Molecular Genetics and Genomics

Chromatin profiling methods, such as ChIP-seq (chromatin immunoprecipitation followed by sequencing), are used to characterize the genomic localization of DNA-associated proteins. While ChIP and ChIP-seq have been used for decades, an orthogonal approach, CUT&Tag (Cleavage Under Targets and Tagmentation), is gaining popularity as an efficient and cost-effective alternative. Although previous comparative studies note discrepancies in signal-to-noise ratios and detection bias at certain genomic regions, many differences between ChIP-seq and CUT&Tag results remain largely unreconciled. Here, we systematically investigate the disagreeing signals captured by these two methods across well annotated genomic regions. We assess multiple histone mark profiles generated by different groups in two cell lines (K562 and MCF-7). Overall, our analysis indicates that compared to ChIP-seq, CUT&Tag may have limited sensitivity in low-GC environments and, as previously observed, increased signal in hyper-accessible chromatin. Notably, within GC-poor regions of active gene bodies and Polycomb-repressed domains, CUT&Tag exhibits a loss of H3K36me3 and H3K27me3 signal, respectively, where occupancy of these histone marks is otherwise expected. Further, active promoters with discrepant H3K4me3 and H3K27ac signal between the two assays differ systematically in GC content and chromatin accessibility. Promoters differentially enriched for CUT&Tag signal relative to ChIP-seq generally show higher, broader GC-content profiles and higher DNase-seq and ATAC-seq signal, while promoters enriched for ChIP-seq signal harbor the opposite characteristics. A local bias for high GC content and/or chromatin accessibility in CUT&Tag may also explain its differing signal patterns at nucleosome-depleted regions (NDRs) compared to ChIP-seq and MNase-seq promoter profiles. Altogether, our results highlight that studies focused on profiling the intensity and structure of histone modification occupancy can be sensitive to potential biases of CUT&Tag to GC content and chromatin accessibility. These characteristics should be accounted for when selecting a chromatin profiling approach, analyzing and interpreting data as well as drawing biological conclusions.

Objective This study aimed to develop and validate a predictive model for sleep disorders in young and middle-aged adults with anemia, providing evidence for early identification, risk stratification, and individualized sleep management. Methods Data in the NHANES were analyzed and randomly divided into development and validation cohorts. LASSO regression was used to screen optimal predictors in the development cohort, followed by univariate and multivariate logistic regression to identify independent predictors and construct a nomogram. Model performance was evaluated using receiver operating characteristic curves, calibration curves, Brier scores, and decision curve analysis. Results The weighted prevalence of sleep disorders among young and middle-aged adults with anemia was 10.19%. Multivariate logistic regression identified seven independent predictors: sex, monocyte count, asthma, overweight, arthritis, congestive heart failure, and hypertension. The model showed good discrimination, with area under the curve values of 0.817 in the development cohort and 0.791 in the validation cohort. Calibration was satisfactory, with Brier scores of 0.074 and 0.075, respectively. Decision curve analysis indicated favorable clinical net benefit in both cohorts. Conclusion The proposed predictive model demonstrated good discrimination, calibration, and may help healthcare providers identify young and middle-aged adults with anemia who are at high risk for sleep disorders.

Tsetse flies are strictly found is sub-Saharan Africa where they are responsible for the transmission and maintenance of African trypanosomiases in humans (HAT) and animals (AAT). Vector control has been recognized as an essential tool to fight against these diseases. Nevertheless, it requires the best possible knowledge of the biology of the targeted population. Population genetics tools can prove very useful to obtain such information but require the use of polymorphic and reliable genetic markers. In this paper we present the development of microsatellite markers using a new high-throughput sequencing based technology (SSRseq). We applied it on two species of tsetse flies from three HAT foci and obtained more accurate results as compared to microsatellite loci developed with classic methods. We could indeed use 9 to 14 SSRseq loci without the several problems generally met with classic microsatellites, as all were located in autosomes, without short allele dominance or stuttering and very few null alleles. SSRseq loci appeared much more polymorphic in tsetse flies as compared to other species (fungi, trees, bees, or fishes), which suggested much higher effective population sizes, much higher mutation rates of the genome or both, suggesting high capacities for evolutionary adaptation. With the 9-14 loci kept, we confirmed the propensity of these flies to move almost freely in the whole zones investigated, and also highlighted the possible evolutionary response of one of the unkept loci regarding vector control devices used in these HAT foci, which will require further studies. We suggest for further population structure studies to use only loci with less than 1% missing data, which proved being a good and fast selection strategy to get the most reliable results.

Ancestry-related genomic variation may contribute to phenotypic diversity. However, its cellular consequences remain difficult to evaluate. To address this, we developed a human induced pluripotent stem cell (hiPSC)-based framework for investigating such phenotypes. We used the Japanese population as a model of Jomon-related and continental East Asian ancestry admixture. Jomon ancestry, estimated using the Jomon allele score (JAS), was associated with clinical measures, including renal-related traits and serum triglycerides in 34,152 Tohoku Medical Megabank participants. Peripheral blood mononuclear cell transcriptomics of donors from the upper and lower extremes of the JAS distribution (n = 10) suggested group-level differences in immune-related gene expression. To test whether the serum triglyceride association coincided with hepatic cellular phenotypes, we differentiated hiPSCs from these donors into hepatocyte-like cells. Neither intracellular triglycerides nor lipid-related gene expression differed between groups. These findings highlight the value of a genetically characterized hiPSC-based framework for investigating ancestry-associated phenotypes across cellular contexts.

Chromosome was "self-assembled" into a complex three-dimensional topology in the nucleus, which provided the structural basis for important biological processes such as gene expression regulation and cell differentiation. The current understanding of the three-dimensional organization of the genome is based on high-throughput sequencing and imaging technologies to visualize the conformation of chromatin in its native environment. This study proposes a new research perspective and technical means to use fusion genes to determine the interaction information and three-dimensional structure of chromosomes. This three-dimensional information provided by the fusion gene spectrum not only shows the universality of the spatial layout of chromosomes, but also increases the interpretability of the occurrence of fusion genes. The chromatin conformation capture data of multiple tissues and periods is significantly correlated with the strength of fusion genes. For example, the frequency of trans-interaction between chromosomes 11 and 17 was the highest, and the frequency of fusion gene occurrence corresponding to KMT2A on chromosome 11 was the highest. Therefore, we infer that this structural universality is a common feature of the human genome, which provides a theoretical basis and data for predicting chromosome fusion events.

In eukaryotic cells, the positive transcription elongation factor b (P-TEFb) plays a critical role in the transition of RNAPII from the paused state to actively transcribing mode. Although three different cyclin (Cyc) T (CycT1, T2a, and T2b) interact with CDK9 to form functional P-TEFb, a majority of P-TEFb complexes are comprised of the CycT1 and CDK9. To analyze the role of the CycT1 subunit of P-TEFb on cellular transcription, we established HEK293T cells lacking CycT1 proteins (CycT1-KO cells). While no apparent growth defects were observed with CYcT1-KO cells, the level of CDK9 was decreased and no compensatory over-expression of CycT2 was observed, suggesting that the reduced level of functional P-TEFb complexes is sufficient for the normal cell growth. Reporter gene assays indicate that NF-kappaB-dependent transcription induced by PMA was impaired in CycT1-KO cells although the function of NF-kappaB (P65) per se was not affected. On the other hand, AP1-dependent transactivation induced by PMA was unaffected in CycT1-KO cells. Moreover, transcription stimulated by JQ1, a strong P-TEFb inducer, was reduced in CycT1-KO cells. Data of biochemical analysis indicate that P-TEFb with CycT1 (P-TEFb (CDK9:CycT1)) was efficiently released from 7SKsnRNP by JQ1 while P-TEFb (CDK9:CycT2) was not affected by JQ1. Transcriptome analysis indicated that the lack of CycT1 had a minor effect on the steady-state transcription. However, expression of JQ1-dependent genes was severely reduced in CycT1-KO cells. Interestingly, referring to gene expression profiles of 33 different types of cancer revealed that 11 out of 13 top JQ1-dependent genes were also highly upregulated in Pancreatic Adenocarcinoma. From these results, we conclude that although many genes can be regulated by both P-TEFb (CDK9:CycT1) and P-TEFb (CDK9:CycT2), CycT1 plays a critical role in regulating highly inducible genes, which are also aberrantly regulated in a particular type of cancer.

Parasitoid wasps develop in or on other arthropods and have evolved a wide range of strategies for exploiting their hosts. Ascogaster reticulata is a solitary egg to larval endoparasitoid wasp belonging to the subfamily Cheloninae of Braconidae. We constructed a genome assembly using long-read sequence data. The assembled genome was 280.5 Mbp, consisting of 209 contigs. The N50 value and GC content ratio were 13.9 Mbp and 30.43%, respectively. The percentage of transposable elements and repetitive regions was 52.43%. The predicted number of genes is 27,261. Merqury revealed that the QV value and completeness of the genome were 61.79 and 99.3917%, respectively. BUSCO analysis detected approximately 99% of the core genes. These results suggest that the genome data were of sufficient quality for use as a reference genome. Therefore, these data can be utilized as a genomic resource for comparative studies within Braconidae and for future investigations into the biology and evolution of Cheloninae parasitoid wasps.

Introduction. Histoplasma capsulatum is an important fungal pathogen of humans, especially in immunocompromised patients, and the World Health Organisation has listed Histoplasma as a High Priority fungal pathogen. The taxonomy of the genus Histoplasma is still under development, but its only species H. capsulatum has at least of seven lineages, and a range of sublineages. Gap Statement. Allelic multilocus sequence typing (MLST) has been very successfully applied for typing and epidemiology of pathogenic bacteria, but its adoption for fungal pathogens is more limited. Genome sequence-based MLST approaches promise much a higher resolution for comparative phylogenetics, but there are only a small number of genome assemblies available for H. capsulatum. Aim. To develop and validate genome-based MLST typing for H. capsulatum. Methodology. A total of 406 genome assemblies were generated from publicly available Illumina short-read sequencing samples. Selected genome assemblies were subsequently used to generate MLST schemes with chewBBACA, based on whole genome sequences (wgMLST), NCBI-obtained annotations (annMLST) and annotations obtained with Funannotate (fanMLST), and used for lineage identification, and comparative analyses of population structure. Results. Genome assemblies were successfully generated for 406 H. capsulatum samples, with BUSCO completeness scores of over 98%. A subset of 60 genome assemblies was used to generate and validate MLST schemes based on the 60 genome assemblies, 5 genome annotations obtained from Genbank, and 13 genome annotations generated using Funannotate, resulting in schemes with > 8,000 markers. Each of the MLST schemes allowed reproducible separation of all 7 lineages and 6 H. capsulatum Suramericanum sublineages, without the need for high-performance clusters or powerful computing equipment. Comparison of the different schemes using tanglegrams and scatter plots showed good reproducibility (r2 values of 0.8-1.0). Comparison of total sizes of genome assemblies showed significant lineage-dependent size differences, with the Suramericanum, LAmB, India, Capsulatum and Mississippiense lineages averaging a total genome size of 30-33 million bp, and the Africa and Ohiense lineages averaging at 39 million bp. Conclusion. Genome sequence- and especially genome annotation-based MLST analyses are a promising approach that can be adapted as a rapid, high-throughput method for genomic clustering and epidemiological tracing of pathogenic fungi.

Abstract Polycystic ovarian syndrome and Type 2 diabetes mellitus are complex multivariate diseases that share well-established clinical pathophysiology, yet the molecular basis of their overlap is unknown. Prior bioinformatics studies have identified common differentially expressed genes between these two conditions. However, they have not accounted for the directionality of the gene expression changes observed, potentially obscuring any biological distinctions they may have. This study applied a direction-aware approach to classify molecular signatures between PCOS and T2DM as concordant or discordant. Transcriptomic analysis of GEO datasets GSE138518 (ovarian granulosa tissue, PCOS) and GSE25724 (pancreatic islet tissue, T2DM) identified 225 and 1,302 DEGs, respectively. Venn diagram analysis showed that only three genes (SLC6A8, RGS4, and SORL1) were shared between PCOS and T2DM from the total of 1,527 genes, and all 3 genes were regulated in opposing directions. Disease gene retrieval from the Comparative Toxicogenomics Database, Online Mendelian Inheritance in Man database, and GeneCards showed 214 shared disease-associated genes, with 311 genes unique to PCOS and 496 genes unique to T2DM. Protein-protein interaction construction using STRING (version 12.0) identified 70 interacting nodes. CytoHubba analysis across 6 scoring methods identified 18 high-confidence hub genes, including INS, BCL2, MTOR, LEP, MFN2, and PIK3CD. Functional enrichment analysis identified biological processes including immune activation, apoptosis, and insulin signaling, which were confirmed as the main pathways in KEGG pathway analysis. These findings show that while PCOS and T2DM share limited DEGs, they share common pathogenic pathways.

Considering the increasing water and labor constraints in rice production, direct-seeded rice is becoming increasingly important. However, direct-seeded rice is vulnerable to water stress and inefficient nutrient use. Nitrogen use efficiency in rice is generally low, and improving nitrogen acquisition and assimilation is important for sustainable productivity. In this study, 48 recombinant inbred lines were evaluated under ammonium and nitrate nutrition in irrigated and rainfed environments across two cropping seasons. Agronomic, physiological, biochemical and molecular responses were assessed. Significant effects of genotype, nitrogen form, environments and their interactions were observed for several traits. Rainfed conditions reduced grain and biological yield compared with irrigated conditions. Ammonium produced a higher mean grain yield than nitrate by approximately 26.2 g m-2 under irrigated conditions and 154.2 g m-2 under rainfed conditions, although the responses varied among genotypes. Biological yield and SPAD values were generally higher with ammonium, whereas total chlorophyll showed season and environment-dependent responses. Glutamine synthetase activity was highest under ammonium treatment, while glutamate synthase activity was higher under nitrate treatment. Grain yield showed strong positive correlations with biological yield, but correlations between grain yield and GS or GOGAT activity were not significant. Expression of OsGln1.2, OsGln2 and OsGlt1 varied according to genotype, tissue and nitrogen treatment. Overall, the results demonstrate that responses to nitrogen forms are genotype- and environment-dependent, and that ammonium may provide a yield advantage under rainfed conditions. Integrating field performance with biochemical and molecular traits may help identify rice genotypes suited to water-limited systems.

Vertical transmission of SARS-CoV-2 is controversial. Owing to the low expression of ACE2 along with TMPRSS2 in placenta, role of secondary receptors becomes significant. In our study we predicted twelve interactors of spike protein expressed in placenta through interactome analysis followed by docking and molecular dynamic simulation. Three interactors of SARS-CoV-2 spike protein were found to be CD147, EGFR and LDLR. Expression at transcriptomic and translational level by real-time PCR and immunohistochemistry respectively was observed in placenta samples obtained from women with COVID exposure risk and compared to control. Elevated protein levels of CD147 (OR=14.667), ACE2 (OR=8.25) and LDLR (OR=1.9) was associated with higher exposure risk. However, the variation in mRNA expression was not statistically significant. Computational study showed that CD147/basigin, EGFR and LDLR stably interact with spike protein outside its primary receptor binding domain. In-vitro binding assay confirmed the physical interaction between spike protein and the placental receptors. In-silico site-directed mutagenesis further identified key potential binding hotspots of spike protein- Tyr28 and Trp64 for CD147, Phe643, Val642, Thr632 for LDLR and Leu8, Phe59, Phe32, Tyr28, Trp64, Trwp152, His245, and Val213 for EGFR. Finally, concluded that CD147, EGFR and LDLR might act as secondary non-canonical cofactors for SARS-CoV-2 spike protein in placenta, thereby increasing its susceptibility and tissue tropism. The study overall gives a better insight into potential placental suscpetibility providing targeted therapeutic opportunities.

Background: Long non protein coding (lnc)RNAs are ubiquitous transcripts known to be major regulators of cellular processes in eukaryotes. In diatoms, specific lncRNA expression is tightly correlated to nutrient fluctuations, which suggests function, but their biological relevance remains to be established. In order to fill this knowledge gap, we sought to design a protocol to generate diatom lncRNA knockout mutants by CRISPR-Cas9 enabling subsequent functional studies to shed light into their mechanistic roles. Since lncRNAs do not contain open reading frames that can be targeted by point mutations for the generation of functional knockouts, we designed a multiplexed CRISPR-Cas9 plasmid harboring two independent gRNA for the specific ablation of lncRNA loci in P. tricornutum. As proof of concept, we used our plasmid-based system to target three phosphate depletion responsive lncRNAs. Results: Using the golden gate based uLoop system, we assembled a single plasmid containing all the necessary components to generate CRISPR-Cas9 large genomic ablations of lncRNAs in P. tricornutum using bacterial conjugation. Three intergenic lncRNAs (Linc91, Linc77 and Linc24) responsive to phosphate fluctuations were selected and independently targeted. Around 32% of the exconjugants presented a monoallelic ablation of the targeted loci and 11% presented a biallelic ablation on the first PCR-screening round. Mutated lines, harboring biallelic lncRNAs ablations for each lncRNA targeted, were successfully generated for each of the targeted lncRNAs. Additionally, a first line of phenotypic analysis revealed significant growth rate differences for one of the mutants ({Delta}Lnc24) under both full and phosphate depleted conditions, suggesting its involvement in cell division regulation under nutrient fluctuations. Conclusions: This work provides a simple and efficient method for the generation of lncRNA knock-out mutants in P. tricornutum. Furthermore, this approach can be easily adapted to other microalgae amenable to transformation, enabling the enhancement of our understanding of these important cellular regulatory transcripts in the marine environment.

Introduction: In both humans and experimental animals, obesity is associated with hypothalamic inflammation and neuronal metabolic dysfunction, which contribute to disrupting body energy homeostasis. Although ciliary neurotrophic factor (CNTF) is a neuroprotective cytokine with recognized anti-obesity effects in peripheral metabolic tissues, its role in obesity-associated hypothalamic dysfunction is still poorly characterized. We investigated whether CNTF exerts any direct effects on mHypoE-N41 (N41) hypothalamic neurons and whether it modulates the effects induced by palmitate (PA), an established model of obesity-associated neuronal metabolic dysfunction. Methods: N41 cells were administered CNTF alone or combined with PA to assess whether CNTF affects PA-induced lipotoxic stress. Gene and protein expression was assessed by qRT-PCR, western blotting, and immunofluorescence analysis. Results: In N41 cells, CNTF activated the STAT3, ERK and AMPK signaling pathways. It also upregulated genes involved in mitochondrial metabolism and fatty acid oxidation, including Ucp2, Cpt1 and Pgc1, as well as the proliferation-associated markers Ki-67 and PCNA. PA treatment induced inflammatory and endoplasmic reticulum stress responses and impaired insulin signaling. In PA-treated N41 cells, CNTF significantly attenuated the expression of pro-inflammatory markers Tnf, Mcp1 and Nfkb, restored insulin-induced AKT phosphorylation, and partially reversed PA-induced changes in the expression of metabolic genes Ucp2 and Cpt1. Conclusions: Collectively, these findings indicate that CNTF exerts direct protective metabolic effects on hypothalamic N41 neurons subjected to lipotoxic stress and supports a role for it in the preservation of neuronal metabolic function under obesogenic conditions.

The phenotype of a focal individual can be affected not only by the individuals own genes (direct genetic effects, DGE) but also by genes of other individuals it interacts with (indirect genetic effects, IGE). Better understanding IGE can help better understand the impact and mechanisms of social effects. Here, we investigated, in laboratory mice, the modulation of IGE by genetic background of the focal individual (social epistasis) and by sex. We focused on IGE arising from Epha4, a gene that has been associated with the individuals own stress-related and social behaviours (i.e. DGE) and the stress-coping behaviour and wound healing of the other mice in the cage (i.e. IGE). Among four different genetic backgrounds for focal mice, significant IGE from Epha4 were detected in CBA females only. A formal model selection analysis was consistent with the modulation of IGE by the similarity of the genetic backgrounds of the focal mouse and its Epha4 cage mate, as well as by sex. Observations of fighting in cages of males with dissimilar genetic backgrounds suggested aggression might underpin these modulatory effects, which prompted to us to expand the conceptual framework describing the mechanisms of social epistasis. Thus, our results strengthen the emerging view that IGE are not fixed properties of social genotypes but can depend on the genetic and sex context of interacting individuals, revealing an underexplored layer of their genetic architecture.

Minor introns are rare but evolutionarily conserved introns in the human genome. Defects in minor spliceosome components have been reported to cause several human diseases. Mutations in ZRSR2 are frequently observed in myelodysplastic syndrome (MDS) patients. However, the precise molecular mechanism by which these mutations cause a loss of ZRSR2 function in minor intron splicing remain elusive. In this study, we characterized the impact of MDS-causing missense mutations in ZRSR2. Most of the mutant ZRSR2 proteins we tested showed different subnuclear localization pattern from that of wild-type ZRSR2 protein. Co-immunoprecipitation (co-IP) analysis revealed that UHM missense mutations disrupted the interaction between ZRSR2 and SF3B complex. We also demonstrated that ZRSR2 preferentially immunoprecipitated minor spliceosome components over major spliceosome components and it directly interacts with SF3B1. Furthermore, co-IP analysis utilizing a variety of ZRSR2 mutants revealed that mutations in the UHM and Zn2 domains significantly attenuated the interaction with SF3B complex. Finally, UHM and Zn2 mutations markedly disrupted the supporting activity of ZRSR2 for minor intron splicing. Our results indicate that disruption of the physical interaction with SF3B complex is one of the hallmarks of MDS-causing mutations of ZRSR2, which results in aberrant splicing of minor introns.

Meteorite impact structures on earth represent unique terrestrial analogues of extreme planetary environments where fractured lithologies, hydrothermal alteration, elevated concentrations of radionuclides, and prolonged water-rock interactions create ecological niches for specialized microbial communities. The Palaeoproterozoic Dhala impact structure in north-central India is characterized by uranium-bearing impactites and possesses a very high concentration of Uranium (up to 99.5 ppm) that makes it a unique natural laboratory to investigate microbial adaptation to radioactive and metal-rich geological environments. In addition, the Dhala structure harbors specialized radiotoxic hydrothermal mineral phases such as coffinite and pitchblende, which show a globally distinct and biologically unexplored niche. The present investigation is the first in-depth, whole-genome metagenomic study of soil and water samples of uranium-rich sites from the Dhala structure, exploring the whole microbial community (Bacteria, Archaea, Eukaryotes, and Viruses) up to the species level present in the samples. The cross-domain metagenomics analysis identified a highly unique and resilient microbiome that consists of bacteria, archaea, eukaryotes, and viruses, coevolved under prolonged radiological and multiple metal stress. Functional metagenomic analysis revealed numerous metabolic adaptations responsible for their survival, homeostasis, and biomineralization in situ. The deep sequencing method further enabled the identification of 58 functional genes directly involved in uranium bioremediation. Furthermore, this study revealed important pathways for radionuclide reduction, cellular efflux, and biotransformation. Novel findings of the present study point out the in-depth evolutionary relations of biosphere-geosphere interaction within an ancient hypervelocity impact-generated ecosystem. In a nutshell, the Dhala extremophiles with novel genetic makeup present an untapped and promising reservoir for the development of eco-friendly, next-generation microbial bioremediation strategies to efficiently tackle the global anthropogenic contamination of uranium and the challenges of nuclear waste management. Thus, the present study establishes Dhala structure as an unparalleled site for environmental geomicrobiology, microbial evolution, and astrobiology.

Genetic variation impacts biological response to chemical and environmental exposures, contributing to differences in resistance or susceptibility. Forward genetic screens of genetically diverse cell populations can be used to identify the precise genetic variants that drive response variation. Cell lines from laboratory mouse genetic reference populations like the Diversity Outbred (DO) population are well powered for genetic mapping. Diverse stem cell panels are especially beneficial for studying early developmental effects of chemical exposures like triphenyl phosphate (TPHP), an organophosphate flame retardant linked to adverse developmental effects, including altered cell cycle in stem cells. Self-renewal and pluripotency are defining properties of stem cells during development; therefore, it is important to understand how chemicals like TPHP may alter these critical characteristics. To better understand the influence of genetic variation on TPHP exposure response, we utilized a genetically diverse panel of DO induced pluripotent stem cells (DO iPSCs). Our analyses characterize interline variation in gene expression through differential gene expression analysis and expression quantitative trait locus (eQTL) mapping. We identified genomic loci that contribute to variation in gene expression following TPHP exposure, including a regulatory hotspot on chromosome 15. Gene set enrichment highlighted several pathways affected by TPHP including lipid metabolism, steroid hormone signaling, and cell cycle. Our study shows that genetic variation modulates the effects of TPHP on gene expression and cell cycle in stem cells. Additionally, our work demonstrates that genetically diverse cell panels like the DO iPSC resource offer a tractable and scalable approach for identifying the genetic determinants of chemical toxicity.

Aim To investigate how repeated historical connections between continental islands and the mainland have shaped the phylogeographic structure and genetic diversity of populations, using the widespread water strider Aquarius paludum as a model. Location Japan Taxon Water strider Aquarius paludum (Insecta: Hemiptera: Gerridae) Methods We analysed mitochondrial (COI and COII) and nuclear (ITS1) DNA sequences from populations across the Japanese archipelago and the Eurasian continent. Phylogenetic and population genetic analyses were used to reconstruct phylogeographic structure and infer demographic history. Results Western Japanese populations exhibited nucleotide diversity comparable to that of continental populations despite their insular distribution. Bayesian phylogenetic analyses identified two major mitochondrial haplogroups (A and B) that frequently co-occurred across the Japanese archipelago. Haplogroup A, which is rare on the Eurasian continent, occurred at high frequency in Japan and showed pronounced isolation by distance, whereas haplogroup B showed only weak but significant isolation by distance and was significantly more frequent in populations closer to the coast. These contrasting spatial patterns within species indicate distinct demographic histories for the two mitochondrial lineages. Main conclusion Repeated colonization from the Eurasian continent and the long-term coexistence of mitochondrial lineages with different demographic histories have shaped the pattern of genetic diversity of A. paludum in the Japanese archipelago. Our findings exemplify how continental islands can accumulate genetic diversity through lineage accumulation and secondary contact, which produces an intraspecific phylogeographic pattern comparable to those observed during an early phase of the taxon cycle between species.

Background: Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related mortality, with an increasing incidence and dismal 10% survival at 5 years despite best available care. PDAC is characterized by low tumour cellularity, recurrent driver mutation alterations, and substantial inter and intra-tumoural heterogeneity. The influence of individual genetic features such as sex and ancestry on tumour heterogeneity remains poorly understood. Methods: We assembled a cohort of PDAC across five datasets for a total of 7,527 tumours. Measures of genomic instability were assessed across sex and ancestry on a global level (SNV density and percent genome altered by copy number events), and at a gene level for recurrent events. KRAS variant frequencies, ploidy and mRNA abundance were also evaluated. Ancestry was assigned using ADMIXTURE from germline genotypes where available, and patient reported otherwise. Results: We observed ancestry-specific mutation patterns in global point mutation density, with higher SNV burden in African PDAC tumours and lower SNV burden in Asian PDAC tumours as compared to European tumours. We also identified an ancestry bias in the driver gene, GNAS, in which driver SNVs in GNAS was detected more frequently in those of Asian ancestry, and less frequently in those of African ancestry. No sex- or ancestry-associations were found for KRAS, but individual KRAS variants showed a trend towards ancestry-related variation. Conclusions: These findings demonstrate that ancestry contributes to the genomic heterogeneity of PDAC, shaping both global and gene-specific mutation patterns. This work underscores the importance of including diverse ancestries in PDAC molecular profiling efforts which may inform the development of tailored molecular diagnostics and targeted therapies.

Chlorella sorokiniana is an industrially important microalga, but its genetic manipulation remains limited by poor transformation efficiency. Although several barriers have been proposed, the role of intracellular nuclease activity has received little attention. Here, we compared the degradation of exogenous double-stranded DNA in whole-cell lysates from transformation-recalcitrant (Chlorella sorokiniana and Tetradesmus obliquus) and transformation-proficient (Phaeodactylum tricornutum, Microchloropsis gaditana, and Nannochloropsis oceanica) microalgae. C. sorokiniana rapidly degraded both linear and plasmid DNA, while T. obliquus exhibited intermediate activity. In contrast, no detectable degradation was observed in the transformation-proficient species. DNA degradation in C. sorokiniana occurred within minutes, and nuclease activity in both recalcitrant species was inhibited by EDTA and Zn2+, consistent with a metal ion-dependent endonuclease. These findings suggest that intracellular nuclease activity may represent an underexplored barrier to exogenous DNA stability during transformation and provide a potential target for improving genetic transformation of recalcitrant microalgae.