bioRxiv Subject Collection: All

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Fecal Hormone Metabolites Concentrations Collected Longitudinally Confirm Spontaneous Polyestry in Mt. Graham Red Squirrels (Tamiasciurus fremonti grahamensis)

The Mount Graham red squirrel (Tamiasciurus fremonti grahamensis, MGRS) is a federally endangered subspecies endemic to southeastern Arizona. Despite the establishment of ex situ conservation programs, reproduction outside the wild has not occurred. We monitored fecal hormone metabolites (FHM) to noninvasively assess estrous cycling and reproductive hormone dynamics in ex situ-managed MGRS. We compared ex situ findings with two in situ (wild) populations. We tracked longitudinal changes in estradiol and progesterone metabolites in three ex situ managed females and evaluated whether ovulation was spontaneous or induced. We detected 22 ovulation events over two years, with timing and regularity consistent with spontaneous polyestrous cycling. Comparative hormone analyses revealed appreciably lower progesterone and corticosteroid metabolite levels in ex situ individuals, whereas estradiol levels were comparable to those of wild counterparts. These findings suggest that maintaining environmental, and social conditions, e.g., lighting, temperature, and conspecific proximity, in ex situ settings is critical and could influence reproductive success. By confirming spontaneous ovulatory cycling in MGRS and demonstrating the utility of FHM as a noninvasive reproductive assessment tool, this study advances ex situ breeding efforts and provides a framework for reproductive monitoring in other endangered taxa.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.05.29.728663v1?rss=1

Wells, S. A., Koprowski, J. L.

Rewiring V-type and K-type enzyme allostery through subunit interface mutations

Allosteric regulation in the heterodimeric enzyme IGPS depends on long-range communication between the effector-binding HisF subunit and the catalytic HisH subunit. This signaling occurs through a densely packed interdomain interface enriched in conserved noncovalent contacts. Here, we use targeted interface mutations to determine how specific interfacial contacts tune the structural and dynamic features that govern allosteric control. The hK181A variant, which disrupts a critical salt bridge with fD98, converts IGPS into a constitutively more active enzyme, increasing basal glutaminase activity and substrate affinity. By contrast, hR18A, which disrupts a secondary salt bridge with fE71, weakens effector-induced activation, revealing functional asymmetry among interfacial interactions. NMR chemical shift perturbation and CPMG relaxation dispersion experiments show that hK181A remodels millisecond-timescale dynamics throughout HisF, consistent with molecular dynamics simulations indicating enhanced sampling of catalytically competent conformations. Network traffic analysis of correlated communication pathways, combined with energetic analysis, further shows that enthalpic and entropic contributions are redistributed to rewire long-range allosteric signaling. Together, these results identify specific interfacial residues as molecular gates that shape the conformational ensemble accessible to IGPS and show how interface reengineering can be used to rationally reprogram allosteric output.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.05.29.728626v1?rss=1

Maschietto, F., Chaudhuri, A., Enny, O., Batista, V. S., Loria, J. P.

A computational framework to model cartilage degeneration induced by mechanoinflammation and cytokine-driven inflammation in post-traumatic osteoarthritis

Knee joint injury is a major risk factor for post-traumatic osteoarthritis (PTOA), often associated with early cartilage degeneration. Mechanical overloading and cytokine-driven inflammation are key drivers of this process, yet the underlying mechanisms and their distinct temporal and spatial contributions to cartilage degradation remain unclear. Here, we present a mechanobiological finite element framework that simulates cartilage degradation through cell-mediated proteolytic activity triggered by mechanoinflammation and cytokine-driven inflammation. The model reproduces experimentally observed depth-dependent loss of collagen and aggrecan, with mechanoinflammation inducing a transient response and cytokine-driven inflammation sustaining prolonged matrix degradation. Sensitivity analysis further shows that mechanoinflammation-driven degradation is governed mainly by protease production per cell, whereas cytokine-driven degradation is more sensitive to the rate of cellular stimulation. Together, this framework provides a mechanistic basis to study proteolytic cartilage degeneration and supports future in silico evaluation of therapeutic strategies aimed at mitigating cartilage degradation in PTOA.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.05.29.728618v1?rss=1

Hamada, M., Eskelinen, A. S. A., Kosonen, J., Hakonen, S., Florea, C., Grodzinsky, A., Korhonen, R. K., Tanska, P.

miDGD: a multi-modal deep generative model predicts miRNA expression from bulk or single-cell mRNA expression

MicroRNAs (miRNAs) are important post-transcriptional regulators, yet their expression is typically unobserved in single-cell and most bulk RNA-seq datasets. We present miDGD, a deep generative decoder model that predicts miRNA abundance directly from gene expression alone. Trained on bulk and single-cell datasets from TCGA, GTEx, and human cell lines, miDGD learned a shared latent representation of matched mRNA and miRNA profiles that organized samples into biologically meaningful clusters reflecting tissue and cancer types. The model reconstructed both tissue-specific and broadly expressed miRNAs, recapitulated known miRNA-target relationships, and showed robust performance in sparse and single-cell data. miDGD outperformed miRSCAPE and recent miRNA activity inference methods, with improved cross-dataset generalization. These results establish a deep generative model as an improved framework for predicting miRNA expression when direct measurements are unavailable.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.05.29.727918v1?rss=1

Zamani, F., Rasmussen, A. M., Schuster, V., Diekema, M. H., Krogh, A., Pedersen, J. S.

Quantifying and Predicting the Difficulty of Multiple Sequence Alignment with AlDiScore

Multiple Sequence Alignment (MSA) constitutes an important and frequent operation in molecular sequence data analysis. There exist numerous tools, algorithms, and criteria to infer an MSA. This plethora of available approaches to MSA may induced an ensemble of divergent MSAs for the same underlying unaligned sequence set. Even a single MSA tool may infer distinct MSAs when varying the input parameters. Hence, when using a diversified set of MSA algorithms and parameterizations, the observed dispersion within an MSA ensemble expresses the difficulty of inferring a robust alignment. We refer to this notion as MSA difficulty. As downstream analyses heavily rely on the MSA, characterizing MSA difficulty for a given unaligned sequence set is critical. Initially, we show that measures of dispersion within diversified MSA ensembles can reliably predict MSA difficulty. We then assess the adequacy of these measures by computing the average reference-based distance between the MSAs in the MSA ensemble and its corresponding structural reference MSA and subsequently comparing this distance to the corresponding reference-free average distance over all MSA pairs in the ensemble. We find that Blackburne and Whelan's dpos alignment metric is most appropriate as its reference-free counterpart most accurately approximates the reference-based difficulty computed on BAliBASE reference data. We therefore use the average pairwise distance measured by dpos to quantify MSA difficulty on a scale from 0 (easy) to 1 (difficult) given an MSA ensemble. Next, we introduce the AlDiScore open-source tool, which uses machine learning to directly and reliably predict reference-free difficulty scores from unaligned sequence sets to completely omit expensive MSA computations. The underlying regression model relies upon a large set of features, including sampling-based measures of transitive consistency. We trained our AlDiScore models on a diverse collection of empirical datasets from BAliBASE, TreeBASE, an published studies. Subsequently, we demonstrate that AlDiScore attains an R2 of 0.89 and of 0.84 on unseen AA and DNA sequence sets extracted from the PANDIT v17 database. Finally, we show that there is no correlation between MSA difficulty and the corresponding phylogenetic difficulty of the respective MSA.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.05.29.727837v1?rss=1

Bodynek, M., Martin-Fernandez, L., Bettisworth, B., Haag, J., Stamatakis, A.

Vegfr3 is required in the Tbx1 expression domain for cardiac outflow tract development.

Gene inactivation in model organisms has identified numerous genes and signaling pathways involved in mammalian cardiac OFT development. Human genetics data have implicated the VEGFR3 gene in OFT development but when and where it is required is unknown. In this study we determined the sensitivity of the developing murine cardiac OFT to reduce Vegfr3 gene dosage and we tested whether its requirement is dependent upon TBX1, a known regulator of Vegfr3 expression in cardiac and lymphatic endothelial cells. We found that in the mouse, a single copy if the Vegfr3 gene was sufficient for normal cardiac OFT development in most cases. Mutation of a single copy of the Tbx1 gene greatly enhanced the sensitivity of OFT development to Vegfr3 dosage reduction and led to the formation of severe OFT anomalies. In addition, deletion of Vegfr3 in the Tbx1 expression domain also led to OFT abnormalities. We used RNAscope to reveal the location of Vegfr3 and Tbx1 transcripts in midterm mouse embryos. This revealed co-localization of these transcripts that was restricted to the aortic sac endothelium, suggesting that the distal OFT is a potential site of genetic interaction between Vegfr3 and TBX1 that is critical for normal OFT development.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.05.29.728669v1?rss=1

Martucciello, S., Bilio, M., Cioffi, S., Cavallaro, M., Baldini, A., Illingworth, E.

Interleukin-11 promotes the colonic epithelial organoid regeneration from mechanical disruption

The intestinal epithelium relies on rapid regeneration to maintain homeostasis after injury, and dysregulation of this process contributes to the pathogenesis of inflammatory bowel disease and colorectal cancer. Interleukin-11 (IL-11), a fibroblast-derived cytokine elevated in these diseases, has well-documented effects on stromal cells, but its direct action on intestinal epithelial cells remains poorly characterized. Here, we used mouse colon organoids as an isolated epithelial system to directly examine the effects of IL-11 on epithelial cells. IL-11 stimulation activated the canonical JAK/STAT3 pathway, as evidenced by increased STAT3 phosphorylation and Socs3 induction in a concentration-dependent manner. In a pipetting-based mechanical disruption model, IL-11 significantly enhanced organoid regeneration, increasing the number recovered. Although mechanical disruption dominated the overall transcriptional landscape, RNA-seq analysis identified coordinated upregulation of STAT3 target genes and proliferation-related pathways specifically in response to IL-11. Pharmacological inhibition of STAT3 attenuated the IL-11-induced promotion of organoid regeneration, confirming the requirement for STAT3-dependent signaling downstream of IL-11. Together, these findings demonstrate that IL-11 directly promotes intestinal epithelial regeneration after mechanical disruption through STAT3-dependent signaling, providing a mechanistic basis for its protective role in acute colonic injury.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.05.29.727830v1?rss=1

Suto, T., Nishina, T., Kashima, M., Suzuki, Y., Kubota, S., GOTO, Y., Yui, S., Nakano, H., Okunishi, K.

Closed-loop auditory stimulation during sleep shapes cortical responses and memory consolidation depending on spindle timing

Sleep spindles contribute to memory consolidation, and acoustic stimulation during sleep can modulate spindle activity. However, it remains unclear whether these effects depend on the timing of stimulation relative to the spindle cycle. Using closed-loop spindle detection, we delivered four variants of pink noise either during ongoing spindles or shortly after spindle offset. In Experiment 1, stimulation delivered during a spindle prolonged its duration, whereas stimulation applied shortly after spindle offset, i.e., within the refractory period, delayed the onset of the subsequent spindle. This pattern was consistent across all sound variants, indicating that the stimulation timing, rather than acoustic properties, drove the observed effects. Experiment 2 examined whether post-spindle stimulation influences memory consolidation. Relative to a no-stimulation nap, post-spindle stimulation reduced post-nap deterioration in motor sequence accuracy but did not significantly affect word-pair recall. EEG analyses revealed that stimulation elicited widespread slow oscillation activity without triggering new spindles, demonstrating that cortical processing of external input persists even during the refractory period. Notably, the motor benefit was greatest in participants with lower spindle density during the baseline adaptation nap. Together, these findings challenge the notion that the post-spindle refractory period is a functionally silent interval. Instead, this period remains receptive to external input, capable of eliciting cortical activity and supporting motor memory consolidation even in the absence of new spindle generation.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.05.29.728629v1?rss=1

Xia, T., Jin, X., Chen, D., Zuo, X., Yao, Y., Zhang, Y., Yuan, T., Zhang, Z., Li, X., Lai, C. S. W., Hu, X.

The Impact of Smoking Status on the Genomic Landscape of Lung Squamous Cell Carcinoma

Purpose: Comprehensive genomic profiling (CGP) has changed the treatment paradigm for non-small cell lung cancer (NSCLC) with the advent of molecularly targeted therapies for actionable genomic alterations (AGA). Despite this, the use of CGP is suboptimal, particularly in squamous cell lung cancer (sqNSCLC), which is more closely associated with smoking exposure and a lack of AGAs. We hypothesized that the prevalence of AGAs is inversely correlated with the chronicity and extent of smoking exposure in patients with sqNSCLC. Experiment Design: We retrospectively evaluated all patients with liquid biopsy testing via Guardant 360CDX or Guardant360 in the context of any sqNSCLC diagnosis at the City of Hope Comprehensive Cancer Center between 10/2020 and 7/2023. Social and clinical histories were evaluated to assess the frequency of AGAs in patients with no or remote smoking history. Results: Of the 56 patients in the initial evaluation, 24% (n=13) were non-smokers or remote smokers (greater than 20 years from cessation). Of these 13 patients, eight (61.5%) harbored AGA. Of these 8 patients, alterations observed included EGFR exon 19 deletion (50%, n=4), MET exon 14 skipping mutation (25%, n=2), EGFR G719S (13%, n=1), EGFR E114K (13%, n=1). Of those patients harboring AGAs that received NCCN-concordant matched targeted therapy, the objective response rate (ORR) with targeted agents was 50% and the clinical benefit rate (CBR) was 83.3%. Conclusion: These data support the use of CGP in sqNSCLC particularly in patients with remote or no smoking exposure.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.05.29.728662v1?rss=1

Garg, S., Salgia, R., Muddasani, R., Antrim, L., Lee, M., Malhotra, J., Nguyen, D., Amini, A., Liu, Y., Sampath, S., Jenkins, C., Rock, A.

Real-time Four-dimensional Imaging and Flow Dynamics Analysis of Cilia-driven Transport in Mammalian Tissues

Quantifying cilia-driven transport in mammalian tissues requires resolving rapid ciliary dynamics while preserving native three-dimensional geometry. Here we combine real-time four-dimensional light-field microscopy with tomographic particle tracking and physics-informed volumetric flow reconstruction to simultaneously image ciliary activity and reconstruct three-dimensional velocity fields in minimally dissected tissues. Applied across brain, respiratory, and reproductive epithelia, the workflow reveals recurrent vortical flow structures and enables quantitative analysis of flow-tissue coupling across individuals.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.06.01.729054v1?rss=1

Porcella, G., Sahin, A. T., Keller, J., Nawroth, J.

FINDER converts zero-background kinetic fingerprinting into area-scalable attomolar biomarker detection

Background constrains analytical sensitivity: surveying larger sensor areas samples more analyte molecules but also accumulates false positives, limiting gains in detection performance. Here we introduce FINDER[-]Fluorogenic INstantaneous Digital Enumeration and Recognition[-]a single-molecule platform that combines kinetic fingerprinting with fluorogenic transient probes for rapid molecular classification under near-zero-background conditions. By suppressing both solution and surface-associated background at micromolar probe concentrations, FINDER classifies individual molecules within seconds-scale observation windows per field of view. This regime allows sensitivity to scale with surveyed sensor area, enabling amplification-free quantification of the miRNA cancer biomarker hsa-miR-16 with an 11 aM detection limit. FINDER further generalizes to HPV16 DNA biomarker detection, two-color RNA/DNA co-profiling, and rapid discrimination of clinically relevant EGFR single-nucleotide variants using multidimensional kinetic filtering. Rapid per-field classification permits tens of fields to be surveyed within minutes. By converting kinetic specificity into area-scalable sensitivity, FINDER enables semi-automated attomolar biomarker counting without amplification in practical workflows.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.06.01.729299v1?rss=1

Walter, N. G., Dai, L., Banerjee, P., Johnson-Buck, A., Blanchard, A., Li, Z.

The protein binding domains of staphylococcal protein A fold independently and form an N- to C-terminal gradient of increasing stability.

Surface factors that contribute to the virulence of Staphylococcus aureus have become therapeutic targets in the treatment of illness associated with this bacterium. Staphylococcal protein A (SpA) is a well-known contributor to S. aureus toxicity and virulence, although relatively little is known about protein A and how its biological function has evolved. SpA is displayed on the surface of the bacterium and contains 5 nearly identical helical ($approx{60}$ aa) domains that bind antibodies with high affinity ($K_dapprox{10}$ nM). The folding free energies of only domains E and B have been determined. In this study we used intrinsic fluorescence detected denaturation to measure the folding thermodynamics of each domain in isolation and in the native multidomain context using a construct that includes the N-terminal half of the mature protein (SpA-N). We also constructed a series of proteins with 1 to 5 repeats of B domain, linked exactly as the five domains of WT SpA are linked. We used nearest neighbor thermodynamic models to explicitly demonstrate that the domains in B domain repeat proteins fold independently. We also showed that the domains in SpA-N fold independently by comparing the folding free energies of domains in isolation and in their multidomain context. Previous dynamic NMR experiments detected highly flexible linkers between domains in 5B, suggesting that the domains of SpA are structurally independent, which is likely responsible for the lack of thermodynamic coupling. Our results also showed a steep increase in domain stability from the N- to C-terminus in SpA-N, from $0.97pm0.05$ to $5.57pm0.28$ kcal/mol. We hypothesize that this stability gradient is related to efficient secretion of protein A.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.05.31.729144v1?rss=1

Hagarman, A., Franch, W. R., Oas, T. G.

Engineering Carbon Nanotube Quantum Well Defects with Recognition Tripeptides for Optical Detection of Extracellular Vesicles in Plasma

Extracellular vesicles (EVs) carry molecular signatures of their originating cells and have thus emerged as promising biomarkers. However, their clinical utility remains limited due to their low abundance and the modest sensitivity of current EV detection methods in complex biological environments. Here, we present a quantum well defect functionalized carbon nanotube sensor coupled with integrin-recognition RGD tripeptide for EV detection in human plasma. Leveraging the abundance of integrins on EV surfaces, we targeted a5b1, aVb1, and aVb3 subtypes. The nanosensor exhibited robust hypsochromic shifts in defect emission upon integrin binding, achieving sub-picomolar detection limits for integrin subunits and quantifying EVs at concentrations as low as 104 EVs/mL for glioblastoma, ovarian cancer, and fibroblast cell-derived EV types. Molecular dynamics simulation indicated that integrin docking at the RGD-coupled quantum defect can substantially reshape the interfacial environments of the quantum defects, explaining the high sensitivity in EV detection in complex biological media. Finally, transmembrane protein analysis validated the expression of surface integrins across the tested EV types. The modular nanosensor construct can be targeted to detect disease-associated EV subpopulations, advancing EV-based diagnostics.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.06.01.729398v1?rss=1

Hwang, I.-J., Kim, J., Patel, A., Zhang, L., Miller, J., Piletsky, S., Clift, C. L., Hisey, C. L., Kim, Y., Kim, M.

The regulatory logic of a dose-dependent developmental fate decision

In canonical developmental patterning, the embryo is exposed to gradients of signaling activators that elicit different cellular responses depending on the activator's concentration. Recent optogenetic studies of terminal ERK signaling downstream of Torso receptor tyrosine kinase in the early Drosophila embryo reveal that even a brief, 5-minute ERK stimulus is sufficient to rescue the development of larval "tail" structures. Here, we reveal components of the molecular network that defines this sensitive developmental fate response. We find that low ERK doses produce sustained Abdominal-B (Abd-B) expression comparable to that of wild-type embryos. Abd-B expression is adjacent to, but non-overlapping with, two other transcriptional repressors: the ERK effector Tailless (Tll) and the gap gene Giant (Gt). Analysis of gene expression patterns in response to optogenetic perturbations suggests that the Tll-dependent repression of gt constitutes the sensitive ERK-responsive step: even low tll expression leads to potent repression of gt in nearby regions, with Abd-B expression arising in a stripe between the tll and gt domains. Our work suggests that the spectrum of phenotypes produced through optogenetic manipulation can be used to define how robust patterning can arise from low doses of inductive signals.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.06.01.729432v1?rss=1

Araten, A. H., Ho, E. K., Toettcher, J. E.

Repurposing of the assembly chaperone PAC1-PAC2 defines a specialized proteasome landscape in mature mammalian sperm

Mature mammalian sperm rely on active proteasomes containing the testis-specific subunit 4s for capacitation, acrosomal exocytosis and fertilization, but how these proteasomes are structurally specialized remains unclear. We determine the endogenous proteasome landscape of mature bovine spermatozoa and testis using cryo-electron microscopy. We resolve five sperm proteasome assemblies: PA200-20S, free 20S, a previously unrecognized PA200-20S-PAC1/2 hybrid, and PAC1/2-20S complexes capped at one or both ends. Unexpectedly, the PAC1/2-bound complexes are mature and POMP-free, selectively incorporated for 4s, lack 2, and adopt a PAC1/2 orientation and gate configuration distinct from known assembly intermediates. Together with elevated proteolytic activity, these features indicate that PAC1/2 is repurposed from a transient assembly chaperone into a stable post-assembly regulator in mature sperm. 4s-containing proteasomes assemble through the canonical PAC1-PAC4/POMP pathway yet may follow {beta}2-{beta}3-{beta}1 incorporation ordering; functionally, 4s incorporation remodels the sCP surface and enhances peptidase activities, definining the spermatoproteasomes as a hyperactive 20S variant. PAC1/2-bound proteasomes are absent from testis, where PA200-20S and 26S proteasomes are present, while 26S proteasomes are nearly lost in mature sperm. Our findings define a sperm-specific, ATP-independent proteasome landscape, expand the functional repertoires of 4s and PAC1/2, and provide a structural framework for proteasome specialization in male fertility.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.05.31.728944v1?rss=1

Cong, Y., Zhou, X., Shen, L., Yuan, X., Chen, C., Wang, T., Li, Z., Peng, J., Lang, X., Ye, X., Chen, T., Chen, K., Su, C., Huang, Z.

Cell fusion after wounding requires plasma membrane damage and endocytosis

Tissue wounds comprise both dead and damaged cells. In epithelial wounds, repair is accomplished by cells at the wound edges, which are themselves often damaged. In the Drosophila pupal notum, wound-adjacent epithelial cells with plasma membrane damage often fuse to form syncytia; when plasma membrane damage is prevented, syncytia do not form. Damaged cells share cytoplasm as soon as milliseconds after wounding, and fusion pores connecting cell membranes form minutes later. A genetic screen reveals that wound-induced fusion requires endocytosis machinery, and dynamin localization indicates that endocytosis preferentially targets plasma membrane removed during fusion. Endocytosis promotes cell fusion by specifically promoting fusion pore expansion, indicated by quantitative analysis of cytoplasmic sharing between cells over time. Without endocytosis-mediated cell fusion, wound healing is slowed. Together, our results support a model of damage-induced cell fusion in which plasma membrane damage initiates fusion pores and endocytosis expands fusion pores, resulting in cellular fusion as an integration of single cell damage with tissue repair.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.05.31.728998v1?rss=1

Hua, J., Krystofiak, E. S., Pumford, A. D., Page-McCaw, A., Hutson, M. S.

Metabolic profiling of cultured erythroblast for the production of transfusion-ready cultured red blood cells

Transfusion-ready red blood cells (RBC) can be cultured ex vivo from hematopoietic progenitors. Despite its promising outlook, a cultured RBC transfusion unit cannot be produced at competitive costs. Large volumes of medium are required to maintain a maximum erythroblast cell density of 1-2.106 cells/mL during the proliferation stage of the culture. To identify the origin of the cell density limitation, we compared the growth support and changes in the cellular metabolomic signature while using different media formulations and feeding regimens. Media that were exposed to an increasing density of erythroblasts (here called spent media) displayed a proportional decrease in their ability to support erythroblast proliferation. A 1:1 combination of spent media with fresh medium (not previously exposed to the cells) restored growth for all tested conditions. Filtering both fresh and spent media with a 3 kDa cut-off filter, and subsequent recombination of the two fractions, indicated that exhaustion of the small molecular weight fraction (<3 kDa) was primarily responsible for growth limitation. We performed targeted and untargeted metabolomics analysis, for both the intra- and extracellular compartments, following seeding in fresh medium (12, 24, 36 h). We observed degradation of nucleosides, depletion of amino acids, and a decrease in intermediates of the glutathione-ascorbate, gamma-glutamyl and cysteine-methionine cycles. The latter compounds suggested an increase in oxidative stress in high density erythroblast cultures. Elimination of nucleosides from the medium led to a lower accumulation of purine salvage intermediates, and a 30% increase in cell productivity. In conclusion, we demonstrate that high-density erythroid cultures are subject to metabolic stress, defining critical constraints for scalable culture expansion.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.06.02.729469v1?rss=1

Gallego-Murillo, J. S., van Lakwijk, I., Yagci, N., Reisz, J. A., Pozo Garcia, V., D'Alessandro, A., van der Wielen, L. A. M., von Lindern, M., Wahl, S. A., Van den akker, E.

ESCRT proteins organise stress-induced organelle contact between endoplasmic reticulum and Golgi

ESCRT proteins remodel membranes at many cell organelles, including the endoplasmic reticulum (ER). Here, we investigate whether ESCRTs in budding yeast participate in stress-induced ER reorganisation. We find that ER stress triggers the formation of tubular ER subdomains that recruit various ESCRT proteins. Recruitment of the major ESCRT-III protein Snf7 is mediated by the ESCRT-associated protein Bro1, a homologue of human ALIX, in a manner that is mechanistically distinct from Bro1 function at endosomes. ESCRT-containing ER subdomains are derived from ceramide-rich ER exit sites and form contacts with the Golgi. Furthermore, Bro1 helps to concentrate the tethering and lipid transfer protein Tcb3, a homologue of human extended synaptotagmins, at these organelle contacts and contributes to cellular fitness when lipid metabolism is perturbed. These results indicate that specialised ER exit sites can be repurposed for contacting the Golgi directly and uncover ESCRTs as organisers of stress-inducible ER-Golgi contacts that help maintain cell homeostasis.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.05.29.728859v1?rss=1

Pajonk, O., Albert, L., Schafer, J. A., de Jager, L., Martin de Hijas, C., Papagiannidis, D., Odehnalova, K., Friemel, N., Esch, B. M., Frohlich, F., Luzarowski, M., Borner, G., Forster, F., Schuck, S.

NECAP antagonizes light-induced Rhodopsin-1 internalization to promote photoreceptor homeostasis

AP-2 is a key mediator of clathrin-mediated endocytosis (CME) that internalizes cargo from the plasma membrane. NECAP proteins physically bind phosphorylated AP-2 during CME, but their roles during endocytosis remain unresolved, with conflicting reports about their function. Here, we report that Drosophila NECAP is dispensable for development, but antagonizes light-dependent Rhodopsin-1 (Rh1) internalization, a process that occurs through AP-2-mediated endocytosis. Specifically, loss of Drosophila NECAP causes excessive light-dependent Rh1 internalization and an age-related retinal degeneration that can be rescued by photoreceptor-specific expression of a wild-type transgenic NECAP. A Drosophila NECAP mutant transgene, equivalent to a canine NECAP1 variant associated with retinal atrophy, failed to rescue the NECAP loss-of-function phenotype in the eye. Furthermore, overexpression of wild-type NECAP suppressed massive Rh1 internalization in a distinct Drosophila model of light-dependent Rh1 endocytosis and retinal degeneration. These results establish NECAP as a negative regulator of light-dependent Rh1 internalization essential for photoreceptor survival.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.06.01.729193v1?rss=1

Huang, H.-W., Tyrlik, M., Huang, P.-W., Yeo, A. I., Kim, A.-R., Perrimon, N., Tseng, C.-Y., Bellen, H. J., Ryoo, H. D.

Anoxia Tolerant DNA Replication is Supported by ATR Kinase in the Annual Killifish Austrofundulus limnaeus

Hypoxia and anoxia are known to suppress cell proliferation due to an increase in replication stress and activation of DNA damage checkpoints. Embryos of the annual killifish Austrofundulus limnaeus show a strong tolerance to extended anoxic exposure, indicating an improved genomic stability under oxygen starvation. Here we investigate the cell cycle regulation of the anoxia tolerant killifish embryonic cell line PSU-AL-WS40NE during anoxic exposure. Live cell imaging confirms continued cell proliferation of WS40NE cells for the first 24 hours of anoxic exposure with minimal cell death. Fluorescent imaging shows that cells begin to accumulate in G1 after the first day in anoxia with a pronounced and rapid entry into the S phase upon reoxygenation. Pharmacological inhibition tests show that this response appears to be reliant more on ATR signaling then ATM, suggesting that increased {gamma}H2AX levels are driven by increased replication stress instead of DNA damage. This conclusion is further supported by an apparent lack of induction of a G2 checkpoint in these cells suggesting that DNA damage during anoxic replication is minimal. Maintaining cellular proliferation during initial exposure to anoxia and accumulating cells in the G1 phase for extended anoxic exposure is likely one way that embryos of the annual killifish are able to survive prolonged anoxia and provides insight into mechanisms that enable cells to proliferate under metabolic stress.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.06.01.729397v1?rss=1

Roth-Carter, R., Helms, E., Saldivar, J. C., Podrabsky, J.

Index-agnostic oblique plane light sheet microscopy of centimetre-scale cleared tissues at subcellular resolution

We present cleared-tissue direct-view oblique plane microscopy (CtDvOPM), which enables optically sectioned subcellular resolution imaging of centimetre-scale tissues at high-throughput over the full range of clearing media refractive indices (n = 1.33-1.56). CtDvOPM can image conventionally-mounted expanded, aqueous or non-aqueous cleared tissue samples at up to 2 m lateral by 14 m axial resolution over a 10 mm x 10 mm x 25 mm sample volume without image tiling, at up to 400 million voxels per second.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.06.01.729284v1?rss=1

Lamb, J. R., Cardoso Mestre, M., Fenwyn Longrin, K., Bhat, P., Stevenson, M., Rhodes, A. D. Y., Gosieniecka, J., Redmond, L. C., Higgins, C. A., Rodriguez-Rodrigues, N., Lancaster, M. A., Manton, J. D.

A self-consistent model for phase separation and active processes in biomolecular condensates

Biomolecular condensates are thought to play a pivotal role in cellular organization by regulating biochemical reactants in space and time. Sustained molecular fluxes across condensate boundaries, together with the participation of phase-separating molecules in active chemical reactions such as ATP hydrolysis, call for a nonequilibrium description. Here, we propose a self-consistent framework in which diffusion--drift dynamics and chemical reactions are coupled through a conditional free energy, defined as the excess contribution to the chemical potential. Self-consistency is achieved by deriving this quantity from the same free-energy functional that governs molecular interactions and phase separation. We apply the framework to a minimal client--scaffold system and investigate how active chemical processes and phase separation interact at steady state. In doing so, our approach recovers the fundamental rules previously identified for the emergence of nonequilibrium steady-state fluxes. The model shows that active reactions involving the scaffold molecules can regulate the phase behavior of the condensate. Moreover, nonequilibrium steady-state fluxes are maximal near the boundary between the phase-separated and homogeneous regimes, suggesting that condensates sustaining molecular transport may operate close to their stability threshold. In the same region, client fluxes are also enhanced, revealing an indirect coupling between scaffold activity and client transport. These results provide a baseline for developing more detailed theories of chemically active condensates.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.06.01.729289v1?rss=1

Di Mambro, M., De Los Rios, P.

Fingertip real contact area scales quadratically with input voltage in electrostatic actuation

Touchscreens have become the dominant interface in consumer electronics, yet interactions with them remain primarily visual. Incorporating haptic feedback that simulates touch sensations could make these interactions more natural and intuitive. Electrostatic actuation, which modulates friction by attracting the finger toward a capacitive surface using an alternating voltage, offers a promising approach. The resulting increase in friction is often attributed to the rise in real contact area; however, direct experimental evidence linking voltage input parameters to real contact area and contact forces remains limited. Here, we use frustrated total internal reflection to directly image the real contact area while simultaneously measuring contact forces during controlled finger sliding under electrostatic actuation. We systematically vary voltage amplitude (75-150 V) and excitation frequency (30-230 Hz) and quantify the changes in contact area and forces as functions of these parameters. Our results reveal a quadratic dependence of real contact area, electrostatic attraction, and tangential force on voltage amplitude, with comparatively small effects of excitation frequency. These findings clarify the respective roles of voltage amplitude and frequency in the electrostatic modulation of finger contact mechanics, providing design guidelines for haptic display design.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.05.31.729125v1?rss=1

Kenanoglu, C. U., Vardar, Y.

Hashi: Bridging Statistical Model Derived 1D Microstate Encodings and Protein 3D Structural Ensembles

The functioning of proteins is intimately linked to the conformational states they sample within the native ensemble. Generating ensembles from a single static structure is therefore a research domain receiving considerable attention. In this application note, we introduce Hashi, a pipeline to rapidly generate realistic structural ensembles from the outputs of the structure-based Wako-Saito-Munoz Eaton (WSME) statistical mechanical model of protein folding. This approach relies on integrating the block WSME model outputs - strings of zeros and ones describing the conformational status of every residue over thousands or millions of microstates each assigned a statistical weight derived from physically grounded energy-entropy terms, and free energy profiles - with the RANCH module of the EOM (ensemble optimization method) from the ATSAS software suite, providing three-dimensional views of the structural ensembles within the model framework. It is applicable to a variety of single-chain monomeric systems with lengths ranging from 30 to 500 residues, including globular and repeat proteins. The generated structural ensembles can also be rank ordered according to their free energies within a given macrostate or a range of reaction coordinate values. Since the statistical weights of the WSME model microstates can be reweighted or calibrated with experiments, the ensembles shed light on not just the folding mechanism but also on the structural excursions that determine function and opening of otherwise buried binding pockets.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.06.01.729173v1?rss=1

Naganathan, A. N., Madhan, H.

Physics-guided design of intrinsically disordered proteins

Intrinsically disordered protein regions (IDPs) are found across the tree of life and characterized by the lack of a stable 3D fold, encoding function through a vast ensemble of conformations. This plasticity makes rational design of IDPs challenging. Physics-based approaches capturing distinct aspects of sequence composition, charge patterning, and molecular interactions have emerged as powerful predictors of ensemble-derived properties. Here, we present a machine learning framework for proteome-scale de novo IDP design by rationally inverting physics-based models. We first program IDPs to tunably sense and respond to diverse biophysical cues and show that IDP ensembles can directly encode complex signal processing, including threshold detection, bandpass filtering, and Boolean-type multi-input logic. We next engineer multicomponent IDP mixtures with tailored emergent condensate properties, including layering and number of phases, compositional specificity, and RNA-dependent remodeling of structure and composition. Finally, we demonstrate designed IDPs that selectively partition into or deplete from biological condensates in living cells. Together, our framework establishes a flexible and scalable strategy for design of ensemble-derived and collective properties in dynamic biomolecules.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.05.29.728696v1?rss=1

Tyagi, N., Boodry, J., Chou, V., Snead, W. T., Shrinivas, K.

Synthetic Fibrous Hydrogels as Minimal Systems to Modulate Cell Migration Modes in 3D

Cell migration in three-dimensional (3D) environments is highly plastic and regulated by extracellular matrix (ECM) cues. Engineered biomaterials provide controllable platforms to investigate how specific matrix signals regulate cell behavior in 3D, yet how defined biochemical signals control migration modes remain unclear. Here, we present tunable fibrous polyisocyanide (PIC) hydrogels functionalized with integrin-binding RGD peptides, cadherin-mimetic HAVDI peptides, or no ligands to direct mesenchymal, hybrid, or amoeboid-like migration of human adipose-derived stem cells without altering matrix mechanics. Using live-cell tracking, 3D displacement microscopy, matrix remodeling analysis, and YAP nuclear localization, we show that ligand identity governs adhesion organization, force transmission, and mechanotransduction. RGD-functionalized matrices promote {beta}1-integrin clustering, extensive matrix remodeling, strong YAP activation and upregulation of migration-related genes. In contrast, non-adhesive matrices limit adhesion formation, resulting in weak force transmission and amoeboid-like behavior. HAVDI-functionalized matrices induce cadherin clustering and heterogeneous cellular responses, indicating that a hybrid migration mode arises from adhesion organization rather than a distinct transcriptional program. Together, these findings demonstrate that ligand identity alone is sufficient to program migration mode in a force-responsive 3D matrix and provide a versatile platform to dissect cell-matrix interactions in complex environments.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.05.29.728729v1?rss=1

Zhang, H., Solis Fernandez, G., Louis, B., Vorsselmans, S., Hofkens, J., Kouwer, P. H. J., Yuan, H., Rocha, S.

Extracting anomalous diffusion parameters from multi-state ensembles of short single molecule trajectories.

Single-molecule tracking measures the stochastic motion of individual biomolecules in the cellular environment. Statistical analysis of trajectory ensembles is required to gain insight into the biophysical nature of mobility states and molecular interactions that they reflect. Mobility states can be parameterized by a generalized diffusion coefficient and anomalous exponent. Experimental constraints such as finite track length and localization precision limit how accurately these parameters can be determined. We compare the performance of analysis methods to recover the input parameters from ensembles of simulated single molecule tracks from different states spanning the range of anomalous diffusive behaviors observed in the cell nucleus. We further develop a framework to quantify error rates in the assignment of mobility states to individual molecules based on recall rates and precision. Our analysis shows that single-track analysis methods are superior to bulk methods in their ability to recover parametric descriptors from mixed populations. The most complete description is obtained by combining outputs from different tools. Our work provides a guide to assess the accuracy of analyses and obtain the most accurate parametric description of experimental single particle tracking data.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.05.30.729014v1?rss=1

Budhathoki, A., Pandey, G., Galeota-Sprung, J., Spille, J.-H.

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.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.05.29.728850v1?rss=1

Sukenik, N., Harris, C. C., Yadav, S., Chavez, M. S., Niman, C. M., Baczewski, L. T., El-Naggar, M. Y.

Single-Cell Electrophysiology Reveals Verapamil's Disruption of Bacterial Membrane Energetics

Verapamil, a clinically used calcium channel blocker, enhances the activity of several tuberculosis antibiotics, but its mechanism of action and physiological effects on bacteria remain unresolved. A central debate concerns whether verapamil primarily inhibits efflux pumps or disrupts membrane energetics. Here, we use Escherichia coli as a model system to quantify single-cell and population-level physiological responses to verapamil with high temporal resolution. Real-time measurements of the rotational speed of individual flagellar motors, a single-cell proxy for the proton motive force (PMF), reveal a heterogeneous response to verapamil: treated cells exhibit either a dose-dependent gradual decrease in PMF, or a rapid collapse of PMF. Although loss of the outer-membrane efflux channel TolC increases growth inhibition by verapamil, it does not alter the rapid PMF disruptions observed at the single-cell level, suggesting that efflux contributes to long-term susceptibility but not to the initial PMF disruption. Independent assays of population-level motility, pH, and membrane-integrity suggest that verapamil may selectively dissipate the electrical component of PMF while leaving intracellular pH largely unchanged. A minimal electrical circuit model captures both steady-state and dynamic behavior. Together, these findings demonstrate that verapamil rapidly and reversibly perturbs bacterial membrane energetics through a mechanism distinct from classical protonophores, helping to reconcile conflicting interpretations of its activity and clarifying how membrane effects may interact with efflux inhibition during antibiotic potentiation.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.05.31.729094v1?rss=1

Biquet-Bisquert, A., Astezan, A., Marmol, M., Voyvodic, P. L., Mohite, N., Pedaci, F., Nord, A. L.

Multimodal dynamics control activity of a glial glutamate transporter

Membrane transporters move polar solutes across lipid bilayers to regulate cellular metabolism, signaling, and drug distribution. These proteins operate via an alternating-access mechanism, cycling between extracellular-, intermediate-, and intracellular-facing conformations. The human excitatory amino acid transporter 1 (EAAT1) protects neurons from excitotoxic damage by mediating the uptake of glutamate and aspartate into glial cells. Defects in EAAT1 function result in numerous pathologies, including epilepsy and ataxia, suggesting that positive modulation of these transporters might ameliorate glutamate neurotoxicity. However, developing EAAT1 activators requires understanding the timing of conformational changes, which remain largely unexplored. Here, we establish an experimental platform that combines single-molecule Forster resonance energy transfer (smFRET) to monitor real-time conformational dynamics, single-transporter activity assays to correlate dynamics with function, and cryogenic electron microscopy (cryoEM) to visualize discrete conformations at high resolution. This platform enables detection of Angstrom-scale movements of single transporter molecules in real time, revealing that EAAT1 intersperses rapid conformational dynamics with long pauses. Slow and fast dynamics can be modulated by substrates, membrane composition, and mutations, and are correlated with the enrichment of specific structural states. We leverage this platform to investigate an EAAT1 mutation associated with severe episodic ataxia and show that it inhibits transport by stabilizing a paused cytoplasm-facing conformation. These results identify multimodal dynamics as an intrinsic, regulatable feature of EAAT1 function and, therefore, a potential therapeutic target. Henceforth, our integrated platform will facilitate investigations of other regulatory factors, including the effects of small-molecule and lipid modulators on the transport cycle.

Date: 2026-06-02
https://www.biorxiv.org/content/10.64898/2026.05.29.728845v1?rss=1

Wu, Q., Ciftci, D., Canul Tec, J., Reyes, N., Gregorio, G., Huang, Y., Boudker, O.