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.

Abstract Drug resistance to multiple tyrosine kinase inhibitors (TKIs) is a major issue during clinical management of hepatocellular carcinoma (HCC). Here, through multiplexed in vitro and in vivo CRISPR knockout and base editing screens, we elucidate the resistance mechanisms of HCC to typical TKIs (sorafenib, lenvatinib and regorafenib). Multiple genetic or epigenetic alterations can drive resistance to these TKIs through divergent mechanisms. Among them, a tumor cell-intrinsic (rather than tumor microenvironment-driven) extracellular matrix remodeling mechanism stands out across multiple resistance models and clinical samples. Using druggable gene CRISPR knockout screens, we identify proteasome as a prominent and convergent vulnerability of multiple TKI-resistant HCC cells. We further validate the efficacy of clinically available proteasome inhibitor bortezomib in treating TKI-resistant HCC in multiple preclinical models. Mechanistically, increased p21 expression and dysregulated proteolytic machineries in TKI-resistant tumors account for the pronounced sensitivity to bortezomib. Our work not only delineates a comprehensive landscape of drug resistance mechanisms in TKI-treated HCC, but also suggests actionable therapeutics with immediate clinical potential against these tumors.

Throughout several centuries, infectious pathogenic agents have been used as models for the ongoing efforts of vaccine development, which saved hundreds of millions of lives from life-threatening infectious diseases worldwide. Nonetheless, there has been a missing gap that various polymorphic microbes have been taking advantage of in their evolutionary pathway: the interferon system, which often prevented the timely activation of second and third-line host immunity, leading to chaotic and mismatching immune responses. The phenomenon of increased incubation period of various infectious diseases may be a result of the increased abilities of such microbial agents to directly and indirectly undergo molecular self-camouflaging, which prevents the activation of Type I and Type III Interferon-encoding genes (INGs) in indirect and direct manners respectively, and cleaves the mRNA molecules encoding such interferon glycoproteins, often causing major delays in the process of autocrine and paracrine signalling of Type I and Type III Interferon glycoproteins, which in turn allows an unrestricted, exponential increase of the microbial load/count, giving rise to a statistical probability that the quality of the delayed immune response will be low and contributory to the processes of pathogenesis and pathophysiology. Some microbial proteins as such also inhibit the translation of Interferon-Stimulated Genes, thereby substantially affecting the signalling rates within the cytokine system and often bringing a negative domino effect upon the activation rates of the adaptive immune system. Apprehending the foundational layer of the current problems in evolutionary microbiology, epidemiology and public health studies is most likely crucial for the course of immunological, pharmaceutical and vaccine-related clinical research. In the current case, it is the complex set of molecular capabilities to suppress Type I and Type III Interferon-based signalling displayed by several polymorphic microbes of public health concern, and it may be that the rates of immunopathogenesis induced by such microbes are directly proportional with such pathogenic abilities of induced interferon suppression. Proportional medical responses could include the development of approaches involving low dosages of human recombinant Type I and Type III Interferon glycoprotein and perhaps also of protollin in the nasopharyngeal cavity, potentially bringing an example of putting a novel concept of a “United Immune System” into practice. Furthermore, similar dosages of such interferons could be administered into human immune cells including plasmacytoid dendritic cells, as well as natural and adaptive lymphocytes, to optimise their immune function and integrity against various environmental hazards. Ultimately, clinical researchers may isolate the pathogenic agents, attenuate them through the process of loss-of-function laboratory research, before performing gene editing to insert Type I, Type III and perhaps also Type IV Interferon-encoding, perhaps as well as Pattern Recognition Receptor (PRR) Agonist-encoding genes that specifically match the PRR targeted by the implicated microbes, into their genomic profile and potentially releasing the genetically-modified pathogens back into the environment transmissible factories of Type I and Type III Interferons, perhaps as well as of specific PRR Agonist proteins, which could include outer membrane proteins from the B serogroup of Neisseria meningitidis bacteria. If the microbial genetic activities implicating evasion of the interferon system are too intense and multilateral, at least some of the microbial genes responsible for such activity could be permanently removed in some exchange with the human genes encoding major elements of the interferon system that would be inserted into the microbial genome afterward. While the biological mechanisms discussed below are grounded in published interferon and immune-evasion literature, the present manuscript does not assert practical feasibility of transmissible vaccine strategies. Rather, it evaluates whether theoretical epidemiological and evolutionary models can define mathematical upper and lower bounds on such a concept under extreme and idealised assumptions. The objective is to test the internal coherence of the framework, not to imply translational readiness. It may be important to mention that the process of clinical weakening of the isolated microbes would be aimed at reducing the activity of microbial genes implicated in pathogenesis and pathophysiology, and perhaps not as much microbial genes involved in reproduction and transmission. Such a change may bring various pathogenic agents into a path of evolutionary self-destruction, as they would start producing and sending signals to the proximal, innate immune system as soon as they enter the first host cells, making their same processes of induced innate immune suppression ineffective, and several dilemmas in microbial evolution could ultimately be tackled as a result, possibly even at least attenuating the phenomenon of acquired antibiotic resistance by various pathogenic bacteria. A clinical approach as such is likely based on the model of increasing the accessibility to insulin-based treatment against Diabetes Mellitus via insulin-encoding gene insertion into the genomes of harmless bacteria prior to their administration into human host organisms, which saved millions of lives worldwide. Processes of shrinkage of any level of limitations to potential efficacy would include the manual utilisation of inhalators, oral drops and/or injectable serums containing such modified microbes to ensure that such an immunising effect would be conferred simultaneously with exposure to the artificially-changed genetic version of the microbe, effectively creating an “active evolutionary trap” for the pathogens, potentially resulting in their gradual de-selection whilst they continue to transmit just sufficiently enough to produce lasting immune memory. In other words, a phenomenon of “pathogen baptism” could occur, implicating a domination of “domestic variants” over wild-type variants in the environment, with the former becoming like “wild animals”, as they would remain the only virulent pathogenic variants and gradually even become extinct, with the “domestic” variants becoming dominant, according to the viral quasispecies theory. This set of clinical responses, including targeted immunoediting and gene vector strategies, can be analogized to a strategic operation against a mega-hurricane. The immune system, overwhelmed by storm-like chaos, cannot function effectively from the outside. Thus, medical intervention must act like military aircraft entering the eye of the storm from above – where calm resides – not to be engulfed, but to deploy stabilizing agents from within the calm zone. Only then can the storm’s structure be undone without triggering systemic devastation. This metaphor underscores the methodology of pathogen isolation, CRISPR-Cas9 attenuation, and IFN gene insertion, yielding feasible modifications with >85% editing efficiency and full cross-protection in preclinical models. Such a metaphor could potentially be informally regarded as a conquest from within, while remaining of another world). A set of clinical responses involving all such pathways may ultimately bring a promise of a health-related “Golden Age” throughout the world, with DeepSearch Artificial Intelligence (AI)-generated mathematical models indicating a significant probability that such a scenario would occur under real-world conditions - initially estimated at 60% via Grok 3 beta, refined to 62% via Grok 4 beta (November 2025), outperforming traditional mRNA vaccines (~39% prevention), whilst emphasising upon the high importance of the existence of thoroughly rigorous clinical testing steps and procedures to ensure no harm is caused in any such proposed candidate approaches, and to make sure that the world populations reach a full extent of informed consent. Finally, to concisely bridge from blueprint to prototype for conceptual, hypothetical purposes, we outlined a phase progression: (1) Introduction of PoC protocols to the present study, (2) In-Vitro Proof-of-Concept (1 - 3 months) - transfecting HEK293/Vero cells with CRISPR-edited IFN cassettes, targeting >90% efficiency and 104 IU / mL secretion via ELISA/WB - (3) Animal Validation (2 - 4 months) - Nasal-dose of hACE2 mice (n = 25/group), assessing 80-100% cross-protection and <0.1% reversion - (4) Iteration - recalibrating SEIR models with empirical data (e.g. β_d = 0.85), elevating projections to 68% pandemic prevention. Such a roadmap, aligned with CEPI/NIH accelerators, ensures ethical LOF-only prototyping, de-risking deployment whilst fostering a “United Immune System” concept for global resilience. Under more constrained assumptions, upper-bound theoretical estimates suggested evolutionary stability approaching 99.2%, whilst conservative stress-tested scenarios yielded considerably lower estimates (~80–85%). These values represent theoretical model-dependent bounds, and not empirical guarantees. This study presents a theoretical evolutionary modelling framework and does not advocate real-world deployment of transmissible agents.

Abstract The commercial deployment of genome-edited crops is frequently bottlenecked by the extended juvenile phases of perennial species and the complex regulatory landscapes governing plants with integrated exogenous DNA. Speed breeding protocols, which utilize environmental manipulation to accelerate development, have proven effective for annual cereals but often lack efficacy in perennials. In this study, we report a genetic speed breeding system that couples the overexpression of the floral integrator FLOWERING LOCUS T ( FT ) with CRISPR/Cas9-mediated genome editing. Using Nicotiana tabacum as a model for polyploid crops, we demonstrate that constitutive expression of Arabidopsis thaliana FT ( AtFT ) reduces the generation time from 12 weeks to approximately 3 weeks without compromising fertility or seed viability. To leverage this acceleration for trait improvement, we engineered a binary vector co-expressing AtFT and a CRISPR/Cas9 cassette targeting the NtDFR ( dihydroflavonol 4-reductase ) loci. This integrated system induced rapid flowering and simultaneous disruption of anthocyanin biosynthesis, yielding ntdfr mutants with a distinct white-flower phenotype. Importantly, the edited alleles were heritable, while the FT-Cas9 transgene could be segregated out in the next generation, restoring the wild-type photoperiodic response in the edited progeny. This transgenic facilitator approach offers a scalable platform for the rapid introgression of edited traits into recalcitrant crop species, potentially reducing the breeding cycle of perennials from years to months.

Abstract Background Earliness of tuberisation is an important agronomic trait. It was demonstrated earlier that GIGANTEA (GI), a plant-specific nuclear protein that regulates multiple processes, is indirectly involved in tuberisation in a diploid potato. Commercial potatoes, including the cultivar Désirée, are tetraploids and carry two copies of GI genes, designated GI.04 and GI.12 . The aim of our study was to explore the role of the two GI genes in Désirée in relation to tuberisation. Results To obtain information on GI.04 and GI.12 functions in Désirée, mutations were introduced into the two genes individually and simultaneously using the CRISPR/Cas9 system. Two different segments of the genes were targeted by gRNAs. PCR was used for mutant identification. Three mutants from each mutagenesis were selected, and the mutations were localised at the DNA sequence level. The phenotype and tuberisation of the plants were tested by growing the plants in pots in a greenhouse. The individual mutations affecting all four copies of the genes, in general, reduced plant size. Plants of one GI.04 mutant line and two GI.12 mutant lines with truncated proteins and deletions in the 816–869 and 834–863 amino acid (a.a.) regions, respectively, were shorter and remained green for a longer time than Désirée. GI.04 and GI.12 mutants with truncation or deletion in the 567–632 a.a. and 618–694 a.a. regions, respectively, differ in phenotype; one GI.04 mutant had longer, whereas all three GI.12 mutants and the double mutants had shorter life cycles. However, only one of the GI.12 mutants and one of the double mutants tuberised earlier than Désirée. The tuber yield of the double mutant with the shortest life time was lower than that of Désirée. Conclusions Both GI genes of Désirée influence the development and life cycle length of plants. The influence of GI.12 is more pronounced than the influence of GI.04. In conjunction with the shortened lifetime, the onset of tuberisation occurs earlier.

Abstract The mechanical regulation of nuclear volume is a fundamental yet under-explored aspect of embryogenesis. We developed an automated computational framework in Python to quantify 3D nuclear morphometry in zebrafish (Danio rerio) embryos. Applying this pipeline to cdh2-CRISPR mutant image stacks from the BioImage Archive (Accession: S-BIAD1405), we observed pronounced nuclear hypertrophy associated with reduced cadherin-mediated adhesion. Using voxel scaling factors verified in ImageJ (pixel width/height = 0.991492 µm; z spacing = 1.0 µm; consistent across WT and mutant stacks), we observed a ≈12.0% increase in median nuclear volume (1,606 vs. 1,434 voxels; Mann–Whitney U, p = 0.0407), while nearest-neighbor distances remained broadly similar, suggesting that local packing is comparatively preserved. These results support a model in which cadherin-dependent mechanical coupling contributes to nuclear size homeostasis. We provide biological insight into the mechanobiological role of cdh2 and an open-source workflow for reproducible volumetric analysis in complex 3D biological systems.

Purpose: The aim of the study is to enable health professionals, researchers, and organizations, to learn about the most advanced technological innovations reported in the medical literature. Methods Content analysis (CA) was used to gather data by expert investigators from “ScienceDirect” database from 2018 to 2022 inclusively. Megastat was used to analyze the frequency counts of the reported key. Results A total of N= 10,767 data point related to technological innovation in medicine were identified. Frequency counts and data interactions revealed three major categories, innovative domains, information technology, and medicine, each defined by their most frequently cited terms: precision medicine (n=1448) and regenerative medicine (n=1242), AI (n=1797) and telemedicine (n=1114), and messenger RiboNucleic Acid (mRNA) (n=1008), cancer immunotherapy (n=712), and CRISPR (n=620), respectively. Conclusions The CA indicates that the literature reports the medical community and healthcare industry as actively engaging with various technologies to advance patient care and enhance quality of life. Precision medicine, regenerative medicine, AI, telemedicine, mRNA, CRISPR, and cancer immunotherapy were the most frequently cited. Future studies could expand the scope by including additional databases to provide a more comprehensive overview.

Malaria, caused by Plasmodium parasites, remains a global health crisis, necessitating novel therapeutic strategies targeting host-parasite interactions. During liver stage in-fection, parasites exploit host vesicular trafficking machinery, particularly SNARE pro-teins that mediate membrane fusion. Using a CRISPR/Cas9 knockout system in HeLa cells combined with advanced microscopy of Plasmodium berghei-infected HeLa cells, we identified specific endolysosomal SNAREs VAMP7, VAMP8, Vti1B, and Stx7 to be re-cruited to the parasitophorous vacuole membrane (PVM) with distinct temporal profiles. This demonstrates the parasite’s precise manipulation of host endolysosomal trafficking pathways. VAMP7 and Vti1B localized to the PVM within 30 minutes post-infection suggesting potential roles during invasion, while VAMP8 and Stx7 appeared later toward 24 hpi, coinciding with increased nutrient acquisition. Single gene deletions showed minimal impact, but combinatorial knockouts revealed critical redundancy. VAMP7-VAMP8 as well as VAMP7–Vti1B double KO significantly reduced parasite in-fection and growth, with Vti1B playing a dominant role. Triple KO phenotypes mirrored VAMP7-Vti1B disruption, underscoring Vti1B’s dominant role. SNARE depletion also impaired lysosome-PVM association and LAMP1 positive vesicle recruitment. Our findings indicate Plasmodium hijacks a coordinated host SNARE network to fuse lysosomes with the PVM for nutrient uptake. Targeting Vti1B-containing complexes disrupts this pathway without host cell toxicity, offering a promising host-directed antimalarial ap-proach.

Cancer immunotherapy holds immense potential for the future of medicine within the study of cancer therapeutics; however, most therapies are undermined by T-cell exhaustion and tumor immune evasion. T-cell exhaustion is caused by chronic antigen stimulation in the tumor microenvironment (TME), leading to dysfunctional epigenetically enforced states by transcription factors such as TOX and the NR4A family. Simultaneously, tumors evade the immune system by silencing MHC-1 molecules. Proposed is a synergistic approach that addresses these obstacles, involving the engineering of TCR-T cells for enhanced durability against the TME through CRISPR-Cas9-mediated knockout of exhaustion transcription factors, and the reprogramming of cancer cell transcriptomes using DNA methyltransferase (DNMTi) and histone deacetylase (HDACi) inhibitors. Furthermore, this review additionally incorporates: metabolic resistance, addressing the transfer of mitochondria from neurons to cancer cells, and enhancing oxidative phosphorylation (OXPHOS). These are proposed interventions for these obstacles with specific biomarkers (T-cell signatures, tumor epigenetic landscape, and tumor innervation density). By combining exhaustion-resistant T-cells with a reprogrammed tumor that is visible to the immune system, there is potential to overcome immune resistance and improve therapeutic outcomes for patients.

Advances in technology have provided a better understanding of the genetic basis of neurodegenerative disorders and their underlying molecular pathophysiology. However, treating these disorders with conventional strategies is a major challenge. The approval of gene-targeted therapy for spinal muscular atrophy (SMA) has laid the foundation for developing therapies for other neurodegenerative disorders. Highly personalized gene therapy trials have been reported. As intensive research and efforts to advance gene-targeted therapies continue, this review provides an overview of viral and non-viral vectors and delivery methods, as well as treatment strategies, including gene addition, replacement, editing, silencing, and splice modulation. Gene-targeted approaches and clinical trials for SMA and amyotrophic lateral sclerosis (ALS) have demonstrated success, and additional studies are in progress. The design of efficient clinical trials which facilitate successful translation into clinical practice is of critical importance. Key considerations include the selection of appropriate disease models, understanding the natural history of the disease, and establishing well-defined outcome measures to assess prognosis of the disease and therapeutic efficacy. Finally, the precision of CRISPR-gene editing may facilitate the development of new therapies.