Chromosome-scale assemblies are increasingly available for non-model organisms, but functional annotation remains limited when deep evolutionary divergence erodes primary amino-acid sequence identity even though protein structural similarity can remain conserved. We present a hybrid annotation framework that decouples gene-model discovery from cross-species similarity assignment by combining Evo2-based ab initio prediction of exon-intron structures with ESM-2 protein-embedding-based structural similarity mapping. Applied to the sea lamprey, the framework derives high- or medium-confidence cross-species similarity assignments for 73,485 Evo2-derived translated protein models, including 35,395 high-confidence calls, and expands the deduplicated structural catalog to 31,286 loci, including 20,871 additions absent from the Ensembl baseline. A joint alignment-structure classification identifies 21,391 structurally supported catalog loci that a fixed human DIAMOND protein search does not confidently assign on its own, including 21,184 loci with no detectable human protein-sequence match and 207 loci with only low-confidence matches in the classical 20-30% amino-acid-identity twilight zone. These rescue-space totals describe catalog loci rather than validated one-to-one human-absent genes. In a single-cell RNA sequencing application, a stricter UTR-aware Ensembl+Evo2 reference improves gene recovery and expands the interpretable feature space of the lamprey immune compartment relative to the Ensembl baseline. This enables more resolved annotation of four transcriptionally defined immune cell states, including VLRA+-associated T-like and VLRB+-associated B-like programs together with oxidative iron-handling and iron-associated VLR-linked states. Together, these results show that structural protein signal often persists beyond the limits of pairwise sequence alignment and that an embedding-based annotation layer can extend that signal to improve downstream comparative and…
Science Journals
Birds and mammals are shrinking and shapeshifting as global temperatures rise. Ecogeographic rules predict that such changes should ease heat stress by increasing surface-area-to-volume ratios, and thus, the capacity for heat exchange. This has led to the hypothesis that body size reductions are driven by thermoregulatory selection or adaptive plasticity, although recent syntheses point to more complex, multifactorial causes. Crucially, recent theoretical models predict that thermoregulatory benefits of smaller body size only emerge at extreme deviations from average phenotypes. Here, we exploit agricultural selection in Japanese quail to directly test this hypothesis, using three breeds spanning extreme differences in body mass, surface area, and relative appendage lengths. Evaporative cooling capacity and the scope for evaporative water loss broadly followed allometric predictions when contrasting small and larger breeds. As expected, this allowed the smallest breed to tolerate higher air temperatures. However, differences in heat tolerance limits between breeds were consistently much smaller than predicted. Additionally, the breadth of thermoneutral zones overlapped in full, and upper critical temperatures were remarkably similar, between breeds. Together, these results show that heat tolerance is only weakly linked to surface-area-to-volume relationships and cannot be explained by size alone. Thus, although smaller bodies may modestly enhance heat dissipation when size variation in a population is substantial, our findings suggest that recent body size reductions and morphological shifts are unlikely to be driven primarily by thermoregulatory benefits.
Biological sequences are known to be not random. Thus, the comparison of in silico restriction fragment distributions of random and biological sequences may be an indicator of this non-randomness. Our analyses show that for most of the tested combinations of restriction enzyme and genome sequence the fragments per Megabase of the biological sequence deviate at least more then 10% from the corresponding random sequence. This deviation goes into both directions, i.e. clearly increased values are as common as clearly decreased values. Although there is no species- or restriction-enzyme-specific effect, a clear impact of the GC content both of the restriction site and of the genome sequence can be seen. In contrast to the random sequences, the genome sequences show distinct peaks in their fragment length distributions, hinting to repetitive elements such as transposons.
Holcosus orcesi, the Orces Blue Whiptail, is a Critically Endangered lizard endemic to the upper Jubones River basin in southern Ecuador. Restricted to a narrow elevational range within semi-arid Andean shrublands, it represents one of the few montane members of a predominantly lowland lineage. Here we present the first high-quality reference genome for H. orcesi, generated using Oxford Nanopore Technologies long-read sequencing. The assembly spans 1.68 Gb across only 91 contigs, with an N50 of 76.2 Mb and a BUSCO completeness of 96.8%, making it among the most contiguous and complete squamate genomes to date. Structural annotation predicted 25,682 genes, of which 85% showed homology to known proteins and 45% were assigned Gene Ontology terms. Repetitive elements accounted for 46.3% of the genome, with LINEs representing the predominant class. This genome provides a foundational resource for future evolutionary, comparative and conservation-genomic research of H. orcesi and other mountain reptiles, enabling studies of population genomics, local adaptation, and genomic erosion in isolated populations. By expanding the genomic representation of tropical montane reptiles, this work helps address longstanding phylogenetic and geographic gaps in global biodiversity genomics and provides a foundation for evidence-based conservation of H. orcesi and related taxa.
RNA-seq experiments routinely identify thousands of differentially expressed genes, but translating these into biological insights and therapeutic hypotheses often requires integrating multiple tools. Existing web platforms such as iDEP, NetworkAnalyst, and GEPIA2 address individual steps, differential expression, network visualization, or TCGA queries, but lack a unified environment spanning raw data processing to clinical and pharmacological interpretation. TransXplorer (https://www.transxplorer.org) is a freely available web platform that addresses this limitation by integrating the complete RNA-seq analytical workflow. It supports processing from raw FASTQ files using HISAT2 or Salmon, as well as direct GEO dataset import with automated metadata handling. Differential expression analysis is implemented via DESeq2, edgeR, and limma-voom, followed by functional enrichment across more than 1,800 species using Bioconductor resources. Batch effects are automatically detected and corrected using a composite of PVCA, kBET, and Silhouette metrics without requiring predefined batch annotations. Downstream analyses include co-expression network construction (WGCNA), protein-protein interaction mapping (STRING), cell-type deconvolution, and transcription factor inference using integrated DoRothEA and TFLink resources. The platform further links gene signatures to drug candidates through DGIdb and OpenTargets and enables survival and tumour-normal comparisons across TCGA cohorts. Application to cardiac endothelial differentiation (GSE151427) and kidney renal papillary cell carcinoma (TCGA-KIRP) datasets demonstrates accurate batch correction, biologically consistent pathway enrichment, recovery of expected cell-type proportions, and identification of clinically relevant genes and drug candidates. TransXplorer is freely available without a login.
Ecological specialization emerges when adaptation to a focal context increases fitness in that context relative to others. Experimental evolution has been widely used to study microbial specialization in abiotic environments but not predatory specialization. Here we demonstrate evolutionary specialization by a bacterial predator and characterize associated diversification of its predation profile. Populations of the bacterium Myxococcus xanthus evolving on single, non-evolving prey species diverged in predatory performance, showing increased performance on their home prey relative to their common ancestor, and relative to foreign prey not encountered during adaptation. Adaptation to the single-prey environments resulted in striking radiation of performance profiles across a diverse panel of foreign prey that was shaped interactively by selection, chance, and indirect effects, with home-prey identity modulating the degree of stochastic indirect diversification. Despite a great diversity of indirect evolutionary effects, correlated evolution was net-positive, yielding positive predatory specialization as the general outcome. Genomic evolution mirrored phenotypic evolution in that degrees of genomic parallelism differed as a function of home-prey identity. These findings show that adaptation to even simple biotic conditions can generate great ecological and behavioral diversity, linking direct selection, deterministic indirect effects of adaptation, stochasticity, and the origins of predator specialization and diversification.
Strong population contractions can leave a persistent genomic legacy that can influence populations long after their demographic recovery. While bottlenecks facilitate the removal of strongly deleterious mutations, the effectiveness of purging may be limited in historically small populations. The Kirtlands warbler (Setophaga kirtlandii) is a rare North American songbird with an ancestrally small population. After narrowly evading extinction, they are one of few species that have been delisted from federal protections in the USA. Despite their recovery, a previous study showed evidence for recent inbreeding and a high burden of deleterious mutations that may have not been purged despite strong bottlenecks. Historical DNA offers a unique opportunity to understand the consequences of recent demographic declines on genetic diversity. Here, we use DNA from over 100-year-old museum specimens to estimate changes in genetic load in the Kirtlands warblers pre- and post-bottleneck. We validate our results with forward-in-time genetic simulations and explore how sample size and missing data can affect estimates. Both empirical data and simulations suggest a reduced ability to purge deleterious mutations in this historically small population. Our simulations also highlight that limited sampling design and data quality can constrain the ability to detect changes.
In modular organisms, where growth and fragmentation blur the boundaries between individuals, the interplay between asexual and sexual reproduction creates complex fitness trade - offs. Life - history theory predicts that resources allocated to one fitness component necessarily reduce investment in others, yet detecting these trade - offs in wild populations of clonal organisms remains challenging. Phenotypic plasticity can enhance survival, yet its influence on reproductive capacity and life - history trade - offs remains poorly understood. Using a fully crossed reciprocal transplant design, we tracked 263 colonies of the branching coral Acropora cervicornis across nine reef sites over 42 months, investigating relationships between fragmentation, morphological plasticity, and the capacity for sexual reproduction. Breakage patterns reflected both environmental and genetic factors. Primary branch breaks created a "double negative" effect - simultaneously more than doubling mortality risk and delaying attainment of a validated reproductive size class by ~40%. Conversely, higher morphological plasticity in surface area - to - volume ratio accelerated sexual maturation up to 6 - fold, counteracting the negative effects of fragmentation. In parallel, a simple demographic model parameterized with published fecundity data estimated that primary breakage reduces expected cumulative reproductive output by ~58%, a result robust across a wide range of parameter assumptions. These results demonstrate a fundamental reproductive trade-off in which asexual reproduction through fragmentation undermines sexual reproductive potential by reducing colony size. Moreover, our findings reveal that fragmentation susceptibility is broadly heritable and subject to selection, and identify a compensatory mechanism through which plasticity enhances fitness beyond immediate survival.
Cliffs are environmentally extreme yet biodiversity-rich ecosystems that harbour specialist plants, many endemic and threatened. Plant persistence in these nutrient-poor substrates may depend on tightly linked soil- and root-associated microbial communities, which remain poorly understood. These interactions may become increasingly important with the global expansion of recreational climbing. While physical climbing impacts on vegetation are documented, potential chemical effects, from the use of climbing chalk (magnesium carbonate), on soil properties and plant-associated microbiota remain unknown. We sampled soils and roots beneath cliff-specialist and generalist plants, and unvegetated soils, across climbed and unclimbed routes in northern, central, and southern Spain. Soil physicochemical properties were quantified, fungal communities were characterized using ITS-metabarcoding, and structural equation modelling was used to disentangle direct and indirect effects. Climbing increased soil pH and altered soil chemical properties, driving shifts in fungal diversity and functional composition in soil and roots. The relative read abundance of root-associated symbiotrophic fungi declined, whereas arbuscular mycorrhizal fungi and pathogens increased in climbed cliffs. Overall effects were consistent, with cliff-specialist plants mediating nutrient and fungal shifts. Our findings show that climbing can reshape cliff soil chemistry and fungal communities, with potential cascading consequences for plant functional performance, nutrient dynamics, and ecosystem resilience.
Accurate and reproducible assessment of foliar disease severity is essential for evaluating the performance of heterogeneous plant communities and understanding host-pathogen interactions. However, traditional visual scoring methods remain subjective, with limited precision, and difficult to scale in large phenotyping experiments. Here, we present a semi-automated image analysis workflow designed to quantify multiple foliar disease symptoms simultaneously on wheat flag leaves sampled from varietal mixtures. The workflow combines three methodological components: (i) a standardized protocol for leaf sampling and imaging, (ii) supervised machine learning segmentation using Random Forest implemented in Ilastik to classify multiple symptoms (powdery mildew and yellow rust), and (iii) a graphical user interface facilitating pipeline deployment by non-specialist operators. To evaluate the influence of image representation on classification performance, four color spaces (RGB, HSV, HLS, LAB) were systematically compared. The approach was validated using images of durum wheat flag leaves collected from a field experiment assessing eight-way varietal mixtures under natural fungal pressure. Cross-validation against manually annotated images demonstrated high segmentation accuracy across all symptom. Comparison among color spaces revealed only minor differences in performance. Overall, this workflow offers a cost-effective, annotation-efficient and reproducible alternative to deep learning approaches, leveraging open-source and actively maintained tools while requiring limited training data and enabling objective, reproducible and scalable disease phenotyping.
Reef restoration practitioners aim to preserve coral genetic diversity by protecting reefs and cultivating diverse genotypes in coral nurseries. However, cryptic genetic lineages in most corals complicate restoration strategies, as the role of between-lineage genetic divergence remains unclear regarding adaptation. In Montastraea cavernosa, researchers have identified cryptic lineages, some strongly segregated by depth. We conducted a ten-week reciprocal transplantation experiment using two cryptic lineages restricted to shallow water (<10m depth), with one lineage more common on nearshore reefs and the other on offshore reefs. We aimed to quantify lineage-specific responses to the environment that explain the genetic and ecological divergence between the two lineages. Surprisingly, the strongest response was not lineage-specific. Instead, both lineages exhibited strong and similar changes in growth and metabolomic profiles, depending on the transplantation habitat. These results suggest that cryptic lineages employ similar mechanisms of adaptation and acclimatization to environmental challenges, despite their genetic distinction.
Allostery enables proteins to couple environmental signals to functional outputs, yet how allosteric mechanisms diversify during evolution remains poorly understood. Here, we address this question in the ubiquitous and functionally diverse arsenic repressor (ArsR) superfamily by integrating information-theoretic bioinformatics, structural characterization of DNA recognition and NMR measurements of fast internal dynamics. We identify conserved residues that define the structural scaffold of ArsR proteins and subfamily-specific positions that encode inducer and DNA specificity. In the persulfide sensor SqrR, the crystal structure of the DNA-bound complex reveals how operator specificity is encoded by a limited set of residues, consistent with sequence-derived predictions functionally validated by in vitro transcription assays across divergent ArsR regulators. We further show that allosteric inhibition of DNA binding in SqrR occurs without large-scale conformational rearrangements and is instead associated with changes in internal dynamics, as previously observed for the zinc sensor CzrA. Together, these results support a model in which conformational entropy preserves allosteric connectivity while relaxing sequence constraints, thereby enabling functional diversification within a protein superfamily.
Avoiding fertilization with genetically incompatible partners, whether too similar or too divergent, is a central challenge for sexually reproducing organisms. Selection can favor mechanisms acting before and after mating, with postmating processes potentially compensating for constraints on premating choice. In the postmating context, female reproductive fluid (FRF) can modulate sperm performance and bias fertilization outcomes, but its contribution to reproductive isolation remains unclear. We tested whether FRF mediates discrimination against heterospecific and related sperm in two naturally hybridizing sister species of swordtails, Xiphophorus birchmanni and X. malinche, that diverge in premating behavior towards heterospecifics. Effects of FRF differed sharply between species. In X. malinche, FRF enhanced the velocity of conspecific sperm relative to heterospecifics, consistent with postmating discrimination against hybridization. In contrast, FRF in X. birchmanni did not favor conspecific sperm. Evidence for inbreeding avoidance was weaker, and we found no indication of a trade-off between discrimination against genetically similar and dissimilar sperm. These results show that female reproductive fluid can serve as a rapidly evolving axis of reproductive isolation through postmating female choice.
Most species are geographically structured, leaving characteristic signatures in neutral regions of the genome. These signatures can be distorted when neutral regions are linked to deleterious mutations. In such regions, purifying selection can reduce genetic diversity through Background Selection (BGS) or, for recessive mutations, increase diversity through Associative Overdominance (AOD). While the effect of BGS and AOD are well characterized in panmictic populations, their effects remain largely unexplored in structured populations. Here, we investigated an Isolation with Migration model using forward simulations across a range of migration, selection, dominance, and recombination parameters. We first used a genotype-based approach to quantify the effects of deleterious mutations on standard summary statistics ({pi}, Dxy, FST, DAFi). We then showed that an Ancestral Recombination Graph-based approach, tracking tree sequences from a sample of one diploid per deme, recovers the same patterns while directly relating genetic variation to the underlying coalescent processes. When recombination is sufficiently low, we found a BGS-driven regime for weakly co-dominant mutations, characterized by lower diversity and increased genetic differentiation (FST). For recessive mutations, we first identified an AOD-driven regime, characterized by increased diversity and lower FST values followed by a transition to a subsequent BGS-driven regime. Genealogies were similarly impacted by deleterious mutations: BGS shrunk coalescent times and produces a shift towards lineage sorting topologies, while AOD stretched coalescent times and produces a shift toward incomplete lineage-sorting topologies. These patterns were weakened by gene flow, with FST and topologies remaining close to expected under neutrality, while diversity and coalescence times remained robust to demography. Our results provide clear evidence of BGS, AOD, and of their transition in a structured model with gene flow…
Susceptibility to viral infection varies widely but is not fully explained by genetics, immune status, or exposure level. We show that time of day strongly influences infection outcome, with up to 100-fold differences in enteric viral burden depending on infection timing. This temporal gating is abolished in mice lacking a functional circadian clock. We identify the antiviral transcription factor IRF1 as a direct target of the circadian transcription factor BMAL1, resulting in rhythmic expression of a basal antiviral gene program prior to infection. Loss of IRF1 eliminates this program and abrogates time-of-day dependent differences in viral replication. This circuit operates within intestinal myeloid cells, establishing a preexisting antiviral state. These findings indicate that the circadian clock programs host susceptibility in the intestine, before infection occurs.
Linkage disequilibrium (LD) makes causal GWAS variants indistinguishable from correlated neighbours; resolving them is the fine-mapping problem, and the challenge is species-specific: humans face dense ancestry-imbalanced LD, yeast and *Arabidopsis* exceptionally long LD, and crop germplasm sparse and fragmented annotations that defeat human-biobank curation pipelines. Bayesian fine-mappers integrate annotations as flat per-variant priors, discarding the relational structure linking variants to tissue-specific eQTLs, pathways and protein-protein interactions. Hierarchical belief propagation (HBP) on a variant-gene-pathway factor graph matches Bayesian baselines at 5-40x speed; an annotation-adaptive complement, graph-augmented fine-mapping (GAFM), wins 27-2 against SuSiE at weak signal and recovers *LDLR*, *APOE*, *LPL*, *GCKR* and *ANGPTL3* at single-variant resolution across four Pan-UK Biobank ancestries. On the 3,000 Rice Genomes grain weight + shape panel, mixture-prior posterior reweightings of GAFM/HBP and their ensemble (GAFM-MX, HBP-MX, ENS) reach 47.6% top-1-PIP exact-position recovery of 21 panel-matched stable QTNs - the highest of any method, exceeding SuSiE (28.6%) and SBayesRC (14.3%) - at 200-700x SuSiE's per-locus speed. Across 692 leads in four species, a non-uniform per-variant prior, not uniform high coverage, lets the graph break LD ties: adding a regulatory-element flag to an otherwise uniform human cache flips HBP narrower than GAFM from 0% to 88% on 321 Pan-UKB leads. These results recast multi-omics fine-mapping as a non-uniform-prior-curation problem rather than a uniform-coverage problem, and reframe post-GWAS analysis as message passing over biological structure rather than weighted regression on flattened annotations.
Rapid connectivity alterations of thalamic nuclei during initial learning of goal-directed behaviour
The thalamus is essential for learning, dynamically engaging with other subcortical and cerebral cortex regions throughout the learning process. Here, the thalamus serves as a critical connector hub and synchroniser within the thalamocortical system of the brain. However, whilst higher order thalamic nuclei are known to be particularly important for this process, the exact contributions of individual higher order and first order thalamic nuclei, alongside their individual involvement with cortical networks and subcortical regions, remains unexplored within the initial phase of learning. In light of this, we analysed fMRI data obtained within a paradigm which is designed to examine initial learning processes within feedback-driven stimulus-response learning, in order to explore thalamic contributions. We investigated dynamic learning-related functional connectivity alterations between various thalamic nuclei with other subcortical regions and cortical networks. Our results show that the initial phase of learning was associated with: (1) decreasing functional connectivity between thalamic nuclei and frontoparietal and cingulo-opercular networks, (2) increasing functional connectivity between thalamic nuclei with default mode and salience networks, (3) decreasing functional connectivity between thalamic nuclei and the putamen, and (4) decreasing functional connectivity amongst higher order thalamic nuclei. Furthermore (5) these dynamic alterations were associated primarily by mediodorsal thalamus. Altogether, these results indicate that higher order thalamic nuclei play a crucial role within initial learning and in the generation of novel goal-directed behaviour. This was demonstrated through enhanced functional connectivity with selected cortical networks which drive goal-directed behaviour, alongside decreased functional connectivity with striatal regions which drive motor selectivity.
The hippocampus is organized along dorsal-ventral and left-right axes, but whether and how these axes interact within defined neuronal populations across behavioral states remains unresolved. Here, we combined within-animal slice electrophysiology with dual-site fiber photometry to compare dorsal and ventral CA1 activity across contralateral hemispheric configurations in mice expressing CaMKII-jGCaMP8s and SynI-jRCaMP1b at distinct longitudinal sites. Ventral CA1 pyramidal neurons exhibited greater intrinsic excitability and stronger AMPAR-mediated synaptic responses than dorsal CA1 neurons. In vivo, CaMKII-defined pyramidal recordings during home cage rest revealed a left-biased event-rate asymmetry within dorsal but not ventral CA1, with no comparable asymmetry in pan-neuronal SynI recordings. Apparent dorsal-ventral differences in spontaneous event rate were therefore configuration-dependent and resolved into a hemispheric, cell-type-specific effect restricted to the CaMKII-defined population. Lead-lag analysis showed that dorsal-ventral temporal coordination was likewise reorganized across configurations and was restricted to pyramidal-cell-biased recordings. During open-field center entries, dorsal CA1 was preferentially recruited before entry across both configurations, whereas non-coordinated entries revealed a relative post-entry suppression of contralateral ventral CA1. Together, these findings suggest that dorsal-ventral CA1 organization cannot be inferred from hemisphere-pooled designs and identify a pyramidal-cell-specific left dorsal CA1 asymmetry as a structural feature that shapes both spontaneous activity and behaviorally driven recruitment along the longitudinal hippocampal axis.
Nature-Based Solutions are increasingly promoted to address current urban challenges. While their potential effects on vector-borne disease risks have been documented, data on Aedes albopictus, a known arbovirus vector, remain limited in France. A previous study showed that urban vegetation moderately increases the abundance of adult mosquitoes of this species, but the monitoring period lasted only six months. Using ovitraps, we monitored Ae. albopictus egg density dynamics over multiple years (2022 to 2024) and analysed its environmental predictors in various urban environments. We included lagged meteorological variables, land cover metrics, and the cumulated egg densities recorded in the previous weeks as environmental predictors. Both parametric (GLMM) and non-parametric (Random Forest) models were fitted to weekly egg counts per trap. Our findings highlight that (i) egg density dynamics were related to how vegetation classes structured the landscape, (ii) growing degree days and cumulated number of eggs recorded in specific lagged time windows were the main contributors to egg density, and (iii) the non-parametric and parametric models performed similarly in terms of prediction accuracy.
Conventional diagonal stride skiing traditionally includes a glide phase, characterised by a period of relatively passive gliding on one ski. While the glide phase may take advantage of low ski-snow friction, it does not exhibit the same whole-cycle mechanical energy fluctuations seen in running or walking on foot. A new sub-technique, known as running style, substantially reduces the glide phase and may alter the role of elastic tissues, making the movement pattern more similar to uphill running on foot in its temporal organisation. We examined knee extensor and plantar flexor muscle-tendon behaviour in eight competitive skiers performing conventional diagonal and running techniques on a treadmill inclined at 10 deg. Using synchronised ultrasonography, 3D kinematics, ski forces and EMG, we quantified gastrocnemius medialis and vastus lateralis fascicle and muscle-tendon unit (MTU) dynamics in both the running (RUN) and conventional (CON) styles. Shorter glide and total cycle durations during RUN shifted MTU peak length and velocity earlier during the kick phase. Fascicles in both muscles operated at similar velocities across techniques, showing MTU-fascicle decoupling. Vastus lateralis fascicles shortened at higher absolute peak velocities than gastrocnemius in both conditions, while normalised velocities were similar. RUN increased preactivation and advanced EMG timing, while integrated EMG during the kick was lower compared to CON. These findings suggest that, despite large shifts in external mechanics between glide-based and more running-like skiing, elastic tissues may help stabilise fascicle behaviour and preserve a similar contractile strategy across muscles and techniques.
How post-mitotic neurons maintain precise transcription factor (TF) levels throughout life remains a fundamental open question. Here, we challenge the prevailing model of positive autoregulation by demonstrating that UNC-3 (Collier/EBF1-4), a dosage-sensitive TF continuously required for cholinergic motor neuron identity in C. elegans, negatively regulates its own expression. Using genetics, biochemistry, and inducible protein depletion, we show this self-repression occurs directly at the transcriptional level and persists beyond development. CRISPR/Cas9 disruption of negative autoregulation causes motor neuron identity and locomotion defects, establishing its functional necessity. Mechanistically, the UNC-3 DNA-binding domain is required and sufficient for self-repression, with an AlphaFold2 screen implicating chromatin factors as interaction partners. Critically, UNC-3 self-repression is continuously counterbalanced by positive input from the HOX cofactor CEH-20/PBX, revealing a dynamic "balancing act" between opposing regulatory inputs that stabilize TF dosage over time. Mutations in the unc-3 ortholog EBF3 cause a neurodevelopmental syndrome, and disease-associated variants disrupt UNC-3 self-repression, revealing a key molecular mechanism underlying the disorder. We propose that negative autoregulation continuously counteracted by positive input represents a broadly applicable principle for maintaining dosage-sensitive TF expression to secure post-mitotic cell identity.
Despite sharing the same genes and the same environment, individuals often develop substantial phenotypic differences. While this pattern has been documented across diverse species and traits, the processes giving rise to this 'stochastic' or non-shared environmental variation remain unclear. Recent mathematical models of development in which phenotypes are gradually constructed may offer some clues. These models show that imperfect environmental cues can generate striking variation in developmental trajectories and adult phenotypes. At the population level, such imperfect cues produce increasing stability of individual differences across ontogeny (e.g. animal personality) and patterned distributions of mature phenotypes (e.g. normal or skewed) that resemble those observed in real organisms. Our paper synthesizes existing models in which stochastic phenotypic variation arises solely as a by-product of mechanisms missing their phenotypic targets because of imperfect cues. We then link these models to related, but independent, mathematical theory exploring the environmental conditions under which stochastic phenotypic variation is favoured by natural selection. Our integration shows that stochastic sampling is often favoured over classic bet-hedging strategies involving non-plastic generalist or specialist strategies. Our findings provide new directions of research on stochastic sampling as a mechanism for adaptive stochastic variation within and across generations.
WD40 domains are major protein-protein interaction (PPI) scaffolds, yet their contributions to fungal pathogenicity remain poorly defined. We systematically analysed 94 canonical WD40 proteins in Cryptococcus neoformans. Conditional knockdown and sporulation identified 36 essential WD40 proteins, while in vitro and in vivo profiling of 103 signature -tagged deletion strains spanning 52 genes uncovered 31 pathogenicity-related WD40 proteins, including epigenetic and post -transcriptional regulators. We identified Wcp1, a dual-domain protein whose WD40-repeat and cyclophilin domains are required for growth at 37{degrees}C under 5% CO2. Its WD40 scaffold and PPIase domain supported CO2/heat tolerance and virulence. Notably, Wcp1 couples these functions to acidic pH adaptation: wcp1{Delta} failed to grow under elevated temperature and CO2 at acidic pH, exhibited enhanced intracellular acidification, reduced macrophage survival and attenuated virulence in Drosophila and mice. Integrated transcriptomic and proteomic analyses place Wcp1 at the centre of intracellular pH homeostasis, coordinating proton transport, metabolic adaptation and stress-buffering networks.
Advancing the utility of plant synthetic biology requires the continued development of protein engineering tools. Self-assembling protein compartments, such as virus-like particles (VLPs), provide versatile scaffolds for synthetic biology. However, few plant-expressed VLPs have demonstrated broad amenability to protein engineering, restricting their applications to specific contexts. Here, the Salmonella typhimurium bacteriophage P22 VLP is explored as a novel protein scaffold for plant synthetic biology, demonstrating its application in a eukaryote for the first time. Through transient expression in the biofactory plant Nicotiana benthamiana, the capacity for P22 VLPs to correctly assemble and selectively encapsulate recombinant protein cargo is demonstrated. The durability of this protein scaffold is explored, through co-encapsulation of multiple cargo protein species and by encapsulation through direct fusion to the P22 coat protein. Finally, the ability to simultaneously program cargo encapsulation and external protein display on P22 VLPs in vivo is demonstrated through SpyTag/SpyCatcher-mediated protein conjugation. This work demonstrates the broad utility of P22 VLPs as nanoscale protein scaffolds for plant synthetic biology. Keywords: protein scaffolds, cargo encapsulation, protein display, SpyTag/SpyCatcher, transient expression, Nicotiana benthamiana.
Dendritic arbor morphology is shaped in part by interactions with neighboring dendrites, and its geometry strongly influences the spatial distribution and strength of synapses. These observations raise the possibility that local dendritic contacts help determine where synapses accumulate and strengthen. Previous work in cultured hippocampal neurons showed that dendrite-dendrite contact sites are non-random and associated with local synaptic clustering. Here we asked whether a different type of dendritic contact, formed between a dendrite and the soma of a neighboring neuron, behaves similarly. Using dissociated hippocampal cultures, immunofluorescence imaging, time-lapse microscopy, quantitative image analysis, stochastic spatial simulations, and minimal quantitative modeling, we identified three recurrent classes of dendrite-soma interactions (DSIs): dendrites crossing directly over a neighboring soma, growing tangentially along the soma perimeter, or contacting the proximal region where a neighboring dendrite emerges from the soma. These interactions were abundant, occurred exclusively between different neurons, and showed substantial structural persistence over several days. Their overall frequency exceeded stochastic predictions across culture densities, and two configurations - proximal and tangential contacts - were selectively enriched above random expectation, whereas soma-crossing contacts were largely consistent with stochastic overlap. DSI composition also changed over development, with proximal contacts becoming progressively more prevalent. At DSI sites, synaptophysin-positive puncta were significantly denser and more intense than on non-interacting dendritic segments, consistent with local enrichment and strengthening of presynaptic specializations. Minimal modeling further indicated that biased formation together with developmental stabilization explains the observed organization better than stochastic geometry alone. These findings identify DSIs a…