bioRxiv Subject Collection: All

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Determining the driving factors shaping genetic architecture of complex traits in recently admixed populations

Understanding the genetic architecture of complex traits in admixed populations remains challenging due to heterogeneous genetic backgrounds and demographic histories. Mischaracterizing admixture can bias genetic association estimates and limit the generalizability of biomedical findings. Here, we systematically evaluate how evolutionary forces&--including admixture, natural selection, and demographic history--jointly shape complex trait architecture and influence genome-wide association study (GWAS) outcomes using a simulation-based framework complemented by empirical analyses. We model five human admixture scenarios and vary the correlation between causal variant effect sizes and selection coefficients to reflect different trait-fitness relationships. This framework enables simulation of complex trait phenotypes with environmental variance, allowing comprehensive assessment of GWAS power and fine-mapping precision across evolutionary contexts. We find that GWAS power is strongly modulated by both genetic architecture and demographic history. Traits with weak coupling between fitness and effect size, such as anthropometric traits, exhibit higher GWAS power than traits under stronger negative selection, including early-onset diseases. Because rare variants contribute substantially to heritability yet are poorly captured by GWAS, bottlenecked populations with fewer rare variants show enhanced power. Despite large differences in GWAS power, fine-mapping precision remains relatively consistent across traits and populations, improving primarily in regions of high recombination. Empirical analyses of diverse cohorts, the All of Us Research Program, support these patterns. Our findings highlight how evolutionary and demographic forces shape the genetic basis of complex traits in admixed populations and underscore the need for tailored study designs to improve GWAS accuracy and fine-mapping performance in diverse cohorts.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.15.694350v1?rss=1

Kim, M. S., Durvasula, A., Zhang, X.

Alzheimer disease risk variant rs11218343 determines functional expression of SORL1 in microglia

Rs11218343 is a non-coding variant of genome-wide significance for sporadic Alzheimer disease (AD) with one of the most protective effects known. It localizes to SORL1, encoding the AD risk factor SORLA. Still, the functional significance of rs11218343 for AD related processes remains unclear. We used iPSC lines from donors, or genome-engineered to carry major and minor rs11218343 alleles, to study the impact of rs11218343 genotype on brain cell activities. We show that rs11218343 uniquely controls functional expression of SORLA in microglia, with incrased receptor expression in the minor protective allele correlating with reduced pro-inflammatory responses. This anti-inflammatory effect is seen in donor iPSC lines but not in SNP-engineered isogenic lines, documenting rs11218343 to be diagnostic but not functional. Our findings corroborate genetically defined expression levels of SORL1 in microglia as a determinant of protection from pro-inflammatory stimulation, a function encoded by a haplotype linked to rs11218343.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.15.694337v1?rss=1

Willnow, T., Gorniak-Walas, M., Telugu, N. S., Rudolph, I.-M., Diecke, S.

Neuroligin-2 is ubiquitinated by Nedd4l to control developmental astrocyte morphogenesis.

Astrocytes of the central nervous system have an intricate and highly branched morphology. Proper development of perisynaptic astrocyte processes is necessary for the tripartite synapse development, maturation, and function. However, how astrocyte morphogenesis is controlled, and the specific molecules orchestrating this development are largely unknown. Previously, we identified Neuroligins (NLs) 1-3 as regulators of astrocyte morphogenesis by forming transcellular adhesions with neuronal neurexins. Here, we found that NL2 plays a non-redundant essential role in astrocyte morphogenesis. Through structure and function studies, we identified a WW-binding motif within the NL2 intracellular domain required for astrocyte morphogenesis. Using cell-specific in vivo proximity labeling by biotinylation (iBioID), we found that each NL displays distinct protein-protein interactions within astrocytes, and NL2 has cell-type specific interactomes in astrocytes versus neurons. By leveraging this interactome, we conducted in vitro and in vivo candidate screens and identified WW domain-containing E3 ubiquitin ligase Nedd4l to control astrocyte morphogenesis. Biochemical assays revealed that Nedd4l ubiquitinates and stabilizes NL2, and this ubiquitination step is required for astrocyte morphogenesis. This study shows that Neuroligins have non-overlapping roles in controlling astrocyte growth and uncovers a molecular mechanism of how NL2 mediates astrocyte morphogenesis.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.15.694023v1?rss=1

Eroglu, C., Sakers, K., Ramirez, J. J., Elazar, N., Nagendren, L., Soderblom, E. J.

Multi-task Intracranial EEG recordings reveal a Comprehensive Role of the Human Dorsal Anterior Insula in High-Level Cognition

The dorsal anterior insula (dAI) is widely recognized as a cornerstone of human cognition. However, its functional characterization remains the subject of considerable debate and competing theories. Embracing a "shift in viewpoint" approach - defining a brain region's function through the observation of the same neural population across diverse experimental contexts - this study used intracranial EEG (iEEG) recordings from a large cohort of patients to investigate the sub-second neural response dynamics of the human dAI across a broad range of cognitive tasks, spanning visual category discrimination, phonological and semantic processing, attentive visual search, and verbal and visuospatial working memory. This within-subject, multi-task iEEG framework offered a robust basis for decoding the structure-function relationship of the dAI and for empirically testing key predictions of prevailing theoretical models concerning its role. Our findings confirmed the dAI's involvement in the Action-Mode Network and shed new light on the large-scale spatiotemporal organization of that network, while providing novel insights into the interplay between cognitive effort and efficiency.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.15.694298v1?rss=1

Chatard, B., Dupin, M., Petton, M., Bontemps, B., Minotti, L., Kahane, P., Rheims, S., Bastin, J., Lachaux, J.-P.

The Inferior Frontal Sulcus, not the Anterior Insula, acts as a late-stage attentional gate for the fast rejection of salient but irrelevant sensory inputs during cognitive tasks

The ability to prioritize behaviorally relevant stimuli is essential for efficient cognition, particularly in distracting environments. The anterior insula (AI), a key node of the salience network, has been proposed to support this selection process by detecting salient events. A similar function, however, has also been attributed to the inferior frontal sulcus (IFS), located at the intersection of the ventral and dorsal attention networks. Here, we directly compared the respective contributions of these regions to attentional gatekeeping - the identification and rejection of attention-grabbing yet task-irrelevant "false positives" to preserve cognitive resources. We recorded intracranial EEG from 63 patients performing an attentive reading task designed to dissociate salience from task relevance. High-Frequency Activity (HFA) responses between 50 Hz and 150 Hz revealed that the IFS was the only region - across extensive sampling of the frontal and insular cortices - to exhibit consistent, subsecond response dynamics compatible with the filtering of false positives. In contrast, the AI did not participate in the analysis or rejection of distractors and showed only sparse, delayed responses to targets, suggesting a role in coordinating or energizing downstream cognitive or motor processes rather than in salience detection per se. These findings indicate that the IFS - rather than the AI - is the primary late-stage node for attentional gatekeeping during cognitive tasks performed under distracting conditions.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.15.694314v1?rss=1

Chatard, B., Dupin, M., Petton, M., Bontemps, B., Minotti, L., Kahane, P., RHEIMS, S., Lachaux, J.-P.

Loose coupling between Ca2+ channels and release sensors as a synaptic correlate of higher order brain function

In the mature neocortex, functionally distinct areas are built by the same archetypes of neurons, but depending on the area, these neurons and their synapses are engaged in very different functions, ranging from lower order processing of sensory information to higher order associations and cognitive functions. We found significant differences in the functional presynaptic nanoarchitectures of the same types of pyramidal neuron synapses, depending on whether they are located in the prefrontal cortex (PFC) or in the primary somatosensory cortex (S1). Synapses in PFC operated with loose microdomain coupling as opposed to tight nanodomain coupling in S1. These differences were associated with significant differences in synaptic timing, efficacy and plasticity between areas. Our data suggest that the mature neocortex uses tuning of synaptic topographies to specialize seemingly identical types of neurons for their required function. They reveal microdomain coupling as a presynaptic structure-function correlate of higher order neocortical functions.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.15.694355v1?rss=1

Schwarze, M., Bornschein, G., Brunner, A., Arshia, A., Brachtendorf, S., Schmidt, H.

Attention to Psuedo-Tone Melodies Enhances Cortical but Not Brainstem Responses in Humans

Auditory selective attention, the ability to focus on specific sounds while ignoring competitors, enables communication in complex soundscapes. Though attention clearly modulates cortical responses to sound, whether and where this modulation occurs in subcortical structures remains disputed. Here, we use electroencephalography to record cortical and subcortical (auditory brainstem responses, ABRs) activity during a selective attention task. Human participants attend to a 3-note melody in one pitch range presented to one ear while ignoring a competing, interleaved melody in a different pitch range played to the other ear (Laffere et al., 2020, 2021). The melodies consist of pitch-evoking pseudo-tones formed by convolving a periodic impulse train with a brief tone pip. These stimuli allow us to measure both ABRs (elicited by each individual tone pip within a pseudo-note) and cortical responses (elicited by the onsets of pseudo-notes) simultaneously. We observed robust ABRs, but no evidence of modulation by attention. Conversely, cortical responses, measured by event related potentials (ERPs), demonstrated attentional modulation of the P1-N1 peak. We conclude that attentional modulation within the brainstem is not measurable in the well-defined peaks of the ABR, which themselves reflect processing up to the input stage to the inferior colliculus.

Date: 2025-12-16
https://www.biorxiv.org/content/10.64898/2025.12.15.694407v1?rss=1

Figarola, V., Li, Y., Tierney, A. T., Dick, F., Noyce, A., Maddox, R. K., Shinn-Cunningham, B. G.

Crowding drives terminal investment in a generalist pest

Apart from genotype and environment, several fitness related traits of individuals are also influenced by the conditions experienced by their parents, i.e., by parental effects. To uncover the underlying physiological or molecular mechanisms, most studies analyze specific drivers in isolation, such as parental age or the degree of crowding experienced by the parents. However, potential interaction effects between different aspects of parental context are poorly understood. We analyzed the combined effect of parental age and population density on the number, fitness and physiology of offspring, in the red flour beetle Tribolium castaneum. Both factors independently reduce fitness, leading us to predict that old parents from high density should produce fewer offspring of the lowest quality. Surprisingly, older parents exposed to crowding produced more eggs than expected. These eggs were larger, had higher survival in an optimal as well as a suboptimal resource, and were provisioned with more lipids. This result is consistent with terminal reproductive investment in response to high density. The interaction between two ecologically relevant contexts -- ageing and crowding -- thus drives terminal investment via nutrient provisioning, perhaps contributing to the success of this generalist pest.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.12.694078v1?rss=1

Govindarajan, S., Patra, D., Agashe, D.

Morphological and molecular characterisation of Orientia tsutsugamushi grown in tick cells

Orientia tsutsugamushi (Ot), the causative agent of scrub typhus, is an obligate intracellular bacterium naturally maintained in Leptotrombidium mites, yet its interactions within arthropod hosts remain poorly understood. Here, we developed two tick cell lines, Ixodes scapularis ISE6 and Rhipicephalus microplus BME/CTVM23, as arthropod models to investigate the intracellular life cycle of Ot strains TA686 and Karp. Both strains efficiently infected and replicated within tick cells, with ISE6 supporting more robust growth. Electron microscopy images revealed that Ot maintains its characteristic cytoplasmic, non-vacuolar location in tick cells and exits infected cells by budding off the surface in a membrane-encased structure. Time-course immunofluorescence imaging demonstrated progressive intracellular replication and dynamic expression of Ot outer membrane autotransporters ScaA and ScaC, with ScaC enriched early in infection and ScaA at later stages. Metabolic labelling using a clickable methionine analog L-homopropargylglycine showed that high ScaA abundance correlated with reduced translational activity, suggesting a link between ScaA abundance and late-stage or extracellular-like developmental states. The subcellular location of Ot in tick cells differs from the characteristic dynein-driven perinuclear clustering observed in mammalian cells, and was not sensitive to disruption of microtubules, indicating a distinct mode of distribution. Together, these findings identify tick cell lines as tractable and biologically relevant arthropod models for studying Ot and dissecting host-microbe interactions.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.15.694307v1?rss=1

Rogowska-van der Molen, M., Gallo, F., Bell-Sakyi, L., Salje, J.

Symbiosis through lysis: prophage activation underlies Lactiplantibacillus plantarum probiotic function

The release of bacterial bioactive molecules across the gut barrier is a crucial yet poorly understood step in microbe-host communication. Here we unravel a phage-driven mechanism that enables this process in a nutritional symbiont. In Lactiplantibacillus plantarum NC8 (LpNC8), we identify a stress-inducible prophage, pp2, that undergoes genotoxic-stress-dependent activation and triggers holin-lysin-mediated lysis. This controlled lytic program produces phage particles together with extracellular, membrane-derived bacterial particles, and is strictly required for the ability of LpNC8 to promote juvenile growth in nutritionally challenged Drosophila melanogaster. Disruption of pp2-dependent lysis abolishes particle release in vitro and eliminates the growth-promoting effect in vivo, without altering bacterial abundance in the gut. The magnitude of phage release correlates with the extent of host growth promotion, indicating that prophage induction contributes to the export of symbiotic factors. These findings reveal prophage-induced lysis as a central and previously unrecognized mechanism by which a beneficial gut bacterium enhances host development under nutritional stress.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.15.694388v1?rss=1

Zhu, C., Manuse, S., Perrier, Q., Akherraz, H., Ramos, C. I., Zhang, M., Grangeasse, C., Leulier, F., Matos, R. C.

Beyond the core: endemic microbiota drive functional and microdiversity differences across salamander populations

Population-specific variation in animal microbiomes is well documented, yet the functional consequences and underlying mechanisms remain poorly understood. To address this, we conducted genome-resolved metagenomic analyses on gut and skin microbiomes from four populations of Pyrenean brook salamanders (Calotriton asper) inhabiting two distinct environments (Pyrenean subalpine brooks and Atlantic montane streams). From paired faecal and skin swab samples, we reconstructed 539 and 43 metagenome-assembled genomes (MAGs), respectively, and examined taxonomic composition, metabolic capacity, and microdiversity across environments. While alpha diversity remained consistent, both gut and skin microbiomes exhibited significant differences in community composition and functional potential between environments. Partitioning the gut microbiome into core, endemic, and marginal fractions revealed a dominant core community, shared across environments and accounting for over 85% of reads, that did not drive functional divergence. Instead, functional differences were primarily shaped by low-abundance, population-specific endemic bacteria. Atlantic salamanders hosted endemic taxa with significantly greater metabolic potential and higher strain-level microdiversity than those at the Pyrenees. These patterns were not explained by dietary differences and may reflect environmental influences such as temperature and nutrient availability. Our findings highlight the relevance of rare, endemic bacteria in driving microbiome function and underscore the power of genome-resolved metagenomics to uncover functional and evolutionary dynamics in wild host-microbe systems.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.14.694248v1?rss=1

Aizpurua, O., Brenner, E., Martin-Bideguren, G., Garin-Barrio, I., Cabido, C., Alberdi, A.

Postnatal increase in MRTF-dependent transcription reduces EGFR activation and proliferation of apical but not basal NSCs

In the ventricular-subventricular zone (V-SVZ), both apical and basal NSCs undergo quiescence and proliferation. The epidermal growth factor receptor (EGFR) and interactions with the extracellular matrix are key regulators of adult NSC proliferation and quiescence, respectively. Here, we show that activation of EGFR significantly declines after the first postnatal weeks in apical NSCs. This decline is accompanied by a shift in serum responsive factor (SRF)-dependent transcription in apical NSCs. Specifically, growth-promoting genes targeted by SRF and ternary complex transcription factors are downregulated, whereas those targeted by SRF and myocardin-related transcription factors (MRTFs), including those involved in the extracellular matrix remodeling, are upregulated. Blocking of MRTFs, whose activity is regulated by the Rho/actin pathway and extracellular matrix interactions, restores EGFR activation and EGFR-dependent proliferation in adult apical NSCs. Thus, transcriptional programs regulated by extracellular cues differentially control EGFR activation and proliferation in neonatal and adult apical NSCs.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.12.693930v1?rss=1

Shen, Y., Baur, K., Irmler, M., Beckers, J., Mandl, C., Hoelzl-Wenig, G., Ciccolini, F.

Glycolysis and hexosamine biosynthesis pathways are key for inflammatory protein maturation and leukocyte adhesion to human aortic valve cells

Inflammation and metabolism reprogramming are hallmarks of calcific aortic valve disease (CAVD). Recent studies link inflammation to hyperglycolysis and calcification in valve interstitial cells (VICs). The metabolism of valve endothelial cells (VECs) has received less attention despite both resident valve cells are exposed to alike inflammatory clues involved in the biosynthesis of pathologically relevant glycoproteins during the early stages of CAVD. On this basis, we investigated the outcomes of glucose metabolism rewiring on glycoprotein maturation and immune cell adhesion in human resident valve cells. Real-time metabolic analysis revealed that basal VECs are more glycolytic than VICs. Also, VECs and VICs exposed to inflammatory stimuli exhibited a distinct rewiring, with VECs shifting to a more energetic metabolism, despite a similar upregulation of glycolytic genes. Blunting glucose metabolism in VICs and VECs inhibited inflammatory routes canonically associated with glycolysis, and the expression of proteins associated to the inflammatory response like interleukin-6 and cyclooxigenase-2. Moreover, Western blot and adhesion assays revealed that glycolysis is necessary for the expression and post-translational modifications of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and the ensuing process of monocyte-VECs adhesion. Notably, inhibition of the hexosamine biosynthetic pathway using DON and of N-glycosylation by tunicamycin, further disrupted adhesion molecule maturation and monocyte-VECs adhesion. In conclusion, glycolysis and its side-branch route the hexosamine biosynthesis pathway are necessary for nutrient-driven post-translational modifications of inflammatory proteins in inflamed valve cells and the subsequent process of monocyte-VECs adhesion that plays a key role in the initial stages of CAVD pathogenesis.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.12.693904v1?rss=1

Sanchez-Bayuela, T., Peral-Rodrigo, M., Lopez, J., Gomez, C., Perez-Riesgo, E., Lopez-Andres, N., Fernandez, N., San Roman, J. A., Sanchez Crespo, M., Garcia-Rodriguez, C.

Retrotransposon Activation in the Aged and Alzheimer's Disease Brain Examined by Nanopore Long-read DNA Sequencing

Background Cellular defenses against retrotransposable elements (RTEs) weaken with age and RTEs have been reported to contribute to Alzheimer's disease (AD) pathogenesis by promoting neuroinflammation. The mechanisms implicated include DNA damage promoted by retrotransposition and interferon system activation by RTE-derived cDNA intermediates. LINE-1 (L1) retrotransposons are of particular interest because they are the only autonomously active RTEs in the human genome. Results To investigate L1 activation and retrotransposition in AD, we performed Nanopore long-read DNA sequencing on six late-onset AD (LOAD) and six age-matched control human prefrontal cortex (PFC) samples. We developed and validated a stringent RTE insertion calling pipeline and identified two high-confidence somatic insertions, one AluY and one L1HS. We estimate that ~1% of cells in the aged PFC have a somatic RTE insertion. AD samples were hypomethylated, and genome-wide analysis of differentially methylated regions (DMRs) supports a process of epigenetic drift in AD. DMR-associated gene sets primarily related to brain function and inflammation. To investigate L1 activation we used CpG methylation as a proxy for L1 expression. We observed decreased methylation at young L1 elements. While most reads overlapping the L1HS promoter were highly methylated (>80% methylated), 7% were <50% methylated, 1% were <25%, and the highly demethylated read fraction increased in AD. L1HS 5' UTR methylation was strongly correlated with RNA expression. Conclusions CpG methylation-mediated repression of young RTEs is compromised in old age - our findings indicate that this is further exacerbated in AD. Amid these failing defenses, we report somatic retrotransposition events in the aging and demented brain.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.12.693943v1?rss=1

Kelsey, M. M. G., Chongtham, A., LaCava, J., Taylor, M. S., Boeke, J. D., Gage, F. H., Seluanov, A., Gorbunova, V., Pereira, A. C., Sedivy, J. M.

RAFTER: a releasing factor tethered RNA editing system for quantifying mRNA translation

Measurement of mRNA translational efficiency has heavily relied on quantifying the amount of polysome-associated RNAs which would not always reflect the efficient translation. To address this, we developed RAFTER, which combined RNA editing enzyme with translation releasing factor to selectively label successfully translated RNAs. The translational efficiency estimated based on RAFTER correlated well with that by Ribo-seq and Polysome-seq, but with greatly simplified workflow, which enables it for single-cell analysis.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.12.693956v1?rss=1

Chen, W., Sun, Z., Dong, P., Yan, H., Wen, X., Li, Y., Jiang, F., Fang, L., Wang, X.

The shifting dynamics of ancestry and culture at a post-Roman crossroads

The collapse of the western Roman Empire in the 5th century created a period of geopolitical upheaval, driven by Barbarians dispersing into the former Empire and reshaping post-Roman communities. While there is now evidence for a major genetic impact of these migrations into specific regions of Europe, it is unknown whether these changes in ancestry were uniform across the continent. We investigate present-day Slovenia, a crucial crossroad connecting the Roman East and West and the gate to Italy during the Langobard invasion. We conducted paleogenomic and isotopic analyses of 410 individuals from 21 sites across Slovenia and Cividale (Italy), establishing a longitudinal transect, spanning eight centuries. During Late Antiquity, despite changes in burial artifacts, kinship practices, and settlement structures reflecting a shift in culture, we find high levels of genetic continuity with the local Late Roman population and reduced mobility. However, demographic turnover began during the 8th century, when communities with northeastern European ancestry and distinct cultural practices entered the region, gradually advancing westward over the span of three centuries, replacing the local populations. This shows that cultural change in post-Roman Europe could be decoupled from genetic change in transit zones, demonstrating a dynamic spatiotemporal process across the continent.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.12.693924v1?rss=1

Vyas, D. N., Koncz, I., Milavec, T., Leskovar, T., Farago, N., Tian, Y., Bausovac, M., Mende, B. G., Francalacci, P., Bavec, U., Bester, H., Borzacconi, A., Brezigar, B., Friedrich, R., Gaspari, A., Giostra, C., Hincak, Z., Karo, S., Kruh, A., Mason, P., Modi, A., Perko, M., Radzeviciute, R., Ratej, R., Saccheri, P., Szecsenyi-Nagy, A., Travan, L., Udovc, K., Vai, S., Zupanek, B., Caramelli, D., Krause, J., Pohl, W., Vida, T., Geary, P. J., Veeramah, K. R.

RBProximity-CLIP Enables Subcellular Mapping of RNA-Binding Protein Interactions at Nucleotide Resolution

RNA-binding proteins (RBPs) enable post-transcriptional gene regulation (PTGR) through specific interactions with RNA molecules, influencing processes ranging from nuclear processing and export to cytoplasmic localization, translation, storage and degradation. A key determinant of PTGR processes is the subcellular compartmentalization of RBPs, which dictates RNA targets they can access and the regulation performed in that environment. To characterize RBP-RNA interactions at subcellular resolution, we developed RBProximity-CLIP. RBProximity-CLIP enables compartment-specific isolation and profiling of individual RBP-RNA interactions by combining APEX2-based proximity labeling and 4-thiouridine-enhanced RNA-protein crosslinking, with sequential RBP- and biotin-affinity purifications. Using this approach, we profiled the RNA targets of three RBPs, AGO2, YBX1, and ELAVL1, across the cytoplasmic, nuclear, and nucleolar compartments, revealing nucleus-specific miRNA-mediated AGO2 targets, as well as subsets of YBX1 and ELAVL1 targets that differ by compartment, yet share identical binding motifs. RBProximity-CLIP enables specific and sensitive detection of compartment-specific RBP-RNA interactomes, thereby providing new insight into spatial gene regulation by RBPs.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.12.693770v1?rss=1

Nowak, I., Polash, A. H., Huynh, H. T., Kaur, M., Lobo, V., Scutenaire, J., Fong, M., Alluhaibi, G., Anastasakis, D. G., Hafner, M., Benhalevy, D., Sarshad, A.

Identification of sodium/myo-inositol transporter 1 as a major determinant of arterial contractility

Background: As the sodium/ myoinositol transporter (SMIT1) is a positive regulator of Kv7.4/7.5 channels in arterial smooth muscle we postulated that altering SMIT1 expression could have a major impact upon vascular reactivity. Consequently, this study aimed to characterise the effects of changes of SMIT1 membrane abundance on vascular tone and the molecular mechanisms involved. Methods: Isometric tension recording on 2nd order mesenteric arteries and left anterior coronary arteries from male and female Wistar Han rats. Whole artery membrane potential recording. Single cell and whole artery antibody-based imaging. Morpholino based protein knockdown of SMIT1. Results: Morpholino-mediated knockdown of SMIT1 enhanced U46619- and methoxamine-mediated contractions of mesenteric artery whilst impairing relaxations to the Kv7 activator ML213, isoprenaline and CGRP. Conversely, augmenting SMIT1 membrane abundance by raising external osmolarity with 150 mM raffinose, impaired receptor-mediated contractions of mesenteric and coronary arteries, augmented relaxations to ML213 and adenosine as well as producing membrane potential hyperpolarisation. Proximity ligation assays revealed that raffinose incubation increased the association of SMIT-Kv7.4/7.5 as well as Kv7.4 and G{beta}{gamma} subunits. The SGK1 inhibitor EMD638683 prevented the raffinose-induced increase in SMIT1 and the anti-contractile effect. Conclusion: Toggling the membrane abundance of SMIT1 had a dramatic effect on the arterial response to vasonstrictors and vasodilators mediated by greater coordination of Kv7 channels and G{beta}{gamma} subunits. This work identified SMT1-Kv7 channel complexes and SGK1 regulation as key modulators of arterial responsiveness.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.12.694071v1?rss=1

Forrester, E. A., Garland, C. J., Barrese, V., Albert, A. P., Greenwood, I. A.

Evaluation of Detection Methods for Wastewater Surveillance of Antimicrobial-Resistant Bacteria from Healthcare Facilities

Carbapenem resistance is an urgent public health threat. Wastewater surveillance could support antimicrobial resistance monitoring at long-term care facilities (LTCFs). Feasibility of wastewater sampling (via composite or passive sampler, and sewer biofilm swabs) for carbapenemase genes (blaKPC, blaVIM, blaOXA-48-like, blaNDM, and blaIMP) detected by qPCR or GeneXpert(R) Carba-R from a LTCF was assessed over 16 months and compared to clinical infections. blaKPC, blaOXA-48-like, and blaVIM were routinely detected in composite wastewater samples (6.4{+/-}0.8 log10 gene copy (GC)/ml (100% of 61 samples), 5.5{+/-}0.8 (98%), and 5.9{+/-}1.3 (34%), respectively), passive samples (8.2{+/-}0.5 log10 GC/g (31% of 55), 6.6{+/-}0.5 (96%), and 6.6{+/-}0.7 (31%)) and sewer biofilm (6.0{+/-}0.7 log10 GC/cm2 of pipe (100% of 17), 5.0{+/-}0.7 (100%), and 5.8{+/-}2.7 (24%)). Resistomes of wastewater and sewer biofilms differed, but both contained blaKPC, blaVIM, and blaIMP. Passive sampling may be a suitable alternative to composite sampling. Wastewater surveillance is a promising addition to carbapenemase monitoring.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.14.694051v1?rss=1

Warren, E., VanDerslice, J., Benson, L. S., Brazelton, W. J., Tanner, W., Lyons, A. K., Whitehill, F., Coulliette-Salmond, A., Fusco, S., Weidhaas, J.

Scratching on French PDO cheese surfaces sheds light on an unexplored microbial genomic and metabolic diversity

Cheeses are fermented dairy products consumed worldwide. Their global diversity results from various local variables, including technological practices, as well as the metabolic activity of diverse microorganisms. In Europe, this typicity is exemplified by Protected Designation of Origin (PDO) cheeses, for which genetic diversity remains largely unexplored. Combining culturomics (n = 373 bacterial genomes) and metagenomic (n = 146 metagenomes), we performed a national-scale survey of the microbial diversity encompassing 44 French PDO cheeses. Taxonomic (bacteria, fungi and viruses) and functional profiling reveal a high diversity in the cheese rind, mainly driven by the cheese technology. We also reconstructed 1,119 bacterial metagenome-assembled genomes (MAGs) encompassing seven phyla, including Actinomycetota, Bacillota, Pseudomonadota and Bacteroidota. Using GTDB as a reference, we identified 221 MAGs encompassing 46 genera, as well as 44 bacterial isolate genomes encompassing eight genera, which represent potentially 81 new species (based on <95% ANI). These species were particularly numerous among the genera Halomonas, Psychrobacter and Brachybacterium. Similar results were observed when compared with the cFMD database. We combined our genomic and metagenomic datasets into a catalog of 26.2 million protein clusters, with 50% of these clusters remaining unassigned to a known function and taxonomy. We illustrated the potential of this resource by searching for methionine gamma-lyase (MGL), an enzyme playing a significant role in cheese flavor. This protein was predominantly found in Pseudoalteromonas, a potentially new MGL-producing genus, Serratia, Pseudomonas, Proteus and Hafnia, and its prevalence varied with cheese technology. Our study provides a substantial genomic resource for food microbiologists and cheesemakers to further explore the biotechnological potential of PDO cheese biodiversity.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.15.694182v1?rss=1

Gardon, H., Tabuteau, S., Irlinger, F., Dugat-Bony, E., Barbe, V., Callon, C., Cantuti Gendre, J., Cruaud, C., Delbes, C., Gavory, F., Loux, V., Mohellibi, N., Neuveglise, C., Renault, P., Rue, O., Theil, S., Aury, J.-M., HERVE, V.

A meta-analysis of environmental sequencing data reveals the global distribution and hidden diversity of marine anaerobic ciliates

Anaerobic protists are diverse, ecologically important members of anoxic microbial communities, acting as grazers, nutrient cyclers, and partners in multi-domain associations, yet remain understudied relative to anaerobic prokaryotes. Ciliates are particularly abundant and diverse in anoxia, but their global diversity and distribution are largely unknown. Here, we conducted a meta-analysis of public 18S rDNA datasets, along with one dataset generated here, to assess the global diversity and ecology of marine anaerobic ciliates. Using a novel pipeline, we processed 2854 samples from 42 studies spanning 19 habitat types. We recovered 3196 anaerobic ciliate amplicon sequence variants (ASVs) across all described lineages. Based on clade-specific divergence thresholds derived from phylogenetic distances, 28.4-46.3% of ASVs qualified as novel. Most sequences belonged to the poorly described plagiopylean family Epalxellidae, suggesting a large reservoir of undescribed diversity in this clade. Community comparisons revealed close phylogenetic similarities between some shallow-water and deep-sea assemblages, suggesting that shared redox conditions may shape communities more than water depth. Our results demonstrate that marine anaerobic ciliates are globally distributed, taxonomically diverse, and rich in novel lineages. This study provides a framework for leveraging environmental sequencing data to better understand the diversity and ecology of neglected protist lineages and under-sampled habitats.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.15.694440v1?rss=1

Schrecengost, A., Frates, E., Al-Haj, A. N., Fulweiler, R. W., Beinart, R. A.

Bacteriophage PAK_P3 genome structuration and dynamics during infection of Pseudomonas aeruginosa reveal specific interactions patterns

Bacteriophages, or phages, are highly abundant and diverse genomic entities that play an important role in microbial ecology, evolution, and horizontal gene transfer. While the dynamic changes in genome organization in cellular organisms have been well described, the 3D folding of phage genomes during the infection of their host is extremely limited. Understanding how phage genomes fold, invade and rearrange the genome of their host to functionally organize the optimal expression of their genes remains unknown. Here, we explore the spatial dynamics of the virulent double-stranded DNA phage PAK_P3 during infection of its host Pseudomonas aeruginosa and reveal how its genome rapidly decondenses to adopt a specific 3D organization that reflects its transcriptional program. Concomitantly, the host genome decondenses as gene expression wanes and transcription induced domains vanish. We also uncover specific and discrete bridging of the host and the phage genomes, showing how the phage genome exploits the spatial genome architecture of its host to succeed in its infection cycle. Our data highlights an unprecedented level of genome folding and gene expression regulation during a viral infection.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.15.694284v1?rss=1

Bignaud, A., Lamy-Besnier, Q., Conti, D., Thierry, A., Girard, F., Misson, P., Koszul, R., Debarbieux, L., Marbouty, M.

The role of heavy metals in the co-selection of plasmid-borne metal and antibiotic resistance genes from industrially contaminated sediments.

A comprehensive understanding of the sources and drivers of antimicrobial resistance is essential for effective antimicrobial stewardship. Co-selection is now recognised as a significant driver of antimicrobial resistance, with established links between heavy metal exposure and the presence of bacteria with antimicrobial resistance. The precise mechanisms that drive this process in the environment are co-resistance, cross-resistance, and co-regulation, but their respective impacts remain largely unexplored. Here, we investigated whether heavy metal contamination in freshwater sediments selects for bacteria harbouring plasmids that carry both metal and antibiotic resistance genes, or genes encoding cross-resistance mechanisms. A diverse set of plasmids was recovered from metal-impacted sites at Lake Macquarie (New South Wales, Australia), which carried resistance genes particularly to copper, zinc, cobalt, cadmium, and arsenic. Two-thirds of these plasmids also carried one or more antibiotic resistance genes, indicating co-selection through the co-resistance mechanism. Functional assessment confirmed that the multi-metal and polymyxin resistance plasmid genotype was linked to the corresponding bacterial phenotype. The metagenome of the metal-impacted sediments was also examined to explore evidence of co-selection, and a broad range of incomplete plasmid sequences containing homologues of both metal- and antibiotic-resistance genes was detected. This study demonstrates the important link between anthropogenic heavy metal contamination and potentially clinically relevant antibiotic resistance genes. It highlights the importance of approaching the management of antimicrobial resistance from a One Health perspective.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.14.694177v1?rss=1

Gillieatt, B. F., Thai, M., Cain, A. K., Zadoks, R. N., Coleman, N. V., Kertesz, M. A.

Genome-scale dissection of phase-variable gene function in Campylobacter jejuni using a stabilized phasotype library

Phase variation (PV) enables bacterial pathogens to rapidly alter their surface structures through reversible mutations in simple sequence repeats, promoting immune evasion and environmental adaptation. In Campylobacter jejuni, the stochastic nature of PV has hindered the systematic functional analysis of phase-variable genes (PVGs). Here, we introduce PV-GenShift, a genome-scale screening platform built on a genetically stabilized library of phase-locked C. jejuni variants. By fixing the ON/OFF states of 15 PVGs, PV-GenShift enables reproducible, high-resolution analysis of phasotypes, defined as unique ON/OFF combinations across multiple PVGs, under defined selective pressures. Using models of human serum exposure, murine colonization, and chicken gut passage, we identified distinct phasotypes associated with serum resistance and with enrichment during mouse colonization, particularly involving capsular polysaccharide modifications such as O-methyl phosphoramidation and methylation. In contrast, chicken gut passage resulted in heterogeneous ON/OFF shifts without a dominant phasotype. These findings highlight the combinatorial impact of PVG expression states on bacterial adaptation and establish PV-GenShift as a broadly applicable framework for dissecting PV-driven phenotypic diversity. This approach provides a scalable strategy for exploring genotype-phenotype relationships and offers insights relevant to vaccine design and targeted therapeutics.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.14.694251v1?rss=1

Yamamoto, S., Lee, K.-i., Kubomura, A., Iyoda, S., Akeda, Y., Shimohata, T., Aikawa, C., Okamura, M., Hojo, F., Osaki, T., Mitobe, J.

A global deep-sea small protein atlas reveals a reservoir of noncanonical antimicrobial peptides

Small proteins encoded by small open reading frames (smORFs; [≤] 100 aa) represent a largely unexplored dimension of microbial diversity, especially in the deep sea. By analyzing 708 metagenomes from five major deep-sea biomes (hadal trenches, cold seeps, hydrothermal vents, abyssal plains, and seamounts), we constructed the Deep-Sea Small Protein Atlas, comprising 88.7 million smORFs with exceptional novelty and strong habitat specificity. Deep-learning predictions identified 5.47 million candidate antimicrobial peptides (c_AMPs), revealing a peptide space far larger and structurally distinct from known AMPs. Deep-sea c_AMPs are longer, enriched in nonpolar and acidic residues, and exhibit low charge and high intrinsic disorder, suggesting non-membranolytic modes of action. We synthesized 131 representative peptides, of which 87% were antimicrobial, with MICs as low as 1.25 M, broad-spectrum antibacterial and even antifungal efficacy, and minimal mammalian cytotoxicity. Transcriptomics, TEM imaging, and peptide-protein modeling showed that representative peptides preserve membrane integrity while disrupting intracellular processes such as translation and metabolism, supporting intracellular, non-lytic mechanisms. This work uncovers a vast reservoir of previously unrecognized deep-sea small proteins and structurally unconventional AMPs, providing a foundational resource for discovering next-generation peptide therapeutics.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.15.694180v1?rss=1

Jiang, Q., Han, Y., Ye, C., Duan, Y., Li, F., Han, Z., Santos-Junior, C. D., Luo, Z.-Q., Dong, X.

Metabolic reshaping drives novel melanin production from tryptophan in a cystic fibrosis Pseudomonas aeruginosa clinical isolate

Pseudomonas aeruginosa is a human opportunistic pathogen, capable of producing a wide range of metabolites, including pyomelanin. This pigment results from alterations in tyrosine catabolism. Melanin synthesis from tryptophan has never been reported in Pseudomonas. In this study, we describe a tryptophan-derived melanin in P. aeruginosa PAH, a strain that was isolated from a fibrocystic patient. PAH produced a brown pigment when grown in LB or L-tryptophan-supplemented media. Structural analysis revealed this pigment was composed by two fractions differing in NaOH solubility: a soluble one consistent of pyomelanin, and an insoluble fraction with a complex structure containing substituted indolic units. A pyomelanin inhibitor enhanced total melanin synthesis, mainly the insoluble fraction, and a tryptophan 2,3-dioxygenase inhibitor decreased pigment formation. Metabolomic profiling identified distinct indolic compounds and low levels of anthranilate in PAH cultures. Genomic and transcriptomic analyses revealed the presence of mutations and downregulation of genes related to pyoverdine biosynthesis. Furthermore, iron supplementation in the culture medium reduced melanin production. Overall, tryptophan arises as a key compound for melanin production in PAH, expanding the diversity of melanins synthesized by this genus. Furthermore, iron deprivation emerges as a critical factor triggering melanin biosynthesis, probably as a survival strategy enabling persistence in the fibrocystic lung environment.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.15.694361v1?rss=1

Appella, M. N. D., Kolender, A. A., Nega, M., Robaldi, S. A., Cassanelli, P. M., Li, N., Liberini, E., Hoijemberg, P., Pelliza, L., Aran, M., Götz, F., Lopez, N. I., Tribelli, P. M.

Modeling the effects of Huntington disease on age-related genes reveals CXXC4 as an epigenetic target to restore health and excitability of Drd1-expressing striatal neurons

Gene profile studies suggest that neurons may face premature aging in neurodegenerative diseases such as Huntington disease (HD). Cells remodel gene expression to resist molecular damage in aging, but how neurons may engage age-related genes to resist HD remains unknown. Here, we found that transcriptional-aging inversion (TAGI) in the Drd1-expressing striatal neurons (Drd1 SNs) of HD knock-in (Hdh) mice shows discrete patterns in the backdrop of a TAG-like (TAGL) signature, and that TAGI persistence as Hdh mice become strongly symptomatic may better explain disease progression compared to TAGL dynamics. We also found that genes affected by 3prime UTR accumulation in aged mouse Drd1 SNs of aged mice are more likely downregulated in aging and deregulated in Hdh mice. Mapping 3prime UTR data in aging on gene dysregulation networks in the Drd1-SNs of weakly symptomatic Hdh mice highlighted a CAG repeat-dependent network of upregulated genes with compensatory potential as suggested by enrichment in development and maintenance factors such as CTCF. This class noticeably contains Atad-5, a PCNA unloader in the DNA repair complex and a modifier of CAG expansion in the plasma of HD patients. This class also contains CXXC4 (IDAX), an epigenetic regulator and repressor of TET2 activity, for which upregulation is lost in strongly symptomatic Hdh mice. CXXC4 reduces the levels of p16INK4a, a cellular senescence marker, in human iPS cell-derived SNs and restores glutamate excitability in human HD iPS cell-derived SNs. Collectively, these data suggest that the resilience capacity of Drd1 SNs against HD involves discrete patterns of age-related genes, where early intervention based on expressing broad-spectrum genes such as CXXC4 may provide novel therapeutic strategies to restore cortico-striatal homeostasis and function.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.12.693965v1?rss=1

Arrieta-Lobo, M., Farina, F., Monteagudo Aboy, T., Mair, M., Mendoza, C., Tran, H., Aaronson, J., Rosinski, J., Ellerby, L., Brouillet, E., Botas, J., Neri, C., Megret, L.

Deliberation is a controllable process governed by desirability and cognitive effort

Humans spend a lifetime making decisions based on incoming sensory information and goals. Prominent theories of perceptual decision-making have described the components of deliberation process, yet they lack a unifying principle that governs how the nervous system tunes the deliberation process across multiple contexts. Desirability (reward) and effort (energy) are major determinants in governing a broad range of human and animal behaviour, such as foraging, walking, and decisions. Here we develop a theory where desirability and cognitive effort tune the control gains that govern deliberation. Several hallmark features of decision behaviour simply emerge from the model, with the deliberation process closely resembling low-dimensional neural dynamics. We also predict and provide a novel mechanistic explanation for ''choking-under-pressure'', where extremely large rewards lead to performance deficits. Our principled framework explains both behavioural and neural phenomena while providing a path to unify disparate fields.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.12.693733v1?rss=1

Calalo, J. A., Sullivan, S. R., Muscara, N. R., Buggeln, J. H., Ngo, T. T., Short, M. M., Carter, M. J., Kurtzer, I. L., Cashaback, J. G. A.

Spatial Polarization-Induced Fluorescence Fluctuation Imaging (SPIFFI) Enables Single-shot Super-Resolution and Multidimensional Imaging

Fluorescence super-resolution microscopy has advanced optical imaging into the nanoscale regime, transforming biological and interdisciplinary research. However, conventional wide-field super-resolution techniques often compromise temporal resolution, thereby limiting the ability to capture rapid and transient biological events in living systems. Here, we introduce spatial polarization-induced fluorescence fluctuation imaging (SPIFFI), a multi-channel polarimetric method that enables single-shot super-resolution imaging and six-dimensional information extraction. By leveraging the inherently smaller point spread function (PSF) under polarized detection and capturing polarization-dependent spatial fluctuations across multiplexed channels, SPIFFI achieves instant resolution enhancement from a single exposure. This capability substantially enhances the feasibility of volumetric live-cell super-resolution imaging. Moreover, SPIFFI images can be seamlessly integrated with existing fluctuation-based methods for further post-processing and resolution improvement. We demonstrate the versatility of SPIFFI through experiments on both fixed and live cells, capturing rapid subcellular dynamics and enabling high-throughput, multidimensional imaging beyond the diffraction limit. SPIFFI thus offers a practical and robust platform for real-time super-resolution imaging in biological research.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.12.693764v1?rss=1

Guo, W., Feletti, L., Radenovic, A.

Quantifying (de)Mixing of Disordered Proteins in Molecular Dynamics Simulations

Biomolecular condensates underpin the spatial and temporal organization of cellular biochemistry in the cell. Their architectures often arises from complex, multicomponent mixtures whose behavior is governed by weak, multivalent interactions, frequently mediated by intrinsically disordered regions (IDRs) of proteins. However, current approaches lack generalizable metrics to determine whether IDRs will mix or segregate within condensates. Here, we show that our domain decomposition method can both accurately determine concentrations in the dense/dilute phases, and provide a continuous metric for characterizing IDR mixing in molecular dynamics simulations. Applying this methodology to 1,963 binary mixtures, we find that mixing at equimolar ratios is rare. Most condensate-forming pairs favoring one dominant scaffold and minority client molecules. We found that hydrophobic IDRs mix promiscuously with most sequences, while the mixing of charged sequences is sensitive to the charge and complementarity of the partner. When applied to experimental protein interaction networks, our simulations successfully distinguish IDR-mediated partitioning from those requiring additional factors such as RNA or site specific binding. Our results provide a foundation for determining condensate composition in complex cellular environments and for designing synthetic IDRs that can infiltrate or modulate biomolecular condensates.

Date: 2025-12-15
https://www.biorxiv.org/content/10.64898/2025.12.12.694022v1?rss=1

Morton, W. S., Vacha, R.