Evolutionary Applications

Background: Spatial organization is increasingly recognized as a key determinant of tumor-immune interactions in head and neck squamous cell carcinoma (HNSCC). The GSE300147 Xenium spatial transcriptomic resource generated by McCord and colleagues established a framework for mapping spatially coordinated T-cell states in HNSCC. However, how tumor-enriched epithelial immune states relate to metabolic, redox, and stress-adaptive transcript programs remains incompletely defined. Methods: A secondary, data-driven reanalysis of GSE300147 was performed, focusing on 17 confirmed HNSCC Xenium sections after exclusion of a non-HNSCC ameloblastoma specimen. A total of 1,148,244 cells were analyzed, including 558,867 EpCAM+ tumor-enriched epithelial cells. Tumor-enriched epithelial cells were classified into Hot, Intermediate, and Cold states using a Composite Hotness framework integrating T-cell inflammatory signature score, checkpoint-associated signaling, CD274 expression, IFN/antigen-presentation signature score (IFN/AP), and tumor-immune proximity. Six metabolic ecosystem states, neighborhood profiling, spatial permutation testing, and an integrated Immune-Metabolic-Redox Ecosystem Score (IMRES) were then applied. Results: Immune activation was spatially heterogeneous across HNSCC sections. Immune-hot tumor-enriched epithelial regions showed not only inflammatory, checkpoint-associated, and antigen-presentation signature scores, but also coordinated metabolic, oxidative-redox, and stress-response transcript programs. IMRES, derived from available immune, metabolic, redox, and stress-response transcript components represented in the Xenium panel, increased progressively from Cold to Intermediate to Hot tumor-enriched epithelial states and was associated with NFE2L2, GDF15, HLA-DRA, CD274, KEAP1, and MDM2. Integrating IMRES with Composite Hotness identified a distinct Hot+IMREShigh ecosystem comprising 106,874 tumor-enriched epithelial cells. This state showed the strongest immune-active and stress-adaptive features and was positioned closer to immune populations than expected by random assignment. An alternative rank-based robustness analysis reproduced the IMRES-associated ecosystem axis and correlated with the original module-based score (Spearman r = 0.597). Conclusions: This secondary reanalysis extends the original spatial T-cell framework by defining a complementary tumor-centered immune-metabolic-redox ecosystem in HNSCC. IMRES provides a transcript-derived framework for identifying Hot+IMREShigh neighborhoods where immune activation, checkpoint signaling, metabolic remodeling, and stress adaptation converge, providing a hypothesis-generating framework for studying immune resistance and therapeutic vulnerability.

Context Pumping stations pose a threat to fish globally through land use change, habitat fragmentation and entrainment risk, with the catadromous and critically endangered European eel particularly impacted. Objectives/methods Establish, model, assess and understand the present-day distribution of European eel and resident fishes in 152 pumping station catchments in a once extensive wetland (The Fens) using eDNA metabarcoding (855 samples over two and half years), with specific focus on anthropogenic influences on hydrological connectivity and habitat quality. A removal survey design maximised confidence in negative results while minimising time and consumable costs. Results Eel occurrence upstream of pumping stations was low (occupancy = 28.3%) and positively associated with catchment area, fish species richness and natural hydrological connectivity (gravity drainage or flooding) and negatively associated with distance from the tidal limit. Fish species richness replaced catchment area and improved model performance, potentially acting as a biotic indicator of habitat quality and connectivity. Pumped catchments with manually operated upstream water transfers had reduced eel presence, potentially linked to the direction of water flow or the timing of operation. By contrast, fish species richness increased in these catchments during summer, suggesting displacement into unsuitable long-term habitats. Physical habitat maintenance had no detectable effect on eel occurrence or fish species richness. Conclusions This study provides the first landscape-scale assessment of European eel distribution and drivers of occurrence in pumped river catchments. The highly novel and comprehensive insights have implications for European eel conservation as well as infrastructure and catchment management, including compliance with legislation (EC Regulation No. 1100/2007).

Elucidating the biological and molecular mechanisms that govern male fertility and mating behavior in mosquitoes is critical for optimizing genetic and sterile insect technique-based vector control strategies. Here, we examined age-related changes in male reproductive capacity in Anopheles culicifacies, using female egg output as an indirect indicator of male fertility. Our results demonstrated that male reproductive age follows a non-linear pattern of fertility. Morphometric analysis from emergence to day 13 post-eclosion revealed a strong correlation between seminal vesicle capacity and female fecundity, suggesting that age-dependent gonadal development directly influences reproductive potential. At the molecular level, we identified AcLITAF6 as a key regulator of male reproductive homeostasis. RNAi-mediated knockdown of AcLITAF6 impaired apoptosis-associated and phagocytic clearance, reduced sperm viability, and decreased female productive outcomes. Conclusively, we reveal a previously unrecognized role of LITAF in sperm quality control and male reproductive fitness, highlighting AcLITAF6 as a potential target for mosquito population suppression strategies.

Squid abundance has been reported to increase globally between 1970 and 2010. This increase has been hypothesized to result from two primary factors: the loss of top predators due to overfishing and rising ocean temperatures. The decline in apex predators may lead to the expansion of squid populations either through reduced predation pressure or diminished competition with juvenile predators. Concurrently, increased temperatures could enhance the somatic growth rates of squid, thereby accelerating their population growth. However, empirically disentangling the impacts of predator loss and temperature on squid biomass remains challenging, especially in a food-web context. In this study, we used a size- and trait-based model of upper trophic levels that resolves the ecosystem structure -- biomass and trophic interactions of fish and squid -- for varying depth, temperature, and secondary production, to investigate two hypotheses of the historical expansion of squid, i.e., the effects of predator depletion from fishing and rising temperatures on squid biomass. Our model reveals that intensified fishing of squid predators -- specifically large demersal fish in shelf systems and large pelagic fish in open oceans -- leads to a slight increase in squid biomass. Conversely, elevated temperatures are associated with a decline in squid biomass. This temperature-driven reduction in biomass is attributed to an increased metabolism of squids beyond the available food supply. If historic overfishing on large marine predators continues to be curtailed, we expect a corresponding reduction in global squid biomass and fisheries potential, which could be further exacerbated by rising temperatures.

Genomic instability is a major driver of tumor evolution, promoting diversification and adaptation while simultaneously increasing the accumulation of deleterious alterations. How tumor populations balance these opposing effects remains poorly understood. Here, we introduce a computational framework that explicitly represents diploid genomes, functional gene classes, point mutations, and chromosome-segregation errors in spatially constrained and well-mixed tumor populations. We identify a viability boundary separating sustained tumor expansion from instability-induced population collapse. Within the viable regime, mutation and selection generate a stable distribution of genomic-instability classes that is accurately captured by an analytical replicator--mutator description. Near the viability boundary, tumor dynamics exhibit prolonged extinction transients and strong sensitivity to stochastic fluctuations, with important differences between solid and liquid architectures. Chromosomal alterations further modify growth by creating transient benefits through increased gene dosage and genetic redundancy, while ultimately increasing genomic fragility. Finally, simulated interventions show that eliminating low-instability subpopulations or increasing the global mutational burden can displace tumors beyond their viability boundary and trigger irreversible collapse. These results identify genome instability as both an evolutionary advantage and an intrinsic vulnerability, providing a quantitative framework for developing therapies that exploit the limits of tumor evolution.

Pancreatic ductal adenocarcinoma (PDAC) harbors a distinct intratumoral microbiome. Yet rigorous characterization of its composition is hampered by pervasive environmental and procedural contamination. Sources of contamination have not been thoroughly explored, and the methods of decontamination have not been sufficiently evaluated in a benchmarking manner. We systematically collected >300 negative control (NCT) samples comprising paraffin from formalin-fixed paraffin-embedded (FFPE) samples, lysis buffer and sterile water over a period of four years processed by different laboratory persons (LP). All samples were sequenced using full-length 16S rRNA gene sequencing with Oxford-Nanopore Technologies. We benchmarked four decontamination methods (restrictive filtering, decontam, SCRuB, and the Nejman et al.-derived (Nj) pipeline) against fresh-frozen tumor samples (FF) from LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) mice, using the abovementioned contamination assessment to calculate a composite score for the assessment. Further, we validated those methods via technical replicates. Microbial profiles of NCT samples were significantly determined by control type, LP, year and season reflecting complex batch effects. The 15 most abundant contaminants spanned well-characterized environmental taxa and human commensals from the oral cavity. The LP processing samples left a significant microbial trace highly contributing to the batch effect. Decontamination benchmarking demonstrated that the Nj method consistently outperformed alternatives in both composite score and inter-replicate concordance. Application of Nj to fresh frozen PDAC samples substantially reduced contaminant burden while preserving putative tumor-associated signals in FF but not FFPE samples. Our results support the adoption of the Nj decontamination approach for future intratumoral microbiome studies in fresh frozen tumor samples.

Rapid evolution allows populations to persist in environments where they would otherwise go extinct. This phenomenon, known as evolutionary rescue, is typically studied in the framework of biological evolution, yet adaptive traits can also arise and spread through cultural evolution. The present study developed a stochastic eco-evolutionary model to compare rescue probabilities through biological and cultural evolution. Transmission bias governed the rescue probability under cultural evolution by setting how readily a rare adaptive trait was copied. Conformity bias suppressed population persistence because a rare trait was the least likely to be copied. Content bias toward the adaptive trait enabled evolutionary rescue when social learning was rapid, but it typically yielded a lower rescue probability than biological evolution. Only anticonformity bias, together with a high social learning rate, exceeded the rescue probability of biological evolution by enabling the adaptive trait to be established more rapidly. These results demonstrate that transmission bias alters the demographic consequences of cultural evolution and highlight the importance of transmission processes in evolutionary rescue theory. Understanding how adaptive behaviours are socially transmitted may also improve predictions of animal population persistence and inform conservation efforts in rapidly changing environments.

Heritable bacterial symbionts are pervasive in terrestrial arthropods, often imposing reproductive manipulations to promote their own spread within host populations. Co-infections are common, potentially allowing symbiont co-infectors to hitchhike through a host population. However, adverse thermal conditions can disrupt these communities, particularly when co-infectors vary in their thermal sensitivity. We used a multi-generation experiment to test whether warm (29 {degrees}C) conditions disrupted spread of heritable symbionts through uninfected populations of the spider, Mermessus fradeorum. We tested two common infection combinations: a single infection with a cytoplasmic incompatibility (CI) inducing Rickettsiella or a feminizing co-infection that included a feminizing Wolbachia, the same Rickettsiella, and up to three apparent hitchhikers (two additional Wolbachia strains and Tisiphia). We initiated replicate populations with 1/3 of one infection type and 2/3 uninfected spiders, evaluating population infection rate over 5 spider generations under different temperature regimes. Under cool (21{degrees}C) conditions, Wolbachia feminization drove co-infection to 88% and Rickettsiella CI drove single infection to 83% of host populations. Vertical transmission for all symbionts was high (97-99%) and hitchhiking symbionts also spread effectively. Under warm conditions, feminization and CI efficacy were reduced, and symbionts suffered variably reduced vertical transmission. Warm conditions ultimately destroyed the co-infecting symbiont consortium and impeded symbiont spread. On its own, though, Rickettsiella was still able to increase, despite reduced strength of CI. We hypothesize that contrasting tensions between feminizing spread of the symbiont consortium versus environmentally driven loss of function and transmission may explain observed patterns of mixed infections in field populations of this spider.

Perennial grains represent a promising frontier for sustainable agriculture, but breeding progress is constrained by the accessibility of genotyping and the difficulty of evaluating complex traits expressed for multiple years after establishment across heterogeneous environments. Phenomic selection may help address these challenges by using inexpensive, scalable, high-dimensional phenotypes collected early in development, although the robustness of such predictions across breeding cycles remains uncertain. Here, we compared genomic selection and phenomic selection across two breeding cycles of Thinopyrum intermedium (intermediate wheatgrass; IWG; Kernza(R)), comprising approximately 2,280 individuals from maternal half-sib families evaluated across multiple field sites and years. We constructed relationship matrices from genomic markers and early-life stage phenomic data, including seed and leaf color (HSV), CropReporter multispectral reflectance and indices, and cycle-specific hyperspectral reflectance sensors. Genomic models provided the strongest predictions on average across all field traits in both cycles. Among phenomic predictors, leaf HSV was consistently the most informative, whereas CropReporter and hyperspectral data showed lower and more trait-dependent performance and seed HSV provided little predictive value. Genomic, leaf HSV, and CropReporter models transferred across breeding cycles with little apparent loss of predictive ability relative to within-cycle validation, demonstrating that their predictive signals were not restricted to a single breeding cycle. Early-life stage leaf HSV emerged as a practical, accessible tool for germplasm thinning and early-stage prioritization in perennial breeding programs. Despite limited similarity among relationship matrices, multi-relationship-matrix models rarely improved prediction beyond the stronger constituent single-relationship-matrix model. Together, these results show that early-life stage phenomic data provide reproducible information about agronomic performance expressed years later, but that predictor complexity and data integration do not guarantee improved prediction.

Ethnopharmacological relevance: Australian Indigenous medicinal plants represent a valuable yet underexplored source of bioactive compounds with potential therapeutic relevance. The Iningai community of Central Queensland has traditionally used native plants to manage conditions associated with inflammation, pain, infection, and general illness. Scientific evaluation of these plants may provide evidence for their customary applications and identify bioactivities relevant to anticancer biodiscovery. Aim of the study: This study evaluated leaf and stem extracts of seven medicinal plants-Pittosporum angustifolium, Alphitonia excelsa, Calytrix microcoma, Geijera parviflora, Melaleuca uncinata, Gossypium australe, and Eucalyptus similis-traditionally used by the Iningai community, focusing on three biological processes relevant to cancer: oxidative stress, inflammation, and cellular proliferation. Materials and methods: Antioxidant activity was assessed using DPPH radical-scavenging and ferric reducing antioxidant power (FRAP) assays. Anti-inflammatory activity was evaluated in lipopolysaccharide (LPS)-stimulated THP-1 macrophage-like cells by profiling IFN-, TNF-, IL-6, IL-12, IL-18, and IL-23. Antiproliferative activity was assessed using MTT-based viability assays in human and murine liver cancer cell lines (Huh7, Hep3B, Hep-55.1c, and A52). Results: The extracts exhibited distinct biological activity profiles. G. parviflora stem and C. microcoma leaf extracts showed the strongest antioxidant activities, whereas P. angustifolium stem exhibited the weakest radical-scavenging capacity. Cytokine responses were extract-specific, with E. similis leaf extract demonstrating broad and pronounced suppression of multiple LPS-induced pro-inflammatory cytokines. Several extracts produced concentration-dependent reductions in liver cancer cell viability, with P. angustifolium stem exhibiting the most consistent and potent antiproliferative activity across the cell lines tested. Notably, strong antioxidant or anti-inflammatory activity did not necessarily correspond with antiproliferative activity. Conclusion: Australian Indigenous medicinal plant extracts demonstrated distinct antioxidant, immunomodulatory, and antiproliferative activities rather than uniform bioactivity across experimental systems. The divergent activities of G. parviflora, C. microcoma, E. similis, and P. angustifolium highlight the importance of integrated biological screening and support the value of Indigenous knowledge-guided biodiscovery. These plants represent promising sources for further investigation of selective bioactive compounds with potential relevance to anticancer drug discovery.

Background Trypanosoma cruzi, the causative agent of Chagas disease, is subdivided into distinct genetic groups known as Discrete Typing Units (DTUs), each with distinct genetic traits that influence epidemiology and transmission dynamics. Several triatomine species serve as potential vectors of T. cruzi in the United States. However, despite the growing number of Chagas disease cases in the country, little is known about the genetic diversity and population structure of T. cruzi in natural vector populations. Methodology/Principal Findings We applied a multilocus metabarcoding approach to improve DTU resolution and characterize the genetic diversity and structure of T. cruzi in triatomines collected across five states of the southern United States. Five single-copy nuclear markers and one mitochondrial marker were amplified and processed by high-throughput sequencing to assess genetic diversity. We recovered 35 nuclear and 15 mitochondrial haplotypes from 70 infected specimens. Overall, genetic diversity was low ({pi} < 0.01 at all nuclear loci), with DTUs TcI and the North American lineage of TcIV detected, TcI being the most prevalent. Geographic structuring was particularly evident in TcI strains, which exhibited a distinctive haplotype profile in Florida populations, potentially linked to the recently revalidated vector species Triatoma ambigua. Mitochondrial introgression from TcIV into TcI suggests inter-DTU genetic exchange in these populations. Multiple haplotypes within individual insects detected across single-copy nuclear markers, support multiclonal infection as common feature of T. cruzi in natural vectors. Conclusions/Significance These findings provide new insights into the genetic landscape and evolution of T. cruzi in the United States. Evolutionary connectivity through mitochondrial introgression and frequent multiclonality highlights the importance of deep sequencing approaches for resolving T. cruzi genetic diversity, with direct implications for understanding for transmission dynamics, disease monitoring and control.

Bulk telomere length measured from tumour sequencing is routinely interpreted as a property of the cancer cells. However, a tumour specimen is a mixture, and the patient who supplies it has a telomere length of their own. Here I re-analyse published pan-cancer telomere estimates and ask how much of a tumour's telomere length is patient-specific. A calibration step comes first. Whole-genome and low-pass estimates recover the known cross-sectional attrition of leukocyte telomeres with age, at 26.6 bp per year in blood normals, whereas whole-exome estimates do not. After adjustment for cancer type, sequencing centre and sex, the exome slope is minus 0.6 bp per year. In 684 blood-normal aliquots sequenced by both assays, the whole-genome estimate declines at 38.9 bp per year, whereas the exome estimate from the same DNA shows no detectable decline. The difference between assays is 41.5 bp per year, with P = 3 x 10^-10. Because exome data constitute 78.6% of the original resource, downstream analyses use only whole-genome and low-pass libraries. Within those data, tumour telomere length tracks the patient's matched-normal telomere length. The Spearman correlation is 0.395 in TCGA, with positive associations in 22 of 23 cancer types. This finding replicates in PCAWG using a different telomere estimator, with a correlation of 0.472 and positive associations in all 24 histologies examined. Adjustment for cancer type, sequencing centre and library type leaves a regression coefficient of 0.385. The association is also stable after adjustment for age, sex, tumour purity, leukocyte fraction, ploidy, sequencing coverage and continental ancestry, with coefficients ranging from 0.406 to 0.429. Pure normal-cell admixture is rejected as the sole explanation. Under a two-compartment mixture model, the coefficient for host telomere length is expected to equal 1 and the host-by-purity interaction to equal minus 1. These restrictions are jointly rejected with P = 0.001. Tumour purity, leukocyte fraction and age each explain only about 1 to 3% of within-cohort variance and do not alter the cross-cancer ranking. By contrast, the between-cohort coefficient is not directly interpretable. Its apparent near one-to-one relationship with tissue-associated telomere length depends strongly on which tissue supplies the matched-normal reference and on the statistical spread of that predictor, falling to 0.44 when organ-matched solid tissue is used. Bulk tumour telomere length is therefore a composite phenotype containing a replicated patient-specific component. Telomere biomarker studies should include matched-normal telomere length as a covariate rather than treating tumour telomere length as exclusively tumour-intrinsic.

Introduction: Gliomas integrate into neural circuits and heighten neuronal excitability, engaging in bidirectional communication whereby neuronal activity promotes tumor growth and proliferation. Aging reshapes the brain microenvironment through extracellular matrix changes, altered secretory factors, and immune dysfunction, creating conditions permissive to tumorigenesis and limiting immunotherapy efficacy in glioblastoma. However, its effect on neuronal excitability and signaling in glioblastoma remains poorly understood. Methods: We developed a novel classification system for glioblastoma by leveraging three classes of DNA methylation-based aging biomarkers: chronological, biological, and mitotic clocks. This approach stratified tumors into accelerated and decelerated epigenetic aging subtypes, which we then characterized at the molecular, functional, and clinical levels using multimodal analyses. Guided by these profiles, we evaluated the in vitro effects of the FDA-approved agents levetiracetam and riluzole, alone and in combination with temozolomide, on U87MG and A172 cell lines. Specifically, we assessed changes in cell viability, apoptosis, and the expression of marker genes related to stemness, neuronal hyperexcitability, and immunosuppression. Results: Tumors with decelerated epigenetic aging showed expression modules and CpG hypomethylation associated with neuronal activity and stemness, and carried significantly worse prognosis. Single-cell and spatial multi-omics analyses revealed enrichment for neurons and malignant neural stem-like cells in these tumors. They also displayed enhanced intercellular communication, driven predominantly by glutamate signaling across the malignant, neuronal, and immune compartments of the tumor microenvironment. In vitro pharmacological inhibition of glutamatergic signaling with levetiracetam and riluzole reduced cell viability, induced apoptosis, and suppressed expression of stemness, neuronal hyperexcitability, and immunosuppression markers. Both agents potentiated the cytotoxic and apoptotic effects of temozolomide, supporting glutamatergic inhibition as a strategy for improving chemosensitivity. Conclusion: By establishing a framework for decoding glioblastoma heterogeneity through epigenetic aging, we identified the glutamatergic pathway as a clinically actionable vulnerability. Our findings suggest that combining anti-glutamatergic therapies with temozolomide exerts synergistic antitumor effects while mitigating adverse chemotherapy-induced phenotypes, such as increased stemness, neuronal hyperexcitability, and immunosuppression, thereby laying the groundwork for novel therapeutic strategies.

The symbiosis with photosynthetic dinoflagellate algae enables corals to build and sustain reef ecosystems. Individual coral polyps hold algal symbionts in their epithelial endoderm cells and lose them under environmental stress, leading to coral bleaching. How the host integrates symbionts into its body plan is not well understood. Here, using a combination of high-resolution imaging, quantitative analysis, and environmental perturbations in the sea anemone Exaiptasia diaphana (Aiptasia) and reef-building coral Pocillopora damicornis, we uncover a spatial organisation of symbionts along the aboral-oral axis of cnidarian polyps that emerges under the long-range translocation of symbionts between host cells through a fluid-filled cavity. The symbiont distribution becomes specifically enriched in the tentacle bud endoderm during Aiptasia polyp morphogenesis. This pattern can form in darkness and with algae-sized inert spheres, suggesting an innate host-intrinsic mechanism. Symbiont-occupied host cells are mechanically constrained within the endoderm and thus unable to rearrange; instead, they go through cycles of symbiont expulsion and re-uptake via the host gastric cavity, with regionally biased rates of these behaviours providing a route to enrich symbionts in the tentacles. Symbiont organisation is remodelled under increased light in adult coral polyps, with a characteristic pattern of reduced tentacle enrichment, lateral clustering and retention in the body column emerging over a timescale of days. Together, our findings reveal that the spatial organisation of symbionts is dynamically regulated in cnidarian host tissues, a capacity that may shape both the establishment of symbiosis and its resilience under environmental change.

Accelerating photoprotective regulation to improve carbon assimilation is a promising strategy to increase crop productivity. Although rapid non-photochemical quenching (NPQ) relaxation has been validated as a target through metabolic engineering, it remains unclear whether conventional breeding has improved this trait. Here, we investigated whether more than a century of soybean breeding enhanced NPQ relaxation alongside light-saturated carbon assimilation and seed traits. We evaluated a historical panel of 24 soybean genotypes across vegetative and reproductive developmental stages by integrating NPQ relaxation, gas exchange parameters, xanthophyll-cycle pigment profiles, expression of key photoprotective genes (VDE, PsbS, and ZEP), seed number and seed weight. NPQ relaxation parameters were not consistently associated with genotype release year, seed number, or seed weight at either developmental stage. The only exception was the amplitude of the rapidly relaxing NPQ component (AqE), which was negatively correlated with all three variables during the reproductive stage. In contrast, genotype release year was positively associated with maximum net CO2 assimilation rate (Amax), maximum carboxylation rate of Rubisco (Vcmax), maximum electron transport rate (Jmax), seed number, and seed weight, while Amax and Vcmax were positively correlated with seed number and seed weight. These findings indicate that the greater photosynthetic capacity of modern genotypes was not accompanied by faster photoprotective response. Thus, photoprotective regulation has not kept pace with gains in photosynthetic capacity under field conditions. We conclude that rapid NPQ relaxation remains an important target for synchronizing photoprotection with the high photosynthetic capacity of modern soybean lines.

Bovine viral diarrhea virus (BVDV) remains an economically important pathogen of cattle despite widespread vaccination. A homozygous CD46-edited Gir heifer (Ginger) was previously shown to have significantly reduced susceptibility to BVDV. The edited allele contains an in-frame six amino acid substitution within the virus-binding domain of the BVDV entry receptor CD46, replacing residues G82QVLAL with A82LPTFS. Here, we investigated whether reduced BVDV susceptibility is maintained when the edited allele is inherited in the heterozygous state. Ginger was artificially inseminated with semen from an unedited Gir bull and produced a healthy heterozygous CD46-edited bull calf (Giraldo). Whole-genome sequencing confirmed the inheritance and structural integrity of Giraldo's edited allele. Compared with Ginger, Giraldo exhibited similarly reduced ex vivo BVDV susceptibility across primary fibroblasts, lymphocytes, and monocytes, despite inheriting a wild-type CD46 allele from the sire. Allele-specific CD46 RNA expression analysis demonstrated expression of both the edited and wild-type CD46 alleles. Thus, the reduced-susceptibility phenotype was not attributable to transcriptional silencing of the wild-type allele. Lentiviral complementation studies in CD46-knockout Madin-Darby bovine kidney (MDBK) cells further demonstrated that this wild-type CD46 allele was competent to support BVDV infection when expressed independently. Together, these findings indicate that the CD46 A82LPTFS allele can confer reduced BVDV susceptibility in the heterozygous state despite expression of a functional wild-type CD46 allele. This result suggests the potential to more rapidly disseminate reduced BVDV susceptibility through conventional breeding using homozygous CD46-edited sires.

Communities exposed to flooding can experience markedly different mental health outcomes, yet conventional resilience indicators capture only part of the social and contextual conditions that may explain this variation. This study develops a multilevel and predictive framework for examining community resilience and depressive symptoms following flood exposure in Indonesia. Data were drawn from 20,303 respondents aged 15 years and older nested within 312 communities in the Indonesia Family Life Survey (IFLS-5). Depressive symptoms were assessed using the 10-item Centre for Epidemiologic Studies Depression Scale (CES-D-10), with Rasch Partial Credit Model calibration used to examine measurement properties. Bayesian multilevel models quantified between-community heterogeneity and assessed how far observable structural resources accounted for this variation. Community resilience was represented through two complementary constructs: structural resilience, based on observable socioeconomic and social-capital resources, and Latent Community Protective Capacity (LCPC), a model-derived proxy for residual contextual variation in depressive-symptom risk. Approximately 6 percent of variation was attributable to between-community differences, while observable structural resources explained only part of this heterogeneity. Structural resilience and LCPC were weakly correlated (r = 0.155). Moderation analyses provided no clear evidence that structural resilience altered the flood-depression association, while LCPC showed a directionally consistent but uncertain buffering pattern. Predictive models incorporating community-level information improved discrimination, with the best-performing model reaching an ROC-AUC of approximately 0.71. The findings suggest that observable resource-based indices provide an incomplete account of community-level mental health vulnerability and that residual contextual measures may provide complementary information, while requiring cautious interpretation and independent validation.

High-phenolic plant genera present a major technical limitation in genomic research. Standard extraction approaches that perform reliably across diverse flora often perform poorly when applied to recalcitrant taxa, producing low DNA yield and integrity incompatible with sequencing requirements. The genus Anigozanthos (Kangaroo paws) from the family Haemodoraceae exemplifies this problem. We identified key physicochemical factors governing extraction failure in this genus and resolved them through targeted modifications to lysis chemistry and contaminant management. The resulting protocol achieved a near threefold improvement in DNA purity, substantially reducing contaminant carry over and consistently yielded high-integrity, long DNA fragments (DIN > 7) across a diverse sample set spanning cultivated and wild material across four diverse genera of Haemodoraceae. We also tested a straightforward purity assessment framework that can be implemented in any standard molecular laboratory, enabling rapid pre-submission quality assessment without the need for specialised equipment. Together these advances open a practical path to genomic characterisation of Anigozanthos that establishes a transferable model for genomic research across Australia ' s chemically complex native flora.

To combat refractory diseases, such as cancer, multitarget-directed ligands (MTDLs) have become an emerging area of research to exploit synthetic lethality (SL) relationships associated with drug resistance. Herein, we present the in silico design of MTDLs for the polypharmaceutical treatment of endometrial adenocarcinoma (EAC) and our discovery of a novel SL in EAC; PTEN loss of function (LOF) and the inhibition of CDK9. We used high-resolution x-ray crystallographic data to chemically engineer, LCI133, to inhibit CDK9, CDK4/6-and AURKA/B kinases. PTEN LOF in EAC results in augmented deregulated transcription and a massive increase in nascent RNA, a phenotype which encodes a high level of apoptotic sensitivity to LCI133 and CDK9 inhibitors. Treatment with LCI133 results in a rapid decline nose-dive in global nRNA, MYC nRNA levels and TS elongation (TE) in PTEN LOF EAC. PTEN LOF is necessary and sufficient to confer sensitivity of EAC cells to LCI133 and other CDK9 inhibitors.

Vaccine effectiveness (VE) estimates can vary widely between years and populations, even for the same vaccine. Estimated VE is known to be sensitive to susceptible depletion and differences in pre-vaccination infection risk between vaccinated and unvaccinated populations. However, how variation in pre-vaccination risk within and between the two groups affects VE estimates over time remains unclear. This uncertainty is especially important given negative VE estimates. We investigated the difference between estimated VE and true vaccine protection considering continuous distributions of pre-vaccination infection risk under three scenarios. When the vaccinated and unvaccinated populations differ in their mean risk, estimated VE can be higher or lower than true vaccine protection. Similar patterns arise when both populations share identical means but different risk distributions. Finally, if infection-derived immunity lasts longer than vaccine protection, annual VE estimates can vary by tens of percentage points between years despite constant true vaccine protection. These theoretical results underscore that VE studies estimate contrasting risk between vaccinated and unvaccinated individuals in a particular time and place, and VE estimates can vary counterintuitively between years and populations even with constant vaccine-induced protection. Explaining variability in estimated VE thus requires a more complete understanding of populations' distributions of infection risk.