Biology subjects

Jain, A.

Publications and source records attributed to Jain, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Interchain base-stacking and spermine bridges govern ssDNA condensation and reentrant transitions

Polyvalent-cation-induced condensation and reentrant transition in double-stranded DNA are classically described by backbone electrostatics. Whether the same principles govern single-stranded DNA, whose solvent-exposed bases can interact within and between chains to form complex phases, is unknown. Here, we employ simulations and experiments to show that the tetravalent cation spermine (Spm4+) drives two equilibrium transitions in poly(dT) and poly(dA). The onset of phase separation, resulting in thermodynamic equilibrium between the dispersed low-density phase and high density coacervate, occurs at a low sequence independent Spm4+ concentration and is primarily driven by charge balance between phosphate groups and Spm4+. Strikingly, the condensed phases are stabilized by at most 1.5 (poly dT) and 2.5 (poly dA) bridging Spm4+ per chain. The ssDNA chains condense to form fluid-like networks without chain collapse. The fluidity of chains in the dense phases is confirmed by fluorescence recovery after photobleaching experiments. Both simulations and experiments show that the range of spermine concentration over which phase coexistence persists is sequence dependent and nearly threefold wider in poly(dA). At higher sequence dependent spermine concentrations, a reentrant phase, characterized by dissolution of the coacervates, emerges. The transition to the reentrant phase occurs when the number of bridging Spm4+ molecules vanishes at high sequence dependent spermine concentration. The combination of experiments and simulations show that the two phase transitions in ssDNA are driven by an interplay of stacking and electrostatic interactions.

biophysics↗

bioRxiv2026

Pathogenic mutations in ATAD3A cause dysregulation of RagC/D-TFEB axis and disrupt lysosomal homeostasis

We previously discovered that a de novo variant p.R528W in ATAD3A, encoding a mitochondrial membrane-anchored protein, causes a human neurological syndrome. While ATAD3A mutations induce aberrant lysosomal expansion accompanied by undigested material in the lysosomes, how mutant ATAD3A disrupts lysosomal homeostasis and whether this contributes to neurodevelopmental defects remain unknown. Here we show that pathogenic ATAD3A p.R528W expression disrupts the mTORC1-TFEB axis as revealed by dysregulation of mTORC1 substrate phosphorylation, TFEB nuclear localization, and CLEAR gene activation associated with lysosomal biogenesis. ATAD3A binds to lysosome-localized Rag C/D GTPases, which constitute a platform for TFEB recruitment, with pathogenic variants increasing this association and thereby decreasing lysosomal localization of Rag GTPases. Importantly, overexpression of RagC or RagD restores TFEB phosphorylation in human cells expressing p.R528W, and RagC-D overexpression or TFEB/Mitf knockdown rescues lysosomal expansion and neurodevelopmental defects in Drosophila. These data indicate that disrupted Rag GTPase recruitment to lysosomes and subsequent aberrant TFEB/Mitf activation contribute to neurodevelopmental and lysosomal phenotypes caused by pathogenic mutations in ATAD3A. Our work reveals a novel role for the mitochondrial resident protein ATAD3A in modulating lysosomal homeostasis through regulation of the mTORC1-TFEB axis, providing a mechanistic link between impaired mitochondrial and lysosomal homeostasis in a neurodevelopmental disorder.

genetics↗

bioRxiv2026

Growth capacity and mortality burden shape microbial persistence under osmotic stress in Bacteroides thetaiotaomicron

Microbial persistence in the gut is central to sustaining beneficial host-microbiota interactions; however, characterizing determinants of persistence in vivo can be challenging due to the complexity of native microbial communities interacting with the host. Here, we used a defined microbial consortium to demonstrate that intrinsic in vitro growth capacity becomes increasingly predictive of in vivo relative abundance as intestinal osmolality increases. In Bacteroides thetaiotaomicron, in vivo abundance was higher than predicted from growth capacity alone, indicating that additional physiological factors contribute to persistence. We identify mortality as one such determinant: adaptation to prolonged osmotic stress markedly reduced cell loss without enhancing growth capacity. This adaptive phenotype was associated with rapid and reversible phase variation in cell surface-associated PUL78/80 rather than fixed de novo mutations, with the PUL78/80-OFF state becoming progressively enriched among surviving cells as mortality increased. Our findings demonstrate that in addition to growth capacity, reduced cell loss can provide a distinct route to microbial persistence during environmental perturbation.

microbiology↗

bioRxiv2026

A passive protein environment offsets RNA folding energetics in cells

RNA structure can be predicted from sequence in dilute solution, but these predictions often fail in cells. Many cellular RNAs keep their in vitro folds, whereas others are substantially less structured. We combined single-molecule FRET with Xenopus oocyte extract to measure RNA folding and duplex formation under cell-like conditions. Extract proteins passively suppressed base pairing: they slowed apparent strand association while leaving duplex dissociation largely unchanged, without sequence specificity or ATP consumption. A competitive binding model captured this activity as an effective folding penalty of ~0.45 kcal/mol per nucleotide. Similar activity occurred in HeLa lysate. Applied to in-cell chemical probing data, this correction stratified reactivity across ~42,000 stem-loops, whereas predictive models established in dilute solution classified nearly all as stably folded. Our results identify a passive protein-mediated mechanism as a key contributor to cellular RNA folding, shifting the balance between paired and unpaired states without changing the underlying base-pairing rules.

biophysics↗

bioRxiv2026

De Novo Design and AlphaFold3 Evaluation of Protein Binders Targeting Specific Sites of MAP4K4

MAP4K4 is a serine/threonine kinase member of the mitogen activated protein kinase family. It acts through the JNK, p38 MAPK, and ERK1/2 pathways and is implicated in cancer proliferation and invasion, TNF--driven insulin resistance, macrophage-mediated inflammation, and cardiomyocyte apoptosis in heart failure. No selective small-molecule inhibitor of MAP4K4 has reached clinical use, and knowledge of the specific epitopes on MAP4K4 is lacking for some existing antibodies, leaving a role for small protein binders directed at specific surface sites. We used a computational pipeline that combines RFdiffusion, ProteinMPNN, and AlphaFold2, as well as BindCraft, an integrated "one-shot" tool, for de novo design of small protein binders (~50-130 residues) to specific MAP4K4 surface hotspots. We also created an automated hotspot determination algorithm that weighs geometry, chemistry, rigidity, and AlphaFold pLDDT. From thousands of candidate sequences, we evaluated 20 of the most promising binders with AlphaFold3 (AF3). The interface predicted template modeling (ipTM) scores ranged from 0.16 to 0.90, with nine candidates having ipTM [≥] 0.80, and five scoring [≥] 0.87. Two binders engage non-overlapping hotspots on opposite faces of MAP4K4, making them a candidate pair for a sandwich assay. BLASTp searches of all designed protein sequences returned only low-significance matches to half of them, indicating that they represent truly novel binding solutions and previously unexplored regions of protein sequence space rather than rediscovered natural motifs. We subjected five complexes spanning the observed AF3 confidence range to 100-ns explicit-solvent molecular dynamics simulation. Interchain contacts were retained throughout, with stability varying substantially between systems. Confidence in the binding specificity of the nine highest-confidence candidates was bolstered by juxtaposition with AF3 evaluations of their interaction with CDK2, a negative-control kinase. This comparison yielded a significant, consistent reduction in ipTM (p = 0.0039) for the control binding partner. ToxinPred2 and AlgPred 2.0 screening suggested that one candidate was a potential allergen and two were potential toxins. These results support that de novo design of small, site-specific protein probes for an underserved disease target is achievable using free, publicly available computational tools and minimal resources, pointing to a greater role for the public and amateur scientists to contribute to biotechnological advancement.

bioengineering↗

bioRxiv2026

The filopodial scaffold polyphosphate dictates cell adhesion-versus-invasion decisions

Inorganic polyphosphate (polyP) is an ancient polymer conserved across all life, serving cell type and location specific functions in every major compartment. Yet its role at the plasma membrane, where it accumulates to peak levels in many primary cells, is largely unknown. Here we identify polyP as a stabilizing component of filopodia, actin based membrane protrusions that govern cell adhesion, contact inhibition, and chemotaxis. Elevating cellular polyP increases filopodial stability and enhances cell adhesion, whereas reducing polyP accelerates filopodial disassembly and promotes cell migration. Mechanistically, we find that polyP acts as a structural filopodial scaffold, recruiting and organizing IRSp53, a membrane curvature inducing protein. We show that metastatic fibroblasts and breast cancer organoids carry markedly reduced and intracellularly reorganized polyP levels relative to their non transformed counterparts. Restoring endogenous polyP via lipid nanoparticle delivery suppresses their invasive phenotypes and reverses prometastatic gene expression signatures, implicating polyP as a primordial tumor suppressor.

cell biology↗

bioRxiv2026

Microbial valerate is associated with CAR T dysbiosis and its supplementation enhances CAR T function in B-cell lymphoma

Anaerobe-depleting antibiotic exposure is associated with inferior progression-free survival after CD19 CAR T-cell therapy in large B-cell lymphoma, yet the cellular mechanisms linking gut dysbiosis to the CAR T-cell product and whether this imprint is reversible have remained undefined. In two independent CAR-T candidate cohorts, low stool valerate at the time of CAR-T eligibility identified a multi-metabolite-deficient dysbiotic gut microbiome state marked by depletion of fiber-fermenting commensals and loss of carbohydrate-fermentation, SCFA-biosynthesis, and amino-acid metabolism pathways. Reanalysis of single-cell RNA sequencing from 42 lymphoma patients stratified by piperacillin-tazobactam/imipenem/meropenem (PIM) exposure revealed that PIM-exposed CAR T-cell products were CD4-skewed, with significantly elevated AP-1/immediate-early gene (IEG) and cellular activation signatures that together predicted inferior progression-free survival. Ex vivo conditioning of CAR T-cells with valerate produced a chromatin and transcription factor program distinct from butyrate or propionate, characterized by KLF/SP/EGR family engagement, KLF4 promoter opening, and broad induction of AP-1/IEG and MHC class II transcripts, whereas butyrate drove broader chromatin remodeling with TBX21/EOMES/NF-{kappa}B gains and KLF2 promoter closure, and propionate induced an NFY-centered program with preferential commitment to low-mitochondrial-content states. Untargeted metabolomics confirmed valerate uptake and mitochondrial {beta}-oxidation in CAR T-cells, while dietary sodium valerate supplementation in meropenem-treated mice bearing A20 lymphoma significantly reduced tumor burden and extended survival compared with CAR T-cells alone. These findings identify stool valerate as a bedside-deployable biomarker of dysbiosis-imprinted CAR T-cell dysfunction and support ex vivo or dietary valerate supplementation as a clinically tractable strategy to improve CAR-T anti-tumor function in patients with disrupted gut microbiomes.

cancer biology↗

bioRxiv2026

Paralemmin 2 is a shared, alternatively-spliced regulator of axon initial segments and paranodal junctions in oligodendrocytes

Nervous system function depends on highly specialized axonal membrane domains. For example, axon initial segments (AIS), nodes of Ranvier, and paranodal junctions are essential for action potential initia-tion and saltatory conduction. These domains use shared molecular machinery converging on the master scaffolding protein AnkyrinG (AnkG). Using endogenous AnkG-TurboID proximity proteomics, we identify Paralemmin 2 (Palm2), a previously uncharacterized CNS protein, as a shared component of the AIS and nodes of Ranvier in neurons, and paranodes in oligodendrocytes. Palm2 localizes transiently to the AIS and nodes during development, but is found at paranodes throughout life, reflecting cell type-specific splicing of a lipid-modified domain both necessary and sufficient for AnkG interaction. Mice lacking oli-godendroglial Palm2 assemble normal paranodes during development, but aged mice lose paranode in-tegrity due to loss of paranodal AnkG and Neurofascin-155. Our findings identify Palm2 as a lipid raft-associated regulator of axonal and oligodendroglial membrane domains and reveal cell type-specific pro-tein variants as a recurring principle for specializing axonal and glial membrane architectures.

neuroscience↗

bioRxiv2026

Kinetic proofreading decouples signal strength and range in paracrine gradient formation

Spatial gradients of signaling molecules pattern multicellular tissues with high precision. The canonical synthesis-diffusion-degradation (SDD) framework imposes a tradeoff on these gradients: ligand-receptor interactions that generate downstream signaling activity are also responsible for consuming the ligand. Correspondingly, at a fixed ligand synthesis rate, raising ligand-receptor affinity increases local signal strength at the expense of spatial range, and lowering it extends range at the expense of strength. Recent live-imaging measurements appear to violate this seemingly fundamental tradeoff, with low-affinity ligands of the epidermal growth factor receptor (EGFR) diffusing farther and driving spatially broader signaling activity compared to high-affinity ligands. Here we explain these observations with a model of multi-step ligand processing at the receptor, and show that the activity-range tradeoff is a consequence of receptor architecture rather than a physical necessity. When receptors process ligand through a multi-step phosphorylation cascade with kinetic-proofreading-like resetting, the states that generate activity decouple from those that consume ligand, and signaling activity and range increase together over a finite window of ligand residence time. This lets cells tune how far a signal travels independently of how strongly it acts through tuning signaling parameters. Realistic EGFR parameters place the low-affinity ligands in this window. Because multi-site phosphorylation and preferential degradation of the active receptor recur across multiple receptor families, kinetic proofreading may be a general strategy for controlling signaling range. Significance StatementCells coordinate by releasing molecules that bind receptors on neighboring cells. For a fixed supply, how strongly a signal acts and how far it spreads are locked together: tight binding gives a strong response but the molecule is captured near its source, while weak binding spreads farther but signals feebly. Yet recent imaging of epidermal growth factor receptor ligands shows the opposite: weak binders activate a broader field of cells. We show this limit reflects how receptors read the signal, not physics. A receptor that processes a bound molecule through several steps, and can release it partway, separates the states that signal from those that destroy it. Cells, and engineers, can then set a signals reach independently of its strength.

biophysics↗

bioRxiv2026

Sustained epigenetic rejuvenation of serially engrafting human iPSC-derived HSCs

Hematopoietic stem cell (HSC) function declines with age, contributing to immunosenescence and inferior transplantation outcomes. Here, we generated iPSC-derived HSCs (iHSCs) from multiple adult donors and performed integrated epigenetic, transcriptional, telomeric, and functional analyses to see if they retain youthful identity across differentiation and serial transplantation. Longitudinal DNA methylation profiling revealed that, independent of donor age, epigenetic age was reset to near zero in iPSCs and remained under seven years across differentiation and transplantation. In contrast, hematopoietic identity was established through a two-phase process: directional remodeling during in vitro differentiation extinguished pluripotency programs and initiated hematopoietic regulatory networks, while long-term engraftment was associated with a second wave of promoter methylation differences that converged toward primary adult HSCs. Notably, methylation at age-associated sites and global entropy remained stable across both phases, and single-cell telomere analysis demonstrated restoration of telomere length in iHSCs compared to primary adult HSCs. Youthful epigenetic features were maintained through secondary transplantation. These findings demonstrate that long-term HSC identity can be achieved independently of epigenetic aging and establish a framework for evaluating rejuvenated stem cell-derived grafts in regenerative medicine.

cell biology↗

bioRxiv2026

Droplet single-cell CRISPR screens identify regulators of T cell-mediated target-cell killing

Cytotoxic CD8 T cells kill target cells through brief cell-cell encounters, but pooled genetic screens cannot readily link perturbations in individual T cells to the fate of the target cells they engage. We developed droplet single-cell CRISPR screening to pair individual primary human CD8 T cells with cancer cells, measure rapid target-cell death, and recover sgRNAs from phenotype-defined droplets. Applied across primary T cells from multiple donors, the platform recovered regulators of T cell receptor signaling, synapse formation, granule exocytosis and cytotoxic differentiation, and identified negative regulators of killing, including established inhibitory nodes such as PTEN, RASA2 and FOXO1, together with AFAP1L2 and components of the mTORC1 pathway. Validation across bispecific engager and TCR-engineered settings showed that selected hits modulate target-cell killing across recognition modalities and tumor models. Unexpectedly, perturbation of RPTOR or RHEB enhanced cytotoxic execution while reducing mTORC1 output, increasing AKT phosphorylation, and attenuating anabolic programs. Transient pharmacologic mTORC1 inhibition reproduced this rapid-killing state and improved antitumor activity after adoptive transfer. These results establish an interaction-resolved pooled genetic strategy for mapping cytotoxicity regulators and reveal that transient modulation of mTORC1 can shift T cells from anabolic growth toward rapid cytotoxic execution to enhance antitumor activity.

immunology↗

bioRxiv2026

Direct Detection and Atomic Modeling of Ligands in Cryo-EM Maps Using Deep Learning

Cryogenic electron microscopy (cryo-EM) has become an increasingly important for structure-based drug discovery by enabling characterization of interactions between macromolecules and small-molecule ligands. However, computational interpretation of ligand density remains challenging, particularly when ligand locations are unknown or local map resolution is limited. Existing methods generally require well-resolved macromolecular structures and predefined binding sites, limiting their applicability during early-stage structure determination. To date, no approach has been able to both reliably detect ligand density and subsequently reconstruct ligand atomic structures directly from experimental cryo-EM maps. Here, we present Emap2lig, a two-stage deep learning framework for automated ligand detection and atomic modeling directly from cryo-EM maps. Emap2lig-Find identifies ligand-associated densities and remains effective for maps at resolutions as low as [~]5 [A]. Emap2lig-Build subsequently uses a diffusion-based generative model to build atomic ligand structures. Together, Emap2lig provides a unified framework for ligand discovery and modeling across a broad range of resolutions.

bioinformatics↗

bioRxiv2026

The effect of cell death on DNA-replication-based estimates of microbial population growth

Inferring bacterial growth rates is fundamental to understanding microbial interactions and community dynamics, but remains difficult in natural settings where time points are limited or organisms are unculturable. In these cases, a widely used method is the origin-to-terminus ratio, or peak-to-trough ratio (PTR), which estimates DNA replication activity from origin and terminus copy numbers. Although PTR is theoretically related to bacterial growth rate, it is frequently benchmarked against population growth rate, which reflects the balance between cell division and loss. Understanding when and why these measures diverge is therefore important for validating PTR-based approaches. To quantify how departures from steady growth affect PTR, population growth rate, and bacterial growth rate, we developed a stochastic, cell-based model that explicitly tracks DNA replication, cell division, and cell death. We show that transient stress-induced mortality and heterogeneous survival can cause PTR to fail to reflect population growth rate while still reflecting bacterial growth rate. We experimentally validated these predictions by exposing \textit{Escherichia coli} to osmotic shock or antibiotics, and measuring population growth rate and DNA replication activity. We further show that initial physiological state, lag before replication resumes, and abrupt growth arrest can alter the relationship between PTR and population growth rate. In the presence of mortality, lag can increase PTR's correspondence with population growth rate while decreasing its correspondence with bacterial growth rate. These results provide a mechanistic and quantitative framework for interpreting PTR across changing growth states, mortality regimes, and environmental perturbations.

microbiology↗

bioRxiv2026

Acute Exposure to Perfluorooctanoic Acid (PFOA) During Cardiomyogenesis disrupts Transcriptional and Electrophysiological Profiles in Differentiated Myocytes

The early developmental environment plays a critical role in the etiology of cardiovascular diseases (CVDs), but underlying molecular mechanisms are poorly understood. Exposure to per and polyfluoroalkyl substances (PFAS) are linked to various CVDs, but effects of developmental PFAS exposures on the human heart remain unclear. Using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM), the objective of this study was to investigate the effects of PFAS exposure during cardiac differentiation on gene expression and function of cardiomyocytes. We exposed two hiPSC lines (one male and one female donor) to perfluorooctanoic acid (PFOA), a common and ubiquitous PFAS (0.05, 0.5, 5, 50, 100, 150, 200 M), followed by assessment of cellular number and pluripotency marker expression. PFOA exposure for 72 hours had no significant effects on hiPSC pluripotency, and modest inhibition of proliferation was observed only at the highest concentration. hiPSCs were then differentiated into ventricular cardiomyocytes in the continued presence or absence of PFOA (0, 0.5, 5, 50 M) using an established small molecules protocol. Optical mapping studies using voltage and calcium-sensitive dyes revealed dose and cell line-specific effects of PFOA on cardiomyocyte voltage and calcium dynamics that were still present 10 days after cessation of exposure. Patch clamping studies demonstrated small but significant reductions in repolarizing IKr currents with 5{micro}M PFOA exposure in cardiomyocytes from both donors. Using RNA-seq, we found that exposure to PFOA led to significant changes in transcriptional pathways related to lipids and lipoproteins in the female hiPSC-CM. In the male hiPSC-CM, we observed significant effects on developmental pathways and calcium homeostasis. Thus, we found that environmentally relevant PFOA exposure during cardiomyocyte differentiation affects the electrophysiological properties and transcriptome of hiPSC-CM even after cessation of exposure, with effects that differ by donor cell line. These findings provide direct experimental evidence that transient developmental exposure to PFOA can durably reprogram human cardiomyocyte function, supporting a developmental origin of PFAS-associated cardiovascular risk. Impact StatementThese studies demonstrate that exposure to environmentally relevant levels of PFOA during the differentiation of hiPSCs into cardiomyocytes alters cardiac gene expression and function, with effects that persist beyond cessation of exposure.

pharmacology and toxicology↗

bioRxiv2026

A Long-lived Avatar for Modeling Age-Related Vascular Disease

BackgroundCurrent microphysiological models do not support long-term investigations into the chronic effects of vascular risk factors and the development of vascular diseases. Prolonged culture frequently leads to cellular senescence and loss of functional integrity, resulting in variability and inconsistency in modeling chronic vascular responses. Here we aimed to develop and sustain a long-term multicellular human vascular avatar, addressing the critical need for long-term disease modeling and drug testing. MethodsTo identify the optimal media for longevity, cell identity and function were assessed by morphology, qPCR, beta-gal staining, ELISA, bulk RNA-seq and single cell RNA-seq analysis. After optimizing the culture media, iPSCs-derived ECs and VSMCs from unaffected and Hutchinson-Gilford Progeria Syndrome (HGPS) donors were grown in Gravitational Lumen Patterning (GLP) Vessel- Chips for 1-6 months to generate a long-lived vascular avatar for the study of vascular aging. ResultsGuided by quantitative morphological analyses and bulk RNAseq profiling, we generated a novel optimized culture media VSL (VEGF, SB431542 as a TGF-{beta} inhibitor, low fetal bovine serum) that enhances the long-term health of vascular endothelial cells (ECs). Furthermore, we modified the VSL formulation (mVSL) by modulating 8Br-cAMP, FGF, PDGF, and a cell viability enhancer HMH1015 levels to enhance EC-VSMC (vascular smooth muscle cell) crosstalk and support long-term cellular viability. Subsequently, we maintained and characterized a human vascular avatar with a three-dimensional extracellular matrix environment and 3D vascular architecture for over 180 days. Finally, we demonstrated that this long-lived human vascular avatar enabled modeling vascular aging using iPSC-derived vascular cells from patients with Hutchinson-Gilford Progeria Syndrome (HGPS). ConclusionsWe have successfully engineered and maintained a human vascular avatar for over 180 days. The vascular avatar provides a robust platform for modeling disease-associated vascular aging and for evaluating therapeutic strategies targeting chronic vascular disorders.

bioengineering↗

bioRxiv2026

A virion protein of an archaeal virus inhibits a Type V BREX defense system in Haloarcula hispanica

Bacteriophage Exclusion (BREX) systems are a diverse family of bacterial and archaeal defense mechanisms defined by two conserved genes encoding a putative alkaline phosphatase BrxZ (PglZ) and an ATPase BrxC (PglY). Here, we characterize a Type V BREX system from the archaeon Haloarcula hispanica. Similar to bacterial Type I BREX systems, host-virus discrimination relies on methylation of specific non-palindromic DNA motifs. Notably, the H. hispanica BREX locus encodes two methyltransferases, BrxX1 and BrxX2, which independently target distinct motifs (GTAYCCG and GACCCC). The system protects H. hispanica against three of seven tested archaeal viruses. However, two related viruses, SH1 and HHIV-2, despite encoding multiple BREX target sites, escape BREX-mediated defense. We demonstrate that a large virion protein, VP1, encoded by these viruses inhibits the BREX system, as mutant viruses carrying partial deletions of VP1 lose resistance to the host defense. Together, these findings provide the first characterization of a Type V BREX system and demonstrate that archaeal viruses can counteract BREX through virion-associated anti-defense proteins.

molecular biology↗

bioRxiv2026

Protein entanglement misfolding determines divergent fates: proteasomal degradation or persistence in near-native misfolded states

A novel class of protein misfolding, involving changes in entanglement status, occurs across the cytosolic proteome of a bacterium and likely occurs in many other organisms. Here, we examine if this class of misfolding has measurable downstream consequences for protein homeostasis. Specifically, we test the hypothesis that proteins that misfold in this way are more likely to be degraded by the ubiquitin-proteasome system immediately after synthesis. We do this by cross-referencing protein structural information with ubiquitin mass spectrometry (Ubq-MS) data from human fibroblast cells. Ubq-MS identifies proteins that have been covalently modified with ubiquitin in a particular pattern and is a cellular signal for that protein to be degraded by the proteasome. We find that nascent proteins with native entanglements, which were previously shown to be twice as likely to misfold, are 93% (95% Confidence Interval: [44%, 160%]) more likely to be tagged with ubiquitin and targeted to the proteasome compared to proteins that do not contain such entanglements. Simulating the folding of these proteins using a coarse-grained model, we find that the ubiquitin-tagged proteins containing native entanglements are four times more likely to misfold than the non-ubiquitinated proteins that are devoid of entanglements. These results indicate that entanglement misfolding, primarily involving a failure to form native entanglements, leads to an increased likelihood that those proteins will be degraded in human cells. Finally, we estimate that approximately one-third of the globular proteome likely misfolds in this way but bypasses proteasomal degradation because their misfolded states are structurally similar to their native ensemble. These consequences for protein degradation are likely common across organisms as entanglement misfolding is inherent to the polymeric nature of proteins.

biophysics↗

bioRxiv2026

A mechanically stable neural probe for percutaneous high-resolution, multichannel recordings in peripheral nerves

The development of neural probes has enabled a deeper understanding and improved treatment for neurological disorders. Microneurography is currently the gold standard for assessing the electrophysiological signature of pain mechanisms in the human peripheral nervous system. However, its clinical utility is limited by the low recording yield and signal-to-noise ratio of single-electrode probes. To overcome these limitations, we developed mechanically robust, multi-electrode probes designed for acute percutaneous insertion and recording in peripheral nerves. The electrical and mechanical stability of these probes was confirmed through repeated insertions in artificial human skin and rat peripheral nerves. In addition, ex vivo and in vivo experiments demonstrated enhanced functional performance, with multi-site recordings enabling the isolation of single-fiber activity. Importantly, our probes can be operated analogously to conventional microneurography needles while substantially increasing the information yield, providing enhanced capabilities for minimally invasive peripheral nerve assessment.

neuroscience↗