Abstract Large-scale pharmacogenomic screens enable systematic searches for genetic determinants of drug sensitivity, but cross–cell-line analyses are confounded by lineage structure and drugfree proliferation rates. We integrate DepMap genome-scale CRISPR dependency profiles with PRISM viability responses and estimate covariate-adjusted associations between gene dependency and a statin selectivity phenotype using cross-fitted double machine learning (DML). The analysis recovers mevalonate-pathway regulators (MVK, UBIAD1) and identifies SLC45A4 as a colorectal-enriched candidate association. To assess robustness, we validate findings in independent AUC datasets, conduct leave-one-drug-out sensitivity analyses, and benchmark against empirical-null drug-bundle and gene-level negative controls. In colorectal lines, the SLC45A4 signal aligns with sterol-biosynthesis-related co-dependency and colorectal-specific shifts in polyunsaturated cholesteryl esters and putrescine in CCLE metabolomics. As positive controls, expected EGFR- and BRAF-matched drug responses were reproduced in independent datasets. These results reduce concern that the SLC45A4 association is explained solely by cross-dataset integration bias and support further experimental testing.

Abstract Background : Inferred gene regulatory networks (GRNs) from single-cell RNA-seq are used to prioritize transcription-factor–target hypotheses, yet edge rankings can be inflated by confounds that are rarely audited systematically. Individual confound classes—technical batch effects, genomic proximity co-expression, and degree-distribution artifacts—have been studied in isolation, but no prior work has conducted a unified audit across all three on the same datasets and inference methods. Results : We present grn_confound_audit, an open-source Python package that implements a unified three-class confound audit covering technical bias (batch, donor, and assay-method leakage), genomic-structural bias (chromosomal proximity inflation), and topological bias (degree-distribution artifacts). Across three Tabula Sapiens tissues and 12 inference methods, the tool reveals that: (i) donor and batch identity are recoverable from edge features at AUC 0.85–0.97; (ii) prior-heavy methods show 2–3× genomic-proximity enrichment that attenuates to 1.15–1.28× under degree-preserving rewiring; (iii) no individual edge reaches FDR ≤ 0.10 under topological null calibration despite strong global separation (z-scores 12–60). The three confound classes are largely orthogonal, and joint filtering retains only ∼28% of candidate edges. Perturbation validation using CRISPR data shows that technically blacklisted edges have 2.7-fold lower perturbation-significant rates. Conclusions : The grn_confound_audit toolkit enables routine multi-class confound diagnostics for any scored GRN edge list, producing per-edge quality indices, standardised reports, and actionable recommendations. We propose that confound auditing should become a standard component of GRN publications alongside accuracy benchmarks.

Abstract Background Inter-patient expression heterogeneity is not merely statistical dispersion but a structured biological signal encoding distinct therapeutic antigen states. Current antigen discovery frameworks over-prioritize mean expression and systematically underinterpret this variance structure, potentially obscuring the most clinically actionable targets. We asked whether inter-patient transcriptomic heterogeneity could be operationalized as a decomposition framework to reveal recurrent, biologically interpretable modes of therapeutic antigen biology. Methods We developed TANK (Tumor Antigen prioritization by variance-based raNKing) as a heterogeneity decomposition framework - not primarily a ranking method, but an approach to resolving recurrent antigen modes from patient-level transcriptomic distributions. TANK was applied across 33 TCGA cancer types (n > 11,000 patients, 60,656 genes). A 10-gene reference panel was predefined based on independent clinical development status. Non-randomness was confirmed against 1,000 random gene set controls (empirical p < 0.0001). External validation was performed in two independent gastric cancer cohorts (GEO GSE26942, n = 217; ACRG GSE66229, n = 400). Single-cell validation was conducted across three cancer types totaling 28,617 annotated tumor epithelial cells. Mode 3 candidates were characterized by survival analysis, immune correlation, and DepMap CRISPR dependency. Beyond the reference panel, CLDN6 was examined as a showcase candidate across the same dimensions. Results TANK resolved four recurrent modes of therapeutic antigen heterogeneity - tumor-restricted rare activation (Mode 1: PRAME), lineage-dependent expression (Mode 2: CLDN18), tumor-enriched heterogeneous expression with functional stratification (Mode 3: MSLN/OLFM4/VSIG1/MUC16), and mean-dominant baseline (Mode 4: ERBB2/EGFR) - each with distinct implications for patient stratification, immune context, and translational modality. Concordance between TANK and MAD confirms the signal reflects a robust variance-associated structure rather than any single metric artifact. Beyond the reference panel, CLDN6 ranked top 0.22% (comparable to FDA-approved CLDN18 at top 0.16%), with significant adverse survival association (p = 0.0049), immune-cold correlates (CD274 r=-0.204, p < 0.0001), and directional CRISPR dependency across gastric (mean=-0.227), colorectal (mean=-0.242), and lung cancer cell lines (mean=-0.288). Conclusions Inter-patient transcriptomic variance defines a previously underutilized axis of antigen biology encoding recurrent therapeutic modes that are systematically inaccessible to mean-based approaches — CV-based ranking recovers 0/9 reference targets and DESeq2 fails to prioritize CLDN18 and CLDN6 within the top 20%, while variance decomposition places both in the top 0.22%. The four-mode framework enables prospective mapping of novel candidates to distinct therapeutic strategies from heterogeneity structure alone, without prior biological knowledge of the candidate. CLDN6 exemplifies this capacity: nominated solely from variance structure, its convergent multi-dimensional evidence positions it as a high-priority oncofetal antigen candidate for patients with limited therapeutic options.

Neurological and mental disorders are among the main causes of disability worldwide, affecting over three billion people and increasing the socioeconomic burden. Advances in molecular genetics and genome engineering have led to gene-targeted therapies that address root causes rather than just symptoms. This review covers current genome-editing tools, including CRISPR/Cas, base editing, and prime editing. The focus is on the benefits of gene editing in the central nervous system, where post-mitotic neurons allow lasting effects after a single treatment. It also discusses emerging delivery platforms such as viral vectors, nanoparticles, and exosome systems, as well as methods to bypass the blood–brain barrier. Recent clinical progress in spinal muscular atrophy, Parkinson’s, Huntington’s, and Alzheimer’s diseases is highlighted, with promising preclinical results for autism, bipolar disorder, epilepsy, and other neurogenetic conditions. The review concludes with regulatory issues, market trends, and ongoing clinical trials, underscoring the potential of gene therapies to transform disease management and provide long-term solutions.

Abstract Purpose Severe combined immunodeficiency(SCID) is a life-threatening primary immunodeficiency disorder. This study aimed to identify novel recombination activating gene 1 ( RAG1 ) variants in a Chinese pedigree and characterize their impact on protein structure and function, providing a genetic basis for preimplantation genetic testing for monogenic (PGT-M) cycle. Methods Potential RAG1 mutations of the probands were screened by whole-exome sequencing (WES) and confirmed by Sanger sequencing. Configuration predictions of the variants were achieved using SWISS-MODEL. PROVEAN, PolyPhen-2 and MutationTaster were used to predict their pathogenicity. Isogenic pre-B cell lines carrying the mutations were established via CRISPR-Cas9 RNP editing. Functional impacts were assessed through Western blotting, proliferation ability, and apoptosis analysis. Results We identified novel compound heterozygous RAG1 variants c.946T>G (p.C316G) and c.1194_1196del (p.L399del) in two affected siblings with typical SCID. Familial genotyping confirmed autosomal recessive inheritance, with each parent as an asymptomatic carrier of one variant. Both mutations were highly conserved and predicted to be pathogenic. Structural modeling revealed disruption of RAG1 secondary and tertiary structure, affecting zinc-binding (p.C316G) and hydrogen-bonding (p.L399del) interactions. Functional studies demonstrated markedly reduced RAG1 protein expression, synergistic impairment of RAG2 expression, and significantly elevated apoptosis in double-mutant pre‑B cells. Further investigation indicated dysregulation of the PI3K/AKT/FOXO1 pathway, evidenced by increased phosphorylation of AKT and FOXO1. Conclusions Our study provides genetic and functional evidence that biallelic RAG1 p.C316G and p.L399del mutations act synergistically to cause SCID through protein destabilization, disruption of RAG1/RAG2 complex integrity, and induction of pre‑B cell apoptosis likely mediated by PI3K/AKT/FOXO1 signaling dysregulation. These findings expand the mutational spectrum of RAG1 and support the clinical application of PGT-M for affected families.

Abstract Photobacterium damselae is a widespread pathogenic bacterium in marine ecosystems, primarily associated with fish skin ulcers. However, infections caused by this bacterium in shrimp are rarely reported. The dominant strain MRY0520, isolated from the hepatopancreas of diseased Litopenaeus vannamei , was identified as Photobacterium damselae subsp. damselae (Phdd) and exhibited high pathogenicity. Elucidating the genetic architecture and infection mechanisms of P. damselae is crucial for the effective prevention and control of related diseases. To characterize its genomic features, whole-genome sequencing and comprehensive functional annotation were performed on the virulent Phdd strain MRY0520, followed by comparative genomic analysis to investigate its evolutionary traits. The genomic sequences have been deposited in GenBank under accession numbers CP113238–CP113240. The genome of strain MRY0520 spans 4,451,849 bp and contains 3,663 coding genes with a combined length of 3,691,779 bp, accounting for 83% of the total genome. It encodes 208 tRNA genes, 62 rRNA genes (including 22 5S rRNA, 20 16S rRNA, and 20 23S rRNA), two CRISPR arrays, and two genomic islands. Comparative analysis revealed that strain MRY0520 exhibits the highest genomic homology with Phdd strain Wu-1. These findings provide a solid theoretical basis for understanding the genomic composition and evolutionary characteristics of MRY0520.

Climate change intensifies challenges for perennial crops like grapevines (Vitis vinifera) and kiwifruit (Actinidia spp.), where prolonged juvenile phases and climate-sensitive flowering hinder rapid breeding progress. This study employs CRISPR/Cas9 to edit key flowering (VvFT1/2) and architecture (VvTB1/AcTB1) genes, accelerating hybrid development for enhanced climate resilience. Using Agrobacterium-mediated transformation, we achieved 85% editing efficiency in embryogenic calli, yielding mutants with 25-35 day earlier flowering and 20% more compact architectures. Hybrids exhibited 40% improved drought tolerance and 30% higher heat stress survival via reduced transpiration and optimized canopy structure. Phenotypic assays under simulated climate scenarios confirmed accelerated generation cycles, shortening breeding timelines from 8-10 years to 2-3 years. RNA-seq revealed upregulated stress-response pathways, underscoring the edits' role in vigor enhancement. These findings demonstrate CRISPR/Cas9's potential to revolutionize horticultural breeding, delivering resilient grapevine-kiwifruit hybrids for sustainable agriculture amid global warming. This framework extends to other perennials, promoting food security.

Abstract The transition from linear reference genomes to graph-based pangenomes, coupled with the rise of artificial intelligence, requires modern biologists to understand highly complex computational structures. However, a significant gap exists between wet-lab biological training and the algorithmic foundations of modern bioinformatics. Here, we present PanGen-AI , a comprehensive, open-source 8-module Python framework designed to bridge this gap. PanGen-AI integrates diverse computational engines, de Bruijn graph sequence assembly, a PyTorch-based 1D convolutional neural network (CNN) for variant impact prediction, Burrows-Wheeler Transform indexing, Needleman-Wunsch alignment, CRISPR-Cas9 design, and 3D protein structure visualization. The framework culminates in an interactive multi-track genome browser that dynamically overlays AI-derived variant saliency maps, CRISPR target sites, and classical gene annotations onto a unified genomic coordinate system. Deployed via a lightweight Streamlit interface, PanGen-AI serves as both a scalable prototyping environment for automated genomic workflows and a translational tool to demystify computational models in mechanobiology and immunometabolism.

Abstract Pollen fertility in flowering plants relies on precise lipid metabolism, yet the enzymatic regulators underlying these processes remain incompletely understood. Here we identify OsGELP87 , a GDSL-type esterase/lipase, as a key regulator of male gametophyte development in rice ( Oryza sativa ). Phylogenetic analysis revealed that OsGELP87 lacks closely related paralogs, suggesting limited functional redundancy. CRISPR–Cas9 genome editing revealed impaired pollen wall architecture, abnormal nuclear morphology, and reduced lipid droplet accumulation, causing reduced male fertility. Complete loss-of-function mutants were not recovered. Transcriptome and qRT–PCR analyses revealed disrupted lipid metabolic networks, including suppression of FAD2 and pollen lipid transfer proteins, together with altered redox regulators. Structural modelling showed that a premature stop codon in exon 3 abolishes conserved C-terminal motifs and predicted palmitoylation sites, likely impairing catalytic function. Functional analyses suggest that OsGELP87 participates in sporopollenin/tryphine assembly during pollen wall formation and triacylglycerol mobilization during pollen germination. Population genomic analysis uncovered five haplotypes with distinct geographic distributions and fertility phenotypes, while no haplotypes carrying non-functional alleles were detected. Together, these results identify OsGELP87 as a non-redundant regulator of lipid-associated cellular processes required for pollen development and provide new insight into how lipid metabolism contributes to reproductive cell differentiation in plants.

Plant diseases severely constrain agricultural productivity, exacerbating food insecurity, economic instability, and environmental degradation. Global trade and climate change further intensify pathogen spread, emergence, and host shifts. While traditional diagnostics and targeted assays, such as PCR and ELISA, improve specificity, they depend on prior knowledge and are limited in detecting novel or mixed infections. High-throughput sequencing (HTS) has emerged as a transformative, unbiased platform that allows comprehensive detection of known and unknown pathogens through metagenomics and transcriptomics. By generating large-scale genomic data, HTS supports pathogen discovery, epidemiological surveillance, quarantine systems, and genome-informed disease management. It underpins advanced strategies, including CRISPR-Cas editing and RNA interference, and accelerates the breeding of resistance. Despite challenges—such as bioinformatics standardization, cost, and data interpretation—HTS, when integrated with classical diagnostics and biological validation, represents a foundational technology for sustainable, proactive plant health management and global phytosanitary resilience.

Abstract Lymph nodes (LNs) constitute a key anatomical sanctuary for HIV. Follicular helper T (Tfh) cells expand early upon infection and represent a principal cellular target for initial viral seeding. Here, we identified the transcription factor BCL6, a Tfh-lineage defining marker, as central in favoring the infection of Tfh cells in LNs during the untreated phase in humans, and for the persistence of the reservoir during ART in non-human primates. In situ and ex vivo analyses of LN from people with HIV (PWH) in absence of antiretroviral therapy (ART) revealed preferential enrichment of viral RNA, total HIV DNA, and intact proviruses within BCL6hi Tfh cells, which also presented significantly lower expression of proteins with antiviral functions (IRF7, MX1, APOBEC3G, pSTAT1). In vitro genetic (genome-wide CRISPR knockouts) and pharmacologic perturbations confirmed that BCL6 enhances the cellular permissiveness of Tfh cells to HIV infection. IL-10 and TGF-β were enriched in LNs from people without HIV (PWoH), and cooperatively induced bona fide BCL6hi Tfh differentiation in vitro, with repressed antiviral pathways. IL-10 and TGF-β blockade limited Tfh differentiation, confirming their contribution to Tfh and LN biology. Human Single Nucleotide Polymorphisms (SNPs) in proximity to genes of the IL-10 and TGF-β pathways were enriched in PWH who controls viremia spontaneously (HIV elite controllers). Importantly, in vivo downmodulation of IL-10 and TGF-β signaling pathways in ART-treated SIV-infected macaques, by using anti–IL-10 and anti–PD-1 therapy, led to reduced frequencies of LN BCL6+ Tfh cells. These Tfh cells expressed significantly higher expression of antiviral machineries, similar to gene signatures found in HIV elite controllers, and resulted in significantly lower SIV reservoir size in LNs. This data highlights that the modulation of the IL-10/TGF-β/BCL6 axis is relevant at early stages upon infection, but also during ART, after the HIV reservoir is already established. In both scenarios it results in higher antiviral machinery and lower HIV seeding and reservoir sizes. Thus, the modulation of these pathways in vivo has potential to alter Tfh biology in LNs leading to HIV reservoir decay, contributing to HIV cure strategies.

Abstract The understanding of genetic basis and potential therapeutic targets for metastatic breast cancer (mBC) remains limited. Here we systematically identify the genetic and epigenetic causes underlying breast cancer metastasis from clinically defined alterations through multiplexed CRISPR knockout and base editing screens in various models. Multiple drivers converge on the down-regulation of SMARCA1, a core component of ISWI chromatin-remodeling complexes, to drive metastasis, largely through potentiating TGFβ signaling. Using druggable gene CRISPR screens, we identify SOD1, a key antioxidant enzyme, as a prominent vulnerability in mBC. We further validate the in vivo efficacy of SOD1 pharmacological inhibitor LCS-1 in treating mBC in multiple preclinical models. The exacerbated SOD1 vulnerability in mBC is due to elevated TGFβ signaling which impinges on mitochondrial homeostasis, thereby resulting in superoxide radical overload and subsequently triggering ferroptosis. Together, our work provides a comprehensive landscape on the genetic basis and informs therapeutic interventions for mBC.

Abstract Background Genomics and transcriptomics workflows require coordinated use of multiple specialized tools, creating technical barriers for many domain scientists. Large language models (LLMs) have shown promise for supporting bioinformatics tasks, but standalone models lack persistent state, autonomous tool use, and reliable multi-step execution. Agentic AI systems, which extend LLMs with planning, tool integration, and iterative execution, may address these limitations, yet systematic evidence of their application in genomics and transcriptomics remains limited. Methods We conducted a systematic review following PRISMA guidelines (PROSPERO: CRD420261292811), searching PubMed, Embase, and Web of Science. Eligible studies included LLM-based agentic systems defined by autonomous multi-step planning, iterative decision-making, and external tool or workflow invocation applied to genomics or transcriptomics tasks with formal performance evaluation. Two independent reviewers (I.R., A.G.) screened 2,932 unique records. Results Ten studies (2024–2026) met inclusion criteria, covering applications such as single-cell RNA-seq annotation, CRISPR guide design, Mendelian randomization, biomarker discovery, and automated bioinformatics workflows. Six systems used single-agent and four multi-agent architectures, all integrating external tools via code execution, retrieval-augmented generation, or domain-specific APIs. GPT-4–family models were the most common backbone (n = 8). Quantitative evaluations (n = 6) reported performance gains of + 1.4 to + 80 percentage points over baseline LLMs or expert comparators, while qualitative assessments (n = 4) showed high agreement with expert benchmarks. Three architectural patterns emerged: multi-agent designs were associated with tasks involving interpretive uncertainty; structural constraints reduced backbone sensitivity more effectively than model upgrades; and excessive iterative self-revision produced diminishing returns. Risk of bias was high in 80% of studies, primarily due to small datasets, lack of external validation, and subjective reference standards. Conclusions Agentic AI systems represent a shift from passive text generation to autonomous analytical orchestration. Be it as it may, the evidence base remains small and methodologically limited, with no system validated outside its originating research group. Future development should prioritize rigorous external benchmarking on real-world datasets, modular and explainable architectures, and coverage of underrepresented domains including variant interpretation and spatial transcriptomics.

Abstract Introduction : MRSA is a global health concern due to its antibiotic resistance and its role in severe, often fatal skin and soft tissue infections in healthcare and community settings. Objective: The study focuses on detecting MRSA and assessing virulence-associated Cas genes in clinical isolates from skin and soft tissue infections. Method Among 100 SSTI patients studied, 20% had Staphylococcus aureus , 55% had other bacteria, and 25% showed no growth based on standard laboratory identification methods. Antimicrobial susceptibility testing by Viteck-2 system was done, and Molecular analysis by PCR showed diverse CRISPR-Cas subtypes. HRM spa genotyping recognised. Results and Discussion: Among the 20 cases, males were more frequently affected than females, with the highest infection rates observed in the 16–20 and 6–10 year age groups, although no significant association with sex or age was found. Antimicrobial susceptibility testing revealed high resistance to oxacillin, confirming the prevalence of MRSA, along with resistance to multiple other antibiotics, indicating widespread multidrug resistance. Molecular analysis showed diverse CRISPR-Cas subtypes, with significant associations between Cas subtypes and resistance to gentamicin and tetracycline. HRM spa typing demonstrated marked genetic diversity, identifying seven clonal complexes with predominant local lineages, highlighting endemic circulation and potential for increased virulence and antimicrobial resistance. Conclusion: The isolates showed high MRSA occurrence (100% oxacillin resistance) and resistance to some antibiotics. CRISPR-Cas analysis discovered genetic heterogeneity, with Cas4 most common, and specific links between Cas subtypes and resistance (gentamicin–Cas4, tetracycline–Cas10).HRM spa genotyping recognized seven clonal complexes, representing both local endemic motion and significant genetic variety, which may influence virulence and resistance patterns. The findings highlight the high MRSA burden, the diverse genetic landscape of S. aureus, and the probable role of CRISPR-Cas in resistance, the necessity for incessant surveillance and informed antimicrobial therapy.

Abstract Genome-wide association studies (GWAS) have identified numerous loci associated with Type 2 Diabetes (T2D), yet translating statistical signals into experimentally testable hypotheses remains a central challenge in post-GWAS biology. The predominance of non-coding regulatory variants complicates target gene assignment and raises uncertainty regarding optimal CRISPR perturbation strategy. Here, we present a structured CRISPR Actionability Framework that integrates genomic context, pancreatic islet enhancer overlap, tissue-specific expression validation, and locus clarity into a quantitative CRISPR Actionability Score (CAS). We applied this framework to ten genome-wide significant T2D loci and assigned modality-aware CRISPR strategies (knockout versus CRISPR interference). CAS values ranged from 4 to 10, enabling tiered prioritization into high, moderate, and lower experimental priority classes. High-priority loci included SLC30A8, TCF7L2, and KCNJ11, which demonstrated strong regulatory or coding evidence combined with islet expression support. By explicitly linking genomic architecture to perturbation modality, this framework provides a transparent and reproducible bridge between statistical genetics and functional genome editing. This approach establishes a scalable template for rational CRISPR target selection in complex disease research.

Abstract Low-temperature plasma provides a chemical-free method for random mutagenesis, however, conventional systems often require bulky equipment and rare gases, which limits their accessibility. Here, we present a Compact Plasma Mutagenesis Instrument (CPMI) that operates in ambient air at 2–8 W, reducing energy consumption while increasing the plasma–sample interaction volume. Mechanistic studies indicate that CPMI induces DNA lesions via reactive oxygen and nitrogen species, leading to base oxidation, strand breaks, and adduct formation. The Application of CPMI to Talaromyces albobiverticillus produced mutant strain CY110. This strain exhibited over 4.5-fold of the wild-type strain extracellular pigment production, significantly decreased citrinin levels, and enhanced antioxidant activity relative to the parental strain. Unlike chemical mutagens or UV irradiation, CPMI generates no toxic byproducts, and unlike genome-editing tools such as CRISPR/Cas, it offers a non-genetically modified organism (GMO) approach suitable for food-grade applications. Collectively, these results establish CPMI as an accessible, energy-efficient, and regulation-compliant mutagenesis platform that complements existing synthetic biology toolkits. Unlike traditional systems, the air-driven CPMI eliminates noble gas reliance, providing a cost-effective and portable platform for industrial microbial optimization.

Abstract Ischemic cardiomyopathy is associated with myocardial injury and increased mortality. During myocardial infarction (MI), necrotic cardiomyocytes release damage-associated molecular patterns (DAMPs) that trigger inflammatory responses, yet the specific cardiac alarmins and downstream mechanisms driving cardiomyocyte apoptosis remain unclear. In this study, we identified endogenous S100 protein isoforms as key cardiac alarmins released during myocardial ischemic injury and elucidated their role in activating IFIT3 overexpression through innate immune signaling pathways. Mining patient biopsy expression data and verifying a rat model of left anterior descending (LAD) artery occlusion, we validated IFIT3 overexpression in both human and rodent cardiac tissues during acute myocardial infarction (AMI). To investigate the functional role of IFIT3, we employed CRISPR-Cas9 gene-editing technology to knock out IFIT3 in AC-16 human cardiac cells and developed a continuous oxygen-glucose deprivation/reperfusion (OGD/R) model to mimic MI at the cellular level. IFIT3 knockout significantly inhibited apoptosis induced by OGD/R and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), as detected by Annexin V-FITC/PI double staining. Mechanistically, we utilized Type I interferons, TLR agonists, and STING agonists to dissect the dominant DAMP signaling pathway, revealing that S100 proteins activate IFIT3 overexpression through the TLR3/TICAM1/IRF3 pathway, thereby promoting cardiomyocyte apoptosis. This research establishes a novel S100-TLR3-IFIT3 signaling axis in the pathophysiology of ischemic cardiomyopathy, providing new mechanistic insights and potential therapeutic targets for myocardial ischemic injury.

Abstract Background In deep-sea hydrothermal vents ecosystems, most animals harbor symbiotic communities supporting their nutrition. This is the case of both shrimps Rimicaris exoculata and Rimicaris chacei , two endemic species of the Mid-Atlantic Ridge (MAR), housing three distinct bacterial symbiotic communities playing a major role in their nutrition and scaling up their immune systems. One is located in the cephalothoracic cavity, the second in the foregut and the last one in the midgut, mostly represented by Candidatus Microvillispirillaceae. However, recent metabarcoding and metagenomics studies reported for the first time the presence of another abundant potential digestive symbiont representing a novel family of Lachnospirales . To date, their role and structuration in the holobiont remain unknown. We combined Fluorescent in situ Hybridization, metabarcoding and genome-resolved metagenomics data to reveal a part of their evolution, contribution to the holobiont functioning and their metabolic potential. For this, we used the raw reads of a recent metabarcoding analysis and Metagenome-Assembled Genomes (MAGs) obtained in a previous study. Results We studied two MAGs reconstructed from TAG and Snake Pit sites (MAR) revealing a novel digestive Lachnospirales family in the midgut. This novel symbiont showed the capacity to degrade host’s chitin, to fix carbon dioxide thanks to secondary pathways, to use oxygen and to encode for flagellar genes implied in host-symbiont recognition. In addition, they harbor CRISPR/cas genes that may be involved in the holobiont defense. Lachnospirales seemed to colonize the ectoperitrophic space, were submitted to elongation without dividing themselves and are acquired post-installation of the juveniles on active sites. Consequently, they share many commonalities with Candidatus Microvillispirillaceae, which they co-occur with. Conclusion Our data suggest that the Lachnospirales would be mixotrophic and would live in syntrophy with Candidatus Microvillispirillaceae. Indeed, they could degrade chitin, allowing Ca. Microvillispirillaceae to use degradation products for their metabolism. As Ca. Microvillispirillaceae, they are acquired post-installation during metamorphosis. Consequently, both symbionts may strongly contribute to the holobiont fitness.

Abstract Low-cost, portable, and high-performance nucleic acid detection technologies are essential for point-of-care diagnosis of infectious diseases. Here, we report an ultralow-cost (~$0.1) magnetofluidic cartridge system featuring a novel tapered cylindrical-like cartridge with stable vertical separation of multiple reagents using a combination of silicone oil and wax layers in a cocktail-like configuration. We systematically investigate the mechanisms that enable shake-proof vertical isolation of multiple immiscible reagent layers, independent of aqueous reagent density, thereby allowing the stable maintenance of arbitrary numbers and arrangements of reagent layers. This innovation facilitates fully automated magnetic bead-based nucleic acid extraction coupled with multiplex PCR or CRISPR‒Cas-assisted isothermal detection in a handheld analyser. The unique reagent isolation in the cartridge overcomes the limitations of previous magnetofluidic systems by physically separating elution and amplification reagents, allowing broader assay compatibility. SARS-CoV-2 and influenza A/B detection reached 0.1 copies/µL sensitivity after 60 minutes (PCR) and 30 minutes (CRISPR). Clinical testing of 33 samples showed 100% concordance with standard methods. This low-cost integrated cartridge system offers a robust platform for rapid, sensitive, and multiplexed nucleic acid testing in resource-limited settings.

Abstract N6-methyladenosine (m6A) is a pervasive RNA modification that modulates transcript stability and translation, yet how m6A deposition is coordinated with transcriptional programs and genetic context during cancer progression remains incompletely understood. Here, we integrate population-resolved m6A profiling with genomics, proteomics and functional modeling to delineate a conserved but selectively reprogrammed epitranscriptomic landscape associated with prostate cancer progression across ancestries. We identify recurrent alteration of ZC3H13, a core m6A regulator, as a central determinant of tumor-specific m6A attenuation and gene-level methylation reprogramming. An m6A regulator-derived risk score robustly stratifies clinical outcomes across multiple independent cohorts. Genetic and in vivo modeling establish ZC3H13 as a causal driver of tumor growth, invasion, and metastasis. Mechanistically, ZC3H13 physically associates with RNA polymerase II and functions as a transcription-coupling scaffold that directs site-specific, co-transcriptional m6A deposition on oncogenic transcripts. This program operates in a genetic context-dependent manner, reinforcing PI3K-AKT signaling in PTEN-intact tumors while sustaining TGF-β/SMAD-driven epithelial-mesenchymal transition following PTEN loss. Collectively, our findings define a transcription-coupled mechanism through which cancer genomes encode epitranscriptomic states and nominate ZC3H13-mediated m6A regulation as a potential genetically-informed therapeutic vulnerability.

The severe acquired respiratory coronavirus–2 (SARS–CoV-2) infection has initiated both acute and chronic COVID–19 disease between 2020 and 2023, currently evolving with other homologous prior coronavirus strains of the Nidoviridae order, which encompasses other prevalent alpha/ beta coronaviruses, but also the Middle East Respiratory Syndrome (MERS-CoV) and SARS-CoV-1, with recent SARS–CoV–2 variants, increasing demands for effective immunogens and therapeutic approaches that will reduce global disease burden and further infection from SARS–CoV-2 affected individuals that may experience post acute sequelae (PASC) or “Long COVID”. Following a worldwide programme of prophylactic vaccination, there is still a dilemma in the efforts to find prophylactic and early therapeutic approaches that would treat novel SARS-CoV-2 variants and prevent future epidemics or pandemics within host human and animal populations, where zoonotic or cross species transfer naturally occurs. Concerns about viral immune escape intersect at a specific point; a gained evolutionary ability of several viruses to co–infect and compete against previous scientific advances since 1796 that remain undetected or asymptomatic during the early stages of infection progressing to symptomatic and severe disease via the double methylation of the 5' end of eukaryotic DNA or RNA-based viral genomes, the 7-MeGpppA2’-O-Me cap, and its double methylation capping process is performed by the activated viral 2’ - O - Methyltransferase (MTase) enzyme, a complex of two viral non-structural proteins (NSPs) joined together through an activation process (NSP10/16) and by N7-Methyltransferase (N7-MTase/NSP14), respectively. Moreover, it was discovered that polymorphic viruses translate NSP1, which prevents the activation of various Pattern Recognition Receptors (PRRs), and consequently, detection of Pathogen-Associated Molecular Patterns (PAMPs) and Damage-Associated Molecular Patterns (DAMPs) alike. NSP1 also silences important interferon-encoding genes (INGs) and interferon-stimulated genes (ISGs), is signalled in a paracrine manner to neighbouring cells, and that induces the apoptosis of host cells, inducing an effect of “trace erase” effect and making the viral infection as immunologically “invisible” as possible during the initial, key stages of viral replication and distribution, all such mechanisms occurring independently of the viruses in cause. Another important viral NSP is NSP14, as it plays two functional roles that are independent of each other; to produce new viral genetic material for the purpose of maintaining the validity of the viral genome as well, and not just transfer a methyl group to the 5’ end of the viral genome. Other viral NSPs share a role with NSP1, 10, 14 and 16 in directly suppressing the activation of PRRs and ISGs, and all such viral proteins help the virus in its process of self-camouflaging against first- and second-line immunity, thereby often severely impacting the quality of the produced adaptive immune responses. The outcome of all such phenomena is the sharp decrease in the host Type I and Type III interferons' (IFNs) rate of synthesis by the host cells, that would usually occur and affect homeostatic cellular pathways, resulting in further viral replication and induced apoptosis. Nonetheless, effects of microbial immune evasion during the development of other viral or carcinogenic pathologies are not widely known. In short, polymorphic viruses developed a proportionate evolutionary response against developed adaptive immune responses, by currently relying on gaps mostly situated in the natural immune system in their process of molecular self-camouflaging. Scientists developed numerous approaches of early treatment that generally showed good success rates and fewer risks of adverse events, and the still early present stages of COVID-19 research should also be taken into consideration whilst filtering for the most appropriate solutions. For example, the administration of recombinant human interferons I and III into the nasal mucosa cellular layer, as key mediators of anti–viral activity, can simulate intracellular infection and stimulate cellular activity in a timely manner, training the innate and adaptive immune system cells to develop and appropriately stimulate an adequate immune response through B and T cells. Another example could involve the treatment of natural and adaptive lymphocytes with a low dose of IFNs I and possibly III, prior to their insertion into the host lymphatic system, possibly alongside additional recruitment of plasmacytoid dendritic cells (pDCs) as further interferon “factories”, all with the purpose of early infection management. It might be that focusing on directly offering the immune system the information about the genetics and protein structure of the pathogen, rather than training its first-line mechanisms to develop faster, excessively increases its specificity, making it reach a level that brings the virus the opportunity to evolve and escape previously-developed host immune mechanisms. With regards to efforts to delay the onset of malignant diseases, approaches of chrono-biological oncotherapies that include a combination of Type I and Type III Interferon-based “immune re-awakening” and low-dose SSRI or SNRI approaches, could display meaningful extents of efficacy, at least in effective delays in the onset of malignant diseases. Such overall approaches could also be considerably effective in efforts to delay and/or even prevent a number of acquired immunodeficiencies (i.e. HIV-1-induced AIDS) and diverse forms of malignant cancer, potentially helping to notably decrease the overall burden of disease worldwide in the long run. It is until the scientific community realises this potentially crucial aspect that large proportions of the world population will probably continue to face serious epidemics and pandemics of respiratory diseases over the coming several decades, evidenced with dengue fever and more recently, monkeypox and possibly avian flu. Of note, it has been indicated that IFN I and / or III display significant immunising, early therapeutic and clinical disease onset-attenuating effects for many other microbial evoked diseases, as well as for a number of oncological diseases. Microbial agents could undergo loss-of-function research upon genes responsible for inducing clinical illness whilst keeping genes responsible for microbial reproduction and transmission at least generally as functional, CRISPR-Cas9 genome editing to have genes encoding proteins suppressive of the host interferon system eliminated prior to human genes encoding Pattern Recognition Receptor activator or agonist proteins, such as outer membrane proteins of Neisseria meningitidis, as well as Type I, Type III and possibly even Type IV Interferons and various ISGs inserted into the microbial genome. Importantly, the present study is theoretical and conceptual in nature and does not advocate for any practical steps or deployment into any real-world context. Such an approach is imagined as a potential prophylactic and early therapeutic method based upon the model of editing genes of harmless bacteria to transform such them into “producers” and “distributors” of human insulin, and could turn several microbial agents into clinically harmless, transmissible “factories” for various key elements of the host interferon system, potentially placing such microbes into a reverse evolutionary path that would be deemed as “natural de-selection”, visibly reducing the average burden of disease and metabolic stresses, which in turn could gradually increase average human and animal lifespans worldwide.

Abstract Prostate adenocarcinoma (PRAD) has become one of the most prevalent malignancies in men worldwide. Although advances in diagnostic techniques and targeted therapies have improved clinical outcomes, a significant subset of patients still develops aggressive, metastatic disease with limited treatment options. At present, identifying reliable biomarkers for early risk stratification remains a critical unmet need in prostate cancer research.This study established a novel genetic signature through multi-omics integration to enhance risk stratification and identify therapeutic targets.Integrated analysis of CRISPR-Cas9 screening and bulk RNA sequencing data identified tumor-dependent genes essential for PRAD cell viability.Pathway enrichment analysis demonstrated critical involvement of these genes in cell cycle regulation and tumor progression.An 11-gene prognostic signature was constructed via LASSO regression, effectively stratifying patients into distinct low-risk and high-risk cohorts.The high-risk cohort demonstrated significantly reduced progression-free survival (PFS; log-rank p < 0.001).Among the hub genes identified, CDC45 exhibited marked upregulation in tumor tissues, which was confirmed by both Western blotting and qRT-PCR. Subsequent functional interrogation established CDC45 as a key driver of tumor proliferation. Collectively, this study not only establishes a robust prognostic signature for PRAD but also nominates CDC45 as a promising therapeutic target, thereby advancing precision oncology strategies.

Abstract The artificially created Liriodendron hybrids, generated through the crossbreeding of L. chinense and L. tulipifera , exhibit enhanced stress resilience and superior growth characteristics. Somatic embryogenesis is a developmental process in which somatic cells undergo dedifferentiation to form embryogenic cells, which subsequently develop into entire plants under controlled conditions. In this study, the conserved transcription factor gene LhAHL15 was successfully cloned, and overexpression as well as CRISPR/Cas9 vectors were constructed to investigate its role in somatic embryogenesis. Subcellular localization analysis revealed that the LhAHL15 protein is localized within the nucleus, suggesting its function as a transcriptional regulator involved in the regulation of plant growth and development. Further examination of the spatiotemporal expression profile of the LhAHL15 promoter demonstrated that transcription of LhAHL15 is initiated during the early stages of somatic embryogenesis and remains active throughout the formation of spherical, heart-shaped, torpedo-shaped, and cotyledonary embryos. These results underscore the importance of LhAHL15 as a key transcriptional regulator in somatic embryogenesis. Notably, overexpression of LhAHL15 in Liriodendron hybrids significantly upregulates the expression of critical genes associated with somatic embryogenesis, including BBM , LEC1 , PIN1 , PLT2 , ARR7 , and ARF12 . This upregulation markedly improved the efficiency of somatic embryogenesis and the induction of embryogenic callus. Flow cytometry analysis revealed that the chromosome number of the AHL15-OE line was approximately twice that of the control, indicating a polyploid state. These findings suggest that overexpression of LhAHL15 may induce chromosome doubling in plants. In summary, our study reveals the pivotal role of LhAHL15 in promoting somatic embryogenesis and inducing polyploidy. This advancement effectively addresses the technical limitations of traditional polyploid breeding approaches, thereby establishing a solid theoretical foundation and providing technical support for the improvement and utilization of Liriodendron hybrids.

Abstract Background: The CD40–TRAF6 signaling axis regulates dendritic cell activation through tightly controlled NF-κB dynamics. The signal amplitude and duration are governed by intracellular negative feedback mechanisms, particularly SOCS1-mediated attenuation. Understanding how structural clustering and genetic perturbations reshape these dynamics requires mechanistic modeling. Methods: We developed the CD40-Immunosome, an interactive systems immunology framework integrating a three-variable ordinary differential equation (ODE) model of TRAF6, NF-κB, and SOCS1 kinetics. The platform implements (i) null-model comparison without feedback, (ii) stochastic Monte Carlo robustness analysis under ±20% parameter perturbations, and (iii) quantitative synergy scoring using area-under the-curve (AUC) metrics. Results: The feedback-enabled model produced transient NF-κB activation, followed by rapid attenuation, whereas the null model exhibited sustained activation consistent with chronic inflammatory dynamics. Monte Carlo simulations (n = 50) confirmed robustness to parameter perturbation (mean peak NF-κB = 1.59 ± 0.27 SD). Simulated SOCS1 deletion increased the cumulative signaling output by ∼ 3.2-fold relative to that of wild-type cells. Furthermore, structural stability simulations prioritized TMEM256 as a candidate modulator under modeled conditions. Conclusion: CD40-Immunosome provides a reproducible computational framework for exploring feedback-regulated immune signaling dynamics. The platform serves as a hypothesis-generating framework to support the mechanistic exploration of receptor clustering, structural perturbations, and targeted immunotherapies.

Abstract Precise targeting of malignant cells remains a central objective in oncology. We developed a programmable intracellular kill-switch that links oncogenic RNA recognition to activation of a lytic effector pathway. The system repurposes a Type III-E CRISPR-associated RNA-activated protease to detect mutation-specific transcripts and trigger cleavage of engineered gasdermin constructs. Activation of the protease liberates the pore-forming gasdermin domain, resulting in membrane permeabilization and cell death. The circuit was assembled and evaluated in Saccharomyces cerevisiae as a eukaryotic model platform. Inducible expression of the full system demonstrated trigger-dependent proteolysis and selective loss of viability. Control strains lacking the target RNA or expressing cleavage-resistant gasdermin variants remained unaffected, confirming mechanistic specificity. These findings establish a modular RNA-responsive cytotoxic framework that operates independently of genome editing and can be retargeted through guide RNA redesign. The work supports further investigation of CRISPR-guided proteases as precision therapeutic tools for transcript-specific cancer targeting.