improving their accessibility for drug-screening strategies. Here, we describe a streamlined protocol for isolating MPs with the Peptergent PDET-1, followed by exchange into His-tagged Peptidiscs for Ni-NTA-based affinity purification. The method comprises membrane isolation, peptide preparation, protein extraction, clarification, and exchange of MPs from Peptergent to Peptidiscs. Application of this workflow yields enriched membrane proteomes compatible with downstream LC-MS/MS analysis, with improved recovery of hydrophobic and multi-pass membrane proteins. Key featuresO_LIDirect extraction and solubilization of membrane proteins in Peptergents C_LIO_LIExchange into His-tagged Peptidiscs enabling affinity purification of MPs C_LIO_LI100% detergent-free workflow compatible with LC-MS/MS analysis C_LIO_LIApplicable to cultured cells and tissue-derived membrane fractions C_LI In BriefWe describe a Peptergent-based workflow for isolating membrane proteins directly from membrane preparations. Proteins are extracted with the Peptergent peptide scaffold (PDET-1) and transferred into His-tagged Peptidisc (HD-43). The water-soluble membrane proteins are enriched by Ni-NTA affinity purification and prepared for bottom-up mass spectrometry, yielding enriched membrane proteomes and dried peptide samples ready for LC-MS analysis Graphical Overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/711971v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@2d5bdforg.highwire.dtl.DTLVardef@1eac5fborg.highwire.dtl.DTLVardef@6c14fcorg.highwire.dtl.DTLVardef@1d5d28f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Distinct Molecular Mechanisms Regulate Feeding State-Dependent CO2 Chemotaxis Plasticity During Different Life Stages in Caenorhabditis elegans

Across developmental stages, animals modulate their behavioural responses to external cues according to intrinsic physiological states. During development, particularly in juvenile stages, nervous systems undergo extensive changes at multiple levels. However, it remains unclear whether nervous systems at different developmental stages employ the same underlying molecular mechanisms to produce equivalent behavioural modulations in response to intrinsic or extrinsic cues. Using the model organism Caenorhabditis elegans, we identify here that animals employ distinct molecular mechanisms to achieve equivalent modulation of CO2-chemosensory behaviour at different developmental stages. Ubiquitin-proteasome-mediated downregulation of the insulin/IGF receptor, DAF-2 by the conserved quality-control ubiquitin ligase CHN-1/CHIP promotes attraction to environmental CO2 during the starvation-induced L1-arrest stage. In contrast, CO2-attaction in dauer animals is independent of CHN-1/CHIP activity. Furthermore, the feeding-induced reversal of CO2-chemotaxis to avoidance during L1-arrest exit requires the insulin/IGF pathway and the conserved CRH-1/CREB1 transcription factor activity in the CO2-sensing BAG neurons. However, induction CO2-avoidance during dauer exit is independent of CRH-1/CREB1 activity. These findings suggest that neural circuits at different life stages may utilize distinct, stage-specific molecular mechanisms to induce identical plasticity in chemosensory behaviour.

neuroscience↗

bioRxiv2026

Differential Maillard Sensitivity Of Exoproteins Favors Keratin Recovery During Sludge Biopolymer Extraction

Proteins are an abundant extracellular polymeric substance (EPS) of activated sludge from wastewater. Hot alkalinization is implemented industrially to recover the EPS from sludge. We sought to assess the feasibility of protein recovery from alkali EPS. We detected a low abundance of bacterial proteins in the EPS. Human keratin was highly abundant and could also be recovered. Keratin was observed as a dominant and integral component of the activated sludge flocs, and it was thus not an extraction artifact. Alkali extraction promoted Maillard reaction between proteins and sugars and removed recoverable peptide signatures. Subsequent chemical modification, along with denaturation, impaired protein binding to ion exchange resins, making bacterial proteins inaccessible to isolation. Keratin has high resistance to Maillard reaction under extraction conditions and thus persists in the EPS. While Maillard modifies bacterial proteins, the resultant product, and possibly even keratin itself, are valuable recoverable byproducts from activated sludge. SynopsisThe different sensitivities of proteins to Maillard reaction determines which proteins dominate alkaline extracellular polymeric substance (EPS) extract from activated sludge, with keratin dominating in alkaline EPS and an integral activated sludge component.

biochemistry↗