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Quo vadis, BGA? A collaborative EDNAP exercise on the challenges and progress in forensic biogeographical ancestry inference.

There is a broad consensus that forensic tests for the prediction of externally visible characteristics (EVC) and analysis of biogeographic ancestry (BGA) of an individual are technically reliable. However, interpretation of the results and population-specific genotype distribution patterns remains challenging. EVC and BGA analyses provide valuable information for population genetics studies and as investigative leads for criminal cases, as well as for historical and contemporary identification tests. However, inaccurate or incorrect predictions, for example, from subjective bias in the interpretations made, have the potential to misdirect police investigations. The legal situation regarding EVC and BGA testing varies by country: ranging from countries where it is explicitly prohibited, to those without specific regulations on biogeographic ancestry prediction, and others that have already enacted laws governing its use. The reluctance to utilize these analyses is not only due to legal restrictions and data protection concerns, but also to initial limited sets of sufficiently comprehensive forensic DNA assays. Forensic BGA marker panels typically contain up to ∼300 SNPs. This relatively small number of genetic markers, along with limited reference population data, complicates the interpretation of results from donors of unknown origin. This paper presents the results of a collaborative EDNAP study, which, for the first time, evaluated the approach to reporting EVC and BGA data between international laboratories. For the study, DNA from nine individuals with self-reported ancestry was collected and analysed using various forensic panels differing in the number and composition of ancestry-informative markers genotyped, comprising: the Precision ID mtDNA Whole Genome Panel, the VISAGE Basic Tool and the VISAGE Enhanced Tool for Appearance and Ancestry Prediction, and the Ion AmpliSeq™ PhenoTrivium Panel. To ensure full data protection, all SNP genotypes and uniparental marker haplotypes obtained were not shared with third parties. Instead, the genetic data were analysed using a range of commonly used population analysis software packages. These analysis outcomes were then distributed to twelve European forensic laboratories (both academic and law enforcement institutions), who were asked to prepare reports based on their interpretation of the phenotypes and ancestry they inferred from the analysis data. A questionnaire sent alongside the genetic information, aimed to evaluate which difficulties were encountered by the participants in processing the BGA analysis data they were given.

Humans

Chemical and sensory profiling of fermented, washed, and artificially flavored coffee beans: Insights into flavour quality, authenticity, and food safety implications.

This study establishes an integrated framework combining chemical profiling, sensory analysis, and molecular mechanism evaluation to compare flavour quality and authenticity among fermented, washed, and artificially flavored coffees. GC&#xa0;&#xd7;&#xa0;GC-TOF-MS and UHPLC-HRMS showed that fermented samples had markedly higher ester and aromatic alcohol levels (total esters 74.5&#xa0;&#xb1;&#xa0;7.8&#xa0;mg&#xa0;kg-1; phenylethanol 27.5&#xa0;&#xb1;&#xa0;3.2&#xa0;mg&#xa0;kg-1, p&#xa0;<&#xa0;0.01), enhancing fruity-floral notes. Washed coffees contained the highest organic acid concentrations (45.2&#xa0;&#xb1;&#xa0;3.8&#xa0;mg&#xa0;kg-1, p&#xa0;<&#xa0;0.01), supporting brightness and umami. Artificially flavored coffees exhibited elevated exogenous aromatics (vanillin 21.5&#xa0;&#xb1;&#xa0;3.1&#xa0;mg&#xa0;kg-1) but significantly fewer Maillard products (p&#xa0;<&#xa0;0.05) and reduced flavour retention (55% after 14 days). Molecular docking revealed higher theoretical binding affinities for naturally generated compounds, suggesting a potential molecular basis for their greater sensory persistence. The framework supports constructing coffee quality fingerprints and verifying flavour authenticity.

Flavoring Agents

An RPA-assisted homogeneous electrochemical DNA sensor for on-site eDNA detection toward early warning of crown-of-thorns starfish outbreaks.

Crown-of-thorns starfish (COTS) outbreaks seriously threaten coral reef ecosystems, while conventional monitoring approaches are time-consuming and often lack sufficient sensitivity for early warning. Existing electrochemical DNA sensors usually require complex electrode-surface immobilization procedures, which can lead to uneven probe distribution, significant steric hindrance, and poor stability. Meanwhile, the low concentration of environmental DNA (eDNA) in marine environments further complicates detection. To overcome these challenges, this study developed a homogeneous electrochemical DNA sensor assisted by recombinase polymerase amplification (RPA) for COTS eDNA detection. Target DNA was first amplified by RPA, and the amplification products were then hybridized in solution with capture probe (CP)-modified magnetic beads (MB) and biotin-labeled signal probe (SP) to form sandwich-structured MB complexes. These complexes were subsequently magnetically enriched and immobilized on the electrode surface for electrochemical signal readout. Under optimized conditions, the sensor displayed a linear response to COTS genomic DNA from 3.77&#xa0;fg/&#x3bc;L to 1&#xa0;ng/&#x3bc;L, with an LOD of 2.02&#xa0;fg/&#x3bc;L and an LOQ of 3.77&#xa0;fg/&#x3bc;L. The sensor was applied to Xisha Islands samples, and the results agreed with droplet digital PCR (ddPCR) (P&#xa0;>&#xa0;0.05), demonstrating its potential for sensitive and reliable on-site COTS eDNA detection.

Animals

Integrative analysis of transcriptome and DNA methylome dynamics during caudal fin regeneration in silver pomfret (Pampus argenteus).

Caudal fin regeneration in teleost fish is a complex, multi-stage process involving coordinated molecular and cellular changes. While the role of epigenetic regulation particularly DNA methylation has been studied in model freshwater species such as zebrafish, its contribution to regeneration in marine teleosts remains largely unexplored. In this study, we integrated transcriptomic and DNA methylomic data to characterize the temporal dynamics of gene expression and methylation during caudal fin regeneration in the silver pomfret (Pampus argenteus). Using RNA-sequencing and reduced representation bisulfite sequencing (RRBS) at three biologically critical time points 1, 3, and 7&#xa0;days post-amputation (dpa), we characterized the spatiotemporal molecular landscape of caudal fin regeneration. These time points capture the key transitional phases of wound healing and inflammation (1 dpa), blastema formation and progenitor proliferation (3 dpa), and regenerative outgrowth with tissue remodeling (7 dpa), enabling robust detection of the major molecular programs underlying epimorphic regeneration. Concurrently, CG-methylome analysis identified thousands of dynamically changing differentially methylated regions (DMRs). A strong global inverse correlation was observed between promoter methylation and gene expression. Integrative analysis pinpointed key regeneration genes (fgf20a, msxb, sox9b) whose expression was associated with dynamic methylation changes in their promoters or gene bodies. We conclude that DNA methylation is a dynamic and key regulatory layer that acts in concert with transcriptional reprogramming to coordinate tissue regeneration, providing new insights into the epigenetic mechanisms underlying complex regenerative processes in teleosts.

Animals

Mitochondrial DNA diversity in Ecuadorian populations: Recurrence of variant 16136 within haplogroup B2.

The identification of lineage-defining variants, frequently found in the coding region of mitochondrial DNA (mtDNA), is essential for refining haplogroup classification. Most mtDNA studies in South American populations have focused on the control region (CR), which has provided important insights into population structure and maternal lineage origins, although information needed for more robust phylogenetic resolution has been neglected. This study investigates the maternal genetic structure of Ecuadorian populations by combining CR and whole mitogenome analyses. Sequences from the mtDNA CR were obtained from 461 individuals (253 Mestizos and 208 Native Americans), while complete mitogenomes were sequenced for 127 individuals to improve phylogenetic resolution by identifying lineage-defining variants present in coding region. Most mtDNA haplogroups in the two population groups analyzed were of Native American origin (A2, B2, B4, C1, D1, D4), with significant differences in the distribution of specific lineages between them. Among Mestizos, African haplogroups (all within the L branches) and Eurasian haplogroups (H, K, R, U) were detected at low frequencies, whereas no African lineages were observed among Native Americans. The results obtained highlighted a heterogeneity within Ecuadorian populations that must be considered when developing mtDNA haplotype databases for forensic purposes. Whole mitogenome sequences enabled the identification of variants that refined haplogroup classifications, provided a more accurate reconstruction of the maternal genetic diversity, and improve the discrimination between Native American and Asian maternal lineages within haplogroup B4b.

Humans

Nanopore-based epigenomic profiling reveals the absence of widespread CpG methylation in the African swine fever virus genome.

DNA methylation is a critical epigenetic mechanism implicated in regulating replication and transcription in DNA viruses. However, the epigenetic landscape of African swine fever virus (ASFV), a large double-stranded DNA virus infecting pigs, remains controversial. Here, we systematically profiled the DNA methylome of the first ASFV strain isolated in Hong Kong (HK_NT_202103) using Oxford Nanopore Technologies (ONT) R10.4.1 sequencing. We employed a paired design: native whole-genome sequencing (WGS) against a methylation-free whole-genome amplification (WGA) control. Using conservative thresholds, we found no evidence of 5-methylcytosine (5mC), especially typical CpG methylation, across the viral genome. Importantly, clear CpG methylation signals were successfully detected in the host genome from WGS data, confirming the functionality of the workflow to detect 5mC at CG sites. While widespread 5mC seems absent, a small number of putative N6-methyladenine (6mA) loci were identified. A specific 6mA candidate exhibited raw ionic current disruptions and gene-level intersection with another ASFV isolate (CAS19-01/2019), although it lacked single-base consensus across different methylation callers or between the two isolates. Although our biological findings are restricted to a single isolate under specific experimental conditions, this study introduces a novel, highly rigorous ONT framework for viral epigenomics research. Furthermore, the absence of ASFV CpG methylation indicates that host CpG-depletion remains a viable strategy for viral metagenomic enrichment. Ultimately, our work offers a critical methodological baseline for ASFV surveillance and highlights the necessity of targeted experimental validation for rare viral modifications.

African Swine Fever Virus

Spatially confined electrochemical strategy with DNA-assembled nanogaps for SNP detection.

Accurate detection of low-abundance single nucleotide polymorphisms (SNPs) against a large excess of homologous wild-type sequences requires both selective molecular recognition and effective transduction of small sequence differences into measurable signals. Here, we report a spatially confined electrochemical strategy that couples sequence-selective recognition with size-dependent mass-transport gating. DNA-hybridization-driven self-assembly of gold nanoparticles (AuNPs) forms a three-dimensional self-assembled electrode (3D-SAE) with a DNA-defined interparticle architecture. Competitive probes (SP/WP) convert single-base recognition into distinct molecular-size states: the SNP-associated pathway preferentially triggers a hybridization chain reaction (HCR), generating bulky AuNP-anchored HCR/methylene blue complexes (Au@HCR/MB) with reduced electrochemical accessibility through the porous 3D-SAE, whereas the wild-type pathway does not trigger HCR and maintains a high-current response from more readily accessible MB-containing species. Thus, sequence recognition is translated into a molecular-size difference and subsequently into an electrochemical signal through differential mass transport. Under buffer conditions, the platform achieved a statistically estimated detection limit of &#x223c;0.47&#x202f;fM and a quantitative range of 1&#x202f;fM-100 pM. It discriminated a 0.1% mutant abundance in a fragmented genomic-DNA background. The downstream signal-transduction chemistry is enzyme-free and isothermal. This work establishes a mechanistical recognition-size-conversion-mass-transport-gating architecture for electrochemical nucleic acid analysis.

Polymorphism, Single Nucleotide

Pervasive hybridization and introgression in Diervilleae (Caprifoliaceae).

Diervilleae (Caprifoliaceae) is a horticulturally important lineage with striking floral diversity and a long history of interspecific crossing, suggesting reticulate evolution. We integrated nuclear SNPs and whole plastome data to reconstruct a phylogenomic backbone for the tribe and to identify hybrids, cultivated accessions, and introgression among lineages. Nuclear and plastid phylogenies consistently recover Weigela and Diervilla as reciprocally monophyletic and resolve four major lineages within Weigela, providing a reproducible framework for revising sectional limits and species boundaries. Cultivated accessions form a well supported clade sister to W. florida and show predominantly W. florida ancestry while retaining contributions from multiple wild lineages, consistent with recurrent crossing, backcrossing, and selection. Analyses of wild populations reveal recurrent hybrids and enable plausible parental combinations to be inferred. Tests across the genome further indicate strong evidence for historical introgression across Diervilleae, with the strongest signals involving W. middendorffiana, W. maximowiczii, and Diervilla. Fossil evidence, divergence time estimation, and paleodistribution modelling together suggest range expansion during the Miocene and Pliocene followed by climate driven contraction, providing a spatiotemporal context for episodic contact, introgression, and the East Asia-North America disjunction.

Hybridization, Genetic

Routine methods misidentify Serratia spp.: Limitations of MALDI-TOF MS revealed by whole-genome sequencing.

Accurate species-level identification within the genus Serratia remains challenging due to extensive phenotypic overlap and high genomic relatedness among closely related and recently described taxa. This study presents an evaluation of routine and genome-based identification approaches applied to clinical Serratia isolates, integrating phenotypic assays, MALDI-TOF MS (Bruker Daltonics), 16S rRNA gene sequencing, and Whole-Genome Sequencing (WGS). A total of 103 isolates collected from a teaching hospital were analyzed. WGS was performed on a subset of isolates. Conventional biochemical methods classified all isolates as Serratia marcescens, whereas MALDI-TOF MS identified 60.1% as S. marcescens, 11.6% as S. ureilytica, and 28.1% just at the genus level. Peak analysis from MALDI-TOF MS revealed specific peaks associated with S. marcescens and S. ureilytica, but limited discriminatory power. WGS of six isolates initially identified as S. ureilytica by MALDI-TOF MS revealed reclassification as Serratia sarumanii (n = 5) and Serratia montpellierensis (n = 1), supported by Average Nucleotide Identity (ANI), Average Amino Acid Identity (AAI), and Digital DNA-DNA Hybridization (dDDH) thresholds. In contrast, 16S rRNA analysis showed limited species-level resolution. Phylogenomic and SNP-based analyses confirmed these classifications with strong support. Overall, this study underscores the critical role of high-resolution genomic approaches for precise species identification and highlights the need for continuous expansion and curation of MALDI-TOF MS reference databases to support reliable clinical diagnostics and epidemiological surveillance of emerging Serratia species.

Spectrometry, Mass, Matrix-Assisted Laser Desorpti

Artificial intelligence for anticancer drug discovery from natural products of macroalgae and sponges: A systematic review.

Marine natural products (MNPs) from macroalgae and marine sponges have inspired clinically important anticancer agents, including the cytarabine pharmacophore and the eribulin scaffold, while cyanobacterial dolastatin chemistry supplies the auristatin payloads of several marine-inspired antibody-drug conjugates (ADCs) such as brentuximab vedotin. Artificial intelligence (AI) methods, encompassing both classical machine learning (ML) with hand-engineered features and modern deep learning (DL) with many-layered neural networks, are increasingly supporting key decisions in natural-product anticancer drug discovery, including bioactivity prediction, target identification, absorption, distribution, metabolism, excretion and toxicity (ADMET) filtering, generative analogue design, and the selection of preclinical candidates. DL architectures relevant to this field include graph neural networks, transformer-based molecular generators, diffusion models for protein-ligand docking, and convolutional networks for mass spectrometry, while classical ML contributes interpretable fingerprint-based bioactivity models and molecular networking for dereplication. This review follows a systematic literature review methodology to organize the landscape of AI methods now applied to MNP anticancer discovery, distinguishing ML and DL approaches where relevant, situating them within the chemical context of macroalgal and sponge-derived oncology leads, and critically examining published case studies, including validation level (computational, in vitro, in vivo, clinical). The principal bottleneck for medical translation has shifted partly from algorithmic capability toward data infrastructure and experimental validation. Sparse, heterogeneous, and taxonomically biased bioactivity records limit what current models can learn and reduce the reliability of AI-prioritized candidates entering the preclinical pipeline. A roadmap is proposed that prioritizes open MNP-specific benchmarks, symbiont-aware modeling, and active learning loops with synthesizability and ADMET constraints. These AI workflows may accelerate the prioritization of marine-derived anticancer leads and support earlier, more evidence-based translational decisions in oncology drug development.

Biological Products

Engineering bubble structures as Cas12a activators for highly sensitive monitoring of WRN helicase function.

The Werner syndrome helicase (WRN) is a critical synthetic lethal target in microsatellite instability cancers, essential for resolving complex genomic structures like replication bubbles and R-loops. However, strategies to simultaneously discriminate WRN activity on DNA versus DNA-RNA substrates in living cells are lacking. Here, we developed a structure-specific CRISPR/Cas12a biosensing strategy to visualize WRN functional activity by engineering bubble-structure probes. These probes were rationally designed to structurally mimic DNA replication bubbles and R-loop associated DNA-RNA hybrids. Upon specific unwinding by WRN, the probes release a sequestered activator strand that triggers Cas12a trans-cleavage, effectively converting the unwinding event into an amplified fluorescent signal. This assay achieves low picomolar sensitivity (LODs: 5.6-6.0 pM) and exceptional selectivity against homologous RecQ helicases. Uniquely, this strategy enables the parallel quantification of WRN activity on both substrate types, providing insights into distinct WRN-mediated pathways for resolving genomic stress. We further demonstrated the strategy's utility by visualizing endogenous WRN dynamics in living cells and profiling the efficacy of small-molecule inhibitors. This work offers a powerful molecular toolkit for dissecting WRN biology and facilitating high-throughput drug screening in targeted cancer therapy.

Werner Syndrome Helicase

Complex evolutionary history of Rosales mediated by extensive incomplete lineage sorting and hybridization.

The angiosperm order Rosales still represents a major challenge for phylogenetic reconstruction. Although its circumscription is now well-defined, phylogenetic relationships among families are still uncertain. Here, we used nuclear, plastid, and mitochondrial genomic data from 33 species representing all nine families to further clarify interfamilial relationships and the group's evolutionary history. We detected significant phylogenetic conflict among the three datasets. Further analyses at the nuclear level identified incomplete lineage sorting (ILS) as the main cause of unstable phylogenetic positions among families. The discordant placements of Rhamnaceae and Elaeagnaceae based on plastid and mitochondrial data are caused by ancient hybridization events, potentially involving differences in organellar inheritance. Our molecular dating confirms earlier suggestions that the ancient rapid diversification of the three Rosaceae subfamilies could be the main reason for the difficulties in resolving their phylogenetic relationships. Our findings provide new insights into the interfamilial relationships of Rosales and demonstrate that the evolutionary history of this order was shaped by ancient and rapid radiation as well as extensive ILS and reticulate evolution. They also suggest that previous attempts to clarify interfamilial relationships in this order were hampered by combining nuclear and organellar sequence data, leading to inconsistent topologies observed across earlier studies.

Phylogeny

Prevalence of Theileria luwenshuni in goats (Capra hircus) on Tarama Island, Okinawa, Japan.

Caprine theileriosis is an economically important tick-borne disease caused by various Theileria species, particularly Theileria lestoquardi, Theileria luwenshuni, and Theileria uilenbergi, in goats (Capra hircus). Goat farming plays an economically and culturally important role on Tarama Island, Okinawa, Japan. Because goats on the island are mainly managed under an extensive grazing system, tick infestation is common. However, Theileria infections have not previously been investigated in goats on Tarama Island. To address this, archived DNA samples prepared from blood collected from 44 goats on Tarama Island were screened using a universal PCR assay targeting 18S rRNA sequences of Theileria and Babesia species. Two DNA samples were positive, and sequencing analysis of the amplicons identified T. luwenshuni. To further investigate the epidemiology of T. luwenshuni on Tarama Island, blood samples were subsequently collected from 96 goats across 19 farms. From each blood sample, a thin blood smear was prepared and genomic DNA was extracted. Microscopic examination of Diff-Quik-stained smears detected intraerythrocytic Theileria-like organisms in 35 (36.5%) goats. In addition, screening of DNA samples using a newly developed T. luwenshuni-specific PCR assay detected 77 (80.2%) positive goats, and the subsequent sequencing analysis confirmed the PCR results. Given that T. luwenshuni can cause severe disease in small ruminants, our findings highlight the importance of managing T. luwenshuni infection in goats on Tarama Island.

Animals

HPV circulating tumor DNA as a potential prognostic and predictive biomarker in head and neck squamous cell carcinoma: a systematic review.

PURPOSE: Human papillomavirus circulating tumor DNA (HPVctDNA) has emerged as a promising prognostic biomarker in HPV-related head and neck squamous cell carcinoma (HNSCC). This systematic review aimed to synthesize current evidence on the diagnostic accuracy and prognostic value of HPVctDNA in HNSCC management. MATERIAL/METHODS: We systematically reviewed a PubMed-indexed database of studies published between January 2012 and September 2025. Eligible studies were assessed for design, primary tumor site and stage, treatment modality, HPVctDNA detection method, diagnostic accuracy (sensitivity and specificity), and reported clinical endpoints. Descriptive syntheses were performed; sensitivity and specificity were standardized to proportions and summarized as median values per group. RESULTS: A total of 60 studies, including 8,234 patients were analyzed, of which 41 (68.3%) focused exclusively on oropharyngeal squamous cell carcinoma (OPSCC) and 17 (28.3%) included mixed HPV-related HNSCC subsites and HPV-positive cancers of unknown primary. The median follow-up across the included studies was 23&#xa0;months. Among the included studies, 19 were retrospective (31.7%) and 33 were prospective (55.0%), with a small proportion of cross-sectional and randomized clinical trials. Overall, 40 (66.7%) evaluated the role of HPVctDNA in a curative setting. Plasma was the most common sample type, analyzed in 55 studies (91.7%), while 5 studies also included saliva. Detection methods varied: 40 employed droplet digital PCR (ddPCR), 16 used quantitative PCR (qPCR) and 4 applied NGS-based assays. Most of these studies (38, 63.3%) evaluated the prognostic utility of HPVctDNA, while only 4 (6.7%) assessed HPVctDNA in a screening or diagnostic setting. Regarding diagnostic accuracy, the median sensitivity across evaluable studies was 91.1%, while the median specificity was 99.4%. In OPSCC-only cohorts, the median sensitivity and specificity were 89.4% and 99.4%, respectively. Dynamic changes in HPVctDNA levels during or after treatment were consistently associated with outcomes: clearance or sustained negativity correlated with higher response rates, improved progression-free survival and overall survival, while persistent positivity or increasing levels predicted disease progression and recurrence. CONCLUSIONS: HPVctDNA demonstrates high diagnostic and prognostic accuracy in HPV-related HNSCC, especially OPSCC, supporting its use for prognosis, treatment monitoring and early detection of recurrence. However, prospective interventional studies are still required to demonstrate that HPVctDNA-guided treatment decisions improve clinical outcomes before routine implementation.

Humans

Diagnostic value of plasma cell-free DNA metagenomic next-generation sequencing in patients with suspected infections and exploration of clinical scenarios-a retrospective study from a single center.

BACKGROUND: Plasma cell-free DNA metagenomic next-generation sequencing (mNGS) is a non-invasive comprehensive method for the etiological diagnosis of various infectious diseases. However, research on the early diagnosis and real-world clinical impact of plasma mNGS in patients with suspected infection are still limited. MATERIALS AND METHODS: This study retrospectively included 140 patients with suspected infections who underwent early plasma mNGS and conventional culture testing. Referring to the clinical diagnosis of infectious diseases, the diagnostic performance of plasma mNGS and culture tests was compared, and the application scenarios and clinical effects of plasma mNGS were evaluated. RESULTS: The positive rate of plasma mNGS was significantly higher than that of culture methods (55.71% vs 25.10%, p&#x2009;<&#x2009;0.001) and blood cultures (55.71% vs 12.86%, p&#x2009;<&#x2009;0.001). Regarding clinical diagnosis, the sensitivity of plasma mNGS was significantly higher than that of culture (58.27% vs 37.80%, p&#x2009;=&#x2009;0.002). The combination of mNGS and culture achieved a higher detection sensitivity (69.29%), especially in patients with multi-site co-infections (73.68%) and blood infections (73.17%). Plasma mNGS demonstrated higher sensitivity in patients with procalcitonin (PCT) index > 5&#x2009;ng/ml or human neutrophil lipocalin (HNL) index > 200&#x2009;ng/ml. In terms of treatment, a total of 69 patients (54.33%) benefited from plasma mNGS. CONCLUSION: This study highlights the significant improvement in pathogen detection performance by combining conventional culture with plasma mNGS detection, especially in patients with multi-site co-infections and blood infections. Early use of plasma mNGS as an adjunct to culture can better guide clinicians to initiate appropriate anti-infective therapy.

Humans

A system-level metastable model of cancer evolution: integrating replication stress, cell cycle deregulation and chromosomal instability.

INTRODUCTION: Cancer cell proliferation occurs within the context of persistent genomic instability. In this review, we propose the RS-CCD-CIN axis as a systems-level framework in which replication stress (RS), cell cycle deregulation (CCD) and chromosomal instability (CIN) form an interdependent triad that shapes tumour evolution. This axis represents a constrained metastable state in which genomic instability is tolerated and buffered. The objective of this review is to synthesize the current understanding of how the RS-CCD-CIN axis contributes to tumour heterogeneity, adaptability and therapy response. DISCUSSION: Evidence indicates that RS, CCD and CIN operate as a dynamic, interconnected network rather than as independent processes. Replication stress induces DNA damage and mutagenesis, while partial checkpoint disruption permits cells with unresolved lesions to proliferate. Chromosomal instability generates both structural and numerical alterations, contributing to intratumoural heterogeneity. Together, these processes facilitate adaptation to environmental and therapeutic pressures. Extrachromosomal DNA, micronuclei formation and cytosolic DNA signalling, including the cGAS-STING pathway, connect genomic instability to adaptive responses and immune modulation. Single-cell and spatial profiling reveal temporal and spatial variability in RS, CCD and CIN states, highlighting the limitations of static biomarkers. Therapeutically, targeting individual components often yields limited durability, whereas approaches that simultaneously perturb multiple aspects of the RS-CCD-CIN axis may improve clinical outcomes. CONCLUSIONS: This review highlights the RS-CCD-CIN axis as a fragile and metastable architecture that supports cancer evolution, while also being susceptible to collapse. A deeper understanding of this interconnected framework may inform the development of therapeutic strategies and enhance the management of resistance.

Humans

Viral replication through phase separation: Cytosolic and nuclear condensates.

Replication of many RNA and DNA viruses occurs within specialized intracellular hubs organized as membraneless biomolecular condensates (BCs) driven by liquid-liquid phase separation. As obligate intracellular parasites, viruses depend on the host cell machinery to complete their replication cycles and therefore actively remodel the intracellular environment to favor viral genome replication, transcription, and assembly. Cytosolic and nuclear phase-separated replication compartments (RC) provide concentrated and dynamic platforms that promote efficient interactions between viral genomes and viral or host proteins essential for infection. The formation of viral replication BCs is typically facilitated by viral proteins enriched in intrinsically disordered regions and low-complexity domains, which enable multivalent interactions with viral nucleic acids and cellular factors. These interactions are mediated by diverse biophysical forces, including hydrophobic and &#x3c0; interactions, hydrogen bonding, molecular crowding, and osmotic effects. Throughout infection, viral BCs remain highly dynamic, allowing continuous exchange of components and functional maturation of replication hubs. Their properties and activities are further regulated by post-translational modifications of viral and host proteins, such as phosphorylation, acetylation, and methylation. In this review, we summarize current evidence supporting liquid-liquid phase separation as a central organizing principle of viral RCs. We focus on representative RNA and DNA viruses that replicate in the cytosol or nucleus, highlighting virus-specific strategies, conserved mechanisms, and the consequences of BC formation for viral replication efficiency, host antiviral responses, and therapeutic intervention.

Phase Separation

First identification and molecular subtyping of Blastocystis spp. in donkeys in Aksaray province, T&#xfc;rkiye.

Blastocystis is a common intestinal protist worldwide that can infect humans and animals. Although its molecular epidemiology in T&#xfc;rkiye is mostly focused primarily on humans and livestock, equids have received limited attention despite their traditional roles and frequent contact with humans and other animals in rural environments. This study aimed to determine the molecular prevalence and subtype (ST) distribution of Blastocystis spp. in donkeys in Aksaray Province, providing the first molecular data on donkeys in T&#xfc;rkiye. A total of 182 fresh fecal samples were collected from donkeys in nine villages within Aksaray province. Genomic DNA was extracted, and the small subunit ribosomal RNA (SSU rRNA) gene fragment of Blastocystis spp. was amplified via PCR analysis. Positive isolates were sequenced bidirectionally for identification and subsequent phylogenetic analysis of Blastocystis in donkeys. The overall molecular prevalence of Blastocystis spp. in donkeys was 4.4% (8/182). The infection rate was higher in young donkeys (under 3&#xa0;years old; 8.33%) than in adults (3&#xa0;years or older; 2.46%). However, this difference was not statistically significant. Sequence analysis of the positive PCR products revealed the presence of one known livestock-specific subtype, ST10. Phylogenetic analysis showed that the ST10 isolates characterized in this study clustered with isolates identified from different hosts. This study provides the first molecular data on Blastocystis presence in donkeys in T&#xfc;rkiye. The exclusive detection of ST10 suggests potential cross-species transmission, likely facilitated by the traditional practice of co-housing donkeys with other animals in confined barns. These findings indicate that donkeys may contribute to Blastocystis transmission, underscoring the importance of a "One Health" approach in future epidemiological surveillance.

Animals