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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2026

Genome-wide fluctuating selection under demographic cycling in Icelandic rock ptarmigan

Understanding how natural populations adapt to continuously shifting environments is central to evolutionary biology. Multi-annual population cycles impose temporally varying selective pressures, yet disentangling the resulting genomic signals from those of sustained directional selection and genetic drift has been challenging due to a lack of high-resolution temporal data. Here we investigate the genomic consequences of demographic cycling in a population of rock ptarmigan (Lagopus muta) from northeastern Iceland. We analyzed whole-genome sequences from 91 individuals sampled over an 11-year period, encompassing two population peaks (2010 and 2018) and two troughs (2007 and 2013). Testing allele frequency change at each SNP against a null model of drift and sampling, we identified candidate loci and grouped them by correlation of their temporal trajectories. Cluster trajectories fluctuated through time, and two were closely correlated with the regional census, one significantly tracking the demographic cycle, while a smaller set changed monotonically in a manner consistent with sustained directional selection. Each cluster contained loci distributed across many chromosomes, indicative of genome-wide responses to selection. The most strongly fluctuating loci overlapped candidate genes of possible functional relevance, most notably CTNNA2 (Catenin Alpha-2), which is associated with startle response and anxiety-like behaviors and may relate to predator avoidance. Our findings indicate that fluctuating selection comprises much of the temporal genomic response in this natural population, and suggest that multi-annual demographic cycles can drive rapid and widespread allele frequency change, highlighting the value of temporal genomics for clarifying the interplay between demography and natural selection in wild populations.

evolutionary biology↗

bioRxiv2026

Endonuclease G governs an antiviral RNA surveillance system in budding yeast

Endonuclease G family DNA/RNA nucleases localize to the mitochondrial intermembrane space and prevent escape of mitochondrial DNA to the cytosol or nucleus. While extramitochondrial Endonuclease G also targets nuclear DNA during cell death, RNA regulatory roles of this family remain enigmatic. Here we discover an RNA surveillance system in Saccharomyces cerevisiae governed by Nuc1, the yeast Endonuclease G homolog. This system limits replication of L-A, an RNA mycovirus that endemically infects this organism. Whereas L-A resides in the cytosol, we find that Nuc1's antiviral function requires its mitochondrial intermembrane space localization. To understand Nuc1's indirect antiviral mechanism we investigated its genetic interactions with REX1 and REX2, RNA exonucleases that control 3' end processing of tRNA and rRNA intermediates in the nucleus. We demonstrate that the nuc1{triangleup} antiviral defect is massively amplified by rex1{triangleup} rex2{triangleup} leading to L-A RNA levels that surpass mature rRNAs and overwhelm the cells. NUC1 concurrently controls a pool of mis-processed tRNA and rRNA species that accumulate in rex1{triangleup} rex2{triangleup} mutants. Linking these mis-processed RNAs with L-A control, we show they are significantly reduced in L-A infected cells, suggesting that L-A depletes them to enhance its replication. Our findings identify a previously unrecognized RNA surveillance function of Nuc1 that prevents aberrant rRNA and tRNA species from promoting replication of the L-A mycovirus.

molecular biology↗

bioRxiv2026

Feedback control via H3K9 methylation and siRNA biogenesis autonomously maintains RNAi heterochromatin through continuous low-level transcription

The logic of RNA-based silencing involves nascent RNA molecules that serve as scaffolds to recruit proteins that repress transcription. Despite transcription and silencing being antagonistic, RNA-based recruitment mechanisms have been widely adopted in organisms ranging from yeast to humans. In fission yeast (Schizosaccharomyces pombe), the nuclear RNAi pathway that establishes heterochromatin at pericentromeric repeats to silence transcription exemplifies this paradox. Current models propose that cells reconcile these opposing activities by confining pericentromeric transcription to S phase while maintaining silencing during the rest of the cell cycle. Here, using strand-specific single-molecule RNA FISH (smFISH), we detect forward and reverse pericentromeric transcripts with high sensitivity and single-cell resolution. We find that repeat-associated transcription is rare, heterogeneous and happens independently of cell-cycle control. Instead, transcript production is governed solely by feedback involving small RNA biogenesis and H3K9 methylation. By combining mathematical modeling with experiments, we show that low levels of H3K9me or siRNAs prevent heterochromatin establishment, whereas high levels of H3K9me cause stochastic switching. Our work reveals that the same RNA-based feedback architecture can sample distinct transcriptional regimes, with feedback strength determining how often cells produce transcripts without any external regulatory inputs.

molecular biology↗

bioRxiv2026

Glutamatergic and Redox Abnormalities in Major Depressive Disorder: Links to Childhood Maltreatment and Perceived Stress

Background: Childhood sexual abuse (CSA) increases the risk for major depressive disorder (MDD), yet the underlying neurobiological mechanisms remain poorly understood. We examined (1) associations of medial prefrontal cortex (mPFC) glutamatergic metabolites and redox balance with childhood maltreatment and MDD; and (2) whether these neurochemical measures predicted future perceived stress. Methods: Using proton and phosphorus 4T magnetic resonance spectroscopy, we quantified glutamate, glutamine, their sum (Glx), and the NAD/NADH ratio within the same mPFC voxel in 71 women across four groups: healthy controls (HC; n=27), current MDD without CSA (n=14), current MDD with CSA (n=14), and remitted MDD with CSA (n=16). Results: Glutamate and Glx levels were lower in both CSA-exposed groups (n=30) compared with HC (n=27), whereas women with MDD without CSA did not differ significantly from HC. Among CSA-exposed women, greater maltreatment severity was associated with lower glutamine, and greater maltreatment multiplicity with lower glutamate and Glx, specifically in women with current MDD. The association between depressive symptom severity and glutamine differed by CSA history. A significant interaction between baseline NAD/NADH ratio and clinical group predicted perceived stress at 6-month follow-up, adjusting for baseline perceived stress. Specifically, lower baseline NAD/NADH ratio predicted greater subsequent perceived stress in MDD without CSA, but not in the other clinical groups. Conclusions: Our findings suggest that mPFC glutamatergic metabolites and redox balance show distinct patterns of association with CSA history, depression status, and subsequent perceived stress, highlighting neurochemical heterogeneity in MDD and molecular markers of risk.

neuroscience↗

bioRxiv2026

E. coli-produced nanobody fusions as a pan-serotype platform for AAV purification, quantification, and detection

Research, production and application of the recombinant adeno-associated virus (rAAV) require versatile tools for rapid and cost-effective advances. We developed fusion constructs with the pan-serotype, AAV-binding VHH-antibody-fragment named nanobody AAVX and devised procedures for AAV purification, quantification, and detection. Starting from an amino acid sequence, we cytosolically expressed in E. coli nanobody AAVX carrying various tags and fusion partners. Very efficient soluble production followed by a one-step immobilized metal-ion affinity chromatography (IMAC) yielded mostly >90% pure protein. Crosslinking AAVX with an agarose matrix enabled efficient purification of intact AAV2 and AAV5 capsids from cell lysates, as assessed by qPCR and ELISA. AAVX also supported effective AAV quantification by ELISA, as capture reagent and, when fused to luciferase, as detection reagent. Using tagged AAVX and streptavidin biosensors, we developed a rapid and label-free quantification assay of AAV capsids by biolayer interferometry (BLI). Finally, immunostaining with an AAVX-eGFP fusion visualized AAV capsids in fixed HEK-293 cells by fluorescence microscopy. Collectively, these results establish nanobody AAVX fusion proteins as versatile and readily customizable tools for AAV research and bioprocessing.

synthetic biology↗

bioRxiv2026

Few-shot regulatory sequence design with a genomic language model

A central challenge in generative biology is translating knowledge learned from natural sequences and experiments into designs with desired functions. In-context learning (ICL) is an emergent capability of large language models that allows them to adapt to new tasks from a few examples without additional training, enabling these models to become general-purpose learners. Here, we show that the genome language model, Evo 2, exhibits generative ICL in biological sequence design. Prompting with just tens of examples conditions Evo 2 to generate novel and diverse regulatory DNA sequences without any additional training. Mechanistic analysis suggests that genomic ICL may originate from induction, similar to phenomena observed in natural language. Reinforcement learning leveraging sequence-to-function models as experiment-derived rewards further strengthens ICL. We first validate this approach by generating enhancers that drive cell-type-specific gene expression in three human cell lines. Extending this approach in vivo, enhancers designed using human regulatory element data selectively label layer 5 extratelencephalic neurons in the mouse cortex with high specificity and stronger activity than existing enhancers. Our study provides evidence of emergent properties in genomic sequence models and introduces a promising approach for improving functional conditioning in generative biology.

bioengineering↗

bioRxiv2026

Anatomically matched spatial comparisons identify Trps1 as a regulator of knee interzone formation

How the interzone forms and gives rise to articular cartilage, ligaments, and synovium is incompletely understood. Using our previously generated spatial and single-cell transcriptomic atlas of the mouse hindlimb, we compared the knee interzone with neighboring tissues and with the ankle interzone at matched positions, and identified Trps1 as a knee interzone-enriched candidate. Pseudotime analysis placed Trps1-high cells in a transitional state between chondrogenic progenitors and matrix-producing chondrocytes, with higher chondrogenic and lower fibroblastic program scores. Targeted Trps1 knockdown in hindlimb explants disrupted joint formation, permitted Col2a1 expression within the interzone, and reduced adjacent cartilage formation. The associated chondrogenic program was conserved in the human embryonic knee, whereas TRPS1 expression in osteoarthritic cartilage was associated with a fibroblastic matrix program. These results support a role for Trps1 in knee interzone development and show that anatomically matched spatial comparison with explant perturbation can nominate and test joint regulators.

developmental biology↗

bioRxiv2026

Salience network expansion as a transdiagnostic neural marker for psychopathology risk in adolescence

Increased cortical surface area of the brain's salience network (SAL) has been observed in depression and appears to emerge during early adolescence. However, similar expansion has also been reported in bipolar disorder (BD), obsessive compulsive disorder (OCD), and other atypicalities of salience network function have been broadly implicated across multiple psychiatric conditions. Whether increased SAL surface area is specific to depression or related more generally with psychopathology remains unclear. Using data collected as part of the Adolescent Brain Cognitive Development (ABCD) Study (baseline: N = 7942; ages 9-10; Year 2: N = 5901; ages 11-12), we quantified individual SAL size and examined associations with dimensional symptoms from the Child Behavior Checklist. SAL surface area showed substantial inter-individual variability, with expansion occurring primarily through encroachment into the default mode (DMN), action-mode (AMN), and frontoparietal (FP) networks. Larger SAL size showed a modest positive association with depressive symptoms in the baseline sample, consistent with prior reports; however, this association did not replicate in the year 2 internal replication sample. Instead, associations with total problems were stronger and replicated across both timepoints, suggesting a subtle, dimensional relationship between SAL organization and general psychopathology burden. These findings suggest that individual differences in SAL organization may represent a broader transdiagnostic neural correlate of dimensional psychopathology rather than a disorder-specific neural signature.

neuroscience↗

bioRxiv2026

On the asymbiotic germination of some New Zealand spider orchids (Corybas).

Corybas is a large and widespread genus of terrestrial orchids, but little has been done to understand their germination. We tested germination of five species of helmet orchid (C. cheesemanii, C. dienemus, C. iridescens, C. macranthus and C. oblongus) on a range of asymbiotic media. Corybas macranthus germinated on four of seven tested media. From other species there was limited germination; for C. cheesemanii germination occurred for only two of five capsules collected concurrently and sown under the same conditions. The failure of filled seeds to germinate under optimal conditions could suggests the presence of seed dormancy. We trialed desiccation and warm moist stratification as methods to break dormancy, however this was unsuccessful.

plant biology↗

bioRxiv2026

Single-nucleus transcriptomics links telencephalic cell signatures to ocean acidification effects on social behaviour in a coral reef fish

Mutualistic interactions are fundamental to the health and stability of reef ecosystems. While elevated CO2 has been implicated in impairments in interaction behaviours, how this environmental change alters cellular states and intercellular communication in the brain remains unresolved. Here, by analysing the single-nucleus RNA profile of ~66,000 telencephalic nuclei from a keystone mutualist, the cleaner wrasse (Labroides dimidiatus), interacting under ambient or elevated CO2 conditions, we identify highly cell type specific neural responses to elevated CO2. We highlight a substantial depletion in Oligodendrocyte Precursor Cells (OPCs), notably linked to oligodendrocyte myelination and neural structure maintenance. Elevated CO2 also elicited targeted transcriptional shifts across discrete cell types, characterised by widespread glutamatergic signalling and altered co-expression networks associated with pH regulation, energetic metabolism, and circadian rhythm, thereby compromising the cleaner's physiologic equilibrium. Additionally, cell-cell communication was altered under elevated CO2, including remodelling in SEMA4 and PTPR signalling pathways within radial glial and OPCs, respectively, revealing network signalling rewiring in response to the changed environment. These findings uncover the high-resolution neural substrates vulnerable to ocean acidification, providing mechanistic insight into how climate-driven environmental changes alter brain function and destabilise complex mutualistic ecological interactions.

molecular biology↗

bioRxiv2026

Identification of TM4SF1 as a novel tumor-associated antigen in biliary tract cancers targetable by CAR-T cell therapy

Background & Aims: Biliary tract cancers (BTCs) are a rare set of genetically heterogenous and aggressive malignancies associated with delayed presentation and poor prognosis. Thus, there is an unmet clinical need for the development of novel therapeutic strategies. Identification of uniquely expressed cell-surface, tumor-associated antigens (TAAs) holds promise in epithelial tumors more generally, as they can serve as ligands for a variety of therapeutics, including CAR-T cell therapy. However, TAA identification in BTCs has been limited by expression in normal liver tissue. Here, we aim to characterize the cell-surface protein transmembrane 4 L six family member 1 (TM4SF1) as a potential BTC TAA targetable by CAR-T therapy. Methods: TM4SF1 expression in BTC relative to normal hepatic/biliary tissue was characterized at the mRNA level by analyzing TCGA datasets and at the protein level by immunohistochemistry of archival primary BTC tissue. Anti-TM4SF1 CAR-T cells were evaluated through in vitro coculture assays against human-derived BTC cell lines and in a cell line-derived xenograft (CDX) model. Results: We find TM4SF1 is significantly upregulated in BTC relative to normal hepatic/biliary tissue and across BTC anatomical subtypes at the mRNA and protein level. To validate TM4SF1 as a targetable TAA in BTC, we show anti-TM4SF1 CAR-T cells demonstrate robust growth inhibition of BTC cell lines in vitro and significant anti-tumor activity against a CDX in vivo. Conclusions: Together, our data provides support for TM4SF1 as a promising TAA in BTCs that can be used for the rational development of targeted therapeutic modalities, including CAR-T cells, in a patient population with significant unmet need.

cancer biology↗

bioRxiv2026

Compost from urban biowaste shapes the bacterial community and its functional traits in the rhizosphere of tomato plants

Industrial compost derived from urban biowaste is a sustainable biofertilizer made from food waste and plant matter. It has long been used as a fertilizer in agriculture and horticulture for its rich nutrient content. However, the composition of the microbiota in industrial biowaste compost has been studied only partially, and the effects of this bacterial community on soil, rhizosphere, and plant root communities have received limited attention. In this study, we conducted community and functional analyses to examine how the microbiota in biowaste compost influences bacterial diversity, community composition, and metabolic potential in tomato plants, compared with plants fertilized with conventional, unsustainable chemical fertilizer. Source contribution analysis tracking bacterial taxa indicated a significant contribution of compost-derived taxa to the soil, rhizosphere, and roots. The community analysis revealed distinct clustering by fertilizer type, with biowaste compost promoting a unique, more diverse rhizosphere community, enriched in Firmicutes species. Functional analysis highlighted increased microbial pathways in the rhizosphere treated with biowaste compost, including enzymes for cellulolytic and xylanolytic activity, nitrogenase, phytase, and 1-ACC deaminase. Overall, food-derived industrial biowaste compost enhances bacterial diversity and the predicted functional and plant growth-promoting potential in the rhizosphere of tomato plants, underscoring its potential as a sustainable biofertilizer that supports soil health, microbial biodiversity, and crop productivity.

plant biology↗

bioRxiv2026

Characterizing risky play-like behaviour in juvenile versus young adult rats

Play is an evolutionarily conserved behaviour that contributes to healthy behavioural and neural development. While animal play is traditionally categorized as locomotor, object, or social play, developmental research in children has highlighted the importance of risky play, defined as voluntary engagement with uncertainty and manageable physical risk. Whether similar forms of behaviour occur in non-human animals remains largely unexplored. The present study aimed to establish an ethological framework for identifying and quantifying risky play-like behaviour in rats. Juvenile and young adult male Lister Hooded rats were repeatedly exposed to a custom-designed environment containing elevated and unstable structures that provided opportunities for climbing, balancing, hanging, jumping and social interaction. Behaviour was quantified using two complementary ethograms: a location-based ethogram describing the animal's position within the apparatus and a risky event ethogram capturing selected behaviours associated with physical challenge. By comparing the behaviour of animals during the natural peak of play behaviour with animals beyond this developmental period, we aimed to evaluate whether engagement with elevated and unstable environments reflects play-related processes rather than environmental complexity alone. This study provides a behavioural framework for investigating risky play-like behaviour in rodents and establishes a foundation for future studies examining its developmental and neurobiological significance.

animal behavior and cognition↗

bioRxiv2026

Two genomes in one mitochondrion: mitochondrial heteroplasmy and mitogenome dynamics in Chlamydomonas reinhardtii

The mitochondrial genome of the model green alga Chlamydomonas reinhardtii has long been described as a 15.8-kb linear molecule capped by inverted telomeres. By leveraging high-accuracy long-read sequencing to overcome limitations associated with conventional genome algorithm biases, we demonstrated that this classical paradigm was incomplete. Our analyses provided no evidence for canonical telomeric structures, instead revealed widespread structural heteroplasmy characterized by the coexistence of two distinct mitochondrial genome configurations. In addition to the historical 15.8-kb configuration (designated as config 2), we identified an expanded 19-kb configuration (config 1) containing 4,001-bp terminal inverted repeats that duplicate the cob and nad4 genes. Single-molecule long-read profiling captured these configurations as a dynamic network of multimeric mitochondrial DNA concatemers bridged with a conserved 17-bp homologous motif. Together, these structural features support a recombination-dependent replication (RDR) model that could enable mitochondrial DNA replication in the absence of conventional telomeric structures and reverse transcriptase-like (rtl) genes. We further identify widespread nuclear mitochondrial DNA segments (NUMTs), which complicate targeted amplification and quantification of these mitogenome configurations. Specifying NUMTs enabled configuration-specific digital PCR measurements across the CC-5325 lineage and additional field-isolated Chlamydomonas strains, revealing substantial variation in the relative abundance of the two mitogenome configurations. These findings revise a four-decade-old model of the C. reinhardtii mitogenome and provided a new framework for investigating mitochondrial genome architecture, structural heteroplasmy, and replication dynamics in photosynthetic eukaryotes.

plant biology↗

bioRxiv2026

A portable C-terminal rheostat calibrates RecE/Redα exonuclease activity for enhanced bacterial recombineering

Recombineering enables precise genome engineering across diverse bacteria, yet efficient tools for large DNA integration remain underdeveloped for Streptomyces and other biotechnologically relevant bacteria. Here we identify a panel of highly active RecET recombinase pairs from actinobacteriophages that mediate efficient homologous recombination in Streptomyces. The lead candidate, Che9cRecET, supports genomic integration of kilobase-scale DNA with only 50bp homology arms and enables accurate insertion of 79kb biosynthetic gene clusters, far outperforming conventional genome editing approaches. Structural and biochemical analyses reveal Che9cRecE as a distinct C3-symmetric toroidal homotrimeric 5'-3' exonuclease, whose extended C-terminal loop acts as a tunable rheostat calibrating exonuclease activity to an optimal window for recombination. We further show this portable C-terminal loop enhances recombineering efficiency across phylogenetically diverse exonucleases including Escherichia coli Redalpha/RecE and Pseudomonas A. Our work uncovers a conserved regulatory mechanism of the RecE/Redalpha exonuclease family and delivers a generalizable strategy for advancing bacterial genome editing.

molecular biology↗

bioRxiv2026

Speed judgments during smooth pursuit reflect low gain efference copy

During smooth pursuit eye movements, the visual system must compensate for self-generated retinal motion to achieve stable perception of motion in the environment. This compensation primarily depends on an efferent copy of motor commands. Systematically under- or overweighting the strength of efference copy should lead to correlations between eye movement velocity and perceived object velocity. However, previous examinations of these trial-by-trial perceptual correlations have produced mixed results. It remains unclear how effectively the brain compensates for eye movement induced retinal motion during pursuit, and what this reveals about the way efference copy influences perception. To address this, we developed a speed perturbation paradigm in which 29 participants tracked a moving target that underwent brief speed changes. On each trial, we recorded eye velocity along with perceptual judgments of the sign of the speed change (faster or slower) and confidence ratings for those judgments. Across participants, perceptual and oculomotor sensitivity were correlated (r =0.53), but trial-by-trial perceptual and oculomotor responses appeared independent. A logistic regression model incorporating efference copy gain to predict perceptual reports from stimulus speed and eye speed revealed systematic under-compensation for eye movements; faster eye movements were associated with lower changes in perceived speed. Confidence ratings were well-calibrated to perceptual accuracy and showed a consistent negative relationship with eye movement speed. Collectively, these results are consistent with systematic under compensation for eye movements through the use of efference copy during motion perception.

physiology↗

bioRxiv2026

Item-specific clustering of high gamma-band activity on theta phase during speech categorization

Theta-gamma phase-amplitude coupling (PAC) has been proposed as a mechanism for separating neural representations in time. Although item-specific PAC has been reported, its role in perception remains unclear. Notably, previous work has shown that theta phase can bias perception, raising the question whether such biases are linked to phase-specific gamma activity. Here, we investigated whether phase-dependent perceptual biases are accompanied by item-specific theta-gamma PAC. We presented ambiguous speech sounds that could be interpreted as either /da/ or /ga/ in patients with epilepsy implanted with intracranial electrocorticography. We found that pre-stimulus theta phase predicted both the neural and perceptual response, with /da/ and /ga/ tuned channels exhibiting gamma activity clustering at distinct theta phases. These findings suggest that neural and perceptual biases stem from phase-specific pre-activation of item-specific neural representations. They demonstrate the generalizability of the theta-gamma code, shaping behavior and enhancing representational capacity.

neuroscience↗

bioRxiv2026

The influence of drug dosing regimen on extinction and adaptation during experimental evolution in Candida albicans

The antifungal azole drug fluconazole (FLC) is frequently prescribed for treating Candida albicans infections. Azole drugs are typically fungistatic; they inhibit cell growth rather than kill susceptible cells within a population. Therefore, fungal populations can adapt to fluconazole through increased drug resistance or increased drug tolerance (the ability of a subpopulation of susceptible cells to grow slowly at inhibitory drug concentrations). To our knowledge, no prior experiments have examined how different factors influence extinction propensity, although previous in vitro studies show that both drug concentration and strain background can influence adaptation. To determine how the drug dosing regimen, namely fluconazole concentration and transfer duration (i.e., the amount of time between transfer into fresh medium), influenced extinction and adaptation in Candida albicans, we conducted a fully factorial series of short in vitro evolution experiments. Dosing regimen significantly influenced extinction: all replicates evolved at all drug concentrations survived the 48 h and 72 h transfer experiments, yet the extinction rate increased sharply from low to high drug concentration with 24 h transfers. Increases in drug tolerance were frequent regardless of the dosing regimen. In contrast, drug resistance and growth rate both tended to decrease following evolution at higher drug concentrations. Fluconazole-evolved replicates exhibited frequent increases in cross-tolerance and decreases in cross-resistance in a panel of other azole drugs. Genome size changes were common in some strain backgrounds evolved to higher fluconazole concentrations, regardless of transfer times. Combined, our results indicate that many experimental factors can influence the trajectory of adaptation, and show that extinction in a fungistatic drug occurs readily at high drug concentrations with rapid transfers.

microbiology↗