bioRxiv Subject Collection: All

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A multi-tiered microDicer with hierarchical blades achieves protein-preserving microdissection down to 10 microns

To study tissue heterogeneity at the sub-millimeter scale, laser capture microdissection (LCM) has been the leading technology for isolating regions of interest (ROI) for downstream molecular profiling. As the ROI approaches cellular dimensions (~10 microns), laser-induced photothermal damage and challenges in capturing microtissues in conventional LCM can compromise protein preservation and quantitative fidelity. This work introduces multi-tiered microDicers, fabricated by two-photon polymerization, to mechanically dissect tissue slices into uniform microtissues down to 10 microns. The hierarchical blade architecture limits instantaneous blade-tissue engagement and lowers the cutting force relative to single-tier designs. For benchmarking, proteomic analysis is performed on ethanol-fixed human squamous cell carcinoma microtissues generated by microDicers and by LCM. Under identical Nanodroplet Processing in One pot for Trace Samples (nanoPOTS) and liquid chromatography-mass spectrometry (LC-MS) conditions, microDicers yield more peptides and proteins than LCM, with the largest gains at 10-20 micron spatial resolution. Confocal imaging shows catapult-associated cavities in LCM-generated microtissues. This material loss, along with membrane-limited protein extraction, likely reduces protein coverage. In contrast, multi-tiered microDicers enables reproducible microdissection down to 10 microns while maintaining high protein coverage. With spatial registration of microtissues under development, microDicers have potential to complement LCM for next-generation spatial proteomic workflows.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.12.694048v1?rss=1

Arif, A., Kumar, R., Kwon, Y., Rodriguez, R., Cordts, S. C., Koppaka, S., Zhu, Y., Pasa-Tolic, L., Tang, S. K. Y.

Cell Barcoding Reveals Lineage-dependent Outcomes in hiPSC Cardiac Differentiation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have potential applications in treating cardiovascular disease but are currently limited in their clinical translation. A primary limitation is the poor clinical scalability of hiPSC-CMs, with the heterogeneity of hiPSC cardiac differentiation significantly contributing to this limitation. We hypothesize that clinical scalability can be improved by tracking and controlling hiPSC clonal heterogeneity, a variable often overlooked in current differentiation approaches. 'Fate priming', wherein clonal lineage identity determines differentiation fate, has been demonstrated in other stem cell differentiation pathways. We investigated fate priming in hiPSC cardiac differentiation using the ClonMapper cell barcoding platform to label, track, and isolate distinct hiPSC lineages from the same cell line. We show that certain hiPSC lineages preferentially differentiate into hiPSC-CMs or non-CMs. After isolating lineages with apparent fate priming, we found significant differences in cardiac differentiation outcomes between these single-clone populations and heterogeneous, multi-clone hiPSC populations. These findings indicate that lineage identity influences hiPSC cardiac differentiation outcomes.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.12.694049v1?rss=1

Sohn, S., Morgan, D., Callahan, C., Dockery, K., Brock, A., Zoldan, J.

Tissue-specific plasticity of DNA methylation across intertidal microhabitats in juvenile mussels (Mytilus californianus)

Epigenetic modifications to DNA are proposed to underpin plastic responses to environmental change, and the manner in which DNA methylation contributes to plasticity likely differs among tissues. However, few studies have investigated tissue-specific DNA methylation responses to ecologically relevant environmental stressors in natural settings. Here, we used reduced representation bisulfite sequencing in foot and gill to examine the influence of in situ microhabitats on DNA methylation in juvenile California mussels (Mytilus californianus), a foundation species with widespread dispersal and little evidence of genetic population structure. We examined mussels from a one-month reciprocal transplant experiment between a cool, wave-exposed and a warm, wave-protected microhabitat. These manipulations, which were previously shown to alter juvenile mussels' heat tolerance, led to significant and highly tissue-specific changes in CpG methylation, including within a number of genes with roles in stress response pathways. Differentially methylated genes were involved in processes including heat shock response, proteolysis, DNA repair, and temperature sensing. In gill, differentially methylated CpGs were more likely to occur in introns relative to other inter- and intragenic features. This study expands on previous research that examined environmentally driven shifts in DNA methylation by documenting plastic and tissue-specific changes in DNA methylation between microhabitats in a natural setting.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.12.693252v1?rss=1

Cai, Q., Bogan, S. N., Tanner, R., Kelley, J. L., Dowd, W. W.

Potential cost and combinatorial effects of commensal gut microbes on survival of individual honey bees

The western honey bee, Apis mellifera, is attracting attention as an experimental model to investigate general interactions between hosts and gut microbiota because of their noteworthy simple and stable bacterial community. Here, we systematically explore the effect of five core bacteria, Snodgrassella alvi, Bifidobacterium asteroides, BombilactobacillusFirm-4, Lactobacillus Firm-5, and Gilliamella apicola, on the longevity of individual honey bees. Generating gnotobiotic bees harboring all 32 different combinations of the core bacteria, we found that bacterial colonization generally reduces the host lifespan. This indicates that despite commensal gut microbes coevolved with the host, their presence is in principle costly for survival of individual bees. Regression analysis including higher-order interactions reveals that bacterial interactions have significant impacts on the host lifespan and show a qualitative trend: 2- and 4-way interactions exhibit positive, while 3- and 5-way interactions are negative. Numerical simulations based on a general Lotka-Volterra model demonstrate that the observed trend is reproduced when competitive interactions dominate in the bacterial community. This study combining empirical and theoretical approaches suggests the general costs of harboring gut microbiota and the stabilization of the bacterial community by higher-order interactions, which will advance our understanding of how commensal gut microbes and their interactions specifically influence host physiology.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.12.694077v1?rss=1

Sato, M., Miyazaki, R.

A 0.6-meter resolution canopy height and structure model for the contiguous United States

Above-ground vertical structure is a critical variable for ecosystem monitoring, carbon accounting, and land management. However, the high cost and limited coverage of airborne lidar hinder its widespread application. To address this, we developed NAIP-CHM, a 0.6-meter resolution canopy height and structure model (CHM) covering the contiguous United States, derived from National Agriculture Imagery Program (NAIP) aerial imagery. Unlike forestry-specific models that exclude human-made features, NAIP-CHM characterizes the full vertical structure of the landscape including vegetation, buildings, and infrastructure. We utilized a U-Net convolutional neural network with attention mechanisms and environmental conditioning, training the model on 18 million lidar-derived training pairs with stratified sampling to ensure robustness in open-canopy ecosystems. Assessing performance against 2.3 million independent samples, the model achieved a root mean square error of 1.57 meters and coefficient of determination (r2) of 0.87. We release the dataset, source code, and cloud-based tools to enable broad application without requiring specialized computational resources.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.12.694075v1?rss=1

Morford, S. L., Allred, B. W., Coons, S. P., Marcozzi, A. A., McCord, S. E., Smith, J. T., Naugle, D. E.

DfE-DB: A systematic database of 3.8 million human decisions across multiple experience-based tasks

Learning from experience is central to human decision making, yet research on experience-based choice remains fragmented across paradigms and disciplines. We present the Decision-from-Experience Database (DfE-DB), a standardized, openly accessible resource comprising 3.8 million trial-level decisions from 11,921 participants across 168 studies and 13 paradigms. By harmonizing raw behavioral data and classifying studies along 13 key design features, the database enables quantitative comparisons previously obscured by heterogeneous task and data structures. Using this resource, we show that choice tendencies--toward higher risk, expected value, or experienced mean--vary substantially across paradigms and are strongly shaped by core design features such as feedback type, outcome structure, stationarity, and sampling. These features explain substantial cross-study variability and reveal underexplored paradigm variants. DfE-DB provides the empirical infrastructure necessary to test the generality of behavioral phenomena and computational models, fostering a more integrated science of decisions from experience.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.12.693971v1?rss=1

Yang, Y., Spektor, M., Thoma, A. I., Hertwig, R., Wulff, D. U.

Projection-specific Routing of Odor Information in the Olfactory Cortex

Sensory processing in the mammalian cortex relies on extensive feedforward and feedback connections, yet how information is routed along these pathways remains poorly understood. Here, we examined the functional properties of feedback and feedforward neurons in the mouse olfactory (piriform) cortex. We selectively labeled neurons projecting to the olfactory bulb (OB, feedback) or medial prefrontal cortex (mPFC, feedforward) and recorded their activity during passive odor exposure and learning of an odor discrimination task. We found that odor identity and reward associations were encoded by OB-projecting ensembles early during odor exposure, whereas mPFC-projecting neurons encoded this information later, aligned with behavioral responses. Moreover, mPFC-projecting neurons maintained a stable representation of valence across days, while OB-projecting neurons exhibited pronounced plasticity. Together, these findings reveal that odor information is selectively routed through feedforward and feedback pathways and suggest that the functional properties of piriform neurons mirror the computational demands of their downstream targets.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.12.694045v1?rss=1

Daste, S., Pham, T. H., Seppo, M., Andre, A., Srinivasan, S., Xiao, J., Sattin, A., Nardin, C., Fellin, T., Franks, K., Dyer, E., Fleischmann, A.

An Interactive Brain Atlas of Knowledge

Biomedical knowledge about the brain increases every day, with a rapidly growing number of scientific publications, datasets, and software tools. While this informational plethora is not merely comprehensible by human beings, recent developments in information science and computational linguistics aim to make this knowledge programmatically accessible by literature mining. However, integrating these semantic methods into neuroimaging standards remains insufficient, hindering researchers from unraveling their full potential. Therefore, we developed the semantic meta-analysis platform The Virtual Brain adapter of semantics (TVBase) that projects biomedical knowledge preserved in over 36 million scientific articles onto a 3D standardized brain. The literature-mining platform SCAIView was used to extract ontologically defined biomedical entities and their associations with brain anatomy from the PubMed database. By querying a specific concept, the association strength with each anatomical term was calculated using entropy. To project the data onto a standardized brain, we created a unique transformation matrix that links over 800 anatomical terms to voxel coordinates of a parcellated standard brain. This novel method of knowledge projection extracts region-specific information about biomedical concepts from the literature to support translational multi-scale approaches to computational neuroscience. The multi-purpose software framework TVBase is openly available as a Python library. It aims for hypothesis-free neuroimaging pattern interpretation, hypothesis generation, and applications in personalized medicine.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.12.694043v1?rss=1

Stefanovski, L., Bülau, K., Martin, L., Langford, C., Palmer, J., Sacks, M., Deger, L., Pille, M., Schirner, M., Meier, J., Neudorfer, C., Horn, A., Solodkin, A., Thirion, B., Hofmann-Apitius, M., Jacobs, M., Tom Kodamullil, A., for the Alzheimer's Disease Neuroimaging Initiative,, Ritter, P.

People report having consistent idiosyncratic diets of imagined sensations when they re-experience the past, and pre-experience the future

To some extent, humans can re-experience the sensations of past events and pre-experience the future. These capacities are inter-related. But there are substantial individual differences. At the extremes, small minorities of people report that they either cannot have imagined experiences at all, or that their imagined sensations are as real to them as their actual experiences of the physical world. We wanted to know if such individual differences are uniform across different types of imagined experience (e.g. vision, audio, taste and smell), or if people generally have idiosyncratic patterns of different types (vision, audio, taste and smell) of imagined experiences. We find that people report having idiosyncratic diets of different types of imagined sensation, characterised by differences in salience. One person might have more salient imagined visual than taste experiences, while another reports the reverse. Moreover, these propensities are consistent across peoples attempts to re-experience the past, and to pre-experience the future, and they predict peoples experience and usage of different types of imagined sensation in their everyday lives.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.12.694050v1?rss=1

Arnold, D. H., Bouyer, L. N., Saurels, B. W., Schwarzkopf, D. S.

Comparative functional profiling of plant ACR3 orthologs reveals metalloid-sensitive trafficking and arsenic efflux capabilities

Arsenic contamination is a pervasive environmental and public health challenge. In plants from microalgae to gymnosperms, members of the ACR3 transporter family mediate arsenic tolerance, yet their evolution, diversity, and regulatory mechanisms remain largely unexplored. Here, ACR3 genes from phylogenetically distant Coccomyxa subellipsoidea (CsACR3), Raphidocelis subcapitata (RsACR3), Chlamydomonas eustigma (CeACR3), Physcomitrium patens (PpACR3), and Picea sitchensis (PsACR3) were cloned, heterologously expressed in budding yeast, and functionally characterized alongside previously reported ACR3 genes from Marchantia polymorpha (MpACR3) and Pteris vittata (PvACR3). Our in silico analysis confirms that the putative proteins share structural and sequence homology with characterized ACR3 transporters, with many family members harboring elongated N-terminal tails unique to plant orthologs. We confirm that the analyzed genes encode functional arsenic efflux transporters conferring distinct levels of arsenite and arsenate resistance in yeast cells. We show that, in yeast cells, CsACR3, RsACR3, and PvACR3 constitutively localize to the plasma membrane, whereas the subcellular localization of CeACR3, MpACR3, PpACR3, and PsACR3 is regulated by metalloid exposure, triggering their endoplasmic reticulum-to-plasma membrane trafficking. Using MpACR3 as a model, we prove that this transporter requires its N-terminal domain for the intracellular retention, which is dynamically alleviated specifically in response to metalloids.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.14.694196v1?rss=1

Zbieralski, K., Mizio, K., Staszewski, J., Janiczek, A., Tomaszewska, P., Wysocki, R., Maciaszczyk-Dziubinska, E., Wawrzycka, D.

The phytolongin AtPhyl2.1 is involved in cell plate formation and root development

SNAREs are critical elements of the membrane trafficking machinery with a wide variety of functionality across this family of proteins. Phytolongins are a recently identified subfamily of longins which possess the typical longin domain but lack a SNARE motif. Phytolongins have an ubiquitous tissue expression in Arabidopsis and are distributed throughout the secretory pathway. We focused on Phytolongin 2.1 (PHYL2.1) which localizes to the endoplasmic reticulum, and observed a strong root growth phenotype in the loss-of-function Atphyl2.1-1 mutant. We demonstrate that whilst cell elongation efficiency was not affected in the mutant, cell division was significantly reduced. The resulting decrease in root length in the Atphyl2.1-1 mutant is explained by a smaller number of cells which then elongate to enable root growth. Root apical meristem architecture of Atphyl2.1-1 and another mutant Atphyl2.1-2 was disturbed and distances from the root quiescent center to the transition zone and the first areas of mis-organized cells were affected in both mutants. Investigation of the SNARE AtKNOLLE revealed significant perturbation of Atphyl2.1-1 cell plate formation in the mis-organised areas. Our results provide a first characterization of the phytolongin AtPHYL2.1 which appears involved in root cell plate formation, root cell division and therefore root development.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.15.694276v1?rss=1

Moreau, P., Wattelet-Boyer, V., Buridan, M., Negroni, Y. L., Mafficini, C., Dittrich-Domergue, F., Maneta-Peyret, L., Breeze, E., Zottini, M., Formentin, E., Filippini, F., Brocard, L.

Leaf variegation in a barley EMS mutant is controlled by two epistatic mutations

Variegation mutants provide valuable insights into chloroplast biogenesis. We characterized a newly identified variegated barley mutant, in which the phenotype is controlled by duplicate dominant epistasis-representing, to our knowledge, the first reported case of digenic control in chloroplast-deficient mutants. The causal loci, Var4 and Var5, were mapped on chromosomes 2H and 3H. We used whole-genome resequencing to identify candidate genes. Our two top candidate genes are an NBR1-like selective autophagy receptor gene and a DNAJ-domain containing gene, respectively. Based on their homology-based functional annotation, both candidates could be implicated in chloroplast proteostasis, regulating protein import, folding, and/or degradation. We propose that mild, independent defects in proteostasis from each mutation act synergistically to surpass a functional threshold, impairing chloroplast development in early leaves while allowing partial recovery in later stages. These findings highlight a novel digenic mechanism underlying variegation and point to proteostasis as a central vulnerability in chloroplast biogenesis.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.15.694392v1?rss=1

Pidon, H., Nagireddy, S. R., Melzer, M., Himmelbach, A., Stein, N.

Network analysis of flowering time genes suggests regulatory changes among SOC1 orthologues in response to cold in Brassica napus

Flowering plants respond to multiple environmental and endogenous cues to determine the timing of their transition from the vegetative to floral state. Most of our knowledge of the gene regulatory network (GRN) controlling flowering time has been derived from the model plant Arabidopsis thaliana. This knowledge needs to be translated to crop plants to support the development of varieties that can be grown in different and rapidly changing climatic conditions. However, due to increased genome complexity and limited prior knowledge, translation into crops is not always straightforward. Here, we present a study of the GRN controlling flowering time in Brassica napus (oilseed rape), an allotetraploid crop that is a close relative of Arabidopsis. Using a comparative transcriptomics approach, we show that the majority of the orthologous gene pairs have similar expression dynamics over development between Arabidopsis and Brassica napus. Some genes, however, have experienced regulatory changes, with flowering time genes in Brassica napus having higher than average differences in their expression profiles from their Arabidopsis orthologues. Despite these differences, the inferred GRN for flowering in Brassica napus exhibits a similar network topology to the network known in Arabidopsis. This is likely due to preferential retention of these genes in higher paralogue numbers, which allows subtle changes in the regulation of individual paralogues, while still conserving the overall regulatory structure through evolution. We discover and present a detailed analysis of one such example where orthologues of SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) have similar expression patterns under normal conditions, but different dynamics under cold temperature conditions, suggesting possible subfunctionalisation among paralogues in response to temperature change.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.16.694548v1?rss=1

Sidhu, G. S., Burrows, S., Woolfenden, H., Wells, R., Morris, R. J.

Both conventionally and organically fertilized tomatoes maintain fruit quality through uncontrolled green peach aphid infestation, with a transcriptional shift towards catabolism

Aphids are a major pest of greenhouse-grown temperate crops, responsible for billions in crop damage yearly. As organic agriculture rapidly grows in popularity, understanding how plants grown under organic systems respond to insect pest pressure may give insights into better management practices and information about the genes of interest for crop improvement. We measured the response of tomato (Solanum lycopersicum) leaf and fruit transcriptome, as well as a few selected metabolites in the mature fruit, to an infestation of the generalist green peach aphid (Myzus persicae). The aphids were introduced approximately halfway through the lifecycle of the plants that were grown under conventional and organic fertilizer regimes. While plants provided with conventional fertilizer experienced greater aphid infestation, neither group suffered a significant loss in total yield or fruit quality. This result is likely a consequence of ample nutrient and water availability. Co-expression network analysis using WGCNA revealed that in leaf tissue, both treatment groups showed a general shift from diverse anabolic processes to catabolism, while fruit tissue experienced relatively minor changes. At the stage of infestation investigated, abscisic acid appeared to be the main phytohormone response. One coexpression network module showed a correlation with both organic fertilizer treatment and aphid infestation; its hub gene (Solyc02g078940.3) may be of interest in exploring unique responses to phloem feeding insect infestation under an organic production regime.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.14.694237v1?rss=1

Labbancz, J., Gustafson, L., Andrews, P., Dhingra, A.

Natural variation in an NLR pair confers thermostable resistance to a devastating bacterial pathogen

Climate change reshapes host-pathogen interactions by increasing pathogen aggressiveness and weakening plant immune responses, particularly under heat stress. Nucleotide-binding leucine-rich repeat (NLR) immune receptors, key players in pathogen recognition, are altered under elevated temperatures in both plants and mammals, posing a major challenge to disease resistance. In Arabidopsis thaliana, the well-described RPS4/RRS1 NLR pair confers resistance to the worldwide devastating phytopathogenic Ralstonia pseudosolanacearum. Here, by combining the exploration of natural genetic variation with genetic mapping, polymorphism analysis, structural modeling, and functional complementation, we demonstrated that the paucity of full thermostable resistance is primarily associated with a unique RPS4/RRS1 haplotype carrying key substitutions in leucine-rich repeat domains. These findings reveal natural genetic diversity as a source of thermostable resistance, highlighting promising opportunities to engineer climate-resilient plants.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.15.694456v1?rss=1

Aoun, N., Singh, M., Chacko, J., Maruta, N., Griffiths, C., Dangla, E., Demont, H., Carrere, S., Gouzy, J., Duflos, R., Zaffuto, M., Marchetti, M., Hancock, A., Bernoux, M., Kobe, B., Deslandes, L., Roux, F., Berthome, R.

Genomic structure, introgression, niche overlap, and morphological variation challenges species delimitation in Thymus sect. Mastichina

Species delimitation remains a major challenge in understanding biodiversity, particularly in recently diverged lineages, where extensive morphological variability complicates taxonomic inference. In this study, we investigate patterns of morphological and ecological variation within Thymus sect. Mastichina, a Mediterranean lineage that includes the endangered species T. albicans. Previous genomic analyses revealed clear differentiation between two major groups corresponding to ploidy levels, as well as additional genetic structure among diploid lineages. Here, we integrate this genomic framework with detailed analyses of floral morphology and ecological niches to assess the correspondence between phenotypic, ecological, and genetic variation. Our results reveal substantial overlap among genetic lineages at both morphological and ecological levels. Although some differentiation is associated with ploidy level, particularly in floral traits, extensive variability within several lineages obscures clear taxonomic boundaries based on discrete morphological characters. By contrast, some diploid lineages (notably the Cadiz and Algarve groups) show lower morphological variability but still both overlap in morphology and ecological niche. We morphologically characterized a recently discovered diploid lineage (Hercynian) that appears to be cryptic with the tetraploid T. mastichina. Patterns of admixture involving this Hercynian lineage, especially with the diploid Donana group, contribute to pronounced phenotypic overlap, whereas no significant effect of tetraploid introgression on diploid morphology was detected. Ecological niche analyses indicate that niche differentiation is primarily associated with ploidy level rather than among diploid lineages, except for the Hercynian group. Overall, our results demonstrate a marked discordance between phenotypic variation and genetic structure, supporting the interpretation of Thymus sect. Mastichina as a complex of cryptic evolutionary lineages. These findings highlight the limitations of morphology-based taxonomy and emphasize the need for an integrative framework incorporating genome size, genomic information, and geographic distribution for species delimitation.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.16.694552v1?rss=1

Nieto-Lugilde, D., del Valle Garcia, J. C., Martin-Carretie, E. M., Doblas, D., Williams-Marland, B., Ortiz Herrera, M. A., Jimenez-Lopez, F. J., Lopez-Tirado, J., Garcia-Cardenas, F. J., Berjano, R.

A viral infection reshapes Arabidopsis water management via root hydraulics, aquaporin downregulation and osmotic adjustment

The effect of plant viruses on root water relations and on how roots and shoots coordinate under infection remains poorly understood. Using a hydroponic Arabidopsis thaliana-Turnip mosaic virus (TuMV) pathosystem, we integrated biometric, anatomical, hydraulic, and gas-exchange measurements to dissect how viral infection reshapes root-shoot water relations. TuMV impaired root development, as reflected by an early plateau of primary root elongation. At the functional level, infected plants exhibited a decrease in root hydraulic conductance per unit root mass, concomitant with transcriptional downregulation of root aquaporin genes. Despite this, the relative contribution of aquaporin-mediated water transport, assessed via sodium azide inhibition, remained unchanged, indicating that the virus downregulates total hydraulic capacity without altering the apoplastic-symplastic partitioning of water flow. Gas-exchange analysis revealed a virus-induced decoupling between stomatal conductance and net CO2 assimilation, resulting in a non-adaptive increase in intrinsic water-use efficiency. This loss of photosynthetic plasticity, combined with shoot-localized osmotic adjustment (more negative leaf osmotic potential and higher relative water content), points to a constrained, suboptimal physiological state. Multivariate analysis confirmed that variation in physiological traits largely drives phenotypic divergence between treatments. Together, these coordinated alterations, reduced root hydraulics, rigid gas-exchange relationships and passive hydraulic matching to a stunted shoot, depict plants locked into a low-performance equilibrium, poorly equipped to compete for water and carbon. This work reveals a systemic hydraulic-photosynthetic reconfiguration that could account for compromises in plant resilience and resource competitiveness.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.15.694488v1?rss=1

Manacorda, C. A., Caceres, P. D., Sutka, M. R., Amodeo, G., Asurmendi, S., Baroli, I.

Contextualizing Pan-Tropical Allometric Models for Biomass Estimation

Allometric Models (AMs) play a central role in monitoring and mitigating climate change as they provide accurate estimation of biomass and carbon sequestered by trees from non-destructive, easy to obtain physical measurements. Unfortunately, practitioners spend considerable effort in researching, qualifying and choosing AMs for specific growth conditions. To overcome this situation Chave et al. (2014) developed a pan-tropical AM with equivalent accuracy to local, site-specific AMs. We ameliorate this result by incorporating contextual information pertaining to growth conditions in a Machine Learning (ML) model, eventually achieving a reduction in Mean Average Error (MAE) of -17% as measured on hold-out data. This breakthrough shall have important impact in applications such as national forest inventories, carbon certifications and calibration of satellite based biomass maps to field data. To complete, we propose a principled method to estimate how much additional error one can expect when applying a given AM to shifting conditions and provide a data-driven safety check to practitioners.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.16.694295v1?rss=1

Diemert, E., Dambreville, A.

Large-scale eQTL analyses in Atlantic salmon reveal persistent dosage compensation 100 million years after genome duplication.

Whole-genome duplication (WGD) through autopolyploidization has played a role in genome evolution across eukaryotes. A major consequence of WGD is the rewiring of gene regulatory networks, partly driven by selection on dosage balance. In multicellular organisms, evidence for dosage balance selection has relied on comparative patterns of duplicate gene retention and expression, with few studies directly examining regulatory architecture after WGD. Here, we analysed a large-scale eQTL dataset from Atlantic salmon (Salmo salar), which experienced a WGD 100 million years ago. We found that trans-regulatory connections were enriched between duplicated regions, indicating long-term conservation of ancestral interchromosomal regulatory interactions. Overall, 230 duplicated genes (5%) shared eQTLs, suggesting conserved regulatory control. Moreover, 16 gene pairs showed compensatory expression effects mediated by a common regulator, consistent with predictions of the dosage balance hypothesis. These gene pairs were significantly enriched in recently rediploidized regions. Our results indicate long-term maintenance of dosage balance after WGD.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.15.694296v1?rss=1

Diblasi, C., Manousi, D., Hazlerigg, D. G., gronvold, L., Barson, N. J., Sandve, S. R., Saitou, M.

HDAC3 inhibition harnesses learning-induced neurobiological mechanisms to enhance signal-in-noise responsivity in auditory cortex and behavior

Auditory learning enables sound-guided behavior and sound-specific enhancements in auditory cortical (AC) processing. Background noise can also alter sound-specific auditory responsivity. Yet, the potential enhancing effects of learning on AC processing in noise is unknown. Pharmacological inhibition of histone deacetylase 3 (HDAC3) by RGFP966 has been shown to improve AC coding when administered during various auditory learning paradigms. Here, adult rats (Sprague-Dawley males) were trained in ideal quiet conditions to learn a tone-reward associative task while treated with RGFP966 (TRAINED+RGFP966, n = 6) to determine if learning-related effects on AC could support tone-signal detection in a later background noise challenge. RGFP966 accelerated sound-reward learning relative to a trained but untreated group of rats (TRAINED, n = 5), though all animals ultimately reached equivalent high levels of performance prior to testing. Training produced a sound-specific enhancement in AC responses evoked by the signal tone, and a sound-general effect that suppressed responses during steady-state noise, relative to untrained rats (NAIVE, n = 7). Effects were strongest when training was with RGFP966 administration and scaled with increasing signal-to-noise (SNR). High levels of background noise abolished sound-specific enhancements in tone-signal evoked AC activity, yet the general suppressive effect to noise was maintained. Remarkably, behavioral responses to the signal tone in different SNRs recapitulated AC patterns of tone-evoked responding. Overall, rapid auditory learning facilitated by RGFP966 yields AC plasticity that can improve signal detection. Learning-induced mechanisms appear to shape cortical decoding mechanisms that selectively enhance representations of signal sounds and suppress the effects of background noise.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.15.694497v1?rss=1

Atesyakar, N., Bieszczad, K.

Perturb-seq identifies TCF7 as a central nexus linking MAPK- and Wnt-driven gene expression

The MAPK pathway is a central signaling cascade whose dysregulation contributes to numerous diseases. While its upstream regulation is well studied, the mechanisms by which MAPK activation leads to diverse transcriptional outcomes remain incompletely understood. To address this shortcoming, we mapped the target gene sets controlled by 22 RAF-inducible transcription factors using targeted Perturb-seq and integrated these data with time-resolved transcriptional profiling. Network reconstruction revealed a topology dominated by two central hubs, EGR1 and FOS, which co-regulate partially overlapping target gene sets. In addition, we uncovered a positive feedback loop between EGR1, a canonical RAF-MAPK effector, and TCF7, a transcription factor typically linked to Wnt signaling. Through this interaction, TCF7 emerges as a nexus that integrates MAPK and Wnt pathway inputs. Together, these findings define the architecture of RAF-MAPK-driven transcriptional regulation and demonstrate how cross-talk between oncogenic signaling pathways can be encoded in transcriptional networks.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.15.694273v1?rss=1

El Kassem, G., Sieber, A., Klinger, B., Uhlitz, F., Steinbrecht, D., van Bentum, M., Hillmer, J., von Schlichting, J., Schaefer, R., Bluethgen, N., Boettcher, M.

Engineering plant tandem kinase immune receptors expands effector recognition profiles

Plant intracellular immune receptors are widely deployed in breeding to protect crops from disease. In addition to nucleotide-binding leucine-rich repeat receptors (NLRs), tandem kinase proteins (TKPs) have recently emerged as an important family of immune receptors within staple cereal food crops, but how TKPs recognize effectors and whether they are amenable to engineering is essentially unknown. Here, we show that the barley and wheat TKPs Rmo2 and Rwt7 recognize different blast fungus effectors via their integrated HMA domains using different protein interfaces with nanomolar binding affinity. Structural analysis pinpointed interface residues that dictate effector recognition and enabled engineering of dual-specificity TKPs. These results establish integrated HMA domains as programmable modules within TKPs for designing new specificities in plant immunity for diseases relevant to global agriculture

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.15.694194v1?rss=1

Yu, D. S., Zdrzalek, R., Katayama, E., Akiyama, H., Daykin, L., Williams, N. J., Goodridge, I., Asuke, S., Banfield, M. J.

Metabolic engineering of a tyrosine-specific phenylpropanoid pathway in plants

While all plants use L-phenylalanine for phenylpropanoid biosynthesis, grasses can also initiate the pathway from L-tyrosine. Curiously, no plant has evolved an exclusive tyrosine-derived route. We generate plants with phenylpropanoid biosynthesis initiated from phenylalanine, tyrosine, or both by expressing a Brachypodium phenylalanine/tyrosine ammonia-lyase (PTAL) in Arabidopsis WT and c4h mutants. Engineering a bifunctional phenylpropanoid pathway in WT plants did not negatively impact growth, while introducing a tyrosine-specific pathway in the c4h mutant could overcome the seedling-lethal phenotype. Interestingly, restored c4h mutants relying solely on the tyrosine route displayed developmental defects linked to the strong overaccumulation of the auxin transport inhibitor cis-cinnamic acid. Our findings suggest that the requirement of this widely overlooked plant metabolite could be the crucial factor for the evolutionary retention of the canonical phenylpropanoid biosynthesis route via L-phenylalanine in plants.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.16.694581v1?rss=1

Van Beirs, C., Bentelspacher, M., Xie, C., Van de Velde, C., Desmet, S., De Wulf, R., Boerjan, W., Barros-Rios, J., Vanholme, B.

Genomic Structural Variation Underlies Cell-Type-Specific Betacyanin Variegation in Chenopodium quinoa

The allotetraploid crop quinoa (Chenopodium quinoa) accumulates red/violet betacyanins, which function as vital stress-mitigating antioxidants. We investigated the genetic basis of red/green variegation observed in the aerial organs of the P0429 accession. We demonstrated that this color mosaic is primarily localized to the epidermal bladder cells (EBCs), with red EBCs accumulating ~50-fold higher betacyanin levels than colorless EBCs. Cell-type-specific RNA-sequencing of EBCs identified the cytochrome P450 gene Cqu0091301 (CYP76AD) as the dominant and rate-limiting factor, exhibiting strong upregulation in red EBCs. This high pigmentation requires a specific structural variation in the P0429 accession: a ~4-kb genomic insertion that restores the full functionality of Cqu0091301, which is otherwise truncated and non-functional in common reference genomes. Genomic analysis reveals that Cqu0091301 is part of a CYP76AD-DODA gene cluster. Notably, expression analysis revealed functional divergence between the quinoa subgenomes, with B-subgenome CYP76AD genes highly dominant in EBCs, while A-subgenome homologs were preferentially expressed in other tissues. Our results establish a clear link between structural genomic variation and cell-type-specific betalain biosynthesis, providing molecular insight into pigment regulation and subgenome specialization in allotetraploid quinoa.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.13.694152v1?rss=1

Zhang, Z., Wang, Y., Hu, X., Yu, T., Feng, Y., Zhang, J., Zhang, T., Feng, G., Zhang, H.

Phenotypic Screening Coupled with AI-Driven Target Deconvolution Identifies α-Terthienyl as a Dual DPP-IV/HSD17β13 Modulator with Efficacy in a Mouse Model of MASLD

Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a highly prevalent condition characterized by fat build-up in the liver and ranges from benign steatosis to progression to metabolic dysfunction-associated steatohepatitis (MASH), fibrosis, cirrhosis and end-stage liver disease including hepatocellular carcinoma, representing a significant cause of chronic liver disease globally.1,2 Current treatment options are limited, primarily relying on lifestyle modifications, highlighting an urgent need for novel therapeutic strategies. Methods: A Cell Painting-style high-content screening phenotypic assay was employed using the PH5CH8 human hepatocyte cell line to identify small molecules capable of modulating induced hepatic steatosis. The Plex Research artificial intelligence (AI) platform was utilized for target deconvolution of the lead hit compound, -terthienyl. In vivo efficacy was assessed in a diet-induced obesity (DIO) C57BL/6J mouse model of MASLD. Biochemical assays and molecular docking simulations were performed to validate predicted target interactions. Results: Phenotypic screening identified 15 chemical probes/drugs that elicit dose-responsive inhibition of steatosis, and 16 that exacerbate steatosis, which could contribute to worsening of the disease clinically. -terthienyl, a plant-derived natural product, was identified as a potent and non-toxic inhibitor of steatosis in PH5CH8 cells with an EC50 of 106 nM. In vivo, -terthienyl administration to diet-induced obesity (60% fat diet) mice significantly reduced hepatic steatosis histologically, improved glucose tolerance, and favorably modulated serum biomarkers including ALT and AST. AI-driven analysis predicted dipeptidyl peptidase 4 (DPP-IV) and 17-beta hydroxysteroid dehydrogenase 13 (HSD17{beta}13) as potential molecular targets of -terthienyl. Biochemical inhibition of DPP-IV was observed and an oxidized -terthienyl analog inhibited HSD17{beta}13. Molecular docking supported these predictions, indicating binding to DPP-IV and HSD17{beta}13. Conclusion: This study demonstrates the successful application of phenotypic screening integrated with AI-driven target deconvolution to identify compounds and drugs that ameliorate or exacerbate hepatic steatosis. -terthienyl was identified as a novel modulator of hepatic steatosis with in vivo efficacy in a MASLD model. Our findings suggest a dual-target mechanism involving DPP-IV and HSD17{beta}13, potentially engaged by the parent compound and its metabolite, respectively, offering a promising polypharmacological approach for MASLD treatment.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.12.693988v1?rss=1

Hoegeman, K. M., Wotring, J. W., Fursmidt, R., Gaetz, J., Khalil, E. M., Selinger, D. W., Schultz, T. L., McCarty, S. M., O'Meara, M. J., Clasby, M. C., Sexton, J. Z.

Molecular profiling of glioblastoma-derived extracellular vesicles identifies small nucleolar RNAs as candidate liquid biomarkers for radiation-induced senescence

Radiation-induced senescence (RIS) in glioblastoma (GBM) is an undesirable cell fate that inhibits tumor cell death and supports resistance and outgrowth. While senescence-targeting drugs are promising adjuvants, their clinical application will require proper patient selection based on post-treatment RIS burden. Current methods to evaluate senescence, however, are tissue based, and given GBM's difficult anatomical location, post-treatment biopsies are impractical. Therefore, novel and less invasive biomarkers for TIS are urgently needed. To this end, we aimed to identify candidate extracellular vesicle (EV) liquid biomarkers for TIS by profiling senescence-associated cargo changes within GBM EVs. Using a panel of GBM patient-derived cell lines, we show that RIS is the primary functional state following radiation exposure and is associated with significant alterations in the cargo of senescent-derived EVs (senEVs). In particular, senEV transcriptomes have an increased abundance of senescence-associated RNA species and enrichment of senescence-associated gene sets. Most striking, however, was that senEVs are most differentiated by the significant enrichment of a panel of snoRNAs. This signature was conserved in 4/5 GBM models of RIS and was validated by qRT-PCR. Further analysis by mass spectrometry revealed that snoRNAs are likely co-packaged with their associating proteins, as senEVs had concurrent increases in these binding partners. Finally, in a preliminary patient cohort comparing plasma EVs obtained prior to surgery to those obtained after completion of their radiation therapy, we identified increased senescence-associated RNA such as CDKN2B and GLB1 and the snoRNA SNORA49 in post-radiation EVs. Altogether, this data suggests that senEV RNA species, and particularly snoRNAs, are a promising analyte for RIS-biomarker development. With further study, this work may open avenues for a companion diagnostic for senotherapeutics.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.12.693499v1?rss=1

De Luca, V., Hansen, N., Digumarti, P., Tang, N., Fink, K., Snipes, G., Pirrotte, P., Berens, M.

Comparative characterization of OncoPro and Wnt-Based media reveals distinct phenotypic and pharmacologic states in patient-derived tumor organoids

Background: Patient-derived tumor organoids (PDTOs) are strongly influenced by culture medium. We compared OncoPro (OP) Tumoroid Culture medium with conventional Wnt/R-spondin medium (Wnt). This recently developed OP medium offers a standardized, serum-free alternative to Wnt-based formulations. Methods: We performed a comparison of OP and Wnt media across 36 PDTO lines from various malignancies. PDTOs were assessed for establishment success, morphology, transcriptomic profiles (bulk RNAseq and fidelity to public single cell (sc) RNAseq databases) and pharmacologic response to a 60-drug panel. Results: Adaptation to OP succeeded in 83% (15/18), whereas de novo establishment favored Wnt (44.4% vs 22.2%). Transcriptomic profiling revealed retained tumor-identity but with different epithelial states: Wnt upregulated proliferation/stemness-associated genes (e.g. LGR5) and OP enriched adhesion-associated genes and inflammatory/TGF-b; programs. In scRNA databases OP signatures preferentially mapped to malignant epithelial compartments in pancreatic cancer, whereas Wnt signatures were linked to non-malignant epithelium. In colon cancer OP signature was correlated with the malignant iCMS3 subtype. Drug screening demonstrated consistent medium-dependent shifts: Wnt-grown organoids were globally more sensitive, particularly to MAPK-axis inhibitors and apoptosis-sensitizers, while OP-grown organoids exhibited relative resistance. Conclusions: Culture medium composition is a key determinant of PDTO phenotype, transcriptome and drug sensitivity. Wnt supports proliferation-associated genes and drug-sensitive states; OP induces adhesion-associated genes and inflammatory-related programs, with broader resistance and high fidelity of the transcriptomic signature in public scRNAseq databases of colon- and pancreatic cancer.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.13.693944v1?rss=1

Seghers, S., Le Compte, M., Rodrigues Fortes, F., Baroen, J., Roeyen, G., Hartman, V., Kumar-Singh, S., Coppens, L., de Maat, M., Komen, N., Van den Broeck, S., Valk, J., Tjalma, W., Hendriks, j. M. H., Van Schil, P., Van Haesendonck, G., Peeters, S., Prenen, H., Deben, C.

ProteoForge: An Imputation-Aware Framework for Differential Proteoform Discovery in Bottom-Up Proteomics

The human genome contains approximately 20,000 protein-coding genes. However, millions of diverse protein variants, called proteoforms, exist. Despite originating from the same gene, proteoforms often have distinct biological roles. In bottom-up proteomics, the aggregation of peptide measurements into protein-level quantities often obscures this information. Existing methods for proteoform deconvolution are limited by their handling of missing data, which can introduce significant bias. To address this we developed ProteoForge, which builds on an imputation-aware statistical model to identify and group co-varying peptides into quantitatively differential proteoforms (dPFs). Benchmarking against existing deconvolution methods demonstrated that ProteoForge provides high accuracy and stability in datasets with high rates of missing values, complex experimental designs, or varying signal strengths. Application of ProteoForge to proteomics data from lung cancer cells under hypoxia revealed extensive proteoform-level regulation hidden by standard protein-level analysis.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.12.694008v1?rss=1

Ergin, E. K., Conrrero, A., Ferguson, K. M., Lange, P. F.

Fine-Grained Structural Classification of Biosynthetic Gene Cluster-Encoded Products

Biosynthetic gene clusters (BGCs) are responsible the biosynthesis of many natural products, including a multitude of effective therapeutics and their precursors. Advances in genomic data collection as well as computational techniques have made it possible to identify BGCs at scale. However, accurately determining the types of BGC-encoded products from genomic content remains elusive. Here, we introduce BGCat (BGC annotation tool), a machine learning method for fine-grained structural classification of BGC-encoded products, leveraging the NPClassifier natural product nomenclature. Our method leverages a pre-trained protein language model for creating meaningful gene representations and a deep neural network for class label prediction. We show the method outperforms state-of-the-art approaches in coarse-grained product classification and is effective for detailed classification. We implement a clustering-based augmentation strategy for BGC-product relationships, addressing a crucial gap in the available datasets. We then introduce the concept of product class profiles (PCPs) of gene cluster families (GCFs), associating each GCF with a probabilisitc distribution of product types and offering a new perspective on GCF functions. Lastly, we use BGCat to provide new product class labels for over 100k BGCs in antiSMASH DB that presently have minimal information about their products.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.12.693975v1?rss=1

Porokhin, V., Mevers, E., van der Hooft, J. J. J., Hassoun, S.

A near-complete lamprey genome illuminates ancestral vertebrate innovations

The origin of vertebrates is characterized by a suite of phenotypic innovations, yet the underlying genetic mechanisms remain poorly understood. As a living representative of jawless vertebrates, the lamprey serves as a pivotal model for investigating the genomic basis of early vertebrate evolution. Here, we report a near telomere-to-telomere (T2T) genome assembly of the reissner lamprey (Lethenteron reissneri), generated from sperm DNA that is not subject to programmed genome rearrangement. The 1.25 Gb assembly resolves over 97% of chromosome sequences into single contigs, enabling reconstruction of previously inaccessible genomic regions. Centromeres display highly diversified, lamprey-specific repeat units and structural architectures, whereas telomeres contain both conserved and lineage-specific satellite repeats. Deep transcriptomic profiling across representative organs and developmental stages substantially improved genome annotation. Comparative genomic analyses revealed a major expansion of 1,562 gene families at the base of the vertebrate lineage, and they are significantly enriched for functions related to neural development, skeletal formation, and cardiac function, echoing the known phenotypic innovations in vertebrates. Notably, the CDH2 gene family underwent vertebrate-specific copy number expansion and acquired conserved cis-regulatory elements (CREs), highlighting its potential role in shaping the early vertebrate circulatory system. Histological analyses and functional knockout experiments demonstrated that one CDH2 paralog (CDH2_H) is essential for lamprey heart development and chamber formation, playing a critical role in the emergence of the closed circulatory system in early vertebrates. This high-quality lamprey genome provides a foundational resource for dissecting the genetic basis of key innovations that shaped vertebrate evolution.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.15.694513v1?rss=1

Du, Z., Wu, H., He, Y., Li, J., Li, J., Lin, L., Lu, J., Zhu, C., Liu, K., Pang, Y., Su, B.