bioRxiv Subject Collection: All

Even though osteoarthritis is considered a disease of the whole joint, integrated analysis of articular calcified cartilage (ACC) behaviour with adjacent tissues remains a persistent limitation. Herein, we performed synchrotron X-ray computed tomography in intact loaded joints and coupled anatomical analysis with digital volume correlation to examine whether microarchitectural ACC features serve as predictive mechano-biomarkers of osteoarthritis. We reveal that male osteoarthritis-prone STR/Ort mice have thicker yet more porous ACC that contains larger but less spherical chondrocyte lacunae than their healthy parental-control CBA mice. This was linked to asymmetrical distribution of tensile and compressive strains generated under physiological loads in the ACC and underlying subchondral bone in tibial epiphyses of STR/Ort mice. Together these data suggest that murine ACC microarchitecture has a mechano-predictive role in age-related osteoarthritis.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.18.752585v1?rss=1

Evans, L. A., Sharma, A., Chen, J., Bourne, L. E., Parmenter, A. L., Brunet, J., Madi, K., Marussi, S., Lee, P. D., Pitsillides, A. A., Staines, K. A.

Hypertriglyceridemia is commonly managed with lipid-lowering drugs such as statins; however, adverse effects, dose intolerance, and financial barriers may limit their use. Therefore, affordable and potentially safer plant-derived alternatives warrant investigation. This study evaluated the lipid-lowering effects of ethanol extracts of Fucus vesiculosus and Phytolacca berry, administered individually or in combination, in mice fed a butter-enriched diet for 12 weeks. Forty-eight mice were randomly assigned to six groups: normal control (Group 1), butter control (Group 2), statin control (Group 3), Fucus vesiculosus extract (Ext-1; Group 4), Phytolacca berry extract (Ext-2; Group 5), and the combined extracts (Group 6). Body weight, organ-to-body-weight ratios, serum lipid parameters, and histopathological changes were assessed. Group 3 showed the lowest final body weight, while Group 6 demonstrated a comparable body-weight trajectory. Organ-to-body-weight ratios varied among groups, with Group 3 generally showing the highest values and Group 6 comparatively lower ratios for several organs. Group 5 had the lowest total cholesterol concentration, followed by Group 3, whereas Group 6 showed cholesterol levels comparable to the normal control and statin groups. Notably, Group 6 exhibited the lowest serum triglyceride concentration (129.9 mg/dL). Histopathological examination revealed relatively mild hepatic alterations and limited vascular plaque formation in Group 6. The combined extracts demonstrated favorable effects on body-weight gain and serum lipid profiles, particularly triglycerides, with comparatively mild histopathological changes. Further mechanistic, dose-response, toxicity, and long-term studies are warranted before clinical application can be considered.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.18.752646v1?rss=1

Hasan, N., Ahsan, N., Ahmed, T. B., Akhand, A. A.

Cardiomyocyte contraction is essential for the pumping action of the heart and deteriorates after myocardial damage, either as a consequence of irreversible cardiomyocyte injury or stiffening of the extracellular matrix, a process described as fibrosis. Cell geometry and substrate stiffness not only influence sarcomere architecture and contractility, they also determine how much work the cardiomyocytes can transfer to their environment. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes provide a model system to examine these effects in a controlled manner, enabling us to mimic key features of the cardiac environment in vitro. Here we show that geometric confinement and substrate stiffness influence different aspects of cardiomyocyte force generation. By combining traction force microscopy and live-cell imaging of hiPSC-derived cardiomyocytes on soft and patterned substrates, we find that geometric confinement leads to longer resting sarcomeres and more pronounced sarcomere shortening on substrates with both physiological (10 kPa) and fibrotic (30 kPa) stiffness. Interestingly, the substrate stiffness itself has little effect on resting sarcomere length, but influences the peak contractile stress. On 10 kPa substrates, confined cells generate higher peak contractile stresses than unconfined cells, whereas this difference is not observed at 30 kPa. Confinement also results in faster mechanical relaxation at 10 kPa, with no detectable difference at 30 kPa. The combination of defined cell geometry and physiological stiffness leads to longer resting sarcomere lengths, more pronounced sarcomere shortening, higher peak contractile stress, and faster relaxation, features associated with a more mature cardiomyocyte phenotype. These findings show that the mechanical consequences of cell geometry depend on substrate stiffness and, therefore, both aspects have to be considered when employing hiPSC-derived cardiomyocytes as a model system in mechanobiology research, disease modeling and drug testing.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.24.754056v1?rss=1

Sinha, M., Rölleke, U., Haag, R., Tiburcy, M., Blumberg, J., Schwarz, U. S., Zimmermann, W., Köster, S.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by repetitive behavior and impaired social interactions. Recent reports from human postmortem brain samples of ASD show transcriptome-wide dysregulations, including changes in differential gene expression and alternative splicing. An important mechanism regulating transcriptome function is the editing of double-stranded RNA by adenosine deaminases acting on RNA (ADARs), which play regulatory roles in neurodevelopment and innate immunity. We explore the ADAR editing changes associated with the use of Selective Serotonin Reuptake Inhibitors (SSRIs) in Arid1b haploinsufficient mouse models. Our results show dynamic changes in site-specific editing rates in autistic mice. We further demonstrate the transcriptome-wide effects of differential ADAR editing in terms of changes in miRNA-mRNA interactions. Moreover, our findings highlight a site-specific change in editing rates for evolutionarily conserved sites in mammals and ASD associated risk genes, such as Gria2. Our analysis of significant biological processes shows the association of differentially edited genes with processes vital for neurotransmission. Overall, our findings imply that the use of SSRIs can induce distinct ADAR editing signatures, potentially contributing to ASD pathology.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.24.753309v1?rss=1

Tariq, A., Piontkivska, H.

Microorganisms are indispensable to terrestrial ecosystems, with their genomic material underpinning critical functions and applications across agriculture, biotechnology, and human health. Although genomic language models have advanced representation learning in DNA sequences, the extensive diversity of microorganisms and imbalanced taxonomic representation in the pretraining corpora pose challenges for effective microbial genomic sequence modeling. Here we present MicroGlot, a taxonomy-informed microbial DNA foundation model pretrained on 3.70 million sequences comprising 378.3 billion nucleotides across 99{,}700 species. MicroGlot encodes the hierarchical relations among taxa through hyperbolic embeddings, incorporating microbial taxonomic knowledge into a sparse mixture-of-experts architecture. Zero-shot evaluation of MicroGlot's layer embeddings demonstrates that the model's representations encode phenotypic traits and taxonomic identity. Comparison with a taxonomy-ablated variant trained under the same pretraining scheme shows that incorporating taxonomic knowledge consistently improves representation quality across the layers of MicroGlot. MicroGlot also combines optimized training techniques with efficient architectural components from modern large language models, achieving leading zero-shot performance across layers and competitive fine-tuning performance with low computational overhead. In a 1000-species set sampled from major cellular domains and viral realms, MicroGlot's routing fingerprints show greater agreement with taxonomic groups than tetranucleotide composition, reflecting taxonomically structured expert routing in multilingual modeling of microbial genomes. Overall, we show that MicroGlot serves as an efficient and effective DNA foundation model for microbial genomic analysis.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.22.753215v1?rss=1

Li, A. Z., Wang, S., Cheng, S., Du, Y., Liu, R.

Relating the drivers of binding affinity to structural features remains difficult because affinity emerges from many weak, context-dependent interactions, while experimental affinity and mutation data are limited. Here, we ask whether a machine-learning model can learn energetically relevant interaction preferences directly from native structures, without affinity or mutation labels. We decompose interfaces into local structural motifs and learn the compatibility of each motif with its surrounding molecular environment. Applied across a protein surface, these models yield target preference maps (TPMs), spatial fields of local interaction compatibility. Trained exclusively on native antibody-antigen structures without affinity or mutation labels, TPMs recover charged, aromatic and backbone-mediated recognition across peptide-protein and other non-antibody interfaces. On an out-of-domain SKEMPI benchmark, wild-type TPM scores discriminated mutation-sensitive positions (AUC 0.643, n=1323), while TPM-based substitution scores ranked alternative amino acids comparably to FoldX and Rosetta Flex {Delta}{Delta}G. These results show that native interface structures can supervise learning of transferable, energetically relevant molecular preferences without direct thermodynamic labels.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.18.752156v1?rss=1

Kienlein, M., Menezes, F., Grass, P., Munker, S., Siebenmorgen, T., Popowicz, G. M.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) poses a significant public health threat, with a fatality rate of 37% and no approved therapeutics. Non-structural protein 10 (nsp10) is an essential cofactor that activates both the nsp14 3'-5' exoribonuclease (ExoN) activity required for RNA proofreading and the nsp16 2'-O-methyltransferase (2'-O-MTase) implicated in viral RNA cap formation. Despite its functional importance, the unbound structure and dynamics of MERS-CoV nsp10 in solution remain uncharacterised. Here we report a near-complete NMR backbone and sidechain assignment and characterise the solution structure and dynamics of the protein by NMR. In contrast to the folded 1 helix observed in crystal structures of coronavirus nsp10, we find that this region is intrinsically disordered in solution, with residues 10-22 undetectable under standard conditions and further evidenced by pH titration, temperature-dependent NMR, and CLEANEX-PM experiments. Analysis of NOE contacts confirmed that the core adopts the conserved coronavirus nsp10 fold, consistent with the AlphaFold-predicted structure. 15N backbone relaxation measurements indicated a rigid, well-ordered core with only localised flexibility, despite a relatively low proportion of secondary structure elements, and CPMG and CEST experiments detected no conformational exchange on the s-ms timescale. Building on this structural and dynamic characterisation, we explored the ligandability of nsp10 by 19F NMR fragment screening. Screening of a 463-compound library identified 23 initial hits (4.97% hit rate), of which 20 were confirmed by 15N SOFAST-HMQC and eight gave quantifiable affinities by MST (Kd 0.5-6.9 mM), the remainder being too weak for reliable determination. Chemical shift perturbations clustered near functional surfaces of the folded core, indicating that MERS-CoV nsp10 is ligandable and providing chemical starting points for antiviral development.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.24.754126v1?rss=1

Dong, D., Kozielski, F. G., Waudby, C. A.

Generating high-quality genome assemblies for small animals with large genomes is complex due to their small body size, DNA contamination, and repetitive elements. Ticks exemplify these complexities, while also being a global health threat to humans, domestic animals, and wildlife. Advances in long-read sequencing platforms now make it feasible to obtain large amounts of raw sequence data from individual specimens, but challenges remain. Key genome workflow challenges include error correction, assembly, transposable element annotation, and chromosome assignment and synteny. Here we examine three individual Dermacentor reticulatus ticks using deep Oxford Nanopore sequencing. Comparing and contrasting bioinformatic tools for raw read and assembly manipulation allows us to identify the parameters that provide a high-quality haploid genome assembly among 38 assemblies. We find Dorado error corrected raw read data from approximately two flow cells are needed, but limiting assembly coverage to 40x produces the highest quality assemblies. Then we examined the effects of optimal workflow parameters on downstream analyses of gene synteny and manual transposable element annotation of the three tick assemblies in comparison to an independent assembly of a fourth D. reticulatus individual from a different country. Gene synteny analysis allows chromosome assignment of scaffolds and transposable element identification was improved markedly with manual curation. Finally, we compared the genetic variation of D. reticulatus across two populations and found similar genetic diversity. Our study provides a clear workflow to obtain high-quality assemblies from Oxford Nanopore sequences and genetic characterization of a species with a large, complex, and repetitive genome.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.18.752137v1?rss=1

Dillon, K. C., Sprong, H., Ronai, I., Choudhary, S., Grant, C., de Paula Baptista, R., Ray, D. A., Glenn, T. C.

Complex traits that span multiple tissues and systems often integrate large numbers of genes across development, physiology, and behavior, making it challenging to identify their essential components. Blood feeding in mosquitoes is one such trait. It is ancestral to the mosquito family, maintained in most species for ~200 million years, and was independently lost in three lineages. These convergent losses offer a natural experiment to discover the genetic, physiological, and neural features required for blood feeding. We assembled high-quality, chromosome-level genomes for seven mosquito species, along with whole-brain tomographic reconstructions. Our study spanned the three known non-blood-feeding lineages (Toxorhynchites rutilus, Topomyia yanbarensis, and Malaya genurostris), blood-feeding relatives, and the variable blood feeder Wyeomyia smithii. Comparing orthologous gene clades, we detected convergent gene loss specific to the three lineages that had lost blood feeding. The losses include the salivary platelet-aggregation inhibitor Aegyptin, blood-activated serine proteases such as Chymotrypsin-1 and 2, and a carboxylesterase expressed in the female fat body and brain glia. The loss of blood feeding also extended to chemosensation. Non-blood feeders lack two odorant-binding protein clades, two ionotropic receptor clades associated with blood-component taste detection, and odorant receptor clades expressed in a discrete, strongly female-biased population of antennal neurons. Female-biased head gene expression was reduced in non-blood feeders. Finally, examination of whole-brain tomographic reconstructions across the species revealed smaller antennal lobes in non-blood-feeding females, consistent with reduced olfactory input. Together, these findings identify a compact set of genes, expression patterns, and brain regions associated with blood feeding, offering an evolutionary entry point for functional dissection of how this complex and dangerous trait is built and dismantled.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.23.753750v1?rss=1

Houri-Zeevi, L., Brand, P., Arnoldi, I., DeFoe, A. E., Balacco, J. R., Shai, N., Makunin, A., Palatini, U., Toma, T., Okazawa, T., Stone, C. M., Schiller, A., Fedrigo, O., Jarvis, E. D., Lawniczak, M. K., Miyagi, I., Gabrieli, P., Vosshall, L. B.

Non-coding variants altering mRNA splicing are increasingly recognized as important cause of Mendelian disorders. Deep intronic variants (DIV) that activate cryptic exons (CEs) cause ~20% inherited retinal diseases (IRD) cases, yet it remains challenging to predict intronic variant pathogenicity accurately, thus we used a high throughput splicing assay (HTSA) to measure the effects of rare deep intronic variants in recessive IRDs with one confirmed pathogenic variant. 640 very rare deep intronic variants were chosen in trans-position with known mutation from 76 patients. We tested them with HTSA, which consists of a split-GFP minigene, separated by an SMN1 gene intron into which the 270bp sequences flanking the DIVs were cloned. The plasmid library was transfected into 293HEK cells, and 48 hours later total RNA was extracted from the transfected cells. RNA transcripts produced by HTSA minigene were amplified by RT-PCR and sequenced. The intron sequences spliced between the two GFP exons were identified and quantified. 98 variants activated CE >100 times more in altered oligo compared to reference oligo and were classified as pathogenic in this experimental setting, validated in longer context, 26 variants were classified as VUS. Interestingly, only 6 from 90 variants (6,6%) were predicted to be pathogenic by in silico algorithms (Splice AI) which leaves room for significant improvement of prediction algorithms. The results of this experiment confirmed diagnosis for 50 of 78 patients (64%). So, 19,4% of deep intronic variants were shown to cause CE activation. This can lead to conclusion that significant portion of undiagnosed patients with recessive IRDs carry pathogenic intronic mutation which causes the disease.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.18.752412v1?rss=1

Weener, M., Valestil, K., Place, E., Mehrotra, S., Navarro, D., Scott, H., Bujakowska, K., Pierce, E.

Eusociality, characterized by overlapping generations, cooperative brood care, and reproductive division of labour, has arisen independently across diverse, phylogenetically distant insect orders, including Hymenoptera (ants, bees, and wasps), Blattodea (termites), and Coleoptera (weevils). While multiple studies have investigated the molecular evolution of sociality from solitary ancestors in Hymenoptera and Blattodea, so far little is known about the evolutionary signatures of social evolution in Coleoptera. Weevils (Curculionidae) provide an ideal system for addressing this question, as they cover the full spectrum of social complexity from parental care, through several origins of facultative eusociality to the only obligately eusocial beetle, Austroplatypus incompertus. We generated genome assemblies for A. incompertus and two facultatively eusocial weevil species, Xylosandrus germanus and Xyleborinus saxesenii, which together with 18 publicly available weevil genomes span two independent evolutionary origins of sociality. Our analyses reveal a genome-wide relaxation of purifying selection with increasing social complexity, which is most pronounced in A. incompertus. We find a significant excess of convergent gene family contractions in lineages where sociality evolved, and no evidence of elevated positive selection. These findings indicate that the molecular mechanisms of social evolution in weevils are primarily characterised by relaxed selection and gene loss, rather than adaptive innovation and gene family expansions. These observations are consistent with sheltering and reduced effective population size playing an important role, a pattern not previously observed in other clades.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.19.752859v1?rss=1

Rinke, S., Bickerstaff, J., Biedermann, P. H. W., Kemena-Rinke, C., Riegler, M., Schebeck, M., Harrison, M. C.

Cytos Multimodal Pooled Screening (CytosMPS) measures CRISPR guide identity, morphology, native 3' transcriptome, and protein abundance and localization in the same fixed cells. Following IL-1{beta} stimulation, cells with higher nuclear NF-{kappa}B show lower phospho-p38/HSP27 and higher NFKBIA/DUSP1 in the same cell, a measurement neither imaging nor transcriptomics alone provides. TGFBR2 and CTNNB1 perturbations produce distinct multimodal phenotypes. Guide representation and perturbation transcriptional responses agree with an orthogonal single-cell transcriptomic platform.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.20.752925v1?rss=1

Lopez, T., Rabalais, J., Song, B., Boddicker, A., Schwartz, A., Honigfort, D., Zhao, J., Kellinger, M., Kruglyak, S.

Biofilm formation by the fungal pathogen Candida albicans is a major source of infection. The biofilm regulator Ume6 forms complexes with partner transcription factors Efg1, Ndt80, and Upc2 to drive expression of biofilm-related genes. Here we present chromatin immunoprecipitation with sequencing (ChIP-seq) data for Ume6-chromatin association in both wild-type and efg1{Delta}/{Delta} ndt80{Delta}/{Delta} upc2{Delta}/{Delta} backgrounds. Ume6 associates with two promoter region classes. For one class, Ume6 association is significantly decreased in the efg1{Delta}/{Delta} ndt80{Delta}/{Delta} upc2{Delta}/{Delta} background. This class includes 577 genes, many with well-established roles in biofilm formation or the related process of filamentation. For the second class, Ume6 association is unperturbed in the triple mutant background. This class includes 534 genes, some with roles in iron homeostasis (e.g., SFU1) and ergosterol synthesis (e.g., ERG11), which may contribute to the impact of Ume6 on ndt80{Delta}/{Delta} mutant azole drug sensitivity. It also includes FLO9, a putative adhesin gene that is required for the emergent biofilm state produced by efg1{Delta}/{Delta} ndt80{Delta}/{Delta} double mutants. The data show that Ume6 binding to ~half of its targets requires known partners, and Ume6 binding to ~half of its targets does not. Ume6 may bind to the latter set of promoter regions independently of any partner, or perhaps with additional partners that have yet to be discovered.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.24.753443v1?rss=1

Do, E., McManus, J., Mitchell, A. P.

Bacterial cultures entering stationary phase (SP) undergo complex physiological alterations, helping the cells to survive when medium resources are exhausted. The onset of various stress responses underlying SP physiology is generally believed to be due to reactions of individual cells to the environmental cues such as starvation, toxic metabolites, etc. The SP physiological state makes bacteria unsuitable for the replication of most bacteriophages; however, some phages are able to infect and multiply in them. We investigated Escherichia coli phage DH23 growth in SP cultures of E. coli MG1655 infected at different hours post inoculation (ages). Under our conditions the cells enter SP at about 6 h, but the phage replication was possible till 11h and then dropped abruptly by 13h of culture aging, indicating an abrupt physiological change to deeper dormancy during SP. The onset of this phage-inhibiting middle-SP dormancy (MSPD) turned out to be mediated by intercellular communication mediated by the middle-SP signal particles (MSP) which are larger than 100 kDa and contain both RNA and DNA. These MSP are inactivated by RNAse or by DNAse, enabling phage growth in 14h-old cultures and lifts the tolerance of such cultures to kanamycin. Moreover, the RNAse or DNAse treatment makes 24h culture spent media suitable for additional cell growth, and cultures initiated in fresh LB supplemented with RNAse reach an OD600 about 1.5 times higher compared to the untreated control. This indicates that MSP signaling influences cell physiology well before the MSPD onset and helps the population cease growth pre-emptively to save about 1/3 of medium resources to support the viability during SP.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.23.753752v1?rss=1

Ivanov, P. A., Timoshina, O. Y., Mureev, S. V., Letarova, M. A., Letarov, A. V.

Pulmonary hypertension (PH) is a progressive and fatal disease characterized by pulmonary vascular remodeling, inflammation, and immune dysregulation. Myeloid-derived suppressor cells (MDSCs) and macrophages contribute to PAH pathobiology; however, the molecular regulators of their pathological activation remain poorly defined. The triggering receptor expressed on myeloid cells 2 (TREM2) is an immunomodulatory receptor that shapes myeloid cell metabolism, survival, and immunosuppressive function, yet its role in PH has not been investigated. Methods: Single-cell RNA sequencing (scRNA-seq) data from human pulmonary artery tissue (GSE210248; n=3 PAH, n=3 donors) were analyzed to characterize TREM2 expression across cell populations. Wild-type (WT) and global TREM2 knockout (TREM2 KO) mice were exposed to chronic hypoxia (10% FiO2, 28 days). Hemodynamic, histological, flow cytometric, and ex vivo functional assessments were performed. TREM2 expression was measured by flow cytometry in circulating MDSCs from PAH patients (n=22) and healthy controls (n=13). Results: TREM2 was markedly enriched in monocyte/macrophage populations in PH pulmonary arteries and TREM2-high immune cells exhibited transcriptional downregulation of chemotaxis programs and upregulation of antigen processing and MHC II presentation pathways. TREM2 deficiency significantly attenuated hypoxia-induced RVSP increase, right ventricular dysfunction, pulmonary inflammation, and vascular remodeling. MDSCs from TREM2 KO mice exerted significantly less suppression of CD4+ and CD8+ T cell proliferation. TREM2 was significantly elevated in circulating MDSCs from PAH patients and showed a directional association with hemodynamic severity. Conclusions: TREM2 is a novel regulator of myeloid-driven immunosuppression and vascular remodeling in PAH and warrants investigation as a therapeutic target and biomarker.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.18.752808v1?rss=1

Oliveira, A. C., da Silva, F., Zhang, Y., Alves, M. D., Pham, A. T., Harris, C. L., Khanfar, S., Abdelnor, R., Alvarez-Castanon, J. V., Philips, C. M., Tarantino, M., Fu, C., Virk, S. T., Ray, K. E., Chen, L., Krause, E. G., de Kloet, A. D., Bryant, A. J.

Solid tumors are known to be refractory to treatment due to a poorly perfused microenvironment that blocks immune cell infiltration and effective drug delivery. Histotripsy is a non-invasive focused ultrasound modality that generates cavitation microbubbles to mechanically disrupt solid tumors, resulting in decreased hypoxia and stimulation of tumor-targeted adaptive immune responses. In this study, we aim to investigate whether post-histotripsy hypoxia reduction and cytotoxic immune cell infiltration are associated with normalization of tumor vasculature and enhanced perfusion. Structural analysis of both treated and contralateral off-target tumors following partial histotripsy revealed significant vascular remodeling, enhanced vascular integrity, and decreased vascular leakage indicative of vascular normalization in multiple in vivo models of melanoma, hepatocellular carcinoma, and pancreatic adenocarcinoma. These effects appeared antigen-specific, as no remodeling was observed in contralateral off-target tumors of discordant pathology. Mechanistically, CXCR3+CD8+ T cells played a role in mediating these vascular changes, as genetic knockout of CD8 and pharmacological antagonism of CXCR3 impaired both remodeling and normalization. Additionally, significant changes in endothelial CXCR4 and Angiopoietin-1 and -2 expressions were observed following histotripsy, indicating their potential involvement in promoting vascular normalization and remodeling. Significant vascular remodeling was accompanied by enhanced tumor perfusion and intratumoral delivery of chemotherapeutic agents and therapeutic monoclonal antibodies. These findings suggest that histotripsy not only creates a more favorable microenvironment for immunotherapy, but also optimizes drug delivery into solid tumors. Combinatorial approaches using histotripsy with chemotherapy or immunotherapy may be a novel therapeutic strategy to overcome current limitations of conventional solid tumor therapies.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.18.752667v1?rss=1

Queen, H., Song, B., Kim, H., McGinnis, R., Liu, J., Sanogo, A., Buglak, K., Karanam, C., Bontrager, A., Worlikar, T., Xu, Z., Ganguly, A., Cho, C. S.

Granulomas are organized immune structures that contribute to host defense against persistent pathogens, yet the mechanisms that coordinate their assembly and protective function remain incompletely understood. Here, using experimental visceral leishmaniasis caused by Leishmania infantum and samples from patients with active disease, we identify the NLRP3 inflammasome as a regulator of protective hepatic granulomatous immunity. Inflammasome-associated mediators were elevated in patients and correlated with systemic inflammation. In mice, L. infantum induced NLRP3 inflammasome activation within hepatic granulomas, while single-cell and spatial transcriptomic analyses revealed enrichment of inflammasome-associated transcription in hepatic macrophages and granuloma-associated regions. NLRP3 deficiency did not prevent granuloma initiation but impaired granuloma expansion, cellular organization, and leukocyte accumulation. This response required Caspase-1/11 and IL-18, but was independent of IL-1{beta}. Loss of NLRP3 also impaired parasite control despite reducing hepatic inflammation and histopathological alterations. Together, our findings identify NLRP3-Caspase-1/11-IL-18 signaling as a mechanism that coordinates the cellular and spatial organization of protective hepatic granulomas, revealing granuloma architecture as a previously unrecognized function of inflammasome-mediated immunity and providing mechanistic insight into host resistance in visceral leishmaniasis, a potentially fatal neglected tropical disease.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.18.752760v1?rss=1

de Oliveira, I. M., Chaves, M. M., Costa-Madeira, J., Barbosa, A. L., Corradi, P. S., de Oliveira, B., Becerra, A. M., Marques, P. H., Rodrigues, T. S., Simizo, A., Lorenzon, L., Batah, S. S., Herrera, A. J., Nakaya, H. I., da Silva, A. M., Gazzinelli, R. T., Cruz, A. K., Takiya, C. M., Fabro, A. T., Menezes, G. B., Costa, C. H., Zamboni, D. S.

Neuroscientific learning theories propose that the brain acquires knowledge by constructing internal world models. Supervised learning, the dominant approach in deep neural networks (DNN), relies on external category labels, making it difficult to reconcile with biological learning. There is an increasing trend towards more biologically valid approaches, such as predictive (minimize future surprise) or contrastive (minimize response to expected, maximize to unexpected inputs) objectives, but these approaches typically rely on pretrained DNN models that vary widely in architecture, size, and hyperparameters, making direct comparisons difficult. Here, we isolate the effect of learning by comparing small, identical networks trained with predictive, contrastive, and supervised learning objectives, as well as local (layer-restricted) vs. global (full backpropagation) learning. We show that brain representations after statistical learning are better modeled by a predictive local target than a supervised or contrastive target, and that during learning, the brain attenuates category-specific representations while retaining predictive ones. We additionally show that predictive objectives explain brain variance that standard supervised DNNs do not, and that this variance is tied to predictive rather than stimulus or mismatch processing. These findings demonstrate a controlled approach for testing the algorithmic basis of learning and identify prediction as a core learning mechanism.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.18.752626v1?rss=1

Gütlin, D., Kittelmann, D., Auksztulewicz, R.

Chronic spatial neglect after right-hemisphere stroke reflects network dysfunction, with recovery influenced by structural connectivity. Music recruits coordinated bi-hemispheric activity and may promote interhemispheric communication after stroke. Following our pilot study in post-stroke aphasia, we asked whether classical music modulates connectivity and behavior in chronic neglect. Three patients completed a crossover protocol comparing two weeks of daily audiovisual classical music with an audiobook control, with neglect, mood, quality-of-life, diffusion MRI, and resting-state high-density EEG measures. All showed right superior longitudinal fasciculus II disconnection with preserved callosal pathways. In N2, line bisection shifted leftward across all five lines after music, together with increased posterior interhemispheric theta-band weighted symbolic mutual information (wSMI), then deteriorated into the pathological range after the audiobook, without mood improvement. N1 showed mixed behavioral effects, with mood and quality of life improving after music. N3 showed no significant spatial improvement and worsened mood after music. These heterogeneous responses motivate larger studies of connectional predictors. The findings support the feasibility of classical music as an adjunct to neglect rehabilitation and suggest that cognitive changes may occur independently of mood, while the audiobook cannot be considered an inert control.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.18.751660v1?rss=1

Zeineldin, R., Takamura, Y., Chea, M., Naccache, L., Bayen, E., Toba, M. N., Bartolomeo, P.

Introduction: Major depressive disorder (MDD) is a highly prevalent and disabling psychiatric disorder. Human neuroimaging studies increasingly frame its neurobiological substrate in terms of alterations of large-scale brain network organization. Resting-state fMRI findings broadly align with this view, yet remaining highly heterogeneous, reflecting both clinical and analytic variability. Predominant approaches largely characterize functional organization in terms of pairwise relationships between regions, which may limit the ability to capture more global features of brain organization associated with depression and its course. Here we use novel methods from Topological Data Analysis to provide new perspectives on these brain-behavior associations Objectives: The main objective of this study was to determine whether whole-brain topological descriptors of functional connectivity capture alterations associated with MDD. In particular, we examined whether these features vary along clinically relevant dimensions of heterogeneity, focusing on illness course (single-episode vs. recurrent MDD) and current antidepressant medication status. To this end, we analyzed the area under the curve (AUC) of the zeroth and first Betti numbers (B and B), which index global network integration and higher-order cycle structure across scales, respectively. Methods: Neuroimaging (resting state fMRI) and phenotypic data were obtained from 1,490 participants (776 individuals with MDD and 714 never-depressed controls) included in the REST-meta-MDD project of the DIRECT consortium. Functional connectivity matrices were computed using Pearson correlations between regions defined by the Power-264 atlas and harmonized across sites using CovBat. Topological Data Analysis was applied to characterize whole-brain network organization across connectivity thresholds using B and B curves and their areas under the curve (AUC). B associated cycle counts and their coarse anatomical configurations were further examined. Group differences and effects of illness course and medication status were assessed using multiple linear and generalized linear regression models. Results: Compared with controls, individuals with MDD showed significantly higher B AUC, indicating altered global integration profiles across connectivity thresholds. B AUC did not differ by illness course, illness duration, or symptom severity. In contrast, B AUC showed a graded increase across illness courses, with the highest values observed in recurrent depression. This effect was driven by an increased number of one-dimensional cycles rather than greater cycle persistence. Medication status further modulated B alterations, with elevated values observed in unmedicated single-episode and medicated recurrent MDD. Anatomical decomposition revealed that higher-order alterations were primarily driven by inter-network configurations spanning multiple large-scale functional systems. Discussion:These findings indicate that MDD is associated with altered multiscale functional organization, combining less compact global integration with an increased prevalence of cross-network hole-defining cycles, with these features varying systematically with illness course and current medication, suggesting that topology-informed measures capture clinically relevant variability in brain organization in depression.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.18.752576v1?rss=1

Castilla-Jimenez, J. F., Diaz-Patino, J. C., Enriquez-Geppert, S., van Tol, M.-J., Barrios, F. A., the DIRECT Consortium,, Alcauter, S.

Face recognition ability varies enormously across humans. Individuals with face-blindness (prosopagnosia) struggle to recognize even close family members while super-recognizers can identify strangers with exceptional accuracy. Where do ANNs fall within the human face recognition spectrum? Here, we administered the standardized tests used to characterize human face recognition ability to a diverse set of models, allowing us to contextualize a model's performance within the distribution of human behavior. We found that the majority 55% of models to be classified as face-blind and that even the best face-trained models do not cross the human threshold to be considered a super-recognizer. Interrogating the internal representations of these models showed that models that performed well on standardized face recognition tasks were more identity-selective and viewpoint-invariant. We also found that low-performing models did contain some identity information in independent representational subspaces. Removing viewpoint-dependent subspace improved face recognition abilities in 49 of the 53 models tested. Targeted unit ablations further identified opposing contributions, with viewpoint-dependent units disrupting identity coding and viewpoint-tolerant units supporting it. Together, our results show that most AI models are face-blind with worse face recognition ability than humans, and demonstrate how differences across AI models can be used generate testable hypotheses about the computational basis of human face recognition in humans which can then be probed in future studies.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.18.750666v1?rss=1

Dudipala, K. R., Martinez-Addiego, F., Rahnev, D., Duchaine, B. C., Ratan Murty, N. A.

MRI is an excellent tool for monitoring functional and structural network reorganization after brain injury and may provide biomarkers for treatment stratification and novel neuromodulatory interventions. In ischemic stroke, functional network changes, particularly within the contralesional hemisphere, and structural integrity, most prominently of the corticospinal tract, have been successfully associated with behavioral deficit and outcome. However, these components are commonly studied separately and at single time points, which does not reflect the highly dynamic nature of brain network plasticity. Here, we characterized structural and functional connectivity (SC-FC) coupling in the healthy mouse brain and longitudinally assessed intra- and inter-hemispheric connections up to 4 weeks after cortical and cortico-striatal stroke. In the healthy brain, intra-hemispheric SC-FC coupling was lateralized in the sensorimotor network (SMN), whereas the default mode network (DMN) showed no significant hemispheric difference. Intra-hemispheric coupling exceeded inter-hemispheric coupling in the SMN. After cortical stroke, regions with high baseline intra-hemispheric SC-FC coupling remained largely stable, whereas initially weakly coupled sensorimotor regions showed delayed increases within the ischemic hemisphere. Contralesional intra-hemispheric SC-FC coupling changed little, but directional inter-hemispheric analyses revealed selective remodeling, including increased contralesional-to-ischemic coupling of primary motor cortex and reduced bidirectional coupling of medial sensorimotor cortex. Cortico-striatal stroke produced fewer longitudinal changes, dominated by reduced inter-hemispheric motor-cortex coupling. These alterations did not simply correspond to regional lesion involvement. Thus, hemisphere- and direction-resolved SC-FC coupling uncovers network- and stroke-model-specific reorganization not detectable when structural and functional connectivity are analyzed independently.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.18.752042v1?rss=1

Mahani, F. S. N., Hoehn, M., Fink, G. R., Aswendt, M.

Rule-based languages have proven effective for modeling systems of interacting structured entities as typically encountered in chemistry and molecular biology. We present PyKappa, a rule-based modeling package written in Python whose interpreted nature enables interactive simulation and analysis, including by agentic AI. The package seeks to broaden the base of developers by utilizing a widely known programming language and serves as an easy-to-deploy teaching tool. Using PyKappa, we conduct a case study of phase separation.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.18.752660v1?rss=1

Alpay, B. A., Lin, A., Damiani, A. C., Fontana, W.

Forster resonance energy transfer (FRET) is commonly used to monitor protein-protein interactions in situ. The high spatiotemporal resolution and facile implementation inside complex molecular environments have spearheaded FRET's widespread adoption in biosensing. Despite these advantages, current FRET biosensors are largely restricted to the detection of the presence/absence of individual inputs and are thus unable to sense several multiplexable inputs simultaneously within complex milieu of biological environments. In this work, we introduce a generalizable strategy to construct genetically encoded protein-based FRET biosensors capable of recognizing multiple inputs following Boolean logic-type (YES/OR/AND) operations. These topologically specified FRET sensors powerfully expand the input capacity in sensing protein-protein interactions while providing a user-programmable platform for monitoring heterogeneous biological activities both in vitro and in living cells.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.24.753966v1?rss=1

Son, H., Hoye, J. W., Khawand, M., Ross, M. L., Gharios, R., DeForest, C. A.

Glucoamylase is essential for industrial starch saccharification, but the limited thermostability and near-neutral pH tolerance of fungal glucoamylases necessitate cooling and acidification of liquefied starch. Here, we developed an artificial intelligence-guided strategy to simultaneously improve the thermostability, pH tolerance, and catalytic activity of glucoamylase from Penicillium oxalicum (PoGA). Two property-specific machine-learning models, CASPE-T and CASPE-A, identified substitutions associated with thermostability and pH tolerance, respectively. Experimental screening identified beneficial substitutions in 11 of 21 CASPE-T and 12 of 22 CASPE-A candidates. Folding-energy-guided recombination integrated the two traits while maintaining structural compatibility. The optimal variant, PoGA T513E/Q305N, exhibited 2.21-fold higher specific activity than the wild type, with half-life extended from 22.3 to 57.9 min at 60 degrees C and from 16.6 to 64.7 min at pH 8.0. Molecular dynamics simulations attributed these improvements to reinforcement of high-occupancy hydrogen-bonding networks, suppression of conformational fluctuations in the linker and carbohydrate-binding module, enhanced long-range dynamic coordination, and preservation of a compact catalytic architecture. At 60 degrees C and pH 6.5 without acidification, PoGA T513E/Q305N produced 219.9 g/L glucose and achieved 89.1% starch conversion, 31.4% higher than the wild type. This work provides an efficient framework for multi-objective enzyme engineering and sustainable starch biorefining.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.24.753989v1?rss=1

QIAO, J., Ma, X., Song, Y., Deng, Q., Ni, X., Liu, G., Meng, S., Shi, F., Deng, L., Cui, H., Li, X.

Skeletal muscle dysfunction is a major contributor to disability and incomplete functional recovery in patients with end-stage knee osteoarthritis (KOA), yet the spatial components of disease-associated molecular remodeling remain poorly understood. Here, we applied spatial transcriptomics to paired skeletal muscle biopsies obtained from the surgical (Sx) and contralateral (Ct) limbs of individuals undergoing total knee arthroplasty (TKA) to define the cellular architecture of periarticular muscle and determine how muscle inflammation susceptibility (MuIS) shapes local transcriptional programs. Integrated analysis of 27,087 spots obtained from 22 muscle histological cross-sections (11 Sx-Ct pairs) revealed seven spatially resolved transcriptional domains corresponding to slow and fast myofiber states, an extracellular matrix/fibroadipogenic-enriched domain, a pericyte/smooth muscle domain, and a satellite cell/myogenic-enriched domain. Despite advanced unilateral disease, the major annotated cellular compartments were similarly represented between Sx and Ct limbs. KOA-associated remodeling was reflected primarily by within-cluster transcriptional changes, with the most informative differences observed in fibroadipogenic, pericyte/smooth muscle, and satellite/myogenic domains. Within Sx, MuIS stratification identified a coordinated transcriptional program characterized by denervation- and regeneration-associated genes and altered contractile and metabolic features. Neighborhood analysis localized denervation-associated signals primarily to fast-myofiber-rich regions, while local adjacency patterns among the examined myofiber, fibroadipogenic, and pericyte/smooth muscle domains were broadly preserved. Our findings provide the first spatial transcriptomic analysis of periarticular skeletal muscle in end-stage KOA and identify MuIS as a distinct local transcriptional phenotype in diseased muscle.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.16.752138v1?rss=1

Boeno, F. P., Graham, Z. A., Su, Q., Stec, M. J., Ferrando, A. A., Lim, W. K., Bamman, M.

Ecdysozoans is one of the most abundant and diverse groups of metazoans and grow through moulting the rigid exoskeleton that provided protection. But the moulting process is often dangerous, resulting in malformations or death. Injuries have been well documented in the calcitic exoskeleton of trilobites, some of which have been attributed to moulting complications when long spines are deformed. Injuries in trilobites are often found in the trunk rather than the cephalon, and abnormal genal spines are rarely documented. Here we examine injuries in eleven specimens of Olenoides serratus from the Burgess Shale (Cambrian, Wuluian) and demonstrate an increased frequency of genal spine malformations compared to other trilobites. The unusual pattern of malformations may be attributed to moulting injuries of the long and delicate genal spines in O. serratus. To moult, trilobites curved ventrally to press the anterior margin of their cephalon into the substrate and open the facial sutures. This process exerted force on the librigenae. To exit the exuvia, the genal spines were forced to bend as the old librigenae dipped ventrally along the anterior edge, resulting in the injured genal spines in O. serratus. A similar process of moulting resulting in injuries may be seen in other early Euarthropods.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.18.752702v1?rss=1

Losso, S. R., Bicknell, R. D. C.

Predicting ligand efficacy is a critical challenge in drug discovery, as a target's functional response is often determined by the way a ligand shifts conformational equilibria between different functional states, a process that is particularly intricate in ion channels. We classify ligands based on the difference of their binding free energies on putative active and inactive conformations, calculated via free energy perturbation (FEP) for 78 protein-ligand pairs across six ion channels from four structural superfamilies: GluA2, GABAAR {rho}1, 3{beta}4 nAChR, 5-HT3AR, TRPML1, and KCNQ2. This approach accurately distinguishes agonists from antagonists across all these ion-channel families with large or subtle structural differences, including at membrane-facing sites, and enables quantitative prediction of maximum response and partial agonism. Importantly, we find that local binding-pocket conformations encode the bound ligand's efficacy even when global channel states are ambiguous. Our results demonstrate that state-specific binding thermodynamics provides a robust framework for leveraging ion channel structures of diverse conformational states to elucidate mechanisms of action and to advance ion-channel drug discovery beyond simple affinity measurements, enabling the identification of new chemical matter with desired functional attributes.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.22.753602v1?rss=1

Vögele, M., Leffler, A. E., Felt, K. C., Denluck, L., Miller, E. B., Chakrapani, S., Wang, L.

Biofilm formation--the establishment of cellular communities within a self-secreted polymer matrix--is a behavior exhibited by almost all species of bacteria. Bacterial biofilms are well described as viscoelastic materials, with properties that grant physical advantages, such as elastic resilience and viscous adaptability. Additionally, many biofilm-forming species produce a subpopulation of highly resilient spores. In the model biofilm- and spore-forming species Bacillus subtilis, sporulation and matrix production are regulated by a common gene pathway, provoking the question of how sporulation influences biofilm viscoelastic properties. Investigating this point will facilitate the management of both salutary and deleterious biofilms, especially through manipulating their establishment and dispersion. Here, we investigate the interplay of sporulation and viscoelastic properties using rheological measurements of B. subtilis biofilms with varied matrix and spore production. We find that, to a significantly greater extent than matrix production, biofilm physical development is strongly altered by activation of sporulation through the spo0A regulation pathway. Spore-deficient strains initially establish physically robust biofilms with linear viscoelastic properties and recovery behavior distinct from spore-producing biofilms. However, spore-deficient biofilms fail to maintain their physical properties over time. In contrast, spore-producing wild-type and matrix-mutant biofilms exhibit more stable physical properties. Our results demonstrate that cell-level gene regulation is associated with macroscopic mechanical transitions in cellular communities, with sporulation granting not only a cellular-level survival advantage, but also greater community-level physical stability.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.23.753867v1?rss=1

Ducharme, N. A., Larkin, J.

Biomolecular condensates are condensed assemblies of biomolecules that form through the process of liquid phase separation. Condensates in biology typically function as membraneless organelles, providing compartmentalization in the absence of a dividing lipid membrane. The molecular make-up of different condensates includes diverse multivalent proteins and nucleic acids as the primary drivers, many of them including significant fractions of intrinsically disordered regions (IDRs). To date, the sequence to phase separation relationship of IDRs has focused largely on one protein at a time, studying single-component condensate formation, or single-component partitioning into condensates. The co-phase separation and mixing of two or more IDRs is considerably more complex as both sequences can vary widely in their self- and cross-interactions, as well as their relative abundance in solution. It is unclear that a rule which predicts how a single sequence behaves will also predict what happens when two sequences are mixed. In this study, we disentangle the influence of sequence from that of composition using a set of 18 LAF-1 RGG variants that keep the same length and amino-acid composition and change only the order of the residues. This lets us vary charge patterning and, to a lesser extent, hydropathy patterning while keeping protein composition fixed. By themselves, the sequences phase separation and single-chain compaction are controlled by their degree of charge and hydropathy patterning. Within single-component condensed phases, each sequence adopts a more extended conformational ensemble, due to a more favorable, selfsolvated environment. We find that mixing two IDRs together into a condensate causes this universal scaling behavior to break, impacted by the relative interactions of the two components and overall composition of the slab. We find two different qualitative behaviors, one characterized by cooperative co-condensation when both sequences are subcritical, and the other by scaffold-client behavior when one sequence is supercritical. The scaffold-client systems generally show a high degree of demixing, while the co-condensing systems are generally quite well-mixed in the dense phase. This is surprising because even in cases where both partners have significantly different patterning parameters, they still mix. Thus, the descriptor that predicts a sequence's behavior alone can help indicate whether it will mix with or separate from a second component, but it does not fully determine the outcome on its own.

Date: 2026-09-24
https://www.biorxiv.org/content/10.64898/2026.09.23.753891v1?rss=1

Singh, A., Dignon, G. L.